Joseph C. Wu

All Publications

• Autologous iPSC-Based Vaccines Elicit Anti-tumor Responses In Vivo. Cell stem cell Kooreman, N. G., Kim, Y., de Almeida, P. E., Termglinchan, V., Diecke, S., Shao, N. Y., Wei, T. T., Yi, H., Dey, D., Nelakanti, R., Brouwer, T. P., Paik, D. T., Sagiv-Barfi, I., Han, A., Quax, P. H., Hamming, J. F., Levy, R., Davis, M. M., Wu, J. C. 2018

  Abstract

  Cancer cells and embryonic tissues share a number of cellular and molecular properties, suggesting that induced pluripotent stem cells (iPSCs) may be harnessed to elicit anti-tumor responses in cancer vaccines. RNA sequencing revealed that human and murine iPSCs express tumor-associated antigens, and we show here a proof of principle for using irradiated iPSCs in autologous anti-tumor vaccines. In a prophylactic setting, iPSC vaccines prevent tumor growth in syngeneic murine breast cancer, mesothelioma, and melanoma models. As an adjuvant, the iPSC vaccine inhibited melanoma recurrence at the resection site and reduced metastatic tumor load, which was associated with fewer Th17 cells and increased CD11b+GR1himyeloid cells. Adoptive transfer of T cells isolated from vaccine-treated tumor-bearing mice inhibited tumor growth in unvaccinated recipients, indicating that the iPSC vaccine promotes an antigen-specific anti-tumor T cell response. Our data suggest an easy, generalizable strategy for multiple types of cancer that could prove highly valuable in clinical immunotherapy.

  View details for PubMedID 29456158

• SETD7 Drives Cardiac Lineage Commitment through Stage-Specific Transcriptional Activation. Cell stem cell Lee, J., Shao, N. Y., Paik, D. T., Wu, H., Guo, H., Termglinchan, V., Churko, J. M., Kim, Y., Kitani, T., Zhao, M. T., Zhang, Y., Wilson, K. D., Karakikes, I., Snyder, M. P., Wu, J. C. 2018; 22 (3): 428–44.e5

  Abstract

  Cardiac development requires coordinated and large-scale rearrangements of the epigenome. The roles and precise mechanisms through which specific epigenetic modifying enzymes control cardiac lineage specification, however, remain unclear. Here we show that the H3K4 methyltransferase SETD7 controls cardiac differentiation by reading H3K36 marks independently of its enzymatic activity. Through chromatin immunoprecipitation sequencing (ChIP-seq), we found that SETD7 targets distinct sets of genes to drive their stage-specific expression during cardiomyocyte differentiation. SETD7 associates with different co-factors at these stages, including SWI/SNF chromatin-remodeling factors during mesodermal formation and the transcription factor NKX2.5 in cardiac progenitors to drive their differentiation. Further analyses revealed that SETD7 binds methylated H3K36 in the bodies of its target genes to facilitate RNA polymerase II (Pol II)-dependent transcription. Moreover, abnormal SETD7 expression impairs functional attributes of terminally differentiated cardiomyocytes. Together, these results reveal how SETD7 acts at sequential steps in cardiac lineage commitment, and they provide insights into crosstalk between dynamic epigenetic marks and chromatin-modifying enzymes.

  View details for PubMedID 29499155

• Global position paper on cardiovascular regenerative medicine: Scientific statement of the transnational alliance for regenerative therapies in cardiovascular syndromes (TACTICS) international group for the comprehensive cardiovascular application of regenerative medicinal products. European heart journal Fernández-Avilés, F., Sanz-Ruiz, R., Climent, A. M., Badimon, L., Bolli, R., Charron, D., Fuster, V., Janssens, S., Kastrup, J., Kim, H., Lüscher, T. F., Martin, J. F., Menasché, P., Simari, R. D., Stone, G. W., Terzic, A., Willerson, J. T., Wu, J. C. 2017

  View details for DOI 10.1093/eurheartj/ehx248

  View details for PubMedID 28575280

• High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells. Science translational medicine Sharma, A., Burridge, P. W., McKeithan, W. L., Serrano, R., Shukla, P., Sayed, N., Churko, J. M., Kitani, T., Wu, H., Holmström, A., Matsa, E., Zhang, Y., Kumar, A., Fan, A. C., Del Álamo, J. C., Wu, S. M., Moslehi, J. J., Mercola, M., Wu, J. C. 2017; 9 (377)

  Abstract

  Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.

  View details for DOI 10.1126/scitranslmed.aaf2584

  View details for PubMedID 28202772

• Molecular and functional resemblance of differentiated cells derived from isogenic human iPSCs and SCNT-derived ESCs. Proceedings of the National Academy of Sciences of the United States of America Zhao, M. T., Chen, H., Liu, Q., Shao, N. Y., Sayed, N., Wo, H. T., Zhang, J. Z., Ong, S. G., Liu, C., Kim, Y., Yang, H., Chour, T., Ma, H., Gutierrez, N. M., Karakikes, I., Mitalipov, S., Snyder, M. P., Wu, J. C. 2017

  Abstract

  Patient-specific pluripotent stem cells (PSCs) can be generated via nuclear reprogramming by transcription factors (i.e., induced pluripotent stem cells, iPSCs) or by somatic cell nuclear transfer (SCNT). However, abnormalities and preclinical application of differentiated cells generated by different reprogramming mechanisms have yet to be evaluated. Here we investigated the molecular and functional features, and drug response of cardiomyocytes (PSC-CMs) and endothelial cells (PSC-ECs) derived from genetically relevant sets of human iPSCs, SCNT-derived embryonic stem cells (nt-ESCs), as well as in vitro fertilization embryo-derived ESCs (IVF-ESCs). We found that differentiated cells derived from isogenic iPSCs and nt-ESCs showed comparable lineage gene expression, cellular heterogeneity, physiological properties, and metabolic functions. Genome-wide transcriptome and DNA methylome analysis indicated that iPSC derivatives (iPSC-CMs and iPSC-ECs) were more similar to isogenic nt-ESC counterparts than those derived from IVF-ESCs. Although iPSCs and nt-ESCs shared the same nuclear DNA and yet carried different sources of mitochondrial DNA, CMs derived from iPSC and nt-ESCs could both recapitulate doxorubicin-induced cardiotoxicity and exhibited insignificant differences on reactive oxygen species generation in response to stress condition. We conclude that molecular and functional characteristics of differentiated cells from human PSCs are primarily attributed to the genetic compositions rather than the reprogramming mechanisms (SCNT vs. iPSCs). Therefore, human iPSCs can replace nt-ESCs as alternatives for generating patient-specific differentiated cells for disease modeling and preclinical drug testing.

  View details for PubMedID 29203658

• iPSC-derived cardiomyocytes reveal abnormal TGF-ß signalling in left ventricular non-compaction cardiomyopathy. Nature cell biology Kodo, K., Ong, S., Jahanbani, F., Termglinchan, V., Hirono, K., Inanloorahatloo, K., Ebert, A. D., Shukla, P., Abilez, O. J., Churko, J. M., Karakikes, I., Jung, G., Ichida, F., Wu, S. M., Snyder, M. P., Bernstein, D., Wu, J. C. 2016; 18 (10): 1031-1042

  Abstract

  Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and this was associated with perturbed transforming growth factor beta (TGF-β) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-β signalling. TBX20 regulates the expression of TGF-β signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-β signalling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.

  View details for DOI 10.1038/ncb3411

  View details for PubMedID 27642787

• A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy CELL Davis, J., Davis, L. C., Correll, R. N., Makarewich, C. A., Schwanekamp, J. A., Moussavi-Harami, F., Wang, D., York, A. J., Wu, H., Houser, S. R., Seidman, C. E., Seidman, J. G., Regnier, M., Metzger, J. M., Wu, J. C., Molkentin, J. D. 2016; 165 (5): 1147-1159

  Abstract

  The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

  View details for DOI 10.1016/j.cell.2016.04.002

  View details for PubMedID 27114035

• Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity NATURE MEDICINE Burridge, P. W., Li, Y. F., Matsa, E., Wu, H., Ong, S., Sharma, A., Holmstrom, A., Chang, A. C., Coronado, M. J., Ebert, A. D., Knowles, J. W., Telli, M. L., Witteles, R. M., Blau, H. M., Bernstein, D., Altman, R. B., Wu, J. C. 2016; 22 (5): 547-556

  Abstract

  Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies, but it causes a dose-related cardiotoxicity that can lead to heart failure in a subset of patients. At present, it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can recapitulate the predilection to DIC of individual patients at the cellular level. hiPSC-CMs derived from individuals with breast cancer who experienced DIC were consistently more sensitive to doxorubicin toxicity than hiPSC-CMs from patients who did not experience DIC, with decreased cell viability, impaired mitochondrial and metabolic function, impaired calcium handling, decreased antioxidant pathway activity, and increased reactive oxygen species production. Taken together, our data indicate that hiPSC-CMs are a suitable platform to identify and characterize the genetic basis and molecular mechanisms of DIC.

  View details for DOI 10.1038/nm.4087

  View details for PubMedID 27089514

• Induced pluripotent stem cells. JAMA Wilson, K. D., Wu, J. C. 2015; 313 (16): 1613-1614

  View details for DOI 10.1001/jama.2015.1846

  View details for PubMedID 25919522

• Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system SCIENCE TRANSLATIONAL MEDICINE Ebert, A. D., Kodo, K., Liang, P., Wu, H., Huber, B. C., Riegler, J., Churko, J., Lee, J., de Almeida, P., Lan, F., Diecke, S., Burridge, P. W., Gold, J. D., Mochly-Rosen, D., Wu, J. C. 2014; 6 (255)

  Abstract

  Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.

  View details for DOI 10.1126/scitranslmed.3009027

  View details for Web of Science ID 000343316800004

• Chemically defined generation of human cardiomyocytes. Nature methods Burridge, P. W., Matsa, E., Shukla, P., Lin, Z. C., Churko, J. M., Ebert, A. D., Lan, F., Diecke, S., Huber, B., Mordwinkin, N. M., Plews, J. R., Abilez, O. J., Cui, B., Gold, J. D., Wu, J. C. 2014; 11 (8): 855-860

  Abstract

  Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.

  View details for DOI 10.1038/nmeth.2999

  View details for PubMedID 24930130

• Global Epigenomic Reconfiguration During Mammalian Brain Development. Science (New York, N.Y.) Lister, R., Mukamel, E. A., Nery, J. R., Urich, M., Puddifoot, C. A., Johnson, N. D., Lucero, J., Huang, Y., Dwork, A. J., Schultz, M. D., Yu, M., Tonti-Filippini, J., Heyn, H., Hu, S., Wu, J. C., Rao, A., Esteller, M., He, C., Haghighi, F. G., Sejnowski, T. J., Behrens, M. M., Ecker, J. R. 2013

  Abstract

  DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Finally, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain, and that CG demethylation at these hmC-poised loci depends on Tet2 activity.

  View details for PubMedID 23828890

• Dyslipidaemia: In vivo genome editing of ANGPTL3: a therapy for atherosclerosis? Nature reviews. Cardiology Rhee, J., Wu, J. C. 2018; 15 (5): 259–60

  View details for PubMedID 29618844

• Omics, Big Data, and Precision Medicine in Cardiovascular Sciences. Circulation research Lau, E., Wu, J. C. 2018; 122 (9): 1165–68

  View details for PubMedID 29700063

• Applications of genetically engineered human pluripotent stem cell reporters in cardiac stem cell biology. Current opinion in biotechnology Zhang, J. Z., Guo, H., Wu, J. C. 2018; 52: 66–73

  Abstract

  The advent of human pluripotent stem cells (hPSCs) has benefited many fields, from regenerative medicine to disease modeling, with an especially profound effect in cardiac research. Coupled with other novel technologies in genome engineering, hPSCs offer a great opportunity to delineate human cardiac lineages, investigate inherited cardiovascular diseases, and assess the safety and efficacy of cell-based therapies. In this review, we provide an overview of methods for generating genetically engineered hPSC reporters and a succinct synopsis of a variety of hPSC reporters, with a particular focus on their applications in cardiac stem cell biology.

  View details for PubMedID 29579626

• Cancer therapy-induced cardiomyopathy: can human induced pluripotent stem cell modelling help prevent it? European heart journal Stack, J. P., Moslehi, J., Sayed, N., Wu, J. C. 2018

  Abstract

  Cardiotoxic effects from cancer therapy are a major cause of morbidity during cancer treatment. Unexpected toxicity can occur during treatment and/or after completion of therapy, into the time of cancer survivorship. While older drugs such as anthracyclines have well-known cardiotoxic effects, newer drugs such as tyrosine kinase inhibitors, proteasome inhibitors, and immunotherapies also can cause diverse cardiovascular and metabolic complications. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly being used as instruments for disease modelling, drug discovery, and mechanistic toxicity studies. Promising results with hiPSC-CM chemotherapy studies are raising hopes for improving cancer therapies through personalized medicine and safer drug development. Here, we review the cardiotoxicity profiles of common chemotherapeutic agents as well as efforts to model them in vitro using hiPSC-CMs.

  View details for PubMedID 29377985

• Prolonged survival of transplanted stem cells after ischaemic injury via the slow release of pro-survival peptides from a collagen matrix Nature Biomedical Engineering Lee, A. S., Inayathullah, ., Lijkwan, . A., Zhao, X., Sun, W., Park, S., Hong, W. X., Parekh, M. B., Malkovskiy, A. V., Lau, E., Qin, X., Pothineni,, . R., Sanchez-Freire, ., Kooreman, N. G., Ebert, A. D., Chan, C. K., Nguyen, P. K., Rajadas, J., Wu, J. C. 2018; 2 (2): 104–13

  Abstract

  Stem-cell-based therapies hold considerable promise for regenerative medicine. However, acute donor-cell death within several weeks after cell delivery remains a critical hurdle for clinical translation. Co-transplantation of stem cells with pro-survival factors can improve cell engraftment, but this strategy has been hampered by the typically short half-lives of the factors and by the use of Matrigel and other scaffolds that are not chemically defined. Here, we report a collagen-dendrimer biomaterial crosslinked with pro-survival peptide analogues that adheres to the extracellular matrix and slowly releases the peptides, significantly prolonging stem cell survival in mouse models of ischaemic injury. The biomaterial can serve as a generic delivery system to improve functional outcomes in cell-replacement therapy.

  View details for DOI 10.1038/s41551-018-0191-4

  View details for PubMedCentralID PMC5927627

• Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling and Precision Medicine A Scientific Statement From the American Heart Association CIRCULATION-CARDIOVASCULAR GENETICS Musunuru, K., Sheikh, F., Gupta, R. M., Houser, S. R., Maher, K. O., Milan, D. J., Terzic, A., Wu, J. C., Amer Heart Assoc Council, Council Cardiovasc Dis Young, Council Cardiovasc Stroke Nursing 2018; 11 (1)

  View details for DOI 10.1161/HCG.0000000000000043

  View details for Web of Science ID 000428988100001

• Human Induced Pluripotent Stem Cell (hiPSC)-Derived Cells to Assess Drug Cardiotoxicity: Opportunities and Problems. Annual review of pharmacology and toxicology Magdy, T., Schuldt, A. J., Wu, J. C., Bernstein, D., Burridge, P. W. 2018; 58: 83–103

  Abstract

  Billions of US dollars are invested every year by the pharmaceutical industry in drug development, with the aim of introducing new drugs that are effective and have minimal side effects. Thirty percent of in-pipeline drugs are excluded in an early phase of preclinical and clinical screening owing to cardiovascular safety concerns, and several lead molecules that pass the early safety screening make it to market but are later withdrawn owing to severe cardiac side effects. Although the current drug safety screening methodologies can identify some cardiotoxic drug candidates, they cannot accurately represent the human heart in many aspects, including genomics, transcriptomics, and patient- or population-specific cardiotoxicity. Despite some limitations, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful and evolving technology that has been shown to recapitulate many attributes of human cardiomyocytes and their drug responses. In this review, we discuss the potential impact of the inclusion of the hiPSC-CM platform in premarket candidate drug screening.

  View details for PubMedID 28992430

• Myocardial perfusion imaging: Lessons learned and work to be done-update. Journal of nuclear cardiology : official publication of the American Society of Nuclear Cardiology Iskandrian, A. E., Dilsizian, V., Garcia, E. V., Beanlands, R. S., Cerqueira, M., Soman, P., Berman, D. S., Cuocolo, A., Einstein, A. J., Morgan, C. J., Hage, F. G., Schelbert, H. R., Bax, J. J., Wu, J. C., Shaw, L. J., Sadeghi, M. M., Tamaki, N., Kaufmann, P. A., Gropler, R., Dorbala, S., Van Decker, W. 2018; 25 (1): 39–52

  Abstract

  As the second term of our commitment to Journal begins, we, the editors, would like to reflect on a few topics that have relevance today. These include prognostication and paradigm shifts; Serial testing: How to handle data? Is the change in perfusion predictive of outcome and which one? Ischemia-guided therapy: fractional flow reserve vs perfusion vs myocardial blood flow; positron emission tomography (PET) imaging using Rubidium-82 vs N-13 ammonia vs F-18 Flurpiridaz; How to differentiate microvascular disease from 3-vessel disease by PET? The imaging scene outside the United States, what are the differences and similarities? Radiation exposure; Special issues with the new cameras? Is attenuation correction needed? Are there normal databases and are these specific to each camera system? And finally, hybrid imaging with single-photon emission tomography or PET combined with computed tomography angiography or coronary calcium score. We hope these topics are of interest to our readers.

  View details for PubMedID 29110288

• Global Overview of the Transnational Alliance for Regenerative Therapies in Cardiovascular Syndromes (TACTICS) Recommendations: A Comprehensive Series of Challenges and Priorities of Cardiovascular Regenerative Medicine. Circulation research Fernández-Avilés, F., Sanz-Ruiz, R., Climent, A. M., Badimon, L., Bolli, R., Charron, D., Fuster, V., Janssens, S., Kastrup, J., Kim, H. S., Lüscher, T. F., Martin, J. F., Menasché, P., Pinto, F. J., Simari, R. D., Stone, G. W., Terzic, A., Willerson, J. T., Wu, J. C. 2018; 122 (2): 199–201

  View details for PubMedID 29348246

• Endothelial deletion of Ino80 disrupts coronary angiogenesis and causes congenital heart disease. Nature communications Rhee, S., Chung, J. I., King, D. A., D'amato, G., Paik, D. T., Duan, A., Chang, A., Nagelberg, D., Sharma, B., Jeong, Y., Diehn, M., Wu, J. C., Morrison, A. J., Red-Horse, K. 2018; 9 (1): 368

  Abstract

  During development, the formation of a mature, well-functioning heart requires transformation of the ventricular wall from a loose trabecular network into a dense compact myocardium at mid-gestation. Failure to compact is associated in humans with congenital diseases such as left ventricular non-compaction (LVNC). The mechanisms regulating myocardial compaction are however still poorly understood. Here, we show that deletion of the Ino80 chromatin remodeler in vascular endothelial cells prevents ventricular compaction in the developing mouse heart. This correlates with defective coronary vascularization, and specific deletion of Ino80 in the two major coronary progenitor tissues-sinus venosus and endocardium-causes intermediate phenotypes. In vitro, endothelial cells promote myocardial expansion independently of blood flow in an Ino80-dependent manner. Ino80 deletion increases the expression of E2F-activated genes and endothelial cell S-phase occupancy. Thus, Ino80 is essential for coronary angiogenesis and allows coronary vessels to support proper compaction of the heart wall.

  View details for PubMedID 29371594

• Comparison of Non-human Primate versus Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Treatment of Myocardial Infarction. Stem cell reports Zhao, X., Chen, H., Xiao, D., Yang, H., Itzhaki, I., Qin, X., Chour, T., Aguirre, A., Lehmann, K., Kim, Y., Shukla, P., Holmström, A., Zhang, J. Z., Zhuge, Y., Ndoye, B. C., Zhao, M., Neofytou, E., Zimmermann, W. H., Jain, M., Wu, J. C. 2018; 10 (2): 422–35

  Abstract

  Non-human primates (NHPs) can serve as a human-like model to study cell therapy using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, whether the efficacy of NHP and human iPSC-CMs is mechanistically similar remains unknown. To examine this, RNU rats received intramyocardial injection of 1 × 107NHP or human iPSC-CMs or the same number of respective fibroblasts or PBS control (n = 9-14/group) at 4 days after 60-min coronary artery occlusion-reperfusion. Cardiac function and left ventricular remodeling were similarly improved in both iPSC-CM-treated groups. To mimic the ischemic environment in the infarcted heart, both cultured NHP and human iPSC-CMs underwent 24-hr hypoxia in vitro. Both cells and media were collected, and similarities in transcriptomic as well as metabolomic profiles were noted between both groups. In conclusion, both NHP and human iPSC-CMs confer similar cardioprotection in a rodent myocardial infarction model through relatively similar mechanisms via promotion of cell survival, angiogenesis, and inhibition of hypertrophy and fibrosis.

  View details for PubMedID 29398480

• Mining Exosomal MicroRNAs from Human-Induced Pluripotent Stem Cells-Derived Cardiomyocytes for Cardiac Regeneration. Methods in molecular biology (Clifton, N.J.) Ong, S. G., Lee, W. H., Zhou, Y., Wu, J. C. 2018; 1733: 127–36

  Abstract

  Myocardial infarction is the leading cause of morbidity and mortality worldwide. Recent advances in cardiac regenerative therapy have allowed for novel modalities in replenishing the damaged myocardium. However, poor long-term engraftment and survival of transplanted cells have largely precluded effective cell replacement. As an alternative to direct cell replacement, the release of paracrine protective factors may be a more plausible effector for cardioprotection which may partially be mediated through secretion of microvesicles, or exosomes, that contribute to cell-cell communication. In this chapter, we describe the isolation of exosomes from induced pluripotent stem cells-derived cardiomyocytes for subsequent microRNA profiling for a better understanding of the biological cargo contained within exosomes.

  View details for PubMedID 29435928

• Radiolabeled Duramycin: Promising Translational Imaging of Myocardial Apoptosis. JACC. Cardiovascular imaging Wu, J. C., Qin, X., Neofytou, E. 2018

  View details for PubMedID 29454760

• Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond. Development (Cambridge, England) Liu, C., Oikonomopoulos, A., Sayed, N., Wu, J. C. 2018; 145 (5)

  Abstract

  The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimicin vivotissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single '4D multi-organ system'. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling.

  View details for PubMedID 29519889

• Induced pluripotent stem cells as a biopharmaceutical factory for extracellular vesicles. European heart journal Nishiga, M., Guo, H., Wu, J. C. 2018

  View details for PubMedID 29547885

• Cardiac Cell Cycle Activation as a Strategy to Improve iPSC-Derived Cardiomyocyte Therapy. Circulation research Rhee, J. W., Wu, J. C. 2018; 122 (1): 14–16

  View details for PubMedID 29301838

• Bioacoustic-enabled patterning of human iPSC-derived cardiomyocytes into 3D cardiac tissue BIOMATERIALS Serpooshan, V., Chen, P., Wu, H., Lee, S., Sharma, A., Hu, D. A., Venkatraman, S., Ganesan, A. V., Usta, O. B., Yarmush, M., Yang, F., Wu, J. C., Demirci, U., Wu, S. M. 2017; 131: 47-57

  Abstract

  The creation of physiologically-relevant human cardiac tissue with defined cell structure and function is essential for a wide variety of therapeutic, diagnostic, and drug screening applications. Here we report a new scalable method using Faraday waves to enable rapid aggregation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into predefined 3D constructs. At packing densities that approximate native myocardium (10(8)-10(9) cells/ml), these hiPSC-CM-derived 3D tissues demonstrate significantly improved cell viability, metabolic activity, and intercellular connection when compared to constructs with random cell distribution. Moreover, the patterned hiPSC-CMs within the constructs exhibit significantly greater levels of contractile stress, beat frequency, and contraction-relaxation rates, suggesting their improved maturation. Our results demonstrate a novel application of Faraday waves to create stem cell-derived 3D cardiac tissue that resembles the cellular architecture of a native heart tissue for diverse basic research and clinical applications.

  View details for DOI 10.1016/j.biomaterials.2017.03.037

  View details for PubMedID 28376365

• Contractile force generation by 3D hiPSC-derived cardiac tissues is enhanced by rapid establishment of cellular interconnection in matrix with muscle-mimicking stiffness BIOMATERIALS Lee, S., Serpooshan, V., Tong, X., Venkatraman, S., Lee, M., Lee, J., Chirikian, O., Wu, J. C., Wu, S. M., Yang, F. 2017; 131: 111-120

  Abstract

  Engineering 3D human cardiac tissues is of great importance for therapeutic and pharmaceutical applications. As cardiac tissue substitutes, extracellular matrix-derived hydrogels have been widely explored. However, they exhibit premature degradation and their stiffness is often orders of magnitude lower than that of native cardiac tissue. There are no reports on establishing interconnected cardiomyocytes in 3D hydrogels at physiologically-relevant cell density and matrix stiffness. Here we bioengineer human cardiac microtissues by encapsulating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in chemically-crosslinked gelatin hydrogels (1.25 × 10(8)/mL) with tunable stiffness and degradation. In comparison to the cells in high stiffness (16 kPa)/slow degrading hydrogels, hiPSC-CMs in low stiffness (2 kPa)/fast degrading and intermediate stiffness (9 kPa)/intermediate degrading hydrogels exhibit increased intercellular network formation, α-actinin and connexin-43 expression, and contraction velocity. Only the 9 kPa microtissues exhibit organized sarcomeric structure and significantly increased contractile stress. This demonstrates that muscle-mimicking stiffness together with robust cellular interconnection contributes to enhancement in sarcomeric organization and contractile function of the engineered cardiac tissue. This study highlights the importance of intercellular connectivity, physiologically-relevant cell density, and matrix stiffness to best support 3D cardiac tissue engineering.

  View details for DOI 10.1016/j.biomaterials.2017.03.039

  View details for PubMedID 28384492

• F-Fluoromaltotriose: A Second Generation PET Tracer Targeting the Maltodextrin Transporter in Bacteria. Journal of nuclear medicine Gowrishankar, G., Hardy, J., Wardak, M., Namavari, M., Reeves, R., Neofytou, E., Srinivasan, A., Wu, J., Contag, C., Gambhir, S. 2017

  Abstract

  Purpose: 6"-(18)F-fluoromaltotriose is a novel positron emission tomography (PET) tracer that can potentially be used to image and localize most bacterial infections, much like 2-deoxy-2-(18)F-fluoro-D-glucose ((18)F-FDG) has been used to image and localize many cancers. However, unlike (18)F-FDG, 6"-(18)F-fluoromaltotriose is not taken up by inflammatory lesions and appears to be specific to bacterial infections by targeting the maltodextrin transporter that is expressed in most Gram-positive and Gram-negative strains of bacteria. Materials and Methods: 6"-(18)F-fluoromaltotriose was synthesized with high radiochemical purity and evaluated in several clinically relevant bacterial strains incultures in vitro and in living mice. Results: 6"-(18)F-fluoromaltotriose was taken up in both Gram-positive and Gram-negative bacterial strains. 6"-[(18)F]-fluoromaltotriose was also able to detect Pseudomonas aeruginosa in a clinically relevant mouse model of wound infection. The utility of 6"-(18)F-fluoromaltotriose to help monitor antibiotic therapies was also evaluated in rats. Conclusion: 6"-(18)F-fluoromaltotriose is a promising new tracer that has significant diagnostic utility, with the potential to change the clinical management of patients suffering from infectious diseases of bacterial origin.

  View details for DOI 10.2967/jnumed.117.191452

  View details for PubMedID 28490473

• 3-Dimensionally Printed, Native-Like Scaffolds for Myocardial Tissue Engineering CIRCULATION RESEARCH Wnorowski, A., Wu, J. C. 2017; 120 (8): 1224-1226

  View details for DOI 10.1161/CIRCRESAHA.117.310862

  View details for PubMedID 28408446

• Patient-Specific iPSC-Derived Endothelial Cells Uncover Pathways that Protect against Pulmonary Hypertension in BMPR2 Mutation Carriers CELL STEM CELL Gu, M., Shao, N., Sa, S., Li, D., Termglinchan, V., Ameen, M., Karakikes, I., Sosa, G., Grubert, F., Lee, J., Cao, A., Taylor, S., Ma, Y., Zhao, Z., Chappell, J., Hamid, R., Austin, E. D., Gold, J. D., Wu, J. C., Snyder, M. P., Rabinovitch, M. 2017; 20 (4): 490-?

  View details for DOI 10.1016/j.stem.2016.08.019

  View details for Web of Science ID 000398350800013

• Induced Pluripotent Stem Cell Model of Pulmonary Arterial Hypertension Reveals Novel Gene Expression and Patient Specificity AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Sa, S., Gu, M., Chappe, J., Shao, N., Ameen, M., Elliott, K. A., Li, D., Grubert, F., Li, C. G., Taylor, S., Cao, A., Ma, Y., Fong, R., Nguyen, L., Wu, J. C., Snyder, M. P., Rabinovitch, M. 2017; 195 (7): 930-941

  View details for DOI 10.1164/rccm.201606-1200OC

  View details for Web of Science ID 000398017200016

• Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons SCIENTIFIC REPORTS Chuang, W., Sharma, A., Shukla, P., Li, G., Mall, M., Rajarajan, K., Abilez, O. J., Hamaguchi, R., Wu, J. C., Wernig, M., Wu, S. M. 2017; 7

  Abstract

  Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.

  View details for DOI 10.1038/srep44840

  View details for PubMedID 28327614

• Human AML-iPSCs Reacquire Leukemic Properties after Differentiation and Model Clonal Variation of Disease. Cell stem cell Chao, M. P., Gentles, A. J., Chatterjee, S., Lan, F., Reinisch, A., Corces, M. R., Xavy, S., Shen, J., Haag, D., Chanda, S., Sinha, R., Morganti, R. M., Nishimura, T., Ameen, M., Wu, H., Wernig, M., Wu, J. C., Majeti, R. 2017; 20 (3): 329-344 e7

  Abstract

  Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia in vivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.

  View details for DOI 10.1016/j.stem.2016.11.018

  View details for PubMedID 28089908

• A Comprehensive TALEN-Based Knockout Library for Generating Human Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases. Circulation research Karakikes, I., Termglinchan, V., Cepeda, D. A., Lee, J., Diecke, S., Hendel, A., Itzhaki, I., Ameen, M., Shrestha, R., Wu, H., Ma, N., Shao, N., Seeger, T., Woo, N. A., Wilson, K. D., Matsa, E., Porteus, M. H., Sebastiano, V., Wu, J. C. 2017

  Abstract

  Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome.The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro.By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development.Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

  View details for DOI 10.1161/CIRCRESAHA.116.309948

  View details for PubMedID 28246128

• Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring. Proceedings of the National Academy of Sciences of the United States of America Cao, Y., Hjort, M., Chen, H., Birey, F., Leal-Ortiz, S. A., Han, C. M., Santiago, J. G., Pasca, S. P., Wu, J. C., Melosh, N. A. 2017

  Abstract

  Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

  View details for DOI 10.1073/pnas.1615375114

  View details for PubMedID 28223521

• Induced pluripotent stem cell technology: a decade of progress NATURE REVIEWS DRUG DISCOVERY Shi, Y., Inoue, H., Wu, J. C., Yamanaka, S. 2017; 16 (2): 115-130

  Abstract

  Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, enormous progress has been made in stem cell biology and regenerative medicine. Human iPSCs have been widely used for disease modelling, drug discovery and cell therapy development. Novel pathological mechanisms have been elucidated, new drugs originating from iPSC screens are in the pipeline and the first clinical trial using human iPSC-derived products has been initiated. In particular, the combination of human iPSC technology with recent developments in gene editing and 3D organoids makes iPSC-based platforms even more powerful in each area of their application, including precision medicine. In this Review, we discuss the progress in applications of iPSC technology that are particularly relevant to drug discovery and regenerative medicine, and consider the remaining challenges and the emerging opportunities in the field.

  View details for DOI 10.1038/nrd.2016.245

  View details for Web of Science ID 000393446800018

  View details for PubMedID 27980341

• Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro. Methods in molecular biology (Clifton, N.J.) Termglinchan, V., Seeger, T., Chen, C., Wu, J. C., Karakikes, I. 2017; 1521: 55-68

  Abstract

  Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

  View details for PubMedID 27910041

• Multiscale technologies for treatment of ischemic cardiomyopathy Nature Nanotechnology Mahmoudi, M., Yu, M., Serpooshan, V., Wu, J. C., Langer, R., Lee, R. T., Karp, J. M., Farokhzad, O. C. 2017; 12: 845–855

  View details for DOI 10.1038/nnano.2017.167

• Imaging Cardiac Stem Cell Therapy Cardiac Regeneration Qin, X., Chen, I. Y., Wu, J. C. Springer. 2017: 241–258

  View details for DOI 10.1007/978-3-319-56106-6_11

• Incremental value of right heart metrics and exercise performance to well-validated risk scores in dilated cardiomyopathy European Heart Journal - Cardiovascular Imaging Moneghetti , K. J., Giraldeau, G., Wheeler, M. T., Kobayashi , Y., Vrtovec, B., Boulate, D., Kuznetsova, T., Schnittger, I., Wu, J. C., Myers, J., Ashely, E., Haddad , F. 2017

  View details for DOI 10.1093/ehjci/jex187

• Photoacoustic Imaging of Embryonic Stem Cell-Derived Cardiomyocytes in Living Hearts with Ultrasensitive Semiconducting Polymer Nanoparticles Advanced Functional Materials Qin, X., Chen, H., Yang, H., Wu, H., Zhao, X., Wang, H., Chour, T., Neofytou, E., Ding, D., Daldrup-Link, H., Heilshorn, S. C., Li, K., Wu, J. C. 2017

  View details for DOI 10.1002/adfm.201704939

• External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas. Stem cells (Dayton, Ohio) Lee, A. S., Tang, C., Hong, W. X., Park, S., Bazalova, M., Nelson, G., Sanchez-Freire, V., Bakerman, I., Zhang, W., Neofytou, E., Connolly, A. J., Chan, C. K., Graves, E. E., Weissman, I. L., Nguyen, P. K., Wu, J. C. 2017

  Abstract

  Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present the evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by utilizing a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced re-seeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1x10(3) higher doses to form new teratomas. We demonstrate that radiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. This article is protected by copyright. All rights reserved.

  View details for PubMedID 28600830

• Patient-Specific Induced Pluripotent Stem Cell-Based Disease Model for Pathogenesis Studies and Clinical Pharmacotherapy. Circulation. Arrhythmia and electrophysiology Li, Y., Sallam, K., Schwartz, P. J., Wu, J. C. 2017; 10 (6)

  View details for PubMedID 28630175

• Autoantibody profiling on a plasmonic nano-gold chip for the early detection of hypertensive heart disease. Proceedings of the National Academy of Sciences of the United States of America Li, X., Kuznetsova, T., Cauwenberghs, N., Wheeler, M., Maecker, H., Wu, J. C., Haddad, F., Dai, H. 2017; 114 (27): 7089–94

  Abstract

  The role of autoimmunity in cardiovascular (CV) diseases has been increasingly recognized. Autoimmunity is most commonly examined by the levels of circulating autoantibodies in clinical practices. Measurement of autoantibodies remains, however, challenging because of the deficiency of reproducible, sensitive, and standardized assays. The lack of multiplexed assays also limits the potential to identify a CV-specific autoantibody profile. To overcome these challenges, we developed a nanotechnology-based plasmonic gold chip for autoantibody profiling. This approach allowed simultaneous detection of 10 CV autoantibodies targeting the structural myocardial proteins, the neurohormonal regulatory proteins, the vascular proteins, and the proteins associated with apoptosis and coagulation. Autoantibodies were measured in four groups of participants across the continuum of hypertensive heart diseases. We observed higher levels of all 10 CV autoantibodies in hypertensive subjects (n= 77) compared with healthy participants (n= 30), and the autoantibodies investigated were related to each other, forming a highly linked network. In addition, we established that autoantibodies to troponin I, annexin-A5, and beta 1-adrenegic receptor best discriminated hypertensive subjects with adverse left ventricular (LV) remodeling or dysfunction (n= 49) from hypertensive subjects with normal LV structure and function (n= 28). By further linking these three significant CV autoantibodies to the innate and growth factors, we revealed a positive but weak association between autoantibodies to troponin I and proinflammatory cytokine IL-18. Overall, we demonstrated that this platform can be used to evaluate autoantibody profiles in hypertensive subjects at risk for heart failure.

  View details for PubMedID 28630342

• Genome-Wide Temporal Profiling of Transcriptome and Open Chromatin of Early Cardiomyocyte Differentiation Derived From hiPSCs and hESCs. Circulation research Liu, Q., Jiang, C., Xu, J., Zhao, M. T., Van Bortle, K., Cheng, X., Wang, G., Chang, H. Y., Wu, J. C., Snyder, M. P. 2017; 121 (4): 376–91

  Abstract

  Recent advances have improved our ability to generate cardiomyocytes from human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). However, our understanding of the transcriptional regulatory networks underlying early stages (ie, from mesoderm to cardiac mesoderm) of cardiomyocyte differentiation remains limited.To characterize transcriptome and chromatin accessibility during early cardiomyocyte differentiation from hiPSCs and hESCs.We profiled the temporal changes in transcriptome and chromatin accessibility at genome-wide levels during cardiomyocyte differentiation derived from 2 hiPSC lines and 2 hESC lines at 4 stages: pluripotent stem cells, mesoderm, cardiac mesoderm, and differentiated cardiomyocytes. Overall, RNA sequencing analysis revealed that transcriptomes during early cardiomyocyte differentiation were highly concordant between hiPSCs and hESCs, and clustering of 4 cell lines within each time point demonstrated that changes in genome-wide chromatin accessibility were similar across hiPSC and hESC cell lines. Weighted gene co-expression network analysis (WGCNA) identified several modules that were strongly correlated with different stages of cardiomyocyte differentiation. Several novel genes were identified with high weighted connectivity within modules and exhibited coexpression patterns with other genes, including noncoding RNA LINC01124 and uncharacterized RNA AK127400 in the module related to the mesoderm stage; E-box-binding homeobox 1 (ZEB1) in the module correlated with postcardiac mesoderm. We further demonstrated that ZEB1 is required for early cardiomyocyte differentiation. In addition, based on integrative analysis of both WGCNA and transcription factor motif enrichment analysis, we determined numerous transcription factors likely to play important roles at different stages during cardiomyocyte differentiation, such as T and eomesodermin (EOMES; mesoderm), lymphoid enhancer-binding factor 1 (LEF1) and mesoderm posterior BHLH transcription factor 1 (MESP1; from mesoderm to cardiac mesoderm), meis homeobox 1 (MEIS1) and GATA-binding protein 4 (GATA4) (postcardiac mesoderm), JUN and FOS families, and MEIS2 (cardiomyocyte).Both hiPSCs and hESCs share similar transcriptional regulatory mechanisms underlying early cardiac differentiation, and our results have revealed transcriptional regulatory networks and new factors (eg, ZEB1) controlling early stages of cardiomyocyte differentiation.

  View details for PubMedID 28663367

• Comparison of Non-Coding RNAs in Exosomes and Functional Efficacy of Human Embryonic Stem Cell- Versus Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Stem cells (Dayton, Ohio) Lee, W. H., Chen, W., Shao, N. Y., Xiao, D., Qin, X., Baker, N., Bae, H. R., Shukla, P., Wu, H., Kodo, K., Ong, S. G., Wu, J. C. 2017

  Abstract

  Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can serve as unlimited cell sources for cardiac regenerative therapy. However, the functional equivalency between human ESC-CMs and iPSC-CMs for cardiac regenerative therapy has not been demonstrated. Here we performed a head-to-head comparison of ESC-CMs and iPSC-CMs in their ability to restore cardiac function in a rat myocardial infarction (MI) model as well as their exosomal secretome.Human ESCs and iPSCs were differentiated into cardiomyocytes using small molecule inhibitors. Fluorescence-activated cell sorting (FACS) analysis confirmed ∼85% and ∼83% of CMs differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T. At a single-cell level, both cell types displayed similar calcium handling and electrophysiological properties, with gene expression comparable to the human fetal heart marked by striated sarcomeres. Sub-acute transplantation of ESC-CMs and iPSC-CMs into nude rats post-MI improved cardiac function, which was associated with increased expression of angiogenic genes in vitro following hypoxia. Profiling of exosomal microRNAs (miRs) and long non-coding RNAs (lncRNAs) revealed that both groups contain an identical repertoire of miRs and lncRNAs, including some that are known to be cardioprotective.We demonstrate for the first time that both ESC-CMs and iPSC-CMs can facilitate comparable cardiac repair. This is advantageous because unlike allogeneic ESC-CMs used in therapy, autologous iPSC-CMs could potentially avoid immune rejection when used for cardiac cell transplantation in the future. This article is protected by copyright. All rights reserved.

  View details for PubMedID 28710827

• Anisotropic microfibrous scaffolds enhance the organization and function of cardiomyocytes derived from induced pluripotent stem cells. Biomaterials science Wanjare, M., Hou, L., Nakayama, K. H., Kim, J. J., Mezak, N. P., Abilez, O. J., Tzatzalos, E., Wu, J. C., Huang, N. F. 2017; 5 (8): 1567–78

  Abstract

  Engineering of myocardial tissue constructs is a promising approach for treatment of coronary heart disease. To engineer myocardial tissues that better mimic the highly ordered physiological arrangement and function of native cardiomyocytes, we generated electrospun microfibrous polycaprolactone scaffolds with either randomly oriented (14 μm fiber diameter) or parallel-aligned (7 μm fiber diameter) microfiber arrangement and co-seeded the scaffolds with human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and endothelial cells (iECs) for up to 12 days after iCM seeding. Here we demonstrated that aligned microfibrous scaffolds induced iCM alignment along the direction of the aligned microfibers after 2 days of iCM seeding, as well as promoted greater iCM maturation by increasing the sarcomeric length and gene expression of myosin heavy chain adult isoform (MYH7), in comparison to randomly oriented scaffolds. Furthermore, the benefit of scaffold anisotropy was evident in the significantly higher maximum contraction velocity of iCMs on the aligned scaffolds, compared to randomly oriented scaffolds, at 12 days of culture. Co-seeding of iCMs with iECs led to reduced contractility, compared to when iCMs were seeded alone. These findings demonstrate a dominant role of scaffold anisotropy in engineering cardiovascular tissues that maintain iCM organization and contractile function.

  View details for PubMedID 28715029

• Navigating the Future of Cardiovascular Drug Development-Leveraging Novel Approaches to Drive Innovation and Drug Discovery: Summary of Findings from the Novel Cardiovascular Therapeutics Conference. Cardiovascular drugs and therapy Povsic, T. J., Scott, R., Mahaffey, K. W., Blaustein, R., Edelberg, J. M., Lefkowitz, M. P., Solomon, S. D., Fox, J. C., Healy, K. E., Khakoo, A. Y., Losordo, D. W., Malik, F. I., Monia, B. P., Montgomery, R. L., Riesmeyer, J., Schwartz, G. G., Zelenkofske, S. L., Wu, J. C., Wasserman, S. M., Roe, M. T. 2017; 31 (4): 445–58

  Abstract

  The need for novel approaches to cardiovascular drug development served as the impetus to convene an open meeting of experts from the pharmaceutical industry and academia to assess the challenges and develop solutions for drug discovery in cardiovascular disease.The Novel Cardiovascular Therapeutics Summit first reviewed recent examples of ongoing or recently completed programs translating basic science observations to targeted drug development, highlighting successes (protein convertase sutilisin/kexin type 9 [PCSK9] and neprilysin inhibition) and targets still under evaluation (cholesteryl ester transfer protein [CETP] inhibition), with the hope of gleaning key lessons to successful drug development in the current era. Participants then reviewed the use of innovative approaches being explored to facilitate rapid and more cost-efficient evaluations of drug candidates in a short timeframe.We summarize observations gleaned from this summit and offer insight into future cardiovascular drug development.The rapid development in genetic and high-throughput drug evaluation technologies, coupled with new approaches to rapidly evaluate potential cardiovascular therapies with in vitro techniques, offer opportunities to identify new drug targets for cardiovascular disease, study new therapies with better efficiency and higher throughput in the preclinical setting, and more rapidly bring the most promising therapies to human testing. However, there must be a critical interface between industry and academia to guide the future of cardiovascular drug development. The shared interest among academic institutions and pharmaceutical companies in developing promising therapies to address unmet clinical needs for patients with cardiovascular disease underlies and guides innovation and discovery platforms that are significantly altering the landscape of cardiovascular drug development.

  View details for PubMedID 28735360

• Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators. eLife Chamberland, S., Yang, H. H., Pan, M. M., Evans, S. W., Guan, S., Chavarha, M., Yang, Y., Salesse, C., Wu, H., Wu, J. C., Clandinin, T. R., Toth, K., Lin, M. Z., St-Pierre, F. 2017; 6

  Abstract

  Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and inDrosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

  View details for PubMedID 28749338

• Combining hiPSCs and Human Genetics: Major Applications in Drug Development. Cell stem cell Zhang, J., Li, H., Trounson, A., Wu, J. C., Nioi, P. 2017; 21 (2): 161–65

  Abstract

  Merging iPSC models and human genetic research has opened up new avenues in understanding disease mechanisms and target biology, which facilitate exciting translation of this research to many areas of drug development. We highlight recent applications of these combined disciplines and discuss remaining challenges and potential solutions.

  View details for PubMedID 28777942

• Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics. Cell reports Kooreman, N. G., de Almeida, P. E., Stack, J. P., Nelakanti, R. V., Diecke, S., Shao, N. Y., Swijnenburg, R. J., Sanchez-Freire, V., Matsa, E., Liu, C., Connolly, A. J., Hamming, J. F., Quax, P. H., Brehm, M. A., Greiner, D. L., Shultz, L. D., Wu, J. C. 2017; 20 (8): 1978–90

  Abstract

  There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an "allogenized" mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.

  View details for PubMedID 28834758

• Multiscale technologies for treatment of ischemic cardiomyopathy. Nature nanotechnology Mahmoudi, M., Yu, M., Serpooshan, V., Wu, J. C., Langer, R., Lee, R. T., Karp, J. M., Farokhzad, O. C. 2017; 12 (9): 845–55

  Abstract

  The adult mammalian heart possesses only limited capacity for innate regeneration and the response to severe injury is dominated by the formation of scar tissue. Current therapy to replace damaged cardiac tissue is limited to cardiac transplantation and thus many patients suffer progressive decay in the heart's pumping capacity to the point of heart failure. Nanostructured systems have the potential to revolutionize both preventive and therapeutic approaches for treating cardiovascular disease. Here, we outline recent advancements in nanotechnology that could be exploited to overcome the major obstacles in the prevention of and therapy for heart disease. We also discuss emerging trends in nanotechnology affecting the cardiovascular field that may offer new hope for patients suffering massive heart attacks.

  View details for PubMedID 28875984

• Incremental value of right heart metrics and exercise performance to well-validated risk scores in dilated cardiomyopathy. European heart journal cardiovascular Imaging Moneghetti, K. J., Giraldeau, G., Wheeler, M. T., Kobayashi, Y., Vrtovec, B., Boulate, D., Kuznetsova, T., Schnittger, I., Wu, J. C., Myers, J., Ashley, E., Haddad, F. 2017

  Abstract

  Risk stratification in heart failure (HF) relies on several established clinical risk scores, however, myocardial deformation, right heart metrics, and exercise performance have not usually been considered. This study sought to assess the incremental value of advanced echocardiographic and cardiopulmonary exercise testing (CPX) parameters to validated risk scores in HF.The Meta-Analysis Global Group in Chronic Heart Failure (MAGGIC) and Metabolic Exercise Test Data Combined with Cardiac and Kidney Indexes (MECKI) scores were applied to 208 ambulatory patients with dilated cardiomyopathy (DCM) who completed echocardiography in conjunction with CPX as part of the Stanford Exercise Testing registry. Patients were followed for the composite end point of death, heart transplant, left ventricular device implantation, and hospitalization for acute HF. Mean age, left ventricular ejection fraction (LVEF), and left ventricular global longitudinal strain (LVGLS) were 47 ± 13 years, 33 ± 13%, and -10.6 ± 4.4%, respectively, while right ventricular free-wall longitudinal strain was -18.8 ± 5.5%. Partial correlation mapping identified strong correlations between LVEF, LVGLS, and LV systolic strain rate, with a moderate correlation between these metrics and peak VO2. Over a median follow up of 5.3 years, the composite end point occurred in 60 patients. Cox proportional hazards identified MAGGIC score [hazard ratio (HR) (2.04 [1.39-3.01], P < 0.01], peak VO2 HR (0.52 [0.28-0.97], P = 0.04), and right atrial volume indexed (RAVI) HR (1.31 [1.07-1.61], P < 0.01) as independent correlates of outcome. RAVI remained an independent correlate when combined with the MECKI score (2.21 [1.59-3.07]), P < 0.01, RAVI, 1.33 [1.06-1.67], P = 0.01).Our study demonstrates that RAVI is complementary to well-validated HF risk scores and highlights the importance of exercise performance in DCM.

  View details for PubMedID 28977353

• Passive Stretch Induces Structural and Functional Maturation of Engineered Heart Muscle as Predicted by Computational Modeling. Stem cells (Dayton, Ohio) Abilez, O. J., Tzatzalos, E., Yang, H., Zhao, M. T., Jung, G., Zöllner, A. M., Tiburcy, M., Riegler, J., Matsa, E., Shukla, P., Zhuge, Y., Chour, T., Chen, V. C., Burridge, P. W., Karakikes, I., Kuhl, E., Bernstein, D., Couture, L. A., Gold, J. D., Zimmermann, W. H., Wu, J. C. 2017

  Abstract

  The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling.Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (hiPSCs, IMR-90 line) were differentiated to human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin(+) flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 x 10(6) hPSC-CMs were mixed with 0.4 x 10(6) human fibroblasts (IMR-90 line) (3:1 ratio) and Type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane (PDMS) reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable to rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, β-adrenergic receptors, and t-tubule protein caveolin-3.Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. This article is protected by copyright. All rights reserved.

  View details for PubMedID 29086457

• Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells. Circulation research Zhao, M. T., Shao, N. Y., Hu, S., Ma, N., Srinivasan, R., Jahanbani, F., Lee, J., Zhang, S. L., Snyder, M. P., Wu, J. C. 2017; 121 (11): 1237–50

  Abstract

  Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart.We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression.We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31+CD144+), cardiac progenitor cells (Sca-1+), fibroblasts (DDR2+), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression.Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases.

  View details for PubMedID 29030344

• GDF-15 (Growth Differentiation Factor 15) Is Associated With Lack of Ventricular Recovery and Mortality After Transcatheter Aortic Valve Replacement. Circulation. Cardiovascular interventions Kim, J. B., Kobayashi, Y., Moneghetti, K. J., Brenner, D. A., O'Malley, R., Schnittger, I., Wu, J. C., Murtagh, G., Beshiri, A., Fischbein, M., Miller, D. C., Liang, D., Yeung, A. C., Haddad, F., Fearon, W. F. 2017; 10 (12)

  Abstract

  Recent data suggest that circulating biomarkers may predict outcome in patients undergoing transcatheter aortic valve replacement (TAVR). We examined the association between inflammatory, myocardial, and renal biomarkers and their role in ventricular recovery and outcome after TAVR.A total of 112 subjects undergoing TAVR were included in the prospective registry. Plasma levels of B-type natriuretic peptide, hs-TnI (high-sensitivity troponin I), CRP (C-reactive protein), GDF-15 (growth differentiation factor 15), GAL-3 (galectin-3), and Cys-C (cystatin-C) were assessed before TAVR and in 100 sex-matched healthy controls. Among echocardiographic parameters, we measured global longitudinal strain, indexed left ventricular mass, and indexed left atrial volume. The TAVR group included 59% male, with an average age of 84 years, and 1-year mortality of 18%. Among biomarkers, we found GDF-15 and CRP to be strongly associated with all-cause mortality (P<0.001). Inclusion of GDF-15 and CRP to the Society of Thoracic Surgeons score significantly improved C index (0.65-0.79; P<0.05) and provided a category-free net reclassification improvement of 106% at 2 years (P=0.01). Among survivors, functional recovery in global longitudinal strain (>15% improvement) and indexed left ventricular mass (>20% decrease) at 1 year occurred in 48% and 22%, respectively. On multivariate logistic regression, lower baseline GDF-15 was associated with improved global longitudinal strain at 1 year (hazard ratio=0.29; P<0.001). Furthermore, improvement in global longitudinal strain at 1 month correlated with lower overall mortality (hazard ratio=0.45; P=0.03).Elevated GDF-15 correlates with lack of reverse remodeling and increased mortality after TAVR and improves risk prediction of mortality when added to the Society of Thoracic Surgeons score.

  View details for PubMedID 29222133

• Simply derived epicardial cells. Nature biomedical engineering Paik, D. T., Wu, J. C. 2017; 1

  Abstract

  A chemically defined protocol requiring no animal-derived components allows for the easier derivation and enduring expansion of epicardial cells from human pluripotent stem cells.

  View details for PubMedID 29354320

• Patient-Specific and Genome-Edited Induced Pluripotent Stem Cell-Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Brugada Syndrome. Journal of the American College of Cardiology Liang, P., Sallam, K., Wu, H., Li, Y., Itzhaki, I., Garg, P., Zhang, Y., Vermglinchan, V., Lan, F., Gu, M., Gong, T., Zhuge, Y., He, C., Ebert, A. D., Sanchez-Freire, V., Churko, J., Hu, S., Sharma, A., Lam, C. K., Scheinman, M. M., Bers, D. M., Wu, J. C. 2016; 68 (19): 2086-2096

  Abstract

  Brugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder.The objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).This study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs.BrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca(2+)) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca(2+) transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression.Patient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca(2+) handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.

  View details for DOI 10.1016/j.jacc.2016.07.779

  View details for PubMedID 27810048

• Magnetic Resonance Imaging of Cardiac Strain Pattern Following Transplantation of Human Tissue Engineered Heart Muscles CIRCULATION-CARDIOVASCULAR IMAGING Qin, X., Riegler, J., Tiburcy, M., Zhao, X., Chour, T., Ndoye, B., Michael Nguyen, M., Adams, J., Ameen, M., Denney, T. S., Yang, P. C., Patricia Nguyen, P., Zimmermann, W. H., Wu, J. C. 2016; 9 (11)

  Abstract

  The use of tissue engineering approaches in combination with exogenously produced cardiomyocytes offers the potential to restore contractile function after myocardial injury. However, current techniques assessing changes in global cardiac performance after such treatments are plagued by relatively low detection ability. Since the treatment is locally performed, this detection could be improved by myocardial strain imaging that measures regional contractility.Tissue engineered heart muscles (EHMs) were generated by casting human embryonic stem cell-derived cardiomyocytes with collagen in preformed molds. EHMs were transplanted (n=12) to cover infarct and border zones of recipient rat hearts 1 month after ischemia reperfusion injury. A control group (n=10) received only sham placement of sutures without EHMs. To assess the efficacy of EHMs, magnetic resonance imaging and ultrasound-based strain imaging were performed before and 4 weeks after transplantation. In addition to strain imaging, global cardiac performance was estimated from cardiac magnetic resonance imaging. Although no significant differences were found for global changes in left ventricular ejection fraction (control -9.6±1.3% versus EHM -6.2±1.9%; P=0.17), regional myocardial strain from tagged magnetic resonance imaging was able to detect preserved systolic function in EHM-treated animals compared with control (control 4.4±1.0% versus EHM 1.0±0.6%; P=0.04). However, ultrasound-based strain failed to detect any significant change (control 2.1±3.0% versus EHM 6.3±2.9%; P=0.46).This study highlights the feasibility of using cardiac strain from tagged magnetic resonance imaging to assess functional changes in rat models following localized regenerative therapies, which may not be detected by conventional measures of global systolic performance.

  View details for DOI 10.1161/CIRCIMAGING.116.004731

  View details for Web of Science ID 000388482500003

  View details for PubMedID 27903535

  View details for PubMedCentralID PMC5378466

• Potential Strategies to Address the Major Clinical Barriers Facing Stem Cell Regenerative Therapy for Cardiovascular Disease: A Review. JAMA cardiology Nguyen, P. K., Neofytou, E., Rhee, J., Wu, J. C. 2016; 1 (8): 953-962

  Abstract

  Although progress continues to be made in the field of stem cell regenerative medicine for the treatment of cardiovascular disease, significant barriers to clinical implementation still exist.To summarize the current barriers to the clinical implementation of stem cell therapy in patients with cardiovascular disease and to discuss potential strategies to overcome them.Information for this review was obtained through a search of PubMed and the Cochrane database for English-language studies published between January 1, 2000, and July 25, 2016. Ten randomized clinical trials and 8 systematic reviews were included.One of the major clinical barriers facing the routine implementation of stem cell therapy in patients with cardiovascular disease is the limited and inconsistent benefit observed thus far. Reasons for this finding are unclear but may be owing to poor cell retention and survival, as suggested by numerous preclinical studies and a small number of human studies incorporating imaging to determine cell fate. Additional studies in humans using imaging to determine cell fate are needed to understand how these factors contribute to the limited efficacy of stem cell therapy. Treatment strategies to address poor cell retention and survival are under investigation and include the following: coadministration of immunosuppressive and prosurvival agents, delivery of cardioprotective factors packaged in exosomes rather than the cells themselves, and use of tissue-engineering strategies to provide structural support for cells. If larger grafts are achieved using these strategies, it will be imperative to carefully monitor for the potential risks of tumorigenicity, immunogenicity, and arrhythmogenicity.Despite important achievements to date, stem cell therapy is not yet ready for routine clinical implementation. Significant research is still needed to address the clinical barriers outlined herein before the next wave of large clinical trials is under way.

  View details for DOI 10.1001/jamacardio.2016.2750

  View details for PubMedID 27579998

• Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy. Proceedings of the National Academy of Sciences of the United States of America Chang, A. C., Ong, S., Lagory, E. L., Kraft, P. E., Giaccia, A. J., Wu, J. C., Blau, H. M. 2016

  Abstract

  Duchenne muscular dystrophy (DMD) is an incurable X-linked genetic disease that is caused by a mutation in the dystrophin gene and affects one in every 3,600 boys. We previously showed that long telomeres protect mice from the lethal cardiac disease seen in humans with the same genetic defect, dystrophin deficiency. By generating the mdx(4cv)/mTR(G2) mouse model with "humanized" telomere lengths, the devastating dilated cardiomyopathy phenotype seen in patients with DMD was recapitulated. Here, we analyze the degenerative sequelae that culminate in heart failure and death in this mouse model. We report progressive telomere shortening in developing mouse cardiomyocytes after postnatal week 1, a time when the cells are no longer dividing. This proliferation-independent telomere shortening is accompanied by an induction of a DNA damage response, evident by p53 activation and increased expression of its target gene p21 in isolated cardiomyocytes. The consequent repression of Pgc1α/β leads to impaired mitochondrial biogenesis, which, in conjunction with the high demands of contraction, leads to increased oxidative stress and decreased mitochondrial membrane potential. As a result, cardiomyocyte respiration and ATP output are severely compromised. Importantly, treatment with a mitochondrial-specific antioxidant before the onset of cardiac dysfunction rescues the metabolic defects. These findings provide evidence for a link between short telomere length and metabolic compromise in the etiology of dilated cardiomyopathy in DMD and identify a window of opportunity for preventive interventions.

  View details for PubMedID 27799523

• Defective Signaling in the JAK-STAT Pathway Tracks with Chronic Inflammation and Cardiovascular Risk in Aging Humans. Cell systems Shen-Orr, S. S., Furman, D., Kidd, B. A., Hadad, F., Lovelace, P., Huang, Y., Rosenberg-Hasson, Y., Mackey, S., Grisar, F. A., Pickman, Y., Maecker, H. T., Chien, Y., Dekker, C. L., Wu, J. C., Butte, A. J., Davis, M. M. 2016; 3 (4): 374-384 e4

  Abstract

  Chronic inflammation, a decline in immune responsiveness, and reduced cardiovascular function are all associated with aging, but the relationships among these phenomena remain unclear. Here, we longitudinally profiled a total of 84 signaling conditions in 91 young and older adults and observed an age-related reduction in cytokine responsiveness within four immune cell lineages, most prominently T cells. The phenotype can be partially explained by elevated baseline levels of phosphorylated STAT (pSTAT) proteins and a different response capacity of naive versus memory T cell subsets to interleukin 6 (IL-6), interferon α (IFN-α), and, to a lesser extent, IL-21 and IFN-γ. Baseline pSTAT levels tracked with circulating levels of C-reactive protein (CRP), and we derived a cytokine response score that negatively correlates with measures of cardiovascular disease, specifically diastolic dysfunction and atherosclerotic burden, outperforming CRP. Thus, we identified an immunological link between inflammation, decreased cell responsiveness in the JAK-STAT pathway, and cardiovascular aging. Targeting chronic inflammation may ameliorate this deficiency in cellular responsiveness and improve cardiovascular function.

  View details for DOI 10.1016/j.cels.2016.09.009

  View details for PubMedID 27746093

• DNA damage-associated biomarkers in studying individual sensitivity to low-dose radiation from cardiovascular imaging. European heart journal Lee, W. H., Nguyen, P. K., Fleischmann, D., Wu, J. C. 2016; 37 (40): 3075-3080

  View details for PubMedID 27272147

• Alternative approaches to generating cardiomyocytes are under development. Nature reviews. Cardiology Chen, I. Y., Matsa, E., Wu, J. C. 2016; 13 (10): 574-?

  View details for DOI 10.1038/nrcardio.2016.130

  View details for PubMedID 27538813

• Adult Stem Cell Therapy and Heart Failure, 2000 to 2016: A Systematic Review. JAMA cardiology Nguyen, P. K., Rhee, J., Wu, J. C. 2016; 1 (7): 831-841

  Abstract

  Stem cell therapy is a promising treatment strategy for patients with heart failure, which accounts for more than 10% of deaths in the United States annually. Despite more than a decade of research, further investigation is still needed to determine whether stem cell regenerative therapy is an effective treatment strategy and can be routinely implemented in clinical practice.To describe the progress in cardiac stem cell regenerative therapy using adult stem cells and to highlight the merits and limitations of clinical trials performed to date.Information for this review was obtained through a search of PubMed and the Cochrane database for English-language studies published between January 1, 2000, and July 26, 2016. Twenty-nine randomized clinical trials and 7 systematic reviews and meta-analyses were included in this review.Although adult stem cells were once believed to have the ability to create new heart tissue, preclinical studies suggest that these cells release cardioprotective paracrine factors that activate endogenous pathways, leading to myocardial repair. Subsequent randomized clinical trials, most of which used autologous bone marrow mononuclear cells, have found only a modest benefit in patients receiving stem cell therapy. The lack of a significant benefit may result from variations in trial methods, discrepancies in reporting, and an overreliance on surrogate end points.Although stem cell therapy for cardiovascular disease is not yet ready for routine clinical application, significant progress continues to be made. Physicians should be aware of the current status of this treatment so that they can better inform their patients who may be in search of alternative therapies.

  View details for DOI 10.1001/jamacardio.2016.2225

  View details for PubMedID 27557438

• Challenging the complementarity of different metrics of left atrial function: insight from a cardiomyopathy-based study. European heart journal cardiovascular Imaging Kobayashi, Y., Moneghetti, K. J., Boralkar, K., Amsallem, M., Tuzovic, M., Liang, D., Yang, P. C., Narayan, S., Kuznetsova, T., Wu, J. C., Schnittger, I., Haddad, F. 2016

  Abstract

  Left ventricular (LV) strain provides incremental values to LV ejection fraction (LVEF) in predicting outcome. We sought to investigate if similar relationship is observed between left atrial (LA) emptying fraction and LA strain.In this study, we selected 50 healthy subjects, 50 patients with dilated, 50 hypertrophic, and 50 infiltrative (light-chain (AL) amyloidosis) cardiomyopathy (CMP). Echocardiographic measures included LVEF and LA emptying fraction as well as LV and LA longitudinal strain (LVLS and LALS). After regression analysis, comparison of least square means of LA strain among aetiologies was performed. Intraclass correlation coefficient (ICC) and coefficient of variation (COV) were used in the assessment of variability and reproducibility of LV and LA metrics. The mean LVLS and all LA metrics were impaired in patients with all CMP compared with healthy subjects. In contrast to the moderate relationship between LVEF and LVLS (r = -0.51, P < 0.001), there was a strong linear relationship between LA emptying fraction and LA strain (r = 0.87, P < 0.001). In multiple regression analysis, total LA strain was associated with LVLS (β = -0.48, P < 0.001), lateral E/e' (β = -0.24, P < 0.001), age (β = -0.21, P < 0.001), and heart rate (β = -0.14, P = 0.02). The least square mean of LA strain adjusted for the parameters was not different among aetiologies (ANOVA P = 0.82). The ICC (>0.77) and COV (<13) were acceptable.In contrast to LV measures, there is a strong linear relationship between volumetric and longitudinal deformation indices of left atrium irrespective of CMP aetiology. Either LA emptying fraction or LA strain could be used as an important parameter in predictive models.

  View details for PubMedID 27638850

• Increased Pyruvate Dehydrogenase Kinase 4 Expression in Lung Pericytes Is Associated with Reduced Endothelial-Pericyte interactions and Small Vessel Loss in Pulmonary Arterial Hypertension AMERICAN JOURNAL OF PATHOLOGY Yuan, K., Shao, N., Hennigs, J. K., Discipulo, M., Orcholski, M. E., Shamskhou, E., Richter, A., Hu, X., Wu, J. C., Perez, V. A. 2016; 186 (9): 2500-2514

  Abstract

  Reduced endothelial-pericyte interactions are linked to progressive small vessel loss in pulmonary arterial hypertension (PAH), but the molecular mechanisms underlying this disease remain poorly understood. To identify relevant gene candidates associated with aberrant pericyte behavior, we performed a transcriptome analysis of patient-derived donor control and PAH lung pericytes followed by functional genomics analysis. Compared with donor control cells, PAH pericytes had significant enrichment of genes involved in various metabolic processes, the top hit being PDK4, a gene coding for an enzyme that suppresses mitochondrial activity in favor of glycolysis. Given reports that link reduced mitochondrial activity with increased PAH cell proliferation, we hypothesized that increased PDK4 is associated with PAH pericyte hyperproliferation and reduced endothelial-pericyte interactions. We found that PDK4 gene and protein expression was significantly elevated in PAH pericytes and correlated with reduced mitochondrial metabolism, higher rates of glycolysis, and hyperproliferation. Importantly, reducing PDK4 levels restored mitochondrial metabolism, reduced cell proliferation, and improved endothelial-pericyte interactions. To our knowledge, this is the first study that documents significant differences in gene expression between human donor control and PAH lung pericytes and the link between mitochondrial dysfunction and aberrant endothelial-pericyte interactions in PAH. Comprehensive characterization of these candidate genes could provide novel therapeutic targets to improve endothelial-pericyte interactions and prevent small vessel loss in PAH.

  View details for DOI 10.1016/j.ajpath.2016.05.016

  View details for Web of Science ID 000382595200025

• Increased Pyruvate Dehydrogenase Kinase 4 Expression in Lung Pericytes Is Associated with Reduced Endothelial-Pericyte Interactions and Small Vessel Loss in Pulmonary Arterial Hypertension. American journal of pathology Yuan, K., Shao, N., Hennigs, J. K., Discipulo, M., Orcholski, M. E., Shamskhou, E., Richter, A., Hu, X., Wu, J. C., de Jesus Perez, V. A. 2016; 186 (9): 2500-2514

  Abstract

  Reduced endothelial-pericyte interactions are linked to progressive small vessel loss in pulmonary arterial hypertension (PAH), but the molecular mechanisms underlying this disease remain poorly understood. To identify relevant gene candidates associated with aberrant pericyte behavior, we performed a transcriptome analysis of patient-derived donor control and PAH lung pericytes followed by functional genomics analysis. Compared with donor control cells, PAH pericytes had significant enrichment of genes involved in various metabolic processes, the top hit being PDK4, a gene coding for an enzyme that suppresses mitochondrial activity in favor of glycolysis. Given reports that link reduced mitochondrial activity with increased PAH cell proliferation, we hypothesized that increased PDK4 is associated with PAH pericyte hyperproliferation and reduced endothelial-pericyte interactions. We found that PDK4 gene and protein expression was significantly elevated in PAH pericytes and correlated with reduced mitochondrial metabolism, higher rates of glycolysis, and hyperproliferation. Importantly, reducing PDK4 levels restored mitochondrial metabolism, reduced cell proliferation, and improved endothelial-pericyte interactions. To our knowledge, this is the first study that documents significant differences in gene expression between human donor control and PAH lung pericytes and the link between mitochondrial dysfunction and aberrant endothelial-pericyte interactions in PAH. Comprehensive characterization of these candidate genes could provide novel therapeutic targets to improve endothelial-pericyte interactions and prevent small vessel loss in PAH.

  View details for DOI 10.1016/j.ajpath.2016.05.016

  View details for PubMedID 27456128

• Transcriptome Profiling of Patient-Specific Human iPSC-Cardiomyocytes Predicts Individual Drug Safety and Efficacy Responses In Vitro. Cell stem cell Matsa, E., Burridge, P. W., Yu, K., Ahrens, J. H., Termglinchan, V., Wu, H., Liu, C., Shukla, P., Sayed, N., Churko, J. M., Shao, N., Woo, N. A., Chao, A. S., Gold, J. D., Karakikes, I., Snyder, M. P., Wu, J. C. 2016; 19 (3): 311-325

  Abstract

  Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patient variation than intra-patient variation, verifying that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.

  View details for DOI 10.1016/j.stem.2016.07.006

  View details for PubMedID 27545504

• HUMAN INDUCED PLURIPOTENT STEM CELLS AS A PLATFORM FOR PERSONALIZED AND PRECISION CARDIOVASCULAR MEDICINE PHYSIOLOGICAL REVIEWS Matsa, E., Ahrens, J. H., Wu, J. C. 2016; 96 (3): 1093-1126

  Abstract

  Human induced pluripotent stem cells (hiPSCs) have revolutionized the field of human disease modeling, with an enormous potential to serve as paradigm shifting platforms for preclinical trials, personalized clinical diagnosis, and drug treatment. In this review, we describe how hiPSCs could transition cardiac healthcare away from simple disease diagnosis to prediction and prevention, bridging the gap between basic and clinical research to bring the best science to every patient.

  View details for DOI 10.1152/physrev.00036.2015

  View details for PubMedID 27335446

• Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant Human Hypoxia-Inducible Factor 1-a (HIF1-a) in an Ovine Model of Acute Myocardial Infarction. Journal of the American Heart Association Hnatiuk, A. P., Ong, S., Olea, F. D., Locatelli, P., Riegler, J., Lee, W. H., Jen, C. H., De Lorenzi, A., Giménez, C. S., Laguens, R., Wu, J. C., Crottogini, A. 2016; 5 (7)

  Abstract

  Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia-inducible factor 1-α (HIF1-α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen-resistant HIF1-α would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI.Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1-α (BMMSC-HIF) were injected in the peri-infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P<0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2-fold (P<0.001) in the presence of markedly decreased end-systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo-treated animals (n=6), neither parameters changed over time. HIF1-α-transfected BMMSCs (BMMSC-HIF) induced angio-/arteriogenesis and decreased apoptosis by HIF1-mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin-1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long-term retention of BMMSC-HIF.Intramyocardial delivery of BMMSC-HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1-mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.

  View details for DOI 10.1161/JAHA.116.003714

  View details for PubMedID 27385426

• Finding Expandable Induced Cardiovascular Progenitor Cells CIRCULATION RESEARCH Chen, I. Y., Wu, J. C. 2016; 119 (1): 16-20

  View details for DOI 10.1161/CIRCRESAHA.116.308679

  View details for PubMedID 27340267

• Distilling complexity to advance cardiac tissue engineering SCIENCE TRANSLATIONAL MEDICINE Ogle, B. M., Bursac, N., Domian, I., Huang, N. F., Menasche, P., Murry, C. E., Pruitt, B., Radisic, M., Wu, J. C., Wu, S. M., Zhang, J., Zimmermann, W., Vunjak-Novakovic, G. 2016; 8 (342)

  Abstract

  The promise of cardiac tissue engineering is in the ability to recapitulate in vitro the functional aspects of a healthy heart and disease pathology as well as to design replacement muscle for clinical therapy. Parts of this promise have been realized; others have not. In a meeting of scientists in this field, five central challenges or "big questions" were articulated that, if addressed, could substantially advance the current state of the art in modeling heart disease and realizing heart repair.

  View details for DOI 10.1126/scitranslmed.aad2304

  View details for PubMedID 27280684

• Effects of cellular origin on differentiation of human induced pluripotent stem cell-derived endothelial cells. JCI insight Hu, S., Zhao, M., Jahanbani, F., Shao, N., Lee, W. H., Chen, H., Snyder, M. P., Wu, J. C. 2016; 1 (8)

  Abstract

  Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC-derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31(+) population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC-ECs maintained a higher CD31(+) population than FB-iPSC-ECs and CPC-iPSC-ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation.

  View details for PubMedID 27398408

• Induced pluripotent stem cells: at the heart of cardiovascular precision medicine NATURE REVIEWS CARDIOLOGY Chen, I. Y., Matsa, E., Wu, J. C. 2016; 13 (6): 333-349

  Abstract

  The advent of human induced pluripotent stem cell (hiPSC) technology has revitalized the efforts in the past decade to realize more fully the potential of human embryonic stem cells for scientific research. Adding to the possibility of generating an unlimited amount of any cell type of interest, hiPSC technology now enables the derivation of cells with patient-specific phenotypes. Given the introduction and implementation of the large-scale Precision Medicine Initiative, hiPSC technology will undoubtedly have a vital role in the advancement of cardiovascular research and medicine. In this Review, we summarize the progress that has been made in the field of hiPSC technology, with particular emphasis on cardiovascular disease modelling and drug development. The growing roles of hiPSC technology in the practice of precision medicine will also be discussed.

  View details for DOI 10.1038/nrcardio.2016.36

  View details for PubMedID 27009425

• Impact and pitfalls of scaling of left ventricular and atrial structure in population-based studies JOURNAL OF HYPERTENSION Kuznetsova, T., Haddad, F., Tikhonoff, V., Kloch-Badelek, M., Ryabikov, A., Knez, J., Malyutina, S., Stolarz-Skrzypek, K., Thijs, L., Schnittger, I., Wu, J. C., Casiglia, E., Narkiewicz, K., Kawecka-Jaszcz, K., Staessen, J. A. 2016; 34 (6): 1186-1194

  Abstract

  Several allometric methods for indexing cardiac structures to body size have been proposed but the optimal way for normalization of cardiac structures is still controversial. We aimed to estimate the allometric exponents that best describe the relationships between cardiac dimensions and body size, propose normative values, and analyze how the different scaling metrics influence the prevalence of left ventricular hypertrophy (LVH) and chambers enlargement as well as predictive models for cardiovascular outcome in the community.We measured left ventricular end-diastolic dimension, end-diastolic volume, left ventricular mass, and left atrial volume in randomly recruited population cohorts (n = 1509; 52.8% women; mean age, 47.8 years).In a healthy subgroup (n = 656), the allometric exponents that described the relationships between left ventricular end-diastolic dimension and body size were 1, 0.5, and 0.33 for body height, body surface area (BSA), and estimated lean body mass, respectively. With regard to left ventricular end-diastolic volume, left ventricular mass, and left atrial volume the allometric exponents for body height were 2.9, 2.7, and 2.0, respectively; for BSA, they ranged from 1.7 to 1.8; for estimated lean body mass all exponents were around 1. These exponents were used to appropriately scale the cardiac dimensions to body size and derived sex-specific cut-off limits for different indexed cardiac dimensions. The hazard ratios of cardiovascular outcome were highest for LVH defined by left ventricular mass/height.Our study resulted in a proposal for thresholds for various indexed cardiac dimensions. Left ventricular mass indexed to height was sensitive in detection of LVH associated with obesity and slightly better predicted outcome.

  View details for DOI 10.1097/HJH.0000000000000922

  View details for PubMedID 27035735

• Translation of Human-Induced Pluripotent Stem Cells From Clinical Trial in a Dish to Precision Medicine JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Sayed, N., Liu, C., Wu, J. C. 2016; 67 (18): 2161-2176

  Abstract

  The prospect of changing the plasticity of terminally differentiated cells toward pluripotency has completely altered the outlook for biomedical research. Human-induced pluripotent stem cells (iPSCs) provide a new source of therapeutic cells free from the ethical issues or immune barriers of human embryonic stem cells. iPSCs also confer considerable advantages over conventional methods of studying human diseases. Since its advent, iPSC technology has expanded with 3 major applications: disease modeling, regenerative therapy, and drug discovery. Here we discuss, in a comprehensive manner, the recent advances in iPSC technology in relation to basic, clinical, and population health.

  View details for DOI 10.1016/j.jacc.2016.01.083

  View details for PubMedID 27151349

• Efficacy of CD34(+) Stem Cell Therapy in Nonischemic Dilated Cardiomyopathy Is Absent in Patients With Diabetes but Preserved in Patients With Insulin Resistance STEM CELLS TRANSLATIONAL MEDICINE Vrtovec, B., Sever, M., Jensterle, M., Poglajen, G., Janez, A., Kravos, N., Zemljic, G., Cukjati, M., Cernelc, P., Haddad, F., Wu, J. C., Jorde, U. P. 2016; 5 (5): 632-638

  Abstract

  : We evaluated the association of diabetes and insulin resistance with the response to cell therapy in patients with nonischemic dilated cardiomyopathy (DCM). A total of 45 outpatients with DCM received granulocyte colony-stimulating factor for 5 days. CD34(+) cells were then collected by apheresis and injected transendocardially. Twelve patients had diabetes mellitus (DM group), 17 had insulin resistance (IR group), and 16 displayed normal glucose metabolism (no-IR group). After stimulation, we found higher numbers of CD34(+) cells in the IR group (94 ± 73 × 10(6) cells per liter) than in the no-IR group (54 ± 35 × 10(6) cells per liter) or DM group (31 ± 20 × 10(6) cells per liter; p = .005). Similarly, apheresis yielded the highest numbers of CD34(+) cells in the IR group (IR group, 216 ± 110 × 10(6) cells; no-IR group, 127 ± 82 × 10(6) cells; DM group, 77 ± 83 × 10(6) cells; p = .002). Six months after cell therapy, we found an increase in left ventricular ejection fraction in the IR group (+5.6% ± 6.9%) and the no-IR group (+4.4% ± 7.2%) but not in the DM group (-0.9% ± 5.4%; p = .035). The N-terminal pro-brain natriuretic peptide levels decreased in the IR and no-IR groups, but not in the DM group (-606 ± 850 pg/ml; -698 ± 1,105 pg/ml; and +238 ± 963 pg/ml, respectively; p = .034). Transendocardial CD34(+) cell therapy appears to be ineffective in DCM patients with diabetes. IR was associated with improved CD34(+) stem cell mobilization and a preserved clinical response to cell therapy.The present study is the first clinical study directly evaluating the effects of altered glucose metabolism on the efficacy of CD34(+) stem cell therapy in patients with nonischemic dilated cardiomyopathy. The results offer critical insights into the physiology of stem cell mobilization in heart failure and possibly an explanation for the often conflicting results obtained with stem cell therapy for heart failure. These results demonstrate that patients with dilated cardiomyopathy and diabetes do not benefit from autologous CD34(+) cell therapy. This finding could serve as a useful tool when selecting heart failure patients for future clinical studies in the field of stem cell therapy.

  View details for DOI 10.5966/sctm.2015-0172

  View details for PubMedID 27025690

• Human-induced pluripotent stem cell approaches to model inborn and acquired metabolic heart diseases. Current opinion in cardiology Chanana, A. M., Rhee, J., Wu, J. C. 2016; 31 (3): 266-274

  Abstract

  The article provides an overview of advances in the induced pluripotent stem cell field to model cardiomyopathies of inherited inborn errors of metabolism and acquired metabolic syndromes in vitro.Several inborn errors of metabolism have been studied using 'disease in a dish' models, including Pompe disease, Danon disease, Fabry disease, and Barth syndrome. Disease phenotypes of complex metabolic syndromes, such as diabetes mellitus and aldehyde dehydrogenase 2 deficiency, have also been observed.Differentiation of patient and disease-specific induced pluripotent stem cell-derived cardiomyocytes has provided the capacity to model deleterious cardiometabolic diseases to understand molecular mechanisms, perform drug screens, and identify novel drug targets.

  View details for DOI 10.1097/HCO.0000000000000277

  View details for PubMedID 27022891

• Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation FASEB JOURNAL Jung, G., Fajardo, G., Ribeiro, A. J., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. 2016; 30 (4): 1464-1479

  Abstract

  Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for uncovering disease mechanisms and assessing drugs for efficacy/toxicity. However, the accuracy with which hiPSC-CMs recapitulate the contractile and remodeling signaling of adult cardiomyocytes is not fully known. We used β-adrenergic receptor (β-AR) signaling as a prototype to determine the evolution of signaling component expression and function during hiPSC-CM maturation. In "early" hiPSC-CMs (less than or equal to d 30), β2-ARs are a primary source of cAMP/PKA signaling. With longer culture, β1-AR signaling increases: from 0% of cAMP generation at d 30 to 56.8 ± 6.6% by d 60. PKA signaling shows a similar increase: 15.7 ± 5.2% (d 30), 49.8 ± 0.5% (d 60), and 71.0 ± 6.1% (d 90). cAMP generation increases 9-fold from d 30 to 60, with enhanced coupling to remodeling pathways (e.g., Akt and Ca(2+)/calmodulin-dependent protein kinase type II) and development of caveolin-mediated signaling compartmentalization. By contrast, cardiotoxicity induced by chronic β-AR stimulation, a major component of heart failure, develops much later: 5% cell death at d 30 vs. 55% at d 90. Moreover, β-AR maturation can be accelerated by biomechanical stimulation. The differential maturation of β-AR functional vs. remodeling signaling in hiPSC-CMs has important implications for their use in disease modeling and drug testing. We propose that assessment of signaling be added to the indices of phenotypic maturation of hiPSC-CMs.-Jung, G., Fajardo, G., Ribeiro, A. J. S., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D.-Q., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.

  View details for DOI 10.1096/fj.15-280982

  View details for Web of Science ID 000372629100009

• Systematic Characterization of Long Noncoding RNAs Reveals the Contrasting Coordination of Cis- and Trans-Molecular Regulation in Human Fetal and Adult Hearts CIRCULATION-CARDIOVASCULAR GENETICS He, C., Hu, H., Wilson, K. D., Wu, H., Feng, J., Xia, S., Churko, J., Qu, K., Chang, H. Y., Wu, J. C. 2016; 9 (2): 110-118

  Abstract

  -The molecular regulation of heart development is regulated by cis- and trans- factors acting on the genome and epigenome. As a class of important regulatory RNAs, the role of long non-coding RNAs (lncRNAs) in human heart development is still poorly understood. Furthermore, factors that interact with lncRNAs in this process are not well characterized.-Utilizing RNA sequencing, we systematically define the contrasting lncRNA expression patterns between fetal and adult heart. We report that lncRNAs up-regulated in adult versus fetal heart have different sequence features and distributions. For example, the adult heart expresses more sense lncRNAs compared to fetal heart. We also report the co-expression of lncRNAs and neighboring coding genes that have important functions in heart development. Importantly, the regulation of lncRNA expression during fetal to adult heart development appears to be due in part to the coordination of specific developmental epigenetic modifications such as H3K4me1 and H3k4me3. The expression of promoter-associated lncRNAs in adult and fetal heart also appears to be related to these epigenetic states. Finally, transcription factor binding analysis suggests that lncRNAs are directly regulating cardiac gene expression during development.-We provide a systematic analysis of lncRNA control of heart development that gives clues to the roles that specific lncRNAs play in fetal and adult hearts.

  View details for DOI 10.1161/CIRCGENETICS.115.001264

  View details for PubMedID 26896382

• Stem cell reprogramming: A 3D boost. Nature materials Abilez, O. J., Wu, J. C. 2016; 15 (3): 259-261

  View details for DOI 10.1038/nmat4583

  View details for PubMedID 26906959

• Comparison of Magnetic Resonance Imaging and Serum Biomarkers for Detection of Human Pluripotent Stem Cell-Derived Teratomas STEM CELL REPORTS Riegler, J., Ebert, A., Qin, X., Shen, Q., Wang, M., Ameen, M., Kodo, K., Ong, S., Lee, W. H., Lee, G., Neofytou, E., Gold, J. D., Connolly, A. J., Wu, J. C. 2016; 6 (2): 176-187

  Abstract

  The use of cells derived from pluripotent stem cells (PSCs) for regenerative therapies confers a considerable risk for neoplastic growth and teratoma formation. Preclinical and clinical assessment of such therapies will require suitable monitoring strategies to understand and mitigate these risks. Here we generated human-induced pluripotent stem cells (iPSCs), selected clones that continued to express reprogramming factors after differentiation into cardiomyocytes, and transplanted these cardiomyocytes into immunocompromised rat hearts post-myocardial infarction. We compared magnetic resonance imaging (MRI), cardiac ultrasound, and serum biomarkers for their ability to delineate teratoma formation and growth. MRI enabled the detection of teratomas with a volume >8 mm(3). A combination of three plasma biomarkers (CEA, AFP, and HCG) was able to detect teratomas with a volume >17 mm(3) and with a sensitivity of more than 87%. Based on our findings, a combination of serum biomarkers with MRI screening may offer the highest sensitivity for teratoma detection and tracking.

  View details for DOI 10.1016/j.stemcr.2015.12.008

  View details for PubMedID 26777057

• Control of Immune Response to Allogeneic Embryonic Stem Cells by CD3 Antibody-Mediated Operational Tolerance Induction. American journal of transplantation Calderon, D., Prot, M., You, S., Marquet, C., Bellamy, V., Bruneval, P., Valette, F., de Almeida, P., Wu, J. C., Pucéat, M., Menasché, P., Chatenoud, L. 2016; 16 (2): 454-467

  Abstract

  Implantation of embryonic stem cells (ESCs) and their differentiated derivatives into allogeneic hosts triggers an immune response that represents a hurdle to clinical application. We established in autoimmunity and in transplantation that CD3 antibody therapy induces a state of immune tolerance. Promising results have been obtained with CD3 antibodies in the clinic. In this study, we tested whether this strategy can prolong the survival of undifferentiated ESCs and their differentiated derivatives in histoincompatible hosts. Recipients of either mouse ESC-derived embryoid bodies (EBs) or cardiac progenitors received a single short tolerogenic regimen of CD3 antibody. In immunocompetent mice, allogeneic EBs and cardiac progenitors were rejected within 20-25 days. Recipients treated with CD3 antibody showed long-term survival of implanted cardiac progenitors or EBs. In due course, EBs became teratomas, the growth of which was self-limited. Regulatory CD4(+)FoxP3(+) T cells and signaling through the PD1/PDL1 pathway played key roles in the CD3 antibody therapeutic effect. Gene profiling emphasized the importance of TGF-β and the inhibitory T cell coreceptor Tim3 to the observed effect. These results demonstrate that CD3 antibody administered alone promotes prolonged survival of allogeneic ESC derivatives and thus could prove useful for enhancing cell engraftment in the absence of chronic immunosuppression.

  View details for DOI 10.1111/ajt.13477

  View details for PubMedID 26492394

• Concise Review: Review and Perspective of Cell Dosage and Routes of Administration From Preclinical and Clinical Studies of Stem Cell Therapy for Heart Disease STEM CELLS TRANSLATIONAL MEDICINE Golpanian, S., Schulman, I. H., Ebert, R. F., Heldman, A. W., Difede, D. L., Yang, P. C., Wu, J. C., Bolli, R., Perin, E. C., Moye, L., Simari, R. D., Wolf, A., Hare, J. M. 2016; 5 (2): 186-191

  Abstract

  : An important stage in the development of any new therapeutic agent is establishment of the optimal dosage and route of administration. This can be particularly challenging when the treatment is a biologic agent that might exert its therapeutic effects via complex or poorly understood mechanisms. Multiple preclinical and clinical studies have shown paradoxical results, with inconsistent findings regarding the relationship between the cell dose and clinical benefit. Such phenomena can, at least in part, be attributed to variations in cell dosing or concentration and the route of administration (ROA). Although clinical trials of cell-based therapy for cardiovascular disease began more than a decade ago, specification of the optimal dosage and ROA has not been established. The present review summarizes what has been learned regarding the optimal cell dosage and ROA from preclinical and clinical studies of stem cell therapy for heart disease and offers a perspective on future directions.Preclinical and clinical studies on cell-based therapy for cardiovascular disease have shown inconsistent results, in part because of variations in study-specific dosages and/or routes of administration (ROA). Future preclinical studies and smaller clinical trials implementing cell-dose and ROA comparisons are warranted before proceeding to pivotal trials.

  View details for DOI 10.5966/sctm.2015-0101

  View details for PubMedID 26683870

• Engineered heart tissues and induced pluripotent stem cells: Macro- and microstructures for disease modeling, drug screening, and translational studies. Advanced drug delivery reviews Tzatzalos, E., Abilez, O. J., Shukla, P., Wu, J. C. 2016; 96: 234-244

  Abstract

  Engineered heart tissue has emerged as a personalized platform for drug screening. With the advent of induced pluripotent stem cell (iPSC) technology, patient-specific stem cells can be developed and expanded into an indefinite source of cells. Subsequent developments in cardiovascular biology have led to efficient differentiation of cardiomyocytes, the force-producing cells of the heart. iPSC-derived cardiomyocytes (iPSC-CMs) have provided potentially limitless quantities of well-characterized, healthy, and disease-specific CMs, which in turn has enabled and driven the generation and scale-up of human physiological and disease-relevant engineered heart tissues. The combined technologies of engineered heart tissue and iPSC-CMs are being used to study diseases and to test drugs, and in the process, have advanced the field of cardiovascular tissue engineering into the field of precision medicine. In this review, we will discuss current developments in engineered heart tissue, including iPSC-CMs as a novel cell source. We examine new research directions that have improved the function of engineered heart tissue by using mechanical or electrical conditioning or the incorporation of non-cardiomyocyte stromal cells. Finally, we discuss how engineered heart tissue can evolve into a powerful tool for therapeutic drug testing.

  View details for DOI 10.1016/j.addr.2015.09.010

  View details for PubMedID 26428619

• Engineered heart tissues and induced pluripotent stem cells: Macro- and microstructures for disease modeling, drug screening, and translational studies. Advanced drug delivery reviews Tzatzalos, E., Abilez, O. J., Shukla, P., Wu, J. C. 2016; 96: 234-244

  Abstract

  Engineered heart tissue has emerged as a personalized platform for drug screening. With the advent of induced pluripotent stem cell (iPSC) technology, patient-specific stem cells can be developed and expanded into an indefinite source of cells. Subsequent developments in cardiovascular biology have led to efficient differentiation of cardiomyocytes, the force-producing cells of the heart. iPSC-derived cardiomyocytes (iPSC-CMs) have provided potentially limitless quantities of well-characterized, healthy, and disease-specific CMs, which in turn has enabled and driven the generation and scale-up of human physiological and disease-relevant engineered heart tissues. The combined technologies of engineered heart tissue and iPSC-CMs are being used to study diseases and to test drugs, and in the process, have advanced the field of cardiovascular tissue engineering into the field of precision medicine. In this review, we will discuss current developments in engineered heart tissue, including iPSC-CMs as a novel cell source. We examine new research directions that have improved the function of engineered heart tissue by using mechanical or electrical conditioning or the incorporation of non-cardiomyocyte stromal cells. Finally, we discuss how engineered heart tissue can evolve into a powerful tool for therapeutic drug testing.

  View details for DOI 10.1016/j.addr.2015.09.010

  View details for PubMedID 26428619

• Modeling Cardiovascular Diseases with Patient-Specific Human Pluripotent Stem Cell-Derived Cardiomyocytes. Methods in molecular biology (Clifton, N.J.) Burridge, P. W., Diecke, S., Matsa, E., Sharma, A., Wu, H., Wu, J. C. 2016; 1353: 119-130

  Abstract

  The generation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs) provides a source of cells that accurately recapitulate the human cardiac pathophysiology. The application of these cells allows for modeling of cardiovascular diseases, providing a novel understanding of human disease mechanisms and assessment of therapies. Here, we describe a stepwise protocol developed in our laboratory for the generation of hiPSCs from patients with a specific disease phenotype, long-term hiPSC culture and cryopreservation, differentiation of hiPSCs to cardiomyocytes, and assessment of disease phenotypes. Our protocol combines a number of innovative tools that include a codon-optimized mini intronic plasmid (CoMiP), chemically defined culture conditions to achieve high efficiencies of reprogramming and differentiation, and calcium imaging for assessment of cardiomyocyte phenotypes. Thus, this protocol provides a complete guide to use a patient cohort on a testable cardiomyocyte platform for pharmacological drug assessment.

  View details for DOI 10.1007/7651_2015_196

  View details for PubMedID 25690476

• 21st Century Cardio-Oncology: Identifying Cardiac Safety Signals in the Era of Personalized Medicine. JACC. Basic to translational science Sheng, C. C., Amiri-Kordestani, L., Palmby, T., Force, T., Hong, C. C., Wu, J. C., Croce, K., Kim, G., Moslehi, J. 2016; 1 (5): 386–98

  Abstract

  Cardiotoxicity is a well-established complication of oncology therapies. Cardiomyopathy resulting from anthracyclines is a classic example. In the past decade, an explosion of novel cancer therapies, often targeted and more specific than traditional therapies, has revolutionized oncology therapy and dramatically changed cancer prognosis. However, some of these therapies have introduced an assortment of cardiovascular (CV) complications. At times, these devastating outcomes have only become apparent after drug approval and have limited the use of potent therapies. There is a growing need for better testing platforms, both for CV toxicity screening, as well as for elucidating mechanisms of cardiotoxicities of approved cancer therapies. This review discusses the utility of nonclinical models (in vitro, in vivo,&in silico) available and highlights recent advancements in modalities like human stem cell-derived cardiomyocytes for developing more comprehensive cardiotoxicity testing and new means of cardioprotection with targeted anticancer therapies.

  View details for PubMedID 28713868

• Emerging Research Directions in Adult Congenital Heart Disease: A Report From an NHLBI/ACHA Working Group. Journal of the American College of Cardiology Gurvitz, M., Burns, K. M., Brindis, R., Broberg, C. S., Daniels, C. J., Fuller, S. M., Honein, M. A., Khairy, P., Kuehl, K. S., Landzberg, M. J., Mahle, W. T., Mann, D. L., Marelli, A., Newburger, J. W., Pearson, G. D., Starling, R. C., Tringali, G. R., Valente, A. M., Wu, J. C., Califf, R. M. 2016; 67 (16): 1956–64

  Abstract

  Congenital heart disease (CHD) is the most common birth defect, affecting about 0.8% of live births. Advances in recent decades have allowed >85% of children with CHD to survive to adulthood, creating a growing population of adults with CHD. Little information exists regarding survival, demographics, late outcomes, and comorbidities in this emerging group, and multiple barriers impede research in adult CHD. The National Heart, Lung, and Blood Institute and the Adult Congenital Heart Association convened a multidisciplinary working group to identify high-impact research questions in adult CHD. This report summarizes the meeting discussions in the broad areas of CHD-related heart failure, vascular disease, and multisystem complications. High-priority subtopics identified included heart failure in tetralogy of Fallot, mechanical circulatory support/transplantation, sudden cardiac death, vascular outcomes in coarctation of the aorta, late outcomes in single-ventricle disease, cognitive and psychiatric issues, and pregnancy.

  View details for PubMedID 27102511

• Cardiac subtype characterization using all-optical action potential imaging. European heart journal Shukla, P., Wu, J. C. 2016

  View details for DOI 10.1093/eurheartj/ehw380

  View details for PubMedID 27593102

• Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs. PloS one Paik, K. J., Maan, Z. N., Zielins, E. R., Duscher, D., Whittam, A. J., Morrison, S. D., Brett, E. A., Ransom, R. C., Hu, M. S., Wu, J. C., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2016; 11 (3)

  Abstract

  The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

  View details for DOI 10.1371/journal.pone.0150927

  View details for PubMedID 26967994

• Fibrosis of the Neonatal Mouse Heart After Cryoinjury Is Accompanied by Wnt Signaling Activation and Epicardial-to-Mesenchymal Transition. Journal of the American Heart Association Mizutani, M., Wu, J. C., Nusse, R. 2016; 5 (3)

  Abstract

  The adult mammalian heart responds to cardiac injury by formation of persistent fibrotic scar that eventually leads to heart failure. In contrast, the neonatal mammalian heart reacts to injury by the development of transient fibrotic tissue that is eventually replaced by regenerated cardiomyocytes. How fibrosis occurs in the neonatal mammalian heart remains unknown. To start elucidating the molecular underpinnings of neonatal cardiac fibrosis, we investigated Wnt signaling in the neonatal heart after cryoinjury.Using expression of the Wnt target gene Axin2 as an indicator of Wnt/β-catenin signaling activation, we discovered that epicardial cells in the ventricles are responsive to Wnt in the uninjured neonatal heart. Lineage-tracing studies of these Wnt-responsive epicardial cells showed that they undergo epithelial-to-mesenchymal transition and infiltrate into the subepicardial space and exhibit fibroblast phenotypes after injury. In addition, we showed that-similar to adult ischemic injury-neonatal cryoinjury results in activation of Wnt signaling in cardiac fibroblasts near injured areas. Furthermore, through in situ hybridization of all 19 Wnt ligands in injured neonatal hearts, we observed upregulation of Wnt ligands (Wnt2b, Wnt5a, and Wnt9a) that had not been implicated in the adult cardiac injury response.These results demonstrate that cryoinjury in neonatal heart leads to the formation of fibrotic tissue that involves Wnt-responsive epicardial cells undergoing epithelial-to-mesenchymal transition to give rise to fibroblasts and activation of Wnt signaling in resident cardiac fibroblasts.

  View details for DOI 10.1161/JAHA.115.002457

  View details for PubMedID 27068625

• Cytokines profile in hypertensive patients with left ventricular remodeling and dysfunction. Journal of the American Society of Hypertension Kuznetsova, T., Haddad, F., Knez, J., Rosenberg-Hasson, Y., Sung, J., Cauwenberghs, N., Thijs, L., Karakikes, I., Maecker, H., Mahaffey, K. W., Wu, J. C., Staessen, J. A. 2015; 9 (12): 975-984 e3

  Abstract

  There is strong evidence that inflammatory mediators play a key role in the progression to heart failure in patients with systemic hypertension (HTN). The present study aimed to identify a set of cytokines that are associated with early left ventricular (LV) remodeling and dysfunction as captured by echocardiography in patients with HTN in a cross-sectional case-control study nested within the FLEMish study on ENvironment, Genes and Health Outcome. We identified three groups of participants from the cohort: normotensive subjects (normotension; n = 30), HTN with normal LV structure and function (HTN [LV-]; n = 30), and HTN with evidence of adverse LV remodeling (HTN [LV+]; n = 50). We measured cytokines using a 63-plex Luminex platform. Using partial least squares-discriminant analysis, we constructed three latent variables from the measured cytokines that explained 35%-45% of the variance between groups. We identified five common cytokines (interleukin 18, monokine induced by gamma interferon, hepatocyte growth factor, epithelial neutrophil-activating peptide 78, and vascular endothelial growth factor D) with a stable signal which had a major impact on the construction of the latent variables. Among these cytokines, after adjustment for confounders, interleukin 18 remained significantly different between HTN participants with and without LV involvement (P = .02). Moreover, granulocyte-macrophage colony-stimulating factor and leptin showed a consistent upward trend in all HTN patients compared with normotensive subjects. In conclusion, in HTN patients with LV remodeling or/and dysfunction, we identified a set of cytokines strongly associated with LV maladaptation. We also found a distinct profile of inflammatory biomarkers that characterize HTN.

  View details for DOI 10.1016/j.jash.2015.10.003

  View details for PubMedID 26565110

• Cytokines profile in hypertensive patients with left ventricular remodeling and dysfunction JOURNAL OF THE AMERICAN SOCIETY OF HYPERTENSION Kuznetsova, T., Haddad, F., Knez, J., Rosenberg-Hasson, Y., Sung, J., Cauwenberghs, N., Thijs, L., Karakikes, I., Maecker, H., Mahaffey, K. W., Wu, J. C., Staessen, J. A. 2015; 9 (12): 975-984

  Abstract

  There is strong evidence that inflammatory mediators play a key role in the progression to heart failure in patients with systemic hypertension (HTN). The present study aimed to identify a set of cytokines that are associated with early left ventricular (LV) remodeling and dysfunction as captured by echocardiography in patients with HTN in a cross-sectional case-control study nested within the FLEMish study on ENvironment, Genes and Health Outcome. We identified three groups of participants from the cohort: normotensive subjects (normotension; n = 30), HTN with normal LV structure and function (HTN [LV-]; n = 30), and HTN with evidence of adverse LV remodeling (HTN [LV+]; n = 50). We measured cytokines using a 63-plex Luminex platform. Using partial least squares-discriminant analysis, we constructed three latent variables from the measured cytokines that explained 35%-45% of the variance between groups. We identified five common cytokines (interleukin 18, monokine induced by gamma interferon, hepatocyte growth factor, epithelial neutrophil-activating peptide 78, and vascular endothelial growth factor D) with a stable signal which had a major impact on the construction of the latent variables. Among these cytokines, after adjustment for confounders, interleukin 18 remained significantly different between HTN participants with and without LV involvement (P = .02). Moreover, granulocyte-macrophage colony-stimulating factor and leptin showed a consistent upward trend in all HTN patients compared with normotensive subjects. In conclusion, in HTN patients with LV remodeling or/and dysfunction, we identified a set of cytokines strongly associated with LV maladaptation. We also found a distinct profile of inflammatory biomarkers that characterize HTN.

  View details for DOI 10.1016/j.jash.2015.10.003

  View details for Web of Science ID 000367214500014

  View details for PubMedID 26565110

• Poorer Left Ventricular Global Longitudinal Strain and Less Tricuspid Regurgitation Predicts Improvement in Left Ventricular Function Following Transcatheter Aortic Valve Replacement Kim, J. B., Brenner, D., Kobayashi, Y., O'Malley, R., Kobayashi, Y., Dao, C., Thu Vu, Yeung, A., Miller, D., Fischbein, M., Wu, J. C., Haddad, F., Fearon, W. F. ELSEVIER SCIENCE INC. 2015: B263–B264

  View details for DOI 10.1016/j.jacc.2015.08.664

  View details for Web of Science ID 000363329000588

• Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells. Current protocols in human genetics Burridge, P. W., Holmström, A., Wu, J. C. 2015; 87: 21 3 1-15

  Abstract

  Since the first discovery that human pluripotent stem cells (hPS cells) can differentiate to cardiomyocytes, efforts have been made to optimize the conditions under which this process occurs. One of the most effective methodologies to optimize this process is reductionist simplification of the medium formula, which eliminates complex animal-derived components to help reveal the precise underlying mechanisms. Here we describe our latest, cost-effective and efficient methodology for the culture of hPS cells in the pluripotent state using a modified variant of chemically defined E8 medium. We provide exact guidelines for cell handling under these conditions, including non-enzymatic EDTA passaging, which have been optimized for subsequent cardiomyocyte differentiation. We describe in depth the latest version of our monolayer chemically defined small molecule differentiation protocol, including metabolic selection-based cardiomyocyte purification and the addition of triiodothyronine to enhance cardiomyocyte maturation. Finally, we describe a method for the dissociation of hPS cell-derived cardiomyocytes, cryopreservation, and thawing. © 2015 by John Wiley & Sons, Inc.

  View details for DOI 10.1002/0471142905.hg2103s87

  View details for PubMedID 26439715

• Human Engineered Heart Muscles Engraft and Survive Long Term in a Rodent Myocardial Infarction Model. Circulation research Riegler, J., Tiburcy, M., Ebert, A., Tzatzalos, E., Raaz, U., Abilez, O. J., Shen, Q., Kooreman, N. G., Neofytou, E., Chen, V. C., Wang, M., Meyer, T., Tsao, P. S., Connolly, A. J., Couture, L. A., Gold, J. D., Zimmermann, W. H., Wu, J. C. 2015; 117 (8): 720-730

  Abstract

  Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure.Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model.We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring.EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

  View details for DOI 10.1161/CIRCRESAHA.115.306985

  View details for PubMedID 26291556

• Tumor-Promoting Effects of Myeloid-Derived Suppressor Cells Are Potentiated by Hypoxia-Induced Expression of miR-210. Cancer research Noman, M. Z., Janji, B., Hu, S., Wu, J. C., Martelli, F., Bronte, V., Chouaib, S. 2015; 75 (18): 3771-3787

  Abstract

  Myeloid-derived suppressor cells (MDSC) contribute significantly to the malignant characters conferred by hypoxic tumor microenvironments. However, selective biomarkers of MDSC function in this critical setting have not been defined. Here, we report that miR-210 expression is elevated by hypoxia-inducible factor-1α (HIF1α) in MDSC localized to tumors, compared with splenic MDSC from tumor-bearing mice. In tumor MDSC, we determined that HIF1α was bound directly to a transcriptionally active hypoxia-response element in the miR-210 proximal promoter. miR-210 overexpression was sufficient to enhance MDSC-mediated T-cell suppression under normoxic conditions, while targeting hypoxia-induced miR-210 was sufficient to decrease MDSC function against T cells. Mechanistic investigations revealed that miR-210 modulated MDSC function by increasing arginase activity and nitric oxide production, without affecting reactive oxygen species, IL6, or IL10 production or expression of PD-L1. In splenic MDSC, miR-210 regulated Arg1, Cxcl12, and IL16 at the levels of both mRNA and protein, the reversal of which under normoxic conditions decreased T-cell-suppressive effects and IFNγ production. Interestingly, miR-210 overexpression or targeting IL16 or CXCL12 enhanced the immunosuppressive activity of MDSC in vivo, resulting in increased tumor growth. Taken together, these results provide a preclinical rationale to explore miR-210 inhibitory oligonucleotides as adjuvants to boost immunotherapeutic responses in cancer patients. Cancer Res; 75(18); 3771-87. ©2015 AACR.

  View details for DOI 10.1158/0008-5472.CAN-15-0405

  View details for PubMedID 26206559

• A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases. Circulation research Wilson, K. D., Shen, P., Fung, E., Karakikes, I., Zhang, A., Inanloorahatloo, K., Odegaard, J., Sallam, K., Davis, R. W., Lui, G. K., Ashley, E. A., Scharfe, C., Wu, J. C. 2015; 117 (7): 603-611

  Abstract

  Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming.We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost.We used double-stranded probes (complementary long padlock probes), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample.Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.

  View details for DOI 10.1161/CIRCRESAHA.115.306723

  View details for PubMedID 26265630

• Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells STEM CELL RESEARCH Chen, V. C., Ye, J., Shukla, P., Hua, G., Chen, D., Lin, Z., Liu, J., Chai, J., Gold, J., Wu, J., Hsu, D., Couture, L. A. 2015; 15 (2): 365-375

  Abstract

  To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(6) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  View details for DOI 10.1016/j.scr.2015.08.002

  View details for PubMedID 26318718

• Microfluidic Single-Cell Analysis of Transplanted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes After Acute Myocardial Infarction. Circulation Ong, S., Huber, B. C., Lee, W. H., Kodo, K., Ebert, A. D., Ma, Y., Nguyen, P. K., Diecke, S., Chen, W., Wu, J. C. 2015; 132 (8): 762-771

  Abstract

  Human induced pluripotent stem cells (iPSCs) are attractive candidates for therapeutic use, with the potential to replace deficient cells and to improve functional recovery in injury or disease settings. Here, we test the hypothesis that human iPSC-derived cardiomyocytes (iPSC-CMs) can secrete cytokines as a molecular basis to attenuate adverse cardiac remodeling after myocardial infarction.Human iPSCs were generated from skin fibroblasts and differentiated in vitro with a small molecule-based protocol. Troponin(+) iPSC-CMs were confirmed by immunohistochemistry, quantitative polymerase chain reaction, fluorescence-activated cell sorting, and electrophysiological measurements. Afterward, 2×10(6) iPSC-CMs derived from a cell line transduced with a vector expressing firefly luciferase and green fluorescent protein were transplanted into adult NOD/SCID mice with acute left anterior descending artery ligation. Control animals received PBS injection. Bioluminescence imaging showed limited engraftment on transplantation into ischemic myocardium. However, magnetic resonance imaging of animals transplanted with iPSC-CMs showed significant functional improvement and attenuated cardiac remodeling compared with PBS-treated control animals. To understand the underlying molecular mechanism, microfluidic single-cell profiling of harvested iPSC-CMs, laser capture microdissection of host myocardium, and in vitro ischemia stimulation were used to demonstrate that the iPSC-CMs could release significant levels of proangiogenic and antiapoptotic factors in the ischemic microenvironment.Transplantation of human iPSC-CMs into an acute mouse myocardial infarction model can improve left ventricular function and attenuate cardiac remodeling. Because of limited engraftment, most of the effects are possibly explained by paracrine activity of these cells.

  View details for DOI 10.1161/CIRCULATIONAHA.114.015231

  View details for PubMedID 26304668

• RNA Sequencing Analysis Detection of a Novel Pathway of Endothelial Dysfunction in Pulmonary Arterial Hypertension AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Rhodes, C. J., Im, H., Cao, A., Hennigs, J. K., Wang, L., Sa, S., Chen, P., Nickel, N. P., Miyagawa, K., Hopper, R. K., Tojais, N. F., Li, C. G., Gu, M., Spiekerkoetter, E., Xian, Z., Chen, R., Zhao, M., Kaschwich, M., del Rosario, P. A., Bernstein, D., Zamanian, R. T., Wu, J. C., Snyder, M. P., Rabinovitch, M. 2015; 192 (3): 356-366

  Abstract

  Pulmonary arterial hypertension is characterized by endothelial dysregulation, but global changes in gene expression have not been related to perturbations in function.RNA sequencing was utilized to discriminate changes in transcriptomes of endothelial cells cultured from lungs of patients with idiopathic pulmonary arterial hypertension vs. controls and to assess the functional significance of major differentially expressed transcripts.The endothelial transcriptomes from seven control and six idiopathic pulmonary arterial hypertension patients' lungs were analyzed. Differentially expressed genes were related to BMPR2 signaling. Those downregulated were assessed for function in cultured cells, and in a transgenic mouse.Fold-differences in ten genes were significant (p<0.05), four increased and six decreased in patients vs.No patient was mutant for BMPR2. However, knockdown of BMPR2 by siRNA in control pulmonary arterial endothelial cells recapitulated six/ten patient-related gene changes, including decreased collagen IV (COL4A1, COL4A2) and ephrinA1 (EFNA1). Reduction of BMPR2 regulated transcripts was related to decreased β-catenin. Reducing COL4A1, COL4A2 and EFNA1 by siRNA inhibited pulmonary endothelial adhesion, migration and tube formation. In mice null for the EFNA1 receptor, EphA2, vs. controls, VEGF receptor blockade and hypoxia caused more severe pulmonary hypertension, judged by elevated right ventricular systolic pressure, right ventricular hypertrophy and loss of small arteries.The novel relationship between BMPR2 dysfunction and reduced expression of endothelial COL4 and EFNA1 may underlie vulnerability to injury in pulmonary arterial hypertension.

  View details for DOI 10.1164/rccm.201408-1528OC

  View details for PubMedID 26030479

• In vivo long-term serial tracking of living mesenchymal stem cells seeded on bioengineered artificial pulmonary valve scaffold in sheeps Gyongyosi, M., Zlabinger, K., Spriersterbach, H., Balkay, L., Trencsenyi, G., Kertesz, I., Emmert, M., Ong, S. G., Wu, J. C., Hoerstrup, S., LifeValve EU Grant Consortium OXFORD UNIV PRESS. 2015: 616

  View details for Web of Science ID 000361205104392

• Assessment of the Radiation Effects of Cardiac CT Angiography Using Protein and Genetic Biomarkers JACC-CARDIOVASCULAR IMAGING Nguyen, P. K., Lee, W. H., Li, Y. F., Hong, W. X., Hu, S., Chan, C., Liang, G., Nguyen, I., Ong, S., Churko, J., Wang, J., Altman, R. B., Fleischmann, D., Wu, J. C. 2015; 8 (8): 873-884

  View details for DOI 10.1016/j.jcmg.2015.04.016

  View details for Web of Science ID 000359895400001

  View details for PubMedID 26210695

• Large Animal Models of Ischemic Cardiomyopathy: Are They Enough to Bridge the Translational Gap? Journal of nuclear cardiology Nguyen, P. K., Wu, J. C. 2015; 22 (4): 666-672

  View details for DOI 10.1007/s12350-015-0078-7

  View details for PubMedID 25777782

• Human Induced Pluripotent Stem Cells Reveal Mitophagy as an Essential Process Against Diabetic Cardiomyopathy Ong, S., Lee, W., Kodo, K., Wu, H., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2015

  View details for Web of Science ID 000374552800219

• Epigenetic Signatures Contribute to the Superior Endothelial Cell Identity in Human Induced Pluripotent Stem Cells Derived From Endothelial Cells Zhao, M., Hu, S., Srinivasan, R., Jahaniani, F., Shao, N., Knowles, D., Lee, W., Swigut, T., Wysocka, J., Snyder, M., Wu, J. LIPPINCOTT WILLIAMS & WILKINS. 2015

  View details for Web of Science ID 000374552800091

• Epigenetic Regulation of Phosphodiesterases 2A and 3A Underlies Compromised ß-Adrenergic Signaling in an iPSC Model of Dilated Cardiomyopathy. Cell stem cell Wu, H., Lee, J., Vincent, L. G., Wang, Q., Gu, M., Lan, F., Churko, J. M., Sallam, K. I., Matsa, E., Sharma, A., Gold, J. D., Engler, A. J., Xiang, Y. K., Bers, D. M., Wu, J. C. 2015; 17 (1): 89-100

  Abstract

  β-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the β-adrenergic agonist isoproterenol induced mature β-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several β-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired β-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.

  View details for DOI 10.1016/j.stem.2015.04.020

  View details for PubMedID 26095046

• MicroRNA-mediated regulation of differentiation and trans-differentiation in stem cells ADVANCED DRUG DELIVERY REVIEWS Ong, S., Lee, W. H., Kodo, K., Wu, J. C. 2015; 88: 3-15

  Abstract

  MicroRNAs (miRNAs) are key components of a broadly conserved post-transcriptional mechanism that controls gene expression by targeting mRNAs. miRNAs regulate diverse biological processes, including the growth and differentiation of stem cells as well as the regulation of both endogenous tissue repair that has critical implications in the development of regenerative medicine approaches. In this review, we first describe key features of miRNA biogenesis and their role in regulating self-renewal, and then discuss the involvement of miRNAs in the determination of cell fate decisions. We highlight the role of miRNAs in the emergent field of reprogramming and trans-differentiation of somatic cells that could further our understanding of miRNA biology and regenerative medicine applications. Finally, we describe potential techniques for proper delivery of miRNAs in target cells.

  View details for DOI 10.1016/j.addr.2015.04.004

  View details for Web of Science ID 000358628200002

• Manganese-Enhanced Magnetic Resonance Imaging Enables In Vivo Confirmation of Peri-Infarct Restoration Following Stem Cell Therapy in a Porcine Ischemia-Reperfusion Model. Journal of the American Heart Association Dash, R., Kim, P. J., Matsuura, Y., Ikeno, F., Metzler, S., Huang, N. F., Lyons, J. K., Nguyen, P. K., Ge, X., Foo, C. W., McConnell, M. V., Wu, J. C., Yeung, A. C., Harnish, P., Yang, P. C. 2015; 4 (7)

  Abstract

  The exact mechanism of stem cell therapy in augmenting the function of ischemic cardiomyopathy is unclear. In this study, we hypothesized that increased viability of the peri-infarct region (PIR) produces restorative benefits after stem cell engraftment. A novel multimodality imaging approach simultaneously assessed myocardial viability (manganese-enhanced magnetic resonance imaging [MEMRI]), myocardial scar (delayed gadolinium enhancement MRI), and transplanted stem cell engraftment (positron emission tomography reporter gene) in the injured porcine hearts.Twelve adult swine underwent ischemia-reperfusion injury. Digital subtraction of MEMRI-negative myocardium (intrainfarct region) from delayed gadolinium enhancement MRI-positive myocardium (PIR and intrainfarct region) clearly delineated the PIR in which the MEMRI-positive signal reflected PIR viability. Human amniotic mesenchymal stem cells (hAMSCs) represent a unique population of immunomodulatory mesodermal stem cells that restored the murine PIR. Immediately following hAMSC delivery, MEMRI demonstrated an increased PIR viability signal compared with control. Direct PIR viability remained higher in hAMSC-treated hearts for >6 weeks. Increased PIR viability correlated with improved regional contractility, left ventricular ejection fraction, infarct size, and hAMSC engraftment, as confirmed by immunocytochemistry. Increased MEMRI and positron emission tomography reporter gene signal in the intrainfarct region and the PIR correlated with sustained functional augmentation (global and regional) within the hAMSC group (mean change, left ventricular ejection fraction: hAMSC 85±60%, control 8±10%; P<0.05) and reduced chamber dilatation (left ventricular end-diastole volume increase: hAMSC 24±8%, control 110±30%; P<0.05).The positron emission tomography reporter gene signal of hAMSC engraftment correlates with the improved MEMRI signal in the PIR. The increased MEMRI signal represents PIR viability and the restorative potential of the injured heart. This in vivo multimodality imaging platform represents a novel, real-time method of tracking PIR viability and stem cell engraftment while providing a mechanistic explanation of the therapeutic efficacy of cardiovascular stem cells.

  View details for DOI 10.1161/JAHA.115.002044

  View details for PubMedID 26215972

• Hurdles to clinical translation of human induced pluripotent stem cells JOURNAL OF CLINICAL INVESTIGATION Neofytou, E., O'Brien, C. G., Couture, L. A., Wu, J. C. 2015; 125 (7): 2551-2557

  Abstract

  Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss potential solutions that could help overcome them. Furthermore, we discuss the use of allogeneic versus autologous stem cell products, including a review of their respective advantages and disadvantages, major clinical requirements, quality standards, time lines, and costs of clinical grade development.

  View details for DOI 10.1172/JCI80575

  View details for Web of Science ID 000357553300002

  View details for PubMedID 26132109

  View details for PubMedCentralID PMC4563685

• Immunologic Network and Response to Intramyocardial CD34(+) Stem Cell Therapy in Patients With Dilated Cardiomyopathy JOURNAL OF CARDIAC FAILURE Haddad, F., Sever, M., Poglajen, G., Lezaic, L., Yang, P., Maecker, H., Davis, M., Kuznetsova, T., Wu, J. C., Vrtovec, B. 2015; 21 (7): 572-582

  Abstract

  Although stem cell therapy (SCT) is emerging as a potential treatment for patients with dilated cardiomyopathy (DCM), clinical response remains variable. Our objective was to determine whether baseline differences in circulating immunologic and nonimmunologic biomarkers may help to identify patients more likely to respond to intramyocardial injection of CD34(+)-based SCT.We enrolled from January 3, 2011 to March 5, 2012 37 patients with longstanding DCM (left ventricular ejection fraction [LVEF] <40%, New York Heart Association functional class III) who underwent peripheral CD34(+) stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) and collection by means of apheresis. CD34(+) cells were labeled with (99m)Tc-hexamethylpropyleneamine oxime to allow assessment of stem cell retention at 18 hours. Response to SCT was predefined as an increase in LVEF of ≥5% at 3 months. The majority (84%) of patients were male with an overall mean LVEF of 27 ± 7% and a median N-terminal pro-B-type natriuretic peptide (NT-proBNP) level of 2,774 pg/mL. Nineteen patients (51%) were responders to SCT. There was no significant difference between responders and nonresponders regarding to age, sex, baseline LVEF, NT-proBNP levels, or 6-minute walking distance. With the use of a partial least squares (PLS) predictive model, we identified 9 baseline factors that were associated with both stem cell response and stem cell retention (mechanistic validation). Among the baseline factors positively associated with both clinical response and stem cell retention were G-CSF, SDF-1, LIF, MCP-1, and MCP-3. Among baseline factors negatively associated with both clinical response and retention were IL-12p70, FASL, ICAM-1, and GGT. A decrease in G-CSF at 3-month follow-up was also observed in responders compared with nonresponders (P = .02).If further validated, baseline immunologic and nonimmunologic biomarkers may help to identify patients with DCM who are more likely to respond to CD34(+)-based SCT.

  View details for DOI 10.1016/j.cardfail.2015.03.011

  View details for PubMedID 25863169

• Exosomes as potential alternatives to stem cell therapy in mediating cardiac regeneration. Circulation research Ong, S., Wu, J. C. 2015; 117 (1): 7-9

  View details for DOI 10.1161/CIRCRESAHA.115.306593

  View details for PubMedID 26089361

• Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Insights Into Molecular, Cellular, and Functional Phenotypes. Circulation research Karakikes, I., Ameen, M., Termglinchan, V., Wu, J. C. 2015; 117 (1): 80-88

  Abstract

  Disease models are essential for understanding cardiovascular disease pathogenesis and developing new therapeutics. The human induced pluripotent stem cell (iPSC) technology has generated significant enthusiasm for its potential application in basic and translational cardiac research. Patient-specific iPSC-derived cardiomyocytes offer an attractive experimental platform to model cardiovascular diseases, study the earliest stages of human development, accelerate predictive drug toxicology tests, and advance potential regenerative therapies. Harnessing the power of iPSC-derived cardiomyocytes could eliminate confounding species-specific and interpersonal variations and ultimately pave the way for the development of personalized medicine for cardiovascular diseases. However, the predictive power of iPSC-derived cardiomyocytes as a valuable model is contingent on comprehensive and rigorous molecular and functional characterization.

  View details for DOI 10.1161/CIRCRESAHA.117.305365

  View details for PubMedID 26089365

• Finding the rhythm of sudden cardiac death: new opportunities using induced pluripotent stem cell-derived cardiomyocytes. Circulation research Sallam, K., Li, Y., Sager, P. T., Houser, S. R., Wu, J. C. 2015; 116 (12): 1989-2004

  Abstract

  Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death.

  View details for DOI 10.1161/CIRCRESAHA.116.304494

  View details for PubMedID 26044252

• Right Heart Score for Predicting Outcome in Idiopathic, Familial, or Drug- and Toxin-Associated Pulmonary Arterial Hypertension. JACC. Cardiovascular imaging Haddad, F., Spruijt, O. A., Denault, A. Y., Mercier, O., Brunner, N., Furman, D., Fadel, E., Bogaard, H. J., Schnittger, I., Vrtovec, B., Wu, J. C., de Jesus Perez, V., Vonk-Noordegraaf, A., Zamanian, R. T. 2015; 8 (6): 627-638

  Abstract

  This study sought to determine whether a simple score combining indexes of right ventricular (RV) function and right atrial (RA) size would offer good discrimination of outcome in patients with pulmonary arterial hypertension (PAH).Identifying a simple score of outcome could simplify risk stratification of patients with PAH and potentially lead to improved tailored monitoring or therapy.We recruited patients from both Stanford University (derivation cohort) and VU University Medical Center (validation cohort). The composite endpoint for the study was death or lung transplantation. A Cox proportional hazard with bootstrap CI adjustment model was used to determine independent correlates of death or transplantation. A predictive score was developed using the beta coefficients of the multivariable models.For the derivation cohort (n = 95), the majority of patients were female (79%), average age was 43 ± 11 years, mean pulmonary arterial pressure was 54 ± 14 mm Hg, and pulmonary vascular resistance index was 25 ± 12 Wood units m(2). Over an average follow-up of 5 years, the composite endpoint occurred in 34 patients, including 26 deaths and 8 patients requiring lung transplant. On multivariable analysis, RV systolic dysfunction grade (hazard ratio [HR]: 3.4 per grade; 95% confidence interval [CI]: 2.0 to 7.8; p < 0.001), severe RA enlargement (HR: 3.0; 95% CI: 1.3 to 8.1; p = 0.009), and systemic blood pressure <110 mm Hg (HR: 3.3; 95% CI: 1.5 to 9.4; p < 0.001) were independently associated with outcome. A right heart (RH) score constructed on the basis of these 3 parameters compared favorably with the National Institutes of Health survival equation (0.88; 95% CI: 0.79 to 0.94 vs. 0.60; 95% CI: 0.49 to 0.71; p < 0.001) but was not statistically different than the REVEAL (Registry to Evaluate Early and Long-Term PAH Disease Management) score c-statistic of 0.80 (95% CI: 0.69 to 0.88) with p = 0.097. In the validation cohort (n = 87), the RH score remained the strongest independent correlate of outcome.In patients with prevalent PAH, a simple RH score may offer good discrimination of long-term outcome.

  View details for DOI 10.1016/j.jcmg.2014.12.029

  View details for PubMedID 25981508

• Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism. European heart journal Gu, M., Mordwinkin, N. M., Kooreman, N. G., Lee, J., Wu, H., Hu, S., Churko, J. M., Diecke, S., Burridge, P. W., He, C., Barron, F. E., Ong, S., Gold, J. D., Wu, J. C. 2015; 36 (13): 806-816

  Abstract

  High-fat diet-induced obesity (DIO) is a major contributor to type II diabetes and micro- and macro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from obese individuals could potentially limit their therapeutic efficacy for PVD. The aim of this study was to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays.Six-week-old C57Bl/6 mice were fed with a normal or high-fat diet. At 24 weeks, iPSCs were generated from tail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayer approach. In vitro functional analysis revealed that iPSC-ECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusion was observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, N(ω)-nitro-l-arginine methyl ester.This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population.

  View details for DOI 10.1093/eurheartj/ehu411

  View details for PubMedID 25368203

• Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy NATURE COMMUNICATIONS Karakikes, I., Stillitano, F., Nonnenmacher, M., Tzimas, C., Sanoudou, D., Termglinchan, V., Kong, C., Rushing, S., Hansen, J., Ceholski, D., Kolokathis, F., Kremastinos, D., Katoulis, A., Ren, L., Cohen, N., Gho, J. M., Tsiapras, D., Vink, A., Wu, J. C., Asselbergs, F. W., Li, R. A., Hulot, J., Kranias, E. G., Hajjar, R. J. 2015; 6

  View details for DOI 10.1038/ncomms7955

  View details for Web of Science ID 000353704700007

  View details for PubMedID 25923014

• Improved Approach for Chondrogenic Differentiation of Human Induced Pluripotent Stem Cells STEM CELL REVIEWS AND REPORTS Nejadnik, H., Diecke, S., Lenkov, O. D., Chapelin, F., Donig, J., Tong, X., Derugin, N., Chan, R. C., Gaur, A., Yang, F., Wu, J. C., Daldrup-Link, H. E. 2015; 11 (2): 242-253

  Abstract

  Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However, current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation, which is required to generate endodermal, ectodermal, and mesodermal cell lineages. We report a new, straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs, which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1, GAG, and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats, magnetic resonance imaging studies showed gradual engraftment, and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs, which could improve cartilage regeneration outcomes in arthritic joints.

  View details for DOI 10.1007/s12015-014-9581-5

  View details for Web of Science ID 000353149700004

  View details for PubMedID 25578634

• Engraftment of Embryonic Stem Cells and Differentiated Progeny by Host Conditioning with Total Lymphoid Irradiation and Regulatory T Cells CELL REPORTS Pan, Y., Leveson-Gower, D. B., de Almeida, P. E., Pierini, A., Baker, J., Florek, M., Nishikii, H., Kim, B., Ke, R., Wu, J. C., Negrin, R. S. 2015; 10 (11): 1793-1802

  Abstract

  Embryonic stem cells (ESCs) hold promise for the treatment of many medical conditions; however, their utility is limited by immune rejection. The objective of our study is to establish tolerance or promote engraftment of transplanted ESCs as well as mature cell populations derived from ESCs. Luciferase (luc(+))-expressing ESCs were utilized to monitor the survival of the ESCs and differentiated progeny in living recipients. Allogeneic recipients conditioned with fractioned total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS) or TLI plus regulatory T cells (Treg) promoted engraftment of ESC allografts after transplantation. Following these treatments, the engraftment of transplanted terminally differentiated endothelial cells derived from ESCs was also significantly enhanced. Our findings provide clinically translatable strategies of inducing tolerance to adoptively transferred ESCs for cell replacement therapy of medical disorders.

  View details for DOI 10.1016/j.celrep.2015.02.050

  View details for Web of Science ID 000351779600001

• Enabling Consistency in Pluripotent Stern Cell-Derived Products for Research and Development and Clinical Applications Through Material Standards STEM CELLS TRANSLATIONAL MEDICINE French, A., Bravery, C., Smith, J., Chandra, A., Archibald, P., Gold, J. D., Artzi, N., Kim, H., Barker, R. W., Meissner, A., Wu, J. C., Knowles, J. C., Williams, D., Garcia-Cardena, G., Sipp, D., Oh, S., Loring, J. F., Rao, M. S., Reeve, B., Wall, I., Carr, A. J., Bure, K., Stacey, G., Karp, J. M., Snyder, E. Y., Brindley, D. A. 2015; 4 (3): 217-223

  Abstract

  SummaryThere is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources. We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RM generation relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application. Undefined variation and product variability after differentiation to the lineage or cell type of interest impede efficient translation and can obscure the evaluation of clinical safety and efficacy. Moreover, in the absence of a consistent population, data generated from in vitro studies could be unreliable and irreproducible. Efforts to devise approaches and tools that facilitate improved consistency of hPSC-derived products, both as development tools and therapeutic products, will aid translation. Standards exist in both written and physical form; however, because many unknown factors persist in the field, premature written standards could inhibit rather than promote innovation and translation. We focused on the derivation of physical standard RMs. We outline the need for RMs and assess the approaches to in-house RM generation for hPSC-derived products, a critical tool for the analysis and control of product variation that can be applied by researchers and developers. We then explore potential routes for the generation of RMs, including both cellular and noncellular materials and novel methods that might provide valuable tools to measure and account for variation. Multiparametric techniques to identify "signatures" for therapeutically relevant cell types, such as neurons and cardiomyocytes that can be derived from hPSCs, would be of significant utility, although physical RMs will be required for clinical purposes.

  View details for DOI 10.5966/sctm.2014-0233

  View details for PubMedID 25650438

• Variable activation of the DNA damage response pathways in patients undergoing single-photon emission computed tomography myocardial perfusion imaging. Circulation. Cardiovascular imaging Lee, W. H., Nguyen, P., Hu, S., Liang, G., Ong, S., Han, L., Sanchez-Freire, V., Lee, A. S., Vasanawala, M., Segall, G., Wu, J. C. 2015; 8 (2)

  Abstract

  Although single-photon emission computed tomography myocardial perfusion imaging (SPECT MPI) has improved the diagnosis and risk stratification of patients with suspected coronary artery disease, it remains a primary source of low-dose radiation exposure for cardiac patients. To determine the biological effects of low-dose radiation from SPECT MPI, we measured the activation of the DNA damage response pathways using quantitative flow cytometry and single-cell gene expression profiling.Blood samples were collected from patients before and after SPECT MPI (n=63). Overall, analysis of all recruited patients showed no marked differences in the phosphorylation of proteins (H2AX, protein 53, and ataxia telangiectasia mutated) after SPECT. The majority of patients also had either downregulated or unchanged expression in DNA damage response genes at both 24 and 48 hours post-SPECT. Interestingly, a small subset of patients with increased phosphorylation had significant upregulation of genes associated with DNA damage, whereas those with no changes in phosphorylation had significant downregulation or no difference, suggesting that some patients may potentially be more sensitive to low-dose radiation exposure.Our findings showed that SPECT MPI resulted in a variable activation of the DNA damage response pathways. Although only a small subset of patients had increased protein phosphorylation and elevated gene expression postimaging, continued care should be taken to reduce radiation exposure to both the patients and operators.

  View details for DOI 10.1161/CIRCIMAGING.114.002851

  View details for PubMedID 25609688

• Variable activation of the DNA damage response pathways in patients undergoing single-photon emission computed tomography myocardial perfusion imaging. Circulation. Cardiovascular imaging Hee Lee, W., Nguyen, P., Hu, S., Liang, G., Ong, S., Han, L., Sanchez-Freire, V., Lee, A. S., Vasanawala, M., Segall, G., Wu, J. C. 2015; 8 (2): e002851

  Abstract

  Although single-photon emission computed tomography myocardial perfusion imaging (SPECT MPI) has improved the diagnosis and risk stratification of patients with suspected coronary artery disease, it remains a primary source of low-dose radiation exposure for cardiac patients. To determine the biological effects of low-dose radiation from SPECT MPI, we measured the activation of the DNA damage response pathways using quantitative flow cytometry and single-cell gene expression profiling.Blood samples were collected from patients before and after SPECT MPI (n=63). Overall, analysis of all recruited patients showed no marked differences in the phosphorylation of proteins (H2AX, protein 53, and ataxia telangiectasia mutated) after SPECT. The majority of patients also had either downregulated or unchanged expression in DNA damage response genes at both 24 and 48 hours post-SPECT. Interestingly, a small subset of patients with increased phosphorylation had significant upregulation of genes associated with DNA damage, whereas those with no changes in phosphorylation had significant downregulation or no difference, suggesting that some patients may potentially be more sensitive to low-dose radiation exposure.Our findings showed that SPECT MPI resulted in a variable activation of the DNA damage response pathways. Although only a small subset of patients had increased protein phosphorylation and elevated gene expression postimaging, continued care should be taken to reduce radiation exposure to both the patients and operators.

  View details for DOI 10.1161/CIRCIMAGING.114.002851

  View details for PubMedID 25609688

  View details for PubMedCentralID PMC4354894

• Intracoronary Transplantation of CD34(+) Cells Is Associated With Improved Myocardial Perfusion in Patients With Nonischemic Dilated Cardiomyopathy. Journal of cardiac failure Lezaic, L., Socan, A., Poglajen, G., Peitl, P. K., Sever, M., Cukjati, M., Cernelc, P., Wu, J. C., Haddad, F., Vrtovec, B. 2015; 21 (2): 145-152

  Abstract

  We investigated the effects of intracoronary transplantation of CD34(+) cells on myocardial perfusion in patients with nonischemic dilated cardiomyopathy (DCM).We enrolled 21 patients with DCM (left ventricular ejection fraction [LVEF] <40%, New York Heart Association functional class III) who underwent peripheral stem cell mobilization with granulocyte-colony stimulating factor (G-CSF). CD34(+) cells were collected by means of apheresis. Patients underwent myocardial perfusion imaging, and CD34(+) cells were injected in the coronary artery supplying viable segments with reduced myocardial perfusion and regional dysfunction. Myocardial perfusion imaging was repeated 6 months later. Clinical response to stem cell therapy was predefined as a change in LVEF >5%. The majority of patients were men (81%) with an overall mean age 53 ± 9 years, LVEF 25 ± 5%, and 6-minute walking distance 354 ± 71 m. Myocardial perfusion defects at rest were observed in 86% of patients and were more common in the left anterior descending territory (50%). At 6 months' follow-up, there was a significant improvement in rest myocardial perfusion scores (6.3 ± 5.8 vs 3.1 ± 4.3; P < .001), LVEF (25 ± 7% vs 29 ± 8%; P = .005), and 6-minute walking distance (354 ± 71 m vs 404 ± 91 m; P < .001). Responders to stem cell therapy had lower summed rest perfusion score at both baseline (3.2 ± 3.0 vs 9.1 ± 6.3; P = .015) and follow-up (1.0 ± 1.5 vs 5.0 ± 5.1; P = .028).CD34(+) cell transplantation may lead to improved myocardial perfusion in patients with nonischemic DCM. Patients with less severe myocardial perfusion defects at baseline may have an increased likelihood to respond to intracoronary CD34(+) cell transplantation.

  View details for DOI 10.1016/j.cardfail.2014.11.005

  View details for PubMedID 25459687

• Bone Marrow Characteristics Associated With Changes in Infarct Size After STEMI: A Biorepository Evaluation From the CCTRN TIME Trial. Circulation research Schutt, R. C., Trachtenberg, B. H., Cooke, J. P., Traverse, J. H., Henry, T. D., Pepine, C. J., Willerson, J. T., Perin, E. C., Ellis, S. G., Zhao, D. X., Bhatnagar, A., Johnstone, B. H., Lai, D., Resende, M., Ebert, R. F., Wu, J. C., Sayre, S. L., Orozco, A., Zierold, C., Simari, R. D., Moyé, L., Cogle, C. R., Taylor, D. A. 2015; 116 (1): 99-107

  Abstract

  Despite significant interest in bone marrow mononuclear cell (BMC) therapy for ischemic heart disease, current techniques have resulted in only modest benefits. However, selected patients have shown improvements after autologous BMC therapy, but the contributing factors are unclear.The purpose of this study was to identify BMC characteristics associated with a reduction in infarct size after ST-segment-elevation-myocardial infarction.This prospective study comprised patients consecutively enrolled in the CCTRN TIME (Cardiovascular Cell Therapy Research Network Timing in Myocardial Infarction Evaluation) trial who agreed to have their BMCs stored and analyzed at the CCTRN Biorepository. Change in infarct size between baseline (3 days after percutaneous coronary intervention) and 6-month follow-up was measured by cardiac MRI. Infarct-size measurements and BMC phenotype and function data were obtained for 101 patients (mean age, 56.5 years; mean screening ejection fraction, 37%; mean baseline cardiac MRI ejection fraction, 45%). At 6 months, 75 patients (74.3%) showed a reduction in infarct size (mean change, -21.0±17.6%). Multiple regression analysis indicated that infarct size reduction was greater in patients who had a larger percentage of CD31(+) BMCs (P=0.046) and in those with faster BMC growth rates in colony-forming unit Hill and endothelial-colony forming cell functional assays (P=0.033 and P=0.032, respectively).This study identified BMC characteristics associated with a better clinical outcome in patients with segment-elevation-myocardial infarction and highlighted the importance of endothelial precursor activity in regenerating infarcted myocardium. Furthermore, it suggests that for these patients with segment-elevation-myocardial infarction, myocardial repair was more dependent on baseline BMC characteristics than on whether the patient underwent intracoronary BMC transplantation.http://www.clinicaltrials.gov. Unique identifier: NCT00684021.

  View details for DOI 10.1161/CIRCRESAHA.116.304710

  View details for PubMedID 25406300

• Guidelines for translational research in heart failure. Journal of cardiovascular translational research Lara-Pezzi, E., Menasché, P., Trouvin, J. H., Badimón, L., Ioannidis, J. P., Wu, J. C., Hill, J. A., Koch, W. J., De Felice, A. F., de Waele, P., Steenwinckel, V., Hajjar, R. J., Zeiher, A. M. 2015; 8 (1): 3–22

  Abstract

  Heart failure (HF) remains a major cause of death and hospitalization worldwide. Despite medical advances, the prognosis of HF remains poor and new therapeutic approaches are urgently needed. The development of new therapies for HF is hindered by inappropriate or incomplete preclinical studies. In these guidelines, we present a number of recommendations to enhance similarity between HF animal models and the human condition in order to reduce the chances of failure in subsequent clinical trials. We propose different approaches to address safety as well as efficacy of new therapeutic products. We also propose that good practice rules are followed from the outset so that the chances of eventual approval by regulatory agencies increase. We hope that these guidelines will help improve the translation of results from animal models to humans and thereby contribute to more successful clinical trials and development of new therapies for HF.

  View details for DOI 10.1007/s12265-015-9606-8

  View details for PubMedID 25604959

• Manganese-Enhanced Magnetic Resonance Imaging Enables In Vivo Confirmation of Peri-Infarct Restoration Following Stem Cell Therapy in a Porcine Ischemia-Reperfusion Model. Journal of the American Heart Association Dash, R., Kim, P. J., Matsuura, Y., Ikeno, F., Metzler, S., Huang, N. F., Lyons, J. K., Nguyen, P. K., Ge, X., Wong Po Foo, C., McConnell, M. V., Wu, J. C., Yeung, A. C., Harnish, P., Yang, P. C. 2015; 4 (7)

  View details for DOI 10.1161/JAHA.115.002044

  View details for PubMedID 26215972

• Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency. Scientific reports Diecke, S., Lu, J., Lee, J., Termglinchan, V., Kooreman, N. G., Burridge, P. W., Ebert, A. D., Churko, J. M., Sharma, A., Kay, M. A., Wu, J. C. 2015; 5: 8081-?

  Abstract

  The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.

  View details for DOI 10.1038/srep08081

  View details for PubMedID 25628230

• Genetic and Epigenetic Regulation of Human Cardiac Reprogramming and Differentiation in Regenerative Medicine ANNUAL REVIEW OF GENETICS, VOL 49 Burridge, P. W., Sharma, A., Wu, J. C. 2015; 49: 461-484

  Abstract

  Regeneration or replacement of lost cardiomyocytes within the heart has the potential to revolutionize cardiovascular medicine. Numerous methodologies have been used to achieve this aim, including the engraftment of bone marrow- and heart-derived cells as well as the identification of modulators of adult cardiomyocyte proliferation. Recently, the conversion of human somatic cells into induced pluripotent stem cells and induced cardiomyocyte-like cells has transformed potential approaches toward this goal, and the engraftment of cardiac progenitors derived from human embryonic stem cells into patients is now feasible. Here we review recent advances in our understanding of the genetic and epigenetic control of human cardiogenesis, cardiac differentiation, and the induced reprogramming of somatic cells to cardiomyocytes. We also cover genetic programs for inducing the proliferation of endogenous cardiomyocytes and discuss the genetic state of cells used in cardiac regenerative medicine.

  View details for DOI 10.1146/annurev-genet-112414-054911

  View details for Web of Science ID 000367291000020

• Genetic and Epigenetic Regulation of Human Cardiac Reprogramming and Differentiation in Regenerative Medicine. Annual review of genetics Burridge, P. W., Sharma, A., Wu, J. C. 2015; 49: 461–84

  Abstract

  Regeneration or replacement of lost cardiomyocytes within the heart has the potential to revolutionize cardiovascular medicine. Numerous methodologies have been used to achieve this aim, including the engraftment of bone marrow- and heart-derived cells as well as the identification of modulators of adult cardiomyocyte proliferation. Recently, the conversion of human somatic cells into induced pluripotent stem cells and induced cardiomyocyte-like cells has transformed potential approaches toward this goal, and the engraftment of cardiac progenitors derived from human embryonic stem cells into patients is now feasible. Here we review recent advances in our understanding of the genetic and epigenetic control of human cardiogenesis, cardiac differentiation, and the induced reprogramming of somatic cells to cardiomyocytes. We also cover genetic programs for inducing the proliferation of endogenous cardiomyocytes and discuss the genetic state of cells used in cardiac regenerative medicine.

  View details for PubMedID 26631515

• Lift NIH restrictions on chimera research. Science (New York, N.Y.) Sharma, A., Sebastiano, V., Scott, C. T., Magnus, D., Koyano-Nakagawa, N., Garry, D. J., Witte, O. N., Nakauchi, H., Wu, J. C., Weissman, I. L., Wu, S. M. 2015; 350 (6261): 640

  View details for PubMedID 26542560

• Reprogramming and transdifferentiation for cardiovascular development and regenerative medicine: where do we stand? EMBO molecular medicine Ebert, A. D., Diecke, S., Chen, I. Y., Wu, J. C. 2015; 7 (9): 1090-1103

  Abstract

  Heart disease remains a leading cause of mortality and a major worldwide healthcare burden. Recent advances in stem cell biology have made it feasible to derive large quantities of cardiomyocytes for disease modeling, drug development, and regenerative medicine. The discoveries of reprogramming and transdifferentiation as novel biological processes have significantly contributed to this paradigm. This review surveys the means by which reprogramming and transdifferentiation can be employed to generate induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and induced cardiomyocytes (iCMs). The application of these patient-specific cardiomyocytes for both in vitro disease modeling and in vivo therapies for various cardiovascular diseases will also be discussed. We propose that, with additional refinement, human disease-specific cardiomyocytes will allow us to significantly advance the understanding of cardiovascular disease mechanisms and accelerate the development of novel therapeutic options.

  View details for DOI 10.15252/emmm.201504395

  View details for PubMedID 26183451

• Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Circulation research Feaster, T. K., Cadar, A. G., Wang, L., Williams, C. H., Chun, Y. W., Hempel, J., Bloodworth, N., Merryman, W. D., Lim, C. C., Wu, J. C., Knollmann, B. C., Hong, C. C. 2015

  Abstract

  The lack of measurable single cell contractility of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery.To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult cardiac myocytes (CMs).Single hiPSC-CMs were cultured for 5 - 7 days on a 0.4 - 0.8 mm thick mattress of undiluted Matrigel ("mattress hiPSC-CM") and compared to hiPSC-CMs maintained on control substrate (<0.1 mm thick 1:60 diluted matrigel, "control hiPSC-CM"). Compared to control hiPSC-CM, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection was comparable to that of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CM were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CM, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity due to a 2-fold increase in sodium current (INa).The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single cell level, which should be valuable to disease modeling, drug discovery and preclinical cardiotoxicity testing.

  View details for PubMedID 26429802

• Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells. PloS one Yang, W., Liu, Y., Slovik, K. J., Wu, J. C., Duncan, S. A., Rader, D. J., Morrisey, E. E. 2015; 10 (8)

  Abstract

  Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and in vitro spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.

  View details for DOI 10.1371/journal.pone.0134995

  View details for PubMedID 26281015

• Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes to understand and test cardiac calcium handling: A glass half full. Journal of molecular and cellular cardiology Hwang, H. S., Kryshtal, D. O., Feaster, T. K., Sánchez-Freire, V., Zhang, J., Kamp, T. J., Hong, C. C., Wu, J. C., Knollmann, B. C. 2015; 89 (Pt B): 379–80

  View details for PubMedID 26695095

• Extracellular Matrix can Recover the Downregulation of Adhesion Molecules after Cell Detachment and Enhance Endothelial Cell Engraftment. Scientific reports He, N., Xu, Y., Du, W., Qi, X., Liang, L., Wang, Y., Feng, G., Fan, Y., Han, Z., Kong, D., Cheng, Z., Wu, J. C., He, Z., Li, Z. 2015; 5: 10902-?

  Abstract

  The low cell engraftment after transplantation limits the successful application of stem cell therapy and the exact pathway leading to acute donor cell death following transplantation is still unknown. Here we investigated if processes involved in cell preparation could initiate downregulation of adhesion-related survival signals, and further affect cell engraftment after transplantation. Human embryonic stem cell-derived endothelial cells (hESC-ECs) were suspended in PBS or Matrigel and kept at 4 °C. Quantitative RT-PCR analysis was used to test the adhesion and apoptosis genes' expression of hESC-ECs. We demonstrated that cell detachment can cause downregulation of cell adhesion and extracellular matrix (ECM) molecules, but no obvious cell anoikis, a form of apoptosis after cell detachment, was observed. The downregulation of adhesion and ECM molecules could be regained in the presence of Matrigel. Finally, we transplanted hESC-ECs into a mouse myocardial ischemia model. When transplanted with Matrigel, the long-term engraftment of hESC-ECs was increased through promoting angiogenesis and inhibiting apoptosis, and this was confirmed by bioluminescence imaging. In conclusion, ECM could rescue the functional genes expression after cell detached from culture dish, and this finding highlights the importance of increasing stem cell engraftment by mimicking stem cell niches through ECM application.

  View details for DOI 10.1038/srep10902

  View details for PubMedID 26039874

• Comparable calcium handling of human iPSC-derived cardiomyocytes generated by multiple laboratories. Journal of molecular and cellular cardiology Hwang, H. S., Kryshtal, D. O., Feaster, T. K., Sánchez-Freire, V., Zhang, J., Kamp, T. J., Hong, C. C., Wu, J. C., Knollmann, B. C. 2015; 85: 79–88

  Abstract

  Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are being increasingly used to model human heart diseases. hiPSC-CMs generated by earlier aggregation-based methods (i.e., embryoid body) often lack functional sarcoplasmic reticulum (SR) Ca stores characteristic of mature mammalian CMs. Newer monolayer-based cardiac differentiation methods (i.e., Matrigel sandwich or small molecule-based differentiation) produce hiPSC-CMs of high purity and yield, but their Ca handling has not been comprehensively investigated. Here, we studied Ca handling and cytosolic Ca buffering properties of hiPSC-CMs generated independently from multiple hiPSC lines at Stanford University, Vanderbilt University and University of Wisconsin-Madison. hiPSC-CMs were cryopreserved at each university. Frozen aliquots were shipped, recovered from cryopreservation, plated at low density and compared 3-5days after plating with acutely-isolated adult rabbit and mouse ventricular CMs. Although hiPSC-CM cell volume was significantly smaller, cell capacitance to cell volume ratio and cytoplasmic Ca buffering were not different from rabbit-CMs. hiPSC-CMs from all three laboratories exhibited robust L-type Ca currents, twitch Ca transients and caffeine-releasable SR Ca stores comparable to adult CMs. Ca transport by sarcoendoplasmic reticulum Ca ATPase (SERCA) and Na/Ca exchanger (NCX) was similar in all hiPSC-CM lines, but slower compared to rabbit-CMs. However, the relative contribution of SERCA and NCX to Ca transport of hiPSC-CMs was comparable to rabbit-CMs. Ca handling maturity of hiPSC-CMs increased from 15 to 21days post-induction. We conclude that hiPSC-CMs generated independently from multiple iPSC lines using monolayer-based methods can be reproducibly recovered from cryopreservation and exhibit comparable and functional SR Ca handling.

  View details for PubMedID 25982839

• Engraftment of Embryonic Stem Cells and Differentiated Progeny by Host Conditioning with Total Lymphoid Irradiation and Regulatory T Cells. Cell reports Pan, Y., Leveson-Gower, D. B., de Almeida, P. E., Pierini, A., Baker, J., Florek, M., Nishikii, H., Kim, B. S., Ke, R., Wu, J. C., Negrin, R. S. 2015

  Abstract

  Embryonic stem cells (ESCs) hold promise for the treatment of many medical conditions; however, their utility is limited by immune rejection. The objective of our study is to establish tolerance or promote engraftment of transplanted ESCs as well as mature cell populations derived from ESCs. Luciferase (luc(+))-expressing ESCs were utilized to monitor the survival of the ESCs and differentiated progeny in living recipients. Allogeneic recipients conditioned with fractioned total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS) or TLI plus regulatory T cells (Treg) promoted engraftment of ESC allografts after transplantation. Following these treatments, the engraftment of transplanted terminally differentiated endothelial cells derived from ESCs was also significantly enhanced. Our findings provide clinically translatable strategies of inducing tolerance to adoptively transferred ESCs for cell replacement therapy of medical disorders.

  View details for DOI 10.1016/j.celrep.2015.02.050

  View details for PubMedID 25801020

• Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency. Scientific reports Diecke, S., Lu, J., Lee, J., Termglinchan, V., Kooreman, N. G., Burridge, P. W., Ebert, A. D., Churko, J. M., Sharma, A., Kay, M. A., Wu, J. C. 2015; 5: 8081-?

  Abstract

  The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.

  View details for DOI 10.1038/srep08081

  View details for PubMedID 25628230

• Teratoma formation: a tool for monitoring pluripotency in stem cell research. Current protocols in stem cell biology Nelakanti, R. V., Kooreman, N. G., Wu, J. C. 2015; 32: 4A 8 1-4A 8 17

  Abstract

  This unit describes protocols for evaluating the pluripotency of embryonic and induced pluripotent stem cells using a teratoma formation assay. Cells are prepared for injection and transplanted into immunodeficient mice at the gastrocnemius muscle, a site well suited for teratoma growth and surgical access. Teratomas that form from the cell transplants are explanted, fixed in paraformaldehyde, and embedded in paraffin. These preserved samples are sectioned, stained, and analyzed. Pluripotency of a cell line is confirmed by whether the teratoma contains tissues derived from each of the embryonic germ layers: endoderm, mesoderm, and ectoderm. Alternatively, explanted and fixed teratomas can be cryopreserved for immunohistochemistry, which allows for more detailed identification of specific tissue types present in the samples. © 2015 by John Wiley & Sons, Inc.

  View details for DOI 10.1002/9780470151808.sc04a08s32

  View details for PubMedID 25640819

• American Journal of Nuclear Medicine and Molecular Imaging: Editorial Board (2015) e-Century Publishing Corporation. American journal of nuclear medicine and molecular imaging Seto, B., Jacobs, P. M., Mach, R. H., Bulte, J. W., Cai, W., Hong, H., Alavi, A., Wu, J. C. 2015; 5 (2): 212-219

  View details for PubMedID 25973342

  View details for PubMedCentralID PMC4396011

• COCATS 4 Task Force 4: Training in Multimodality Imaging. Journal of the American College of Cardiology Narula, J., Chandrashekhar, Y. S., Dilsizian, V., Garcia, M. J., Kramer, C. M., Malik, S., Ryan, T., Sen, S., Wu, J. C. 2015

  View details for DOI 10.1016/j.jacc.2015.03.024

  View details for PubMedID 25777648

• Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy. Nature communications Karakikes, I., Stillitano, F., Nonnenmacher, M., Tzimas, C., Sanoudou, D., Termglinchan, V., Kong, C., Rushing, S., Hansen, J., Ceholski, D., Kolokathis, F., Kremastinos, D., Katoulis, A., Ren, L., Cohen, N., Gho, J. M., Tsiapras, D., Vink, A., Wu, J. C., Asselbergs, F. W., Li, R. A., Hulot, J., Kranias, E. G., Hajjar, R. J. 2015; 6: 6955-?

  Abstract

  A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca(2+) handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies.

  View details for DOI 10.1038/ncomms7955

  View details for PubMedID 25923014

• [Pyr-1]-Apelin-13 delivery via nano-liposomal encapsulation attenuates pressure overload-induced cardiac dysfunction BIOMATERIALS Serpooshan, V., Sivanesan, S., Huang, X., Mahmoudi, M., Malkovskiy, A. V., Zhao, M., Inayathullah, M., Wagh, D., Zhang, X. J., Metzler, S., Bernstein, D., Wu, J. C., Ruiz-Lozano, P., Rajadas, J. 2015; 37: 289-298

  Abstract

  Nanoparticle-mediated sustained delivery of therapeutics is one of the highly effective and increasingly utilized applications of nanomedicine. Here, we report the development and application of a drug delivery system consisting of polyethylene glycol (PEG)-conjugated liposomal nanoparticles as an efficient in vivo delivery approach for [Pyr1]-apelin-13 polypeptide. Apelin is an adipokine that regulates a variety of biological functions including cardiac hypertrophy and hypertrophy-induced heart failure. The clinical use of apelin has been greatly impaired by its remarkably short half-life in circulation. Here, we investigate whether [Pyr1]-apelin-13 encapsulation in liposome nanocarriers, conjugated with PEG polymer on their surface, can prolong apelin stability in the blood stream and potentiate apelin beneficial effects in cardiac function. Atomic force microscopy and dynamic light scattering were used to assess the structure and size distribution of drug-laden nanoparticles. [Pyr1]-apelin-13 encapsulation in PEGylated liposomal nanocarriers resulted in sustained and extended drug release both in vitro and in vivo. Moreover, intraperitoneal injection of [Pyr1]-apelin-13 nanocarriers in a mouse model of pressure-overload induced heart failure demonstrated a sustainable long-term effect of [Pyr1]-apelin-13 in preventing cardiac dysfunction. We concluded that this engineered nanocarrier system can serve as a delivery platform for treating heart injuries through sustained bioavailability of cardioprotective therapeutics.

  View details for DOI 10.1016/j.biomaterials.2014.08.045

  View details for PubMedID 25443792

• The Presence of Electromechanical Mismatch In Nonischemic Dilated Cardiomyopathy Is Associated With Ventricular Repolarization Instability JOURNAL OF CARDIAC FAILURE Mikuz, U., Poglajen, G., Fister, M., Starc, V., Wu, J. C., Hsia, H., Haddad, F., Vrtovec, B. 2014; 20 (12): 891-898

  Abstract

  We analyzed electromechanical mismatch (EMM) and its relationship to ventricular repolarization in patients with non-ischemic dilated cardiomyopathy (DCM).In 39 DCM patients with LVEF<40% and NHYA class ≥III, electroanatomical mapping was used to quantify areas of EMM. High resolution ECG was used to measure heart rate variability (HRV) and QT variability (QTVI). EMM was present in 22 patients (56%, Group 1), whereas 17 patients presented no mismatched segments (44%, Group 2). The groups did not differ in age (56±10 years in Group 1 vs. 57±7 years in Group 2, P=0.82), sex (male: 82% vs. 94%, P=0.40), LVEF (27±8% vs. 25±6%, P=0.18), or NT-proBNP (2350 pg/ml vs. 2831 pg/ml, P=0.32). Although heart rate and HRV were similar in both groups (rate: 80±20 bpm in Group 1 vs. 74±19 bpm in Group 2, P=0.47; SDNN: 106±79 vs. 88±115, P=0.61), we found significantly higher QTVI values in patients from Group 1 (-1.15±0.46 vs. -1.62±0.51 in Group 2, P=0.005). In patients with ICDs, ventricular arrhythmias recorded within 1 year prior enrollment were more frequent in Group 1 than Group 2 (58% vs. 13%, P=0.02).EMM is present in majority of patients with DCM and is associated with ventricular repolarization instability.clinicaltrials.gov: NCT01350310.

  View details for DOI 10.1016/j.cardfail.2014.10.002

  View details for PubMedID 25305502

• VEGF-C and aortic cardiomyocytes guide coronary artery stem development JOURNAL OF CLINICAL INVESTIGATION Chen, H. I., Poduri, A., Numi, H., Kivela, R., Saharinen, P., McKay, A. S., Raftrey, B., Churko, J., Tian, X., Zhou, B., Wu, J. C., Alitalo, K., Red-Horse, K. 2014; 124 (11): 4899-4914

  Abstract

  Coronary arteries (CAs) stem from the aorta at 2 highly stereotyped locations, deviations from which can cause myocardial ischemia and death. CA stems form during embryogenesis when peritruncal blood vessels encircle the cardiac outflow tract and invade the aorta, but the underlying patterning mechanisms are poorly understood. Here, using murine models, we demonstrated that VEGF-C-deficient hearts have severely hypoplastic peritruncal vessels, resulting in delayed and abnormally positioned CA stems. We observed that VEGF-C is widely expressed in the outflow tract, while cardiomyocytes develop specifically within the aorta at stem sites where they surround maturing CAs in both mouse and human hearts. Mice heterozygous for islet 1 (Isl1) exhibited decreased aortic cardiomyocytes and abnormally low CA stems. In hearts with outflow tract rotation defects, misplaced stems were associated with shifted aortic cardiomyocytes, and myocardium induced ectopic connections with the pulmonary artery in culture. These data support a model in which CA stem development first requires VEGF-C to stimulate vessel growth around the outflow tract. Then, aortic cardiomyocytes facilitate interactions between peritruncal vessels and the aorta. Derangement of either step can lead to mispatterned CA stems. Studying this niche for cardiomyocyte development, and its relationship with CAs, has the potential to identify methods for stimulating vascular regrowth as a treatment for cardiovascular disease.

  View details for DOI 10.1172/JCI77483

  View details for Web of Science ID 000344203300026

• The effects of levosimendan on renal function early after heart transplantation: results from a pilot randomized trial CLINICAL TRANSPLANTATION Knezevic, I., Poglajen, G., Hrovat, E., Oman, A., Pintar, T., Wu, J. C., Vrtovec, B., Haddad, F. 2014; 28 (10): 1105-1111

  Abstract

  We evaluated the effects of a levosimendan (LS)-based strategy compared with standard inotropic therapy on renal function in heart transplantation.Using a randomized study design, 94 patients were assigned to LS-based therapy or standard inotropic support. At the time of transplantation, the groups did not differ in age, gender, heart failure etiology, hemodynamic profile, LVEF, or comorbidities. While there were no differences in serum creatinine (sCr) or eGFR between groups at baseline, patients in the LS group had a greater increase in their relative eGFR (62% vs. 12%, p = 0.002) and a lower incidence of acute kidney injury (AKI) (28% vs. 6%, p = 0.01) during the first post-transplant week. On logistic regression analysis, correlates of AKI were randomization to LS therapy (OR = 0.21 [0.09-0.62], p = 0.01), baseline renal dysfunction (OR = 3.9 [1.1-13.6], p = 0.032), and diabetes mellitus (OR = 4.2 [1.1-16.5], p = 0.038). However, LS was associated with a greater need for additional norepinephrine therapy (40 [85%] vs. 15 [31%], p < 0.001) and a trend toward longer intensive care unit stay (9.5 ± 9.0 d vs. 7.0 ± 6.0 d, p = 0.13).In patients undergoing heart transplantation, levosimendan-based strategy may be associated with better renal function when compared to standard therapy.

  View details for DOI 10.1111/ctr.12424

  View details for Web of Science ID 000344186200007

  View details for PubMedID 25053182

• Cardiac tissue slice transplantation as a model to assess tissue-engineered graft thickness, survival, and function. Circulation Riegler, J., Gillich, A., Shen, Q., Gold, J. D., Wu, J. C. 2014; 130 (11): S77-86

  Abstract

  Cell therapies offer the potential to improve cardiac function after myocardial infarction. Although injection of single-cell suspensions has proven safe, cell retention and survival rates are low. Tissue-engineered grafts allow cell delivery with minimal initial cell loss and mechanical support to the heart. However, graft performance cannot be easily compared, and optimal construct thickness, vascularization, and survival kinetics are unknown.Cardiac tissue slices (CTS) were generated by sectioning mouse hearts (n=40) expressing firefly luciferase and green fluorescent protein into slices of defined size and thickness using a vibrating blade microtome. Bioluminescence imaging of CTS transplanted onto hearts of immunodeficient mice demonstrated survival of ≤30% of transplanted cells. Cardiac slice perfusion was re-established within 3 days, likely through anastomosis of pre-existing vessels with the host vasculature and invasion of vessels from the host. Immunofluorescence showed a peak in cell death 3 days after transplantation and a gradual decline thereafter. MRI revealed preservation of contractile function and an improved ejection fraction 1 month after transplantation of CTS (28±2% CTS versus 22±2% control; P=0.05). Importantly, this effect was specific to CTS because transplantation of skeletal muscle tissue slices led to faster dilative remodeling and higher animal mortality.In summary, this is the first study to use CTS as a benchmark to validate and model tissue-engineered graft studies. CTS transplantation improved cell survival, established reperfusion, and enhanced cardiac function after myocardial infarction. These findings also confirm that dilative remodeling can be attenuated by topical transplantation of CTS but not skeletal muscle tissue grafts.

  View details for DOI 10.1161/CIRCULATIONAHA.113.007920

  View details for PubMedID 25200059

• Cardiac Tissue Slice Transplantation as a Model to Assess Tissue-Engineered Graft Thickness, Survival, and Function CIRCULATION Riegler, J., Gillich, A., Shen, Q., Gold, J. D., Wu, J. C. 2014; 130 (11): S77-?

  Abstract

  Cell therapies offer the potential to improve cardiac function after myocardial infarction. Although injection of single-cell suspensions has proven safe, cell retention and survival rates are low. Tissue-engineered grafts allow cell delivery with minimal initial cell loss and mechanical support to the heart. However, graft performance cannot be easily compared, and optimal construct thickness, vascularization, and survival kinetics are unknown.Cardiac tissue slices (CTS) were generated by sectioning mouse hearts (n=40) expressing firefly luciferase and green fluorescent protein into slices of defined size and thickness using a vibrating blade microtome. Bioluminescence imaging of CTS transplanted onto hearts of immunodeficient mice demonstrated survival of ≤30% of transplanted cells. Cardiac slice perfusion was re-established within 3 days, likely through anastomosis of pre-existing vessels with the host vasculature and invasion of vessels from the host. Immunofluorescence showed a peak in cell death 3 days after transplantation and a gradual decline thereafter. MRI revealed preservation of contractile function and an improved ejection fraction 1 month after transplantation of CTS (28±2% CTS versus 22±2% control; P=0.05). Importantly, this effect was specific to CTS because transplantation of skeletal muscle tissue slices led to faster dilative remodeling and higher animal mortality.In summary, this is the first study to use CTS as a benchmark to validate and model tissue-engineered graft studies. CTS transplantation improved cell survival, established reperfusion, and enhanced cardiac function after myocardial infarction. These findings also confirm that dilative remodeling can be attenuated by topical transplantation of CTS but not skeletal muscle tissue grafts.

  View details for DOI 10.1161/CIRCULATIONAHA.113.007920

  View details for Web of Science ID 000345283800011

• Cross Talk of Combined Gene and Cell Therapy in Ischemic Heart Disease Role of Exosomal MicroRNA Transfer CIRCULATION Ong, S., Lee, W. H., Huang, M., Dey, D., Kodo, K., Sanchez-Freire, V., Gold, J. D., Wu, J. C. 2014; 130 (11): S60-?

  Abstract

  Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs.After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes.In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.

  View details for DOI 10.1161/CIRCULATIONAHA.113.007917

  View details for Web of Science ID 000345283800009

• Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer. Circulation Ong, S., Lee, W. H., Huang, M., Dey, D., Kodo, K., Sanchez-Freire, V., Gold, J. D., Wu, J. C. 2014; 130 (11): S60-9

  Abstract

  Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs.After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes.In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.

  View details for DOI 10.1161/CIRCULATIONAHA.113.007917

  View details for PubMedID 25200057

• Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform. Circulation research Sharma, A., Marceau, C., Hamaguchi, R., Burridge, P. W., Rajarajan, K., Churko, J. M., Wu, H., Sallam, K. I., Matsa, E., Sturzu, A. C., Che, Y., Ebert, A., Diecke, S., Liang, P., Red-Horse, K., Carette, J. E., Wu, S. M., Wu, J. C. 2014; 115 (6): 556-566

  Abstract

  Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

  View details for DOI 10.1161/CIRCRESAHA.115.303810

  View details for PubMedID 25015077

• Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus b3-induced myocarditis and antiviral drug screening platform. Circulation research Sharma, A., Marceau, C., Hamaguchi, R., Burridge, P. W., Rajarajan, K., Churko, J. M., Wu, H., Sallam, K. I., Matsa, E., Sturzu, A. C., Che, Y., Ebert, A., Diecke, S., Liang, P., Red-Horse, K., Carette, J. E., Wu, S. M., Wu, J. C. 2014; 115 (6): 556-566

  Abstract

  Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection.This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy.hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonβ1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonβ1 treatment.This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

  View details for DOI 10.1161/CIRCRESAHA.115.303810

  View details for PubMedID 25015077

  View details for PubMedCentralID PMC4149868

• Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Recapitulates Long QT Phenotype for Drug Testing. Journal of the American College of Cardiology Wang, Y., Liang, P., Lan, F., Wu, H., Lisowski, L., Gu, M., Hu, S., Kay, M. A., Urnov, F. D., Shinnawi, R., Gold, J. D., Gepstein, L., Wu, J. C. 2014; 64 (5): 451-459

  Abstract

  Human induced pluripotent stem cells (iPSCs) play an important role in disease modeling and drug testing. However, the current methods are time-consuming and lack an isogenic control.This study sought to establish an efficient technology to generate human PSC-based disease models with isogenic control.The ion channel genes KCNQ1 and KCNH2 with dominant negative mutations causing long QT syndrome types 1 and 2, respectively, were stably integrated into a safe harbor AAVS1 locus using zinc finger nuclease technology.Patch-clamp recording revealed that the edited iPSC-derived cardiomyocytes (iPSC-CMs) displayed characteristic long QT syndrome phenotype and significant prolongation of the action potential duration compared with the unedited control cells. Finally, addition of nifedipine (L-type calcium channel blocker) or pinacidil (KATP-channel opener) shortened the action potential duration of iPSC-CMs, confirming the validity of isogenic iPSC lines for drug testing in the future.Our study demonstrates that iPSC-CM-based disease models can be rapidly generated by overexpression of dominant negative gene mutants.

  View details for DOI 10.1016/j.jacc.2014.04.057

  View details for PubMedID 25082577

• Effect of human donor cell source on differentiation and function of cardiac induced pluripotent stem cells. Journal of the American College of Cardiology Sanchez-Freire, V., Lee, A. S., Hu, S., Abilez, O. J., Liang, P., Lan, F., Huber, B. C., Ong, S., Hong, W. X., Huang, M., Wu, J. C. 2014; 64 (5): 436-448

  Abstract

  Human-induced pluripotent stem cells (iPSCs) are a potentially unlimited source for generation of cardiomyocytes (iPSC-CMs). However, current protocols for iPSC-CM derivation face several challenges, including variability in somatic cell sources and inconsistencies in cardiac differentiation efficiency.This study aimed to assess the effect of epigenetic memory on differentiation and function of iPSC-CMs generated from somatic cell sources of cardiac versus noncardiac origins.Cardiac progenitor cells (CPCs) and skin fibroblasts from the same donors were reprogrammed into iPSCs and differentiated into iPSC-CMs via embryoid body and monolayer-based differentiation protocols.Differentiation efficiency was found to be higher in CPC-derived iPSC-CMs (CPC-iPSC-CMs) than in fibroblast-derived iPSC-CMs (Fib-iPSC-CMs). Gene expression analysis during cardiac differentiation demonstrated up-regulation of cardiac transcription factors in CPC-iPSC-CMs, including NKX2-5, MESP1, ISL1, HAND2, MYOCD, MEF2C, and GATA4. Epigenetic assessment revealed higher methylation in the promoter region of NKX2-5 in Fib-iPSC-CMs compared with CPC-iPSC-CMs. Epigenetic differences were found to dissipate with increased cell passaging, and a battery of in vitro assays revealed no significant differences in their morphological and electrophysiological properties at early passage. Finally, cell delivery into a small animal myocardial infarction model indicated that CPC-iPSC-CMs and Fib-iPSC-CMs possess comparable therapeutic capabilities in improving functional recovery in vivo.This is the first study to compare differentiation of iPSC-CMs from human CPCs versus human fibroblasts from the same donors. The authors demonstrate that although epigenetic memory improves differentiation efficiency of cardiac versus noncardiac somatic cell sources in vitro, it does not contribute to improved functional outcome in vivo.

  View details for DOI 10.1016/j.jacc.2014.04.056

  View details for PubMedID 25082575

• Effects of Transendocardial CD34(+) Cell Transplantation in Patients With Ischemic Cardiomyopathy CIRCULATION-CARDIOVASCULAR INTERVENTIONS Poglajen, G., Sever, M., Cukjati, M., Cernelc, P., Knezevic, I., Zemljic, G., Haddad, F., Wu, J. C., Vrtovec, B. 2014; 7 (4): 552-559

  Abstract

  We investigated the effects of transendocardial CD34(+) cell transplantation in patients with ischemic cardiomyopathy.In a prospective crossover study, we enrolled 33 patients with ischemic cardiomyopathy with New York Heart Association class III and left ventricular ejection fraction <40%. In phase 1, patients were treated with medical therapy for 6 months. Thereafter, all patients underwent transendocardial CD34(+) cell transplantation. Peripheral blood CD34(+) cells were mobilized by granulocyte colony stimulating factor, collected via apheresis, and injected transendocardially in the areas of hibernating myocardium. Patients were followed up for 6 months after the procedure (phase 2). Two patients died during phase 1 and none during phase 2. The remaining 31 patients were 85% men, aged 57±6 years. In phase 1, we found no change in left ventricular ejection fraction (from 25.2±6.2% to 27.1±6.6%; P=0.23), N-terminal pro B-type natriuretic peptide (from 3322±3411 to 3672±5165 pg/mL; P=0.75) or 6-minute walk distance (from 373±68 to 411±116 m; P=0.17). In contrast, in phase 2 there was an improvement in left ventricular ejection fraction (from 27.1±6.6% to 34.9±10.9%; P=0.001), increase in 6-minute walk distance (from 411±116 to 496±113 m; P=0.001), and a decrease in N-terminal pro B-type natriuretic peptide (from 3672±5165 to 1488±1847 pg/mL; P=0.04). The average number of injected CD34(+) cells was 90.6±7.5×10(6). Higher doses of CD34(+) cells and a more diffuse distribution of transendocardial cell injections were associated with better clinical response.Transendocardial CD34(+) cell transplantation may be associated with improved left ventricular function, decreased N-terminal pro B-type natriuretic peptide levels, and better exercise capacity in patients with ischemic cardiomyopathy. These effects seem to be particularly pronounced in patients receiving diffusely distributed cell injections and high-dose cell therapy.http://www.clinicaltrials.gov. Unique identifier: NCT01350310.

  View details for DOI 10.1161/CIRCINTERVENTIONS.114.001436

  View details for PubMedID 25097199

• A human pluripotent stem cell surface N-glycoproteome resource reveals markers, extracellular epitopes, and drug targets. Stem cell reports Boheler, K. R., Bhattacharya, S., Kropp, E. M., Chuppa, S., Riordon, D. R., Bausch-Fluck, D., Burridge, P. W., Wu, J. C., Wersto, R. P., Chan, G. C., Rao, S., Wollscheid, B., Gundry, R. L. 2014; 3 (1): 185-203

  Abstract

  Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures.

  View details for DOI 10.1016/j.stemcr.2014.05.002

  View details for PubMedID 25068131

• Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy. Science translational medicine Asimaki, A., Kapoor, S., Plovie, E., Karin Arndt, A., Adams, E., Liu, Z., James, C. A., Judge, D. P., Calkins, H., Churko, J., Wu, J. C., MacRae, C. A., Kléber, A. G., Saffitz, J. E. 2014; 6 (240): 240ra74-?

  Abstract

  Arrhythmogenic cardiomyopathy (ACM) is characterized by frequent cardiac arrhythmias. To elucidate the underlying mechanisms and discover potential chemical modifiers, we created a zebrafish model of ACM with cardiac myocyte-specific expression of the human 2057del2 mutation in the gene encoding plakoglobin. A high-throughput screen identified SB216763 as a suppressor of the disease phenotype. Early SB216763 therapy prevented heart failure and reduced mortality in the fish model. Zebrafish ventricular myocytes that expressed 2057del2 plakoglobin exhibited 70 to 80% reductions in INa and IK1 current densities, which were normalized by SB216763. Neonatal rat ventricular myocytes that expressed 2057del2 plakoglobin recapitulated pathobiological features seen in patients with ACM, all of which were reversed or prevented by SB216763. The reverse remodeling observed with SB216763 involved marked subcellular redistribution of plakoglobin, connexin 43, and Nav1.5, but without changes in their total cellular content, implicating a defect in protein trafficking to intercalated discs. In further support of this mechanism, we observed SB216763-reversible, abnormal subcellular distribution of SAP97 (a protein known to mediate forward trafficking of Nav1.5 and Kir2.1) in rat cardiac myocytes expressing 2057del2 plakoglobin and in cardiac myocytes derived from induced pluripotent stem cells from two ACM probands with plakophilin-2 mutations. These observations pinpoint aberrant trafficking of intercalated disc proteins as a central mechanism in ACM myocyte injury and electrical abnormalities.

  View details for DOI 10.1126/scitranslmed.3008008

  View details for PubMedID 24920660

• Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy. Science translational medicine Asimaki, A., Kapoor, S., Plovie, E., Karin Arndt, A., Adams, E., Liu, Z., James, C. A., Judge, D. P., Calkins, H., Churko, J., Wu, J. C., MacRae, C. A., Kléber, A. G., Saffitz, J. E. 2014; 6 (240): 240ra74-?

  View details for DOI 10.1126/scitranslmed.3008008

  View details for PubMedID 24920660

• Human Stem Cells for Modeling Heart Disease and for Drug Discovery SCIENCE TRANSLATIONAL MEDICINE Matsa, E., Burridge, P. W., Wu, J. C. 2014; 6 (239)

  Abstract

  A major research focus in the field of cardiovascular medicine is the prospect of using stem cells and progenitor cells for cardiac regeneration. With the advent of induced pluripotent stem cell (iPSC) technology, major efforts are also underway to use iPSCs to model heart disease, to screen for new drugs, and to test candidate drugs for cardiotoxicity. Here, we discuss recent advances in the exciting fields of stem cells and cardiovascular disease.

  View details for DOI 10.1126/scitranslmed.3008921

  View details for Web of Science ID 000337184200004

• Human-induced pluripotent stem cell models of inherited cardiomyopathies. Current opinion in cardiology Karakikes, I., Termglinchan, V., Wu, J. C. 2014; 29 (3): 214-219

  Abstract

  This article provides an overview of the latest advances in in-vitro modeling of inherited cardiomyopathies using human-induced pluripotent stem cells (iPSCs).Inherited cardiomyopathies have been recently modeled by generating iPSCs from patients harboring mutations in genes associated with the pathogenesis of hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy/dysplasia.Patient-specific iPSCs and their differentiated cardiomyocytes (induced pluripotent stem cell-derived cardiomyocytes) now provide a novel model to study the underlying molecular mechanism of the pathogenesis of familial cardiomyopathies as well as for in-vitro drug screening and drug discovery.

  View details for DOI 10.1097/HCO.0000000000000049

  View details for PubMedID 24576884

• Development of a Scalable Suspension Culture for Cardiac Differentiation From Human Pluripotent Stem Cells 17th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT) Chen, V. C., Ye, J., Hua, G., Liu, F., Chen, D., Lin, B., Chai, J., Shukla, P., Wu, J., Hsu, D., Couture, L. A. NATURE PUBLISHING GROUP. 2014: S204–S205

  View details for Web of Science ID 000337231300522

• Stem Cell Imaging: From Bench to Bedside CELL STEM CELL Nguyen, P. K., Riegler, J., Wu, J. C. 2014; 14 (4): 431-444

  Abstract

  Although cellular therapies hold great promise for the treatment of human disease, results from several initial clinical trials have not shown a level of efficacy required for their use as a first line therapy. Here we discuss how in vivo molecular imaging has helped identify barriers to clinical translation and potential strategies that may contribute to successful transplantation and improved outcomes, with a focus on cardiovascular and neurological diseases. We conclude with a perspective on the future role of molecular imaging in defining safety and efficacy for clinical implementation of stem cell therapies.

  View details for DOI 10.1016/j.stem.2014.03.009

  View details for Web of Science ID 000334766400008

  View details for PubMedID 24702995

• Relationship between Echocardiographic and Magnetic Resonance Derived Measures of Right Ventricular Size and Function in Patients with Pulmonary Hypertension. Journal of the American Society of Echocardiography Shiran, H., Zamanian, R. T., McConnell, M. V., Liang, D. H., Dash, R., Heidary, S., Sudini, N. L., Wu, J. C., Haddad, F., Yang, P. C. 2014; 27 (4): 405-412

  Abstract

  Transthoracic echocardiographic (TTE) imaging is the mainstay of clinical practice for evaluating right ventricular (RV) size and function, but its accuracy in patients with pulmonary hypertension has not been well validated.Magnetic resonance imaging (MRI) and TTE images were retrospectively reviewed in 40 consecutive patients with pulmonary hypertension. RV and left ventricular volumes and ejection fractions were calculated using MRI. TTE areas and indices of RV ejection fraction (RVEF) were compared.The average age was 42 ± 12 years, with a majority of women (85%). There was a wide range of mean pulmonary arterial pressures (27-81 mm Hg) and RV end-diastolic volumes (111-576 mL), RVEFs (8%-67 %), and left ventricular ejection fractions (26%-72%) by MRI. There was a strong association between TTE and MRI-derived parameters: RV end-diastolic area (by TTE imaging) and RV end-diastolic volume (by MRI), R(2) = 0.78 (P < .001); RV fractional area change by TTE imaging and RVEF by MRI, R(2) = 0.76 (P < .001); and tricuspid annular plane systolic excursion by TTE imaging and RVEF by MRI, R(2) = 0.64 (P < .001). By receiver operating characteristic curve analysis, an RV fractional area change < 25% provided excellent discrimination of moderate systolic dysfunction (RVEF < 35%), with an area under the curve of 0.97 (P < .001). An RV end-diastolic area index of 18 cm(2)/m(2) provided excellent discrimination for moderate RV enlargement (area under the curve, 0.89; P < .001).Echocardiographic estimates of RV volume (by RV end-diastolic area) and function (by RV fractional area change and tricuspid annular plane systolic excursion) offer good approximations of RV size and function in patients with pulmonary hypertension and allow the accurate discrimination of normal from abnormal.

  View details for DOI 10.1016/j.echo.2013.12.011

  View details for PubMedID 24444659

• Myocardial Scar Burden and Response to Stem Cell Therapyin Heart Failure Patients Zemljic, G., Poglajen, G., Mikuz, U., Sever, M., Frljak, S., Mali, P., Haddad, E., Wu, J. C., Vrtovec, B. ELSEVIER SCIENCE INC. 2014: S275

  View details for DOI 10.1016/j.healun.2014.01.730

  View details for Web of Science ID 000333866700753

• DIABETES IMPAIRS CD34+STEM CELL MOBILIZATION IN PATIENTS WITH CHRONIC HEART FAILURE Poglajen, G., Sever, M., Mali, P., Haddad, F., Wu, J. C., Vrtovec, B. ELSEVIER SCIENCE INC. 2014: A863

  View details for DOI 10.1016/S0735-1097(14)60863-9

  View details for Web of Science ID 000359579101521

• MYOCARDIAL SCAR BURDEN DOES NOT PREDICT CLINICAL RESPONSE TO CD34+STEM CELL TRANSPLANTATION IN PATIENTS WITH CHRONIC HEART FAILURE Zemljic, G., Poglajen, G., Mikuz, U., Sever, M., Frljak, S., Mali, P., Haddad, F., Wu, J. C. ELSEVIER SCIENCE INC. 2014: A927

  View details for DOI 10.1016/S0735-1097(14)60927-X

  View details for Web of Science ID 000359579101585

• Modeling Inherited Cardiac Disorders - A Cell Is Worth a Thousand Genes CIRCULATION JOURNAL Sallam, K., Kodo, K., Wu, J. C. 2014; 78 (4): 784-794

  View details for DOI 10.1253/circj.CJ-14-0182

  View details for Web of Science ID 000333792900001

• Modeling inherited cardiac disorders. Circulation journal Sallam, K., Kodo, K., Wu, J. C. 2014; 78 (4): 784-794

  Abstract

  Advances in the understanding and treatment of cardiac disorders have been thwarted by the inability to study beating human cardiac cells in vitro. Induced pluripotent stem cells (iPSCs) bypass this hurdle by enabling the creation of patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). These cells provide a unique platform to study cardiac diseases in vitro, especially hereditary cardiac conditions. To date, iPSC-CMs have been used to successfully model arrhythmic disorders, showing excellent recapitulation of cardiac channel function and electrophysiologic features of long QT syndrome types 1, 2, 3, and 8, and catecholaminergic polymorphic ventricular tachycardia (CPVT). Similarly, iPSC-CM models of dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) have shown robust correlation of predicted morphologic, contractile, and electrical phenotypes. In addition, iPSC-CMs have shown some features of the respective phenotypes for arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), LEOPARD syndrome, Pompe's disease, and Friedriech's ataxia. In this review, we examine the progress of utilizing iPSC-CMs as a model for cardiac conditions and analyze the potential for the platform in furthering the biology and treatment of cardiac disorders.  

  View details for PubMedID 24632794

• Stem cells and cardiovascular drug development--reply. JAMA Mordwinkin, N. M., Lee, A. S., Wu, J. C. 2014; 311 (10): 1070-1071

  View details for DOI 10.1001/jama.2014.634

  View details for PubMedID 24618976

• Construction and validation of nano gold tripods for molecular imaging of living subjects. Journal of the American Chemical Society Cheng, K., Kothapalli, S., Liu, H., Koh, A. L., Jokerst, J. V., Jiang, H., Yang, M., Li, J., Levi, J., Wu, J. C., Gambhir, S. S., Cheng, Z. 2014; 136 (9): 3560-3571

  Abstract

  Anisotropic colloidal hybrid nanoparticles exhibit superior optical and physical properties compared to their counterparts with regular architectures. We herein developed a controlled, stepwise strategy to build novel, anisotropic, branched, gold nanoarchitectures (Au-tripods) with predetermined composition and morphology for bioimaging. The resultant Au-tripods with size less than 20 nm showed great promise as contrast agents for in vivo photoacoustic imaging (PAI). We further identified Au-tripods with two possible configurations as high-absorbance nanomaterials from various gold multipods using a numerical simulation analysis. The PAI signals were linearly correlated with their concentrations after subcutaneous injection. The in vivo biodistribution of Au-tripods favorable for molecular imaging was confirmed using small animal positron emission tomography (PET). Intravenous administration of cyclic Arg-Gly-Asp-d-Phe-Cys (RGDfC) peptide conjugated Au-tripods (RGD-Au-tripods) to U87MG tumor-bearing mice showed PAI contrasts in tumors almost 3-fold higher than for the blocking group. PAI results correlated well with the corresponding PET images. Quantitative biodistribution data revealed that 7.9% ID/g of RGD-Au-tripods had accumulated in the U87MG tumor after 24 h post-injection. A pilot mouse toxicology study confirmed that no evidence of significant acute or systemic toxicity was observed in histopathological examination. Our study suggests that Au-tripods can be reliably synthesized through stringently controlled chemical synthesis and could serve as a new generation of platform with high selectivity and sensitivity for multimodality molecular imaging.

  View details for DOI 10.1021/ja412001e

  View details for PubMedID 24495038

• Tracking gene and cell fate for therapeutic gain NATURE MATERIALS Kooreman, N. G., Ransohoff, J. D., Wu, J. C. 2014; 13 (2): 106-109

  View details for DOI 10.1038/nmat3868

  View details for Web of Science ID 000330182700006

  View details for PubMedID 24452344

• Predicting the Future With Stem Cells CIRCULATION Mohsin, S., Wu, J. C., Sussman, M. A. 2014; 129 (2): 136-138

  View details for DOI 10.1161/CIRCULATIONAHA.113.007045

  View details for PubMedID 24249719

• Cardiac stem cell biology: glimpse of the past, present, and future. Circulation research Matsa, E., Sallam, K., Wu, J. C. 2014; 114 (1): 21-27

  Abstract

  Cardiac regeneration strategies and de novo generation of cardiomyocytes have long been significant areas of research interest in cardiovascular medicine. In this review, we outline a variety of common cell sources and methods used to regenerate cardiomyocytes and highlight the important role that key Circulation Research articles have played in this flourishing field.

  View details for DOI 10.1161/CIRCRESAHA.113.302895

  View details for PubMedID 24385505

• Multi-cellular interactions sustain long-term contractility of human pluripotent stem cell-derived cardiomyocytes. American journal of translational research Burridge, P. W., Metzler, S. A., Nakayama, K. H., Abilez, O. J., Simmons, C. S., Bruce, M. A., Matsuura, Y., Kim, P., Wu, J. C., Butte, M., Huang, N. F., Yang, P. C. 2014; 6 (6): 724-735

  Abstract

  Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca(2+) ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases.

  View details for PubMedID 25628783

• In vivo tomographic cardiac imaging: positron emission tomography and magnetic resonance imaging MANUAL OF RESEARCH TECHNIQUES IN CARDIOVASCULAR MEDICINE Huber, B. C., Nguyen, P. K., Wu, J. C., Ardehali, H., Bolli, R., Losordo, D. W. 2014: 287–94

  View details for Web of Science ID 000360465900035

• VEGF-C and aortic cardiomyocytes guide coronary artery stem development. The Journal of clinical investigation Chen, H. I., Poduri, A., Numi, H., Kivela, R., Saharinen, P., McKay, A. S., Raftrey, B., Churko, J., Tian, X., Zhou, B., Wu, J. C., Alitalo, K., Red-Horse, K. 2014

  Abstract

  Coronary arteries (CAs) stem from the aorta at 2 highly stereotyped locations, deviations from which can cause myocardial ischemia and death. CA stems form during embryogenesis when peritruncal blood vessels encircle the cardiac outflow tract and invade the aorta, but the underlying patterning mechanisms are poorly understood. Here, using murine models, we demonstrated that VEGF-C-deficient hearts have severely hypoplastic peritruncal vessels, resulting in delayed and abnormally positioned CA stems. We observed that VEGF-C is widely expressed in the outflow tract, while cardiomyocytes develop specifically within the aorta at stem sites where they surround maturing CAs in both mouse and human hearts. Mice heterozygous for islet 1 (Isl1) exhibited decreased aortic cardiomyocytes and abnormally low CA stems. In hearts with outflow tract rotation defects, misplaced stems were associated with shifted aortic cardiomyocytes, and myocardium induced ectopic connections with the pulmonary artery in culture. These data support a model in which CA stem development first requires VEGF-C to stimulate vessel growth around the outflow tract. Then, aortic cardiomyocytes facilitate interactions between peritruncal vessels and the aorta. Derangement of either step can lead to mispatterned CA stems. Studying this niche for cardiomyocyte development, and its relationship with CAs, has the potential to identify methods for stimulating vascular regrowth as a treatment for cardiovascular disease.

  View details for DOI 10.1172/JCI77483

  View details for PubMedID 25271623

• Short Hairpin RNA Gene Silencing of Prolyl Hydroxylase-2 with a Minicircle Vector Improves Neovascularization of Hindlimb Ischemia HUMAN GENE THERAPY Lijkwan, M. A., Hellingman, A. A., Bos, E. J., van der Bogt, K. E., Huang, M., Kooreman, N. G., de Vries, M. R., Peters, H. A., Robbins, R. C., Hamming, J. F., Quax, P. H., Wu, J. C. 2014; 25 (1): 41-49

  Abstract

  Abstract In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.

  View details for DOI 10.1089/hum.2013.110

  View details for PubMedID 24090375

• Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions. PloS one Durruthy-Durruthy, J., Briggs, S. F., Awe, J., Ramathal, C. Y., Karumbayaram, S., Lee, P. C., Heidmann, J. D., Clark, A., Karakikes, I., Loh, K. M., Wu, J. C., Hoffman, A. R., Byrne, J., Reijo Pera, R. A., Sebastiano, V. 2014; 9 (4)

  View details for DOI 10.1371/journal.pone.0094231

  View details for PubMedID 24718618

• Second Generation Codon Optimized Minicircle (CoMiC) for Nonviral Reprogramming of Human Adult Fibroblasts. Methods in molecular biology (Clifton, N.J.) Diecke, S., Lisowski, L., Kooreman, N. G., Wu, J. C. 2014; 1181: 1-13

  Abstract

  The ability to induce pluripotency in somatic cells is one of the most important scientific achievements in the fields of stem cell research and regenerative medicine. This technique allows researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful tool for disease modeling and drug screening approaches. However, using viruses for the delivery of reprogramming genes and transcription factors may result in integration into the host genome and cause random mutations within the target cell, thus limiting the use of these cells for downstream applications. To overcome this limitation, various non-integrating techniques, including Sendai virus, mRNA, minicircle, and plasmid-based methods, have recently been developed. Utilizing a newly developed codon optimized 4-in-1 minicircle (CoMiC), we were able to reprogram human adult fibroblasts using chemically defined media and without the need for feeder cells.

  View details for DOI 10.1007/978-1-4939-1047-2_1

  View details for PubMedID 25070322

• Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions. PloS one Durruthy-Durruthy, J., Briggs, S. F., Awe, J., Ramathal, C. Y., Karumbayaram, S., Lee, P. C., Heidmann, J. D., Clark, A., Karakikes, I., Loh, K. M., Wu, J. C., Hoffman, A. R., Byrne, J., Reijo Pera, R. A., Sebastiano, V. 2014; 9 (4)

  Abstract

  Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

  View details for DOI 10.1371/journal.pone.0094231

  View details for PubMedID 24718618

• On-line visualization of ischemic burden during repetitive ischemia/reperfusion. JACC. Cardiovascular imaging Pavo, N., Emmert, M. Y., Giricz, Z., Varga, Z. V., Ankersmit, H. J., Maurer, G., Hoerstrup, S. P., Ferdinandy, P., Wu, J. C., Gyöngyösi, M. 2014; 7 (9): 956–58

  View details for PubMedID 25212802

• High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry. Journal of visualized experiments : JoVE Bhattacharya, S., Burridge, P. W., Kropp, E. M., Chuppa, S. L., Kwok, W., Wu, J. C., Boheler, K. R., Gundry, R. L. 2014: 52010-?

  Abstract

  There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle "in a dish" for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

  View details for DOI 10.3791/52010

  View details for PubMedID 25286293

• Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance. Nature communications de Almeida, P. E., Meyer, E. H., Kooreman, N. G., Diecke, S., Dey, D., Sanchez-Freire, V., Hu, S., Ebert, A., Odegaard, J., Mordwinkin, N. M., Brouwer, T. P., Lo, D., Montoro, D. T., Longaker, M. T., Negrin, R. S., Wu, J. C. 2014; 5: 3903-?

  View details for DOI 10.1038/ncomms4903

  View details for PubMedID 24875164

• Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance. Nature communications de Almeida, P. E., Meyer, E. H., Kooreman, N. G., Diecke, S., Dey, D., Sanchez-Freire, V., Hu, S., Ebert, A., Odegaard, J., Mordwinkin, N. M., Brouwer, T. P., Lo, D., Montoro, D. T., Longaker, M. T., Negrin, R. S., Wu, J. C. 2014; 5: 3903-?

  Abstract

  The exact nature of the immune response elicited by autologous-induced pluripotent stem cell (iPSC) progeny is still not well understood. Here we show in murine models that autologous iPSC-derived endothelial cells (iECs) elicit an immune response that resembles the one against a comparable somatic cell, the aortic endothelial cell (AEC). These cells exhibit long-term survival in vivo and prompt a tolerogenic immune response characterized by elevated IL-10 expression. In contrast, undifferentiated iPSCs elicit a very different immune response with high lymphocytic infiltration and elevated IFN-γ, granzyme-B and perforin intragraft. Furthermore, the clonal structure of infiltrating T cells from iEC grafts is statistically indistinguishable from that of AECs, but is different from that of undifferentiated iPSC grafts. Taken together, our results indicate that the differentiation of iPSCs results in a loss of immunogenicity and leads to the induction of tolerance, despite expected antigen expression differences between iPSC-derived versus original somatic cells.

  View details for DOI 10.1038/ncomms4903

  View details for PubMedID 24875164

• Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening STEM CELL RESEARCH & THERAPY Sharma, A., Wu, J. C., Wu, S. M. 2013; 4

  View details for DOI 10.1186/scrt380

  View details for Web of Science ID 000329186900001

  View details for PubMedID 24476344

• Elevated Right Ventricular Operant Diastolic Elastance Strongly Predicts Increased Risk of Mortality Following Heart Transplantation Rhee, J., Sudini, N. L., Pham, M., Fearon, W., Lee, D., Beygui, R. E., Montoya, J. G., Wu, J. C., Vrtovec, B., Hunt, S. A., Haddad, F. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000332162907319

• Induced Pluripotent Stem Cell-Derived Endothelial Cells From Diet-Induced Obesity Mice Exhibit Decreased Vascular Function in a Murine Model of Hindlimb Ischemia Mordwinkin, N. M., Gu, M., Kooreman, N. G., Hu, S., Churko, J. M., Diecke, S., Burridge, P. W., He, C., Lee, J., Gold, J. D., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000332162906118

• In Vivo Crosstalk Between Transplanted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Host Myocardial Cells Ong, S., Huber, B. C., Ebert, A., Kodo, K., Gold, J. D., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000332162906220

• Human Cardiomyocytes Generated From Muliple iPS Cell Lines Exhibit Mature Calcium Handling Hwang, H., Kryshtal, D. O., Sanchez-Freire, V., Feaster, T. K., Zhang, J., Kamp, T. J., Hong, C. C., Wu, J. C., Knollmann, B. C. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000332162906253

• Effects of Transendocardial CD34+Stem Cell Transplantation in Patients With Ischemic Cardiomyopathy Poglajen, G., Sever, M., Mali, P., Haddad, F., Wu, J. C., Vrtovec, B. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000332162906350

• Patient-specific stem cells and cardiovascular drug discovery. JAMA-the journal of the American Medical Association Mordwinkin, N. M., Lee, A. S., Wu, J. C. 2013; 310 (19): 2039-2040

  View details for DOI 10.1001/jama.2013.282409

  View details for PubMedID 24240927

• Costimulation-Adhesion Blockade is Superior to Cyclosporine A and Prednisone Immunosuppressive Therapy for Preventing Rejection of Differentiated Human Embryonic Stem Cells Following Transplantation. Stem cells Huber, B. C., Ransohoff, J. D., Ransohoff, K. J., Riegler, J., Ebert, A., Kodo, K., Gong, Y., Sanchez-Freire, V., Dey, D., Kooreman, N. G., Diecke, S., Zhang, W. Y., Odegaard, J., Hu, S., Gold, J. D., Robbins, R. C., Wu, J. C. 2013; 31 (11): 2354-2363

  Abstract

  Rationale: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction. Objective: To test the hypothesis that a short-course, dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents. Methods and Results: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein, and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury, as assessed by magnetic resonance imaging. Mechanistically, costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile. Conclusions: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment, costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism. Stem Cells 2013;31:2354-2363.

  View details for DOI 10.1002/stem.1501

  View details for PubMedID 24038578

• Imaging cardiac stem cell transplantation using radionuclide labeling techniques: clinical applications and future directions. Methodist DeBakey cardiovascular journal Lezaic, L., Haddad, F., Vrtovec, B., Wu, J. C. 2013; 9 (4): 218-222

  Abstract

  Stem cell therapy is emerging as a potential new therapy for patients with advanced heart failure. In recent years, advances in molecular imaging have allowed monitoring of stem cell homing and survival. In this review article, we will discuss the clinical application and future directions of stem cell imaging in advanced heart failure.

  View details for PubMedID 24298314

• CD34(+) Stem Cell Therapy in Nonischemic Dilated Cardiomyopathy Patients. Clinical pharmacology & therapeutics Vrtovec, B., Poglajen, G., Sever, M., Lezaic, L., Socan, A., Haddad, F., Wu, J. C. 2013; 94 (4): 452-458

  Abstract

  Recent trends indicate that patients with nonischemic dilated cardiomyopathy represent the largest subpopulation of heart failure patients with a significant need for alternative treatment modalities. Similar to patients with ischemic cardiomyopathy, patients with nonischemic dilated cardiomyopathy have been found to have myocardial regions with flow abnormalities, which may represent targets for neoangiogenic therapies. CD34(+) stem cells might contribute to the formation of new blood vessels from existing vascular structures in ischemic tissues by the direct incorporation of injected cells into the newly developing vasculature or by the production and secretion of angiogenic cytokines. This review summarizes the long-term clinical effects and potential underlying mechanisms of CD34(+) cell therapy in patients with nonischemic dilated cardiomyopathy.Clinical Pharmacology & Therapeutics (2013); 94 4, 452-458. doi:10.1038/clpt.2013.134.

  View details for DOI 10.1038/clpt.2013.134

  View details for PubMedID 23903668

• Noninvasive imaging of hypoxia-inducible factor-1a gene therapy for myocardial ischemia. Human gene therapy methods Chen, I. Y., Gheysens, O., Li, Z., Rasooly, J. A., Wang, Q., Paulmurugan, R., Rosenberg, J., Rodriguez-Porcel, M., Willmann, J. K., Wang, D. S., Contag, C. H., Robbins, R. C., Wu, J. C., Gambhir, S. S. 2013; 24 (5): 279-288

  View details for DOI 10.1089/hgtb.2013.028

  View details for PubMedID 23937265

• Noninvasive imaging of hypoxia-inducible factor-1a gene therapy for myocardial ischemia. Human gene therapy methods Chen, I. Y., Gheysens, O., Li, Z., Rasooly, J. A., Wang, Q., Paulmurugan, R., Rosenberg, J., Rodriguez-Porcel, M., Willmann, J. K., Wang, D. S., Contag, C. H., Robbins, R. C., Wu, J. C., Gambhir, S. S. 2013; 24 (5): 279-288

  Abstract

  Abstract Hypoxia-inducible factor-1 alpha (HIF-1α) gene therapy holds great promise for the treatment of myocardial ischemia. Both preclinical and clinical evaluations of this therapy are underway and can benefit from a vector strategy that allows noninvasive assessment of HIF-1α expression as an objective measure of gene delivery. We have developed a novel bidirectional plasmid vector (pcTnT-HIF-1α-VP2-TSTA-fluc), which employs the cardiac troponin T (cTnT) promoter in conjunction with a two-step transcriptional amplification (TSTA) system to drive the linked expression of a recombinant HIF-1α gene (HIF-1α-VP2) and the firefly luciferase gene (fluc). The firefly luciferase (FLuc) activity serves as a surrogate for HIF-1α-VP2 expression, and can be noninvasively assessed in mice using bioluminescence imaging after vector delivery. Transfection of cultured HL-1 cardiomyocytes with pcTnT-HIF-1α-VP2-TSTA-fluc led to a strong correlation between FLuc and HIF-1α-dependent vascular endothelial growth factor expression (r(2)=0.88). Intramyocardial delivery of pcTnT-HIF-1α-VP2-TSTA-fluc into infarcted mouse myocardium led to persistent HIF-1α-VP2 expression for 4 weeks, even though it improved neither CD31+ microvessel density nor echocardiographically determined left ventricular systolic function. These results lend support to recent findings of suboptimal efficacy associated with plasmid-mediated HIF-1α therapy. The imaging techniques developed herein should be useful for further optimizing HIF-1α-VP2 therapy in preclinical models of myocardial ischemia.

  View details for DOI 10.1089/hgtb.2013.028

  View details for PubMedID 23937265

• Screening drug-induced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays. Circulation Navarrete, E. G., Liang, P., Lan, F., Sanchez-Freire, V., Simmons, C., Gong, T., Sharma, A., Burridge, P. W., Patlolla, B., Lee, A. S., Wu, H., Beygui, R. E., Wu, S. M., Robbins, R. C., Bers, D. M., Wu, J. C. 2013; 128 (11): S3-13

  Abstract

  Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

  View details for DOI 10.1161/CIRCULATIONAHA.112.000570

  View details for PubMedID 24030418

• Screening drug-induced arrhythmia events using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays. Circulation Navarrete, E. G., Liang, P., Lan, F., Sanchez-Freire, V., Simmons, C., Gong, T., Sharma, A., Burridge, P. W., Patlolla, B., Lee, A. S., Wu, H., Beygui, R. E., Wu, S. M., Robbins, R. C., Bers, D. M., Wu, J. C. 2013; 128 (11): S3-13

  Abstract

  Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

  View details for DOI 10.1161/CIRCULATIONAHA.112.000570

  View details for PubMedID 24030418

  View details for PubMedCentralID PMC3855862

• Comparison of transendocardial and intracoronary CD34+ cell transplantation in patients with nonischemic dilated cardiomyopathy. Circulation Vrtovec, B., Poglajen, G., Lezaic, L., Sever, M., Socan, A., Domanovic, D., Cernelc, P., Torre-Amione, G., Haddad, F., Wu, J. C. 2013; 128 (11): S42-9

  Abstract

  In an open-label blinded study, we compared intracoronary and transendocardial CD34(+) cell transplantation in patients with nonischemic dilated cardiomyopathy.Of the 40 patients with dilated cardiomyopathy, 20 were randomized to receive intracoronary injection and 20 received transendocardial CD34(+) cell delivery. In both groups, CD34(+) cells were mobilized by filgrastim, collected via apheresis, and labeled with technetium-99m radioisotope for single-photon emission computed tomographic imaging. In the intracoronary group, cells were injected intracoronarily in the artery supplying segments of greater perfusion defect on myocardial perfusion scintigraphy. In the transendocardial group, electroanatomic mapping was used to identify viable but dysfunctional myocardium, and transendocardial cell injections were performed. Nuclear single-photon emission computed tomographic imaging for quantification of myocardial retention was performed 18 hours thereafter. At baseline, groups did not differ in age, sex, left ventricular ejection fraction, or N-terminal pro-brain natriuretic peptide levels. The number of CD34(+) cells was also comparable (105 ± 31 × 10(6) in the transendocardial group versus 103 ± 27 × 10(6) in the intracoronary group, P=0.62). At 18 hours after procedure, myocardial retention was higher in the transendocardial group (19.2 ± 4.8%) than in the intracoronary group (4.4 ± 1.2%, P<0.01). At 6 months, left ventricular ejection fraction improved more in the transendocardial group (+8.1 ± 4.3%) than in the intracoronary group (+4.2 ± 2.3%, P=0.03). The same pattern was observed for the 6-minute walk test distance (+125 ± 33 m in the transendocardial group versus +86 ± 13 m in the intracoronary group, P=0.03) and N-terminal pro-brain natriuretic peptide (-628 ± 211 versus -315 ± 133 pg/mL, P=0.04).In patients with dilated cardiomyopathy, transendocardial CD34(+) cell transplantation is associated with higher myocardial retention rates and greater improvement in ventricular function, N-terminal pro-brain natriuretic peptide, and exercise capacity compared with intracoronary route.http://www.clinicaltrials.gov. Unique identifier: NCT01350310.

  View details for DOI 10.1161/CIRCULATIONAHA.112.000230

  View details for PubMedID 24030420

• Development of Poly(ß-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA. ACS nano Keeney, M., Ong, S., Padilla, A., Yao, Z., Goodman, S., Wu, J. C., Yang, F. 2013; 7 (8): 7241-7250

  Abstract

  Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy.

  View details for DOI 10.1021/nn402657d

  View details for PubMedID 23837668

• Cortical bone-derived stem cells: a novel class of cells for myocardial protection. Circulation research Ong, S., Wu, J. C. 2013; 113 (5): 480-483

  View details for DOI 10.1161/CIRCRESAHA.113.302083

  View details for PubMedID 23948579

• Global Epigenomic Reconfiguration During Mammalian Brain Development SCIENCE Lister, R., Mukamel, E. A., Nery, J. R., Urich, M., Puddifoot, C. A., Johnson, N. D., Lucero, J., Huang, Y., Dwork, A. J., Schultz, M. D., Yu, M., Tonti-Filippini, J., Heyn, H., Hu, S., Wu, J. C., Rao, A., Esteller, M., He, C., Haghighi, F. G., Sejnowski, T. J., Behrens, M. M., Ecker, J. R. 2013; 341 (6146): 629-?

  Abstract

  DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Finally, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain, and that CG demethylation at these hmC-poised loci depends on Tet2 activity.

  View details for DOI 10.1126/science.1237905

  View details for Web of Science ID 000322884800030

• Molecular imaging: The key to advancing cardiac stem cell therapy TRENDS IN CARDIOVASCULAR MEDICINE Chen, I. Y., Wu, J. C. 2013; 23 (6): 201-210

  Abstract

  Cardiac stem cell therapy continues to hold promise for the treatment of ischemic heart disease despite the fact that early promising pre-clinical findings have yet to be translated into consistent clinical success. The latest human studies have collectively identified a pressing need to better understand stem cell behavior in humans and called for more incorporation of noninvasive imaging techniques into the design and evaluation of human stem cell therapy trials. This review discusses the various molecular imaging techniques validated to date for studying stem cells in living subjects, with a particular emphasis on their utilities in assessing the acute retention and the long-term survival of transplanted stem cells. These imaging techniques will be essential for advancing cardiac stem cell therapy by providing the means to both guide ongoing optimization and predict treatment response in humans.

  View details for DOI 10.1016/j.tcm.2012.12.003

  View details for Web of Science ID 000322147600003

  View details for PubMedID 23561794

• Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nature medicine Lee, A. S., Tang, C., Rao, M. S., Weissman, I. L., Wu, J. C. 2013; 19 (8): 998-1004

  Abstract

  Human pluripotent stem cells (PSCs) are a leading candidate for cell-based therapies because of their capacity for unlimited self renewal and pluripotent differentiation. These advances have recently culminated in the first-in-human PSC clinical trials by Geron, Advanced Cell Technology and the Kobe Center for Developmental Biology for the treatment of spinal cord injury and macular degeneration. Despite their therapeutic promise, a crucial hurdle for the clinical implementation of human PSCs is their potential to form tumors in vivo. In this Perspective, we present an overview of the mechanisms underlying the tumorigenic risk of human PSC-based therapies and discuss current advances in addressing these challenges.

  View details for DOI 10.1038/nm.3267

  View details for PubMedID 23921754

• Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, C. K., Lindau, P., Jiang, W., Chen, J. Y., Zhang, L. F., Chen, C., Seita, J., Sahoo, D., Kim, J., Lee, A., Park, S., Nag, D., Gong, Y., Kulkarni, S., Luppen, C. A., Theologis, A. A., Wan, D. C., DeBoer, A., Seo, E. Y., Vincent-Tompkins, J. D., Loh, K., Walmsley, G. G., Kraft, D. L., Wu, J. C., Longaker, M. T., Weissman, I. L. 2013; 110 (31): 12643-12648

  Abstract

  Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

  View details for DOI 10.1073/pnas.1310212110

  View details for PubMedID 23858471

• Ex vivo acidic preconditioning enhances bone marrow ckit cell therapeutic potential via increased CXCR4 expression EUROPEAN HEART JOURNAL Cencioni, C., Melchionna, R., Straino, S., Romani, M., Cappuzzello, C., Annese, V., Wu, J. C., Pompilio, G., Santoni, A., Gaetano, C., Napolitano, M., Capogrossi, M. C. 2013; 34 (26): 2007–16

  Abstract

  The chemokine receptor CXCR4 modulates endothelial progenitor cell migration, homing, and differentiation, and plays a key role in cardiovascular regeneration. Here we examined the effect of ex vivo acidic preconditioning (AP) on CXCR4 expression and on the regenerative potential of mouse bone marrow (BM) ckit(+) cells.Acidic preconditioning was achieved by exposing BM ckit(+) cells to hypercarbic acidosis (pH 7.0) for 24 h; control cells were kept at pH 7.4. Acidic preconditioning enhanced CXCR4 and stromal cell-derived factor 1 (SDF-1) mRNA levels, as well as CXCR4 phosphorylation. Acidic preconditioning ability to modulate CXCR4 expression depended on cytosolic calcium [Ca(2+)]i mobilization and on nitric oxide (NO), as determined by [Ca(2+)]i buffering with BAPTA, and by treatment with the NO donor (DETA/NO) and the NO synthase inhibitor (L-NAME). Further, AP increased SDF-1-driven chemotaxis, transendothelial migration, and differentiation toward the endothelial lineage in vitro. In a mouse model of hindlimb ischaemia, control and AP ckit(+) cells were transplanted into the ischaemic muscle; AP cells accelerated blood flow recovery, increased capillary, and arteriole number as well as the number of regenerating muscle fibres vs. control. These effects were abolished by treating AP cells with L-NAME.Acidic preconditioning represents a novel strategy to enhance BM ckit(+) cell therapeutic potential via NO-dependent increase in CXCR4 expression.

  View details for DOI 10.1093/eurheartj/ehr219

  View details for Web of Science ID 000321831900014

  View details for PubMedID 21784762

  View details for PubMedCentralID PMC3703307

• The Role of SIRT6 Protein in Aging and Reprogramming of Human Induced Pluripotent Stem Cells. journal of biological chemistry Sharma, A., Diecke, S., Zhang, W. Y., Lan, F., He, C., Mordwinkin, N. M., Chua, K. F., Wu, J. C. 2013; 288 (25): 18439-18447

  Abstract

  Aging is known to be the single most important risk factor for multiple diseases. Sirtuin-6, or SIRT6, has recently been identified as a critical regulator of transcription, genome stability, telomere integrity, DNA repair, and metabolic homeostasis. A knockout mouse model of SIRT6 has displayed dramatic phenotypes of accelerated aging. In keeping with its role in aging, we demonstrated that human dermal fibroblasts (HDFs) from older subjects were more resistant to reprogramming by classic Yamanaka factors than those from young subjects, but the addition of SIRT6 during reprogramming substantially improved such efficiency in older HDFs. Despite the importance of SIRT6, little is known about the molecular mechanism of its regulation. We show for the first time post-transcriptional regulation of SIRT6 by miR-766 and inverse correlation in the expression of this microRNA in HDFs from different age groups. Our results suggest that SIRT6 regulates miR-766 transcription via a feedback regulatory loop, which has implications for the modulation of SIRT6 expression in reprogramming of aging cells.

  View details for DOI 10.1074/jbc.M112.405928

  View details for PubMedID 23653361

• Overview of high throughput sequencing technologies to elucidate molecular pathways in cardiovascular diseases. Circulation research Churko, J. M., Mantalas, G. L., Snyder, M. P., Wu, J. C. 2013; 112 (12): 1613-1623

  Abstract

  High throughput sequencing technologies have become essential in studies on genomics, epigenomics, and transcriptomics. Although sequencing information has traditionally been elucidated using a low throughput technique called Sanger sequencing, high throughput sequencing technologies are capable of sequencing multiple DNA molecules in parallel, enabling hundreds of millions of DNA molecules to be sequenced at a time. This advantage allows high throughput sequencing to be used to create large data sets, generating more comprehensive insights into the cellular genomic and transcriptomic signatures of various diseases and developmental stages. Within high throughput sequencing technologies, whole exome sequencing can be used to identify novel variants and other mutations that may underlie many genetic cardiac disorders, whereas RNA sequencing can be used to analyze how the transcriptome changes. Chromatin immunoprecipitation sequencing and methylation sequencing can be used to identify epigenetic changes, whereas ribosome sequencing can be used to determine which mRNA transcripts are actively being translated. In this review, we will outline the differences in various sequencing modalities and examine the main sequencing platforms on the market in terms of their relative read depths, speeds, and costs. Finally, we will discuss the development of future sequencing platforms and how these new technologies may improve on current sequencing platforms. Ultimately, these sequencing technologies will be instrumental in further delineating how the cardiovascular system develops and how perturbations in DNA and RNA can lead to cardiovascular disease.

  View details for DOI 10.1161/CIRCRESAHA.113.300939

  View details for PubMedID 23743227

• Stem cell isolation: Differential stickiness. Nature materials Abilez, O. J., Wu, J. C. 2013; 12 (6): 474-476

  View details for DOI 10.1038/nmat3664

  View details for PubMedID 23695740

• Dissecting the molecular relationship among various cardiogenic progenitor cells. Circulation research Dey, D., Han, L., Bauer, M., Sanada, F., Oikonomopoulos, A., Hosoda, T., Unno, K., de Almeida, P., Leri, A., Wu, J. C. 2013; 112 (9): 1253-1262

  Abstract

  Multiple progenitors derived from the heart and bone marrow (BM) have been used for cardiac repair. Despite this, not much is known about the molecular identity and relationship among these progenitors. To develop a robust stem cell therapy for the heart, it is critical to understand the molecular identity of the multiple cardiogenic progenitor cells.This study is the first report of high-throughput transcriptional profiling of cardiogenic progenitor cells carried out on an identical platform.Microarray-based transcriptional profiling was carried out for 3 cardiac (ckit(+), Sca1(+), and side population) and 2 BM (ckit(+) and mesenchymal stem cell) progenitors, obtained from age- and sex-matched wild-type C57BL/6 mice. Analysis indicated that cardiac-derived ckit(+) population was very distinct from Sca1(+) and side population cells in the downregulation of genes encoding for cell-cell and cell-matrix adhesion proteins, and in the upregulation of developmental genes. Significant enrichment of transcripts involved in DNA replication and repair was observed in BM-derived progenitors. The BM ckit(+) cells seemed to have the least correlation with the other progenitors, with enrichment of immature neutrophil-specific molecules.Our study indicates that cardiac ckit(+) cells represent the most primitive population in the rodent heart. Primitive cells of cardiac versus BM origin differ significantly with respect to stemness and cardiac lineage-specific genes, and molecules involved in DNA replication and repair. The detailed molecular profile of progenitors reported here will serve as a useful reference to determine the molecular identity of progenitors used in future preclinical and clinical studies.

  View details for DOI 10.1161/CIRCRESAHA.112.300779

  View details for PubMedID 23463815

• Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity. Circulation Liang, P., Lan, F., Lee, A. S., Gong, T., Sanchez-Freire, V., Wang, Y., Diecke, S., Sallam, K., Knowles, J. W., Wang, P. J., Nguyen, P. K., Bers, D. M., Robbins, R. C., Wu, J. C. 2013; 127 (16): 1677-1691

  Abstract

  Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations.We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays.

  View details for DOI 10.1161/CIRCULATIONAHA.113.001883

  View details for PubMedID 23519760

• Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity. Circulation Liang, P., Lan, F., Lee, A. S., Gong, T., Sanchez-Freire, V., Wang, Y., Diecke, S., Sallam, K., Knowles, J. W., Wang, P. J., Nguyen, P. K., Bers, D. M., Robbins, R. C., Wu, J. C. 2013; 127 (16): 1677-1691

  View details for DOI 10.1161/CIRCULATIONAHA.113.001883

  View details for PubMedID 23519760

• Clinical and Echocardiographic Presentation of Rejection Episodes Following Heart Transplantation 33rd Annual Meeting and Scientific Sessions of the International-Society-for-Heart-and-Lung-Transplantation Sudini, N. L., Huo, J., Pan, S., Montoya, J., Leon, S., Vu, T., Beygui, R. E., Vrtovec, B., Wu, J. C., Pham, M., KUSH, K., Berry, G., Hunt, S., Haddad, F. ELSEVIER SCIENCE INC. 2013: S254–S254

  View details for Web of Science ID 000316712100696

• Clinical Effects of CD34+Cell Transplantation in Non-Ischemic Dilated Cardiomyopathy Correlate with Myocardial Cell Engraftment Vrtovec, B., Poglajen, G., Lezaic, L., Sever, M., Haddad, F., Wu, J. C. ELSEVIER SCIENCE INC. 2013: S96

  View details for DOI 10.1016/j.healun.2013.01.1006

  View details for Web of Science ID 000316712100239

• SUSTAINED RESTORATION OF LV FUNCTION IN A PORCINE ISCHEMIA-REPERFUSION INJURY MODEL USING HUMAN PLACENTAL MESENCHYMAL STEM CELLS AND MANGANESE-ENHANCED MRI 62nd Annual Scientific Session of the American-College-of-Cardiology Dash, R., Kim, P., Matsuura, Y., Ikeno, F., Lyons, J., Ge, X., Metzler, S., Huang, N., Nguyen, P., Wu, J. C., Cooke, J., Luiz-Rozano, P., Robbins, R., McConnell, M., Yeung, A., Harnish, P., Yang, P. ELSEVIER SCIENCE INC. 2013: E1142–E1142

  View details for Web of Science ID 000316555201247

• Enhanced A beta(1-40) Production in Endothelial Cells Stimulated with Fibrillar A beta(1-42) PLOS ONE Rajadas, J., Sun, W., Li, H., Inayathullah, M., Cereghetti, D., Tan, A., Coelho, V. d., Chrest, F. J., Kusiak, J. W., Smith, W. W., Taub, D., Wu, J. C., Rifkind, J. M. 2013; 8 (3)

  Abstract

  Amyloid accumulation in the brain of Alzheimer's patients results from altered processing of the 39- to 43-amino acid amyloid β protein (Aβ). The mechanisms for the elevated amyloid (Aβ(1-42)) are considered to be over-expression of the amyloid precursor protein (APP), enhanced cleavage of APP to Aβ, and decreased clearance of Aβ from the central nervous system (CNS). We report herein studies of Aβ stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving Aβ(1-42) fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of Aβ(1-40) by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that Aβ(1-40) accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar Aβ(1-42) treatment involving eNOS upregulation and increased autophagy are also reported.

  View details for DOI 10.1371/journal.pone.0058194

  View details for Web of Science ID 000318334500058

  View details for PubMedCentralID PMC3591408

• Effective Delivery of Stem Cells Using an Extracellular Matrix Patch Results in Increased Cell Survival and Proliferation and Reduced Scarring in Skin Wound Healing TISSUE ENGINEERING PART A Lam, M. T., Nauta, A., Meyer, N. P., Wu, J. C., Longaker, M. T. 2013; 19 (5-6): 738-747

  Abstract

  Wound healing is one of the most complex biological processes and occurs in all tissues and organs of the body. In humans, fibrotic tissue, or scar, hinders function and is aesthetically unappealing. Stem cell therapy offers a promising new technique for aiding in wound healing; however, current findings show that stem cells typically die and/or migrate from the wound site, greatly decreasing efficacy of the treatment. Here, we demonstrate effectiveness of a stem cell therapy for improving wound healing in the skin and reducing scarring by introducing stem cells using a natural patch material. Adipose-derived stromal cells were introduced to excisional wounds created in mice using a nonimmunogenic extracellular matrix (ECM) patch material derived from porcine small-intestine submucosa (SIS). The SIS served as an attractive delivery vehicle because of its natural ECM components, including its collagen fiber network, providing the stem cells with a familiar structure. Experimental groups consisted of wounds with stem cell-seeded patches removed at different time points after wounding to determine an optimal treatment protocol. Stem cells delivered alone to skin wounds did not survive post-transplantation as evidenced by bioluminescence in vivo imaging. In contrast, delivery with the patch enabled a significant increase in stem cell proliferation and survival. Wound healing rates were moderately improved by treatment with stem cells on the patch; however, areas of fibrosis, indicating scarring, were significantly reduced in wounds treated with the stem cells on the patch compared to untreated wounds.

  View details for DOI 10.1089/ten.tea.2012.0480

  View details for PubMedID 23072446

• MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2 STEM CELLS Hu, S., Wilson, K. D., Ghosh, Z., Han, L., Wang, Y., Lan, F., Ransohoff, K. J., Burridge, P., Wu, J. C. 2013; 31 (2): 259-268

  Abstract

  MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302, which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as "KMOS3") when compared to using four factors ("KMOS"). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.

  View details for DOI 10.1002/stem.1278

  View details for PubMedID 23136034

• Immunogenicity of Pluripotent Stem Cells and Their Derivatives CIRCULATION RESEARCH de Almeida, P. E., Ransohoff, J. D., Nahid, A., Wu, J. C. 2013; 112 (3): 549-561

  Abstract

  The ability of pluripotent stem cells to self-renew and differentiate into all somatic cell types brings great prospects to regenerative medicine and human health. However, before clinical applications, much translational research is necessary to ensure that their therapeutic progenies are functional and nontumorigenic, that they are stable and do not dedifferentiate, and that they do not elicit immune responses that could threaten their survival in vivo. For this, an in-depth understanding of their biology, genetic, and epigenetic make-up and of their antigenic repertoire is critical for predicting their immunogenicity and for developing strategies needed to assure successful long-term engraftment. Recently, the expectation that reprogrammed somatic cells would provide an autologous cell therapy for personalized medicine has been questioned. Induced pluripotent stem cells display several genetic and epigenetic abnormalities that could promote tumorigenicity and immunogenicity in vivo. Understanding the persistence and effects of these abnormalities in induced pluripotent stem cell derivatives is critical to allow clinicians to predict graft fate after transplantation, and to take requisite measures to prevent immune rejection. With clinical trials of pluripotent stem cell therapy on the horizon, the importance of understanding immunologic barriers and devising safe, effective strategies to bypass them is further underscored. This approach to overcome immunologic barriers to stem cell therapy can take advantage of the validated knowledge acquired from decades of hematopoietic stem cell transplantation.

  View details for DOI 10.1161/CIRCRESAHA.111.249243

  View details for PubMedID 23371903

• Advances in nanotechnology for the management of coronary artery disease TRENDS IN CARDIOVASCULAR MEDICINE Rhee, J., Wu, J. C. 2013; 23 (2): 39-45

  Abstract

  Nanotechnology holds tremendous potential to advance the current treatment of coronary artery disease. Nanotechnology may assist medical therapies by providing a safe and efficacious delivery platform for a variety of drugs aimed at modulating lipid disorders, decreasing inflammation and angiogenesis within atherosclerotic plaques, and preventing plaque thrombosis. Nanotechnology may improve coronary stent applications by promoting endothelial recovery on a stent surface utilizing bio-mimetic nanofibrous scaffolds, and also by preventing in-stent restenosis using nanoparticle-based delivery of drugs that are decoupled from stents. Additionally, nanotechnology may enhance tissue-engineered graft materials for application in coronary artery bypass grafting by facilitating cellular infiltration and remodeling of a graft matrix.

  View details for DOI 10.1016/j.tcm.2012.08.009

  View details for PubMedID 23245913

• Sacrificial layer technique for axial force post assay of immature cardiomyocytes BIOMEDICAL MICRODEVICES Taylor, R. E., Kim, K., Sun, N., Park, S., Sim, J. Y., Fajardo, G., Bernstein, D., Wu, J. C., Pruitt, B. L. 2013; 15 (1): 171-181

  Abstract

  Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease, and offer potential for patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function, and to gain insight into cardiac growth and disease, tissue engineers must quantify the contractile forces of these single cells. Currently, axial contractile forces of isolated adult heart cells can only be measured by two-point methods such as carbon fiber techniques, which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificial support layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput, is functionally analogous to current gold-standard axial force assays for adult heart cells, and prescribes elongated cell shapes without protein patterning. Finally, we calibrate these force posts with piezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far better than that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culture platforms will enable improved cell placement and the complex suspension of cells across 3D constructs.

  View details for DOI 10.1007/s10544-012-9710-3

  View details for PubMedID 23007494

• A Review of Human Pluripotent Stem Cell-Derived Cardiomyocytes for High-Throughput Drug Discovery, Cardiotoxicity Screening, and Publication Standards JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH Mordwinkin, N. M., Burridge, P. W., Wu, J. C. 2013; 6 (1): 22-30

  Abstract

  Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human-induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.

  View details for DOI 10.1007/s12265-012-9423-2

  View details for PubMedID 23229562

• Effects of Intracoronary CD34(+) Stem Cell Transplantation in Nonischemic Dilated Cardiomyopathy Patients 5-Year Follow-Up CIRCULATION RESEARCH Vrtovec, B., Poglajen, G., Lezaic, L., Sever, M., Domanovic, D., Cernelc, P., Socan, A., Schrepfer, S., Torre-Amione, G., Haddad, F., Wu, J. C. 2013; 112 (1): 165-173

  Abstract

  CD34+ transplantation in dilated cardiomyopathy was associated with short-term improvement in left ventricular ejection fraction and exercise tolerance.We investigated long-term effects of intracoronary CD34+ cell transplantation in dilated cardiomyopathy and the relationship between intramyocardial cell homing and clinical response.Of 110 dilated cardiomyopathy patients, 55 were randomized to receive CD34+ stem cell transplantation (SC group) and 55 received no cell therapy (controls). In the SC group, CD34+ cells were mobilized by granulocyte colony-stimulating factor and collected via apheresis. Patients underwent myocardial scintigraphy and cells were injected in the artery supplying segments with the greatest perfusion defect. At baseline, 2 groups did not differ in age, sex, left ventricular ejection fraction, or N-terminal B-type natriuretic peptide levels. At 5 years, stem cell therapy was associated with increased left ventricular ejection fraction (from 24.3 ± 6.5% to 30.0 ± 5.1%; P=0.02), increased 6-minute walk distance (from 344 ± 90 m to 477 ± 130 m; P<0.001), and decreased N-terminal B-type natriuretic peptide (from 2322 ± 1234 pg/mL to 1011 ± 893 pg/mL; P<0.01). Left ventricular ejection fraction improvement was more significant in patients with higher myocardial homing of injected cells. During follow-up, 27 (25%) patients died and 9 (8%) underwent heart transplantation. Of the 27 deaths, 13 were attributed to pump failure and 14 were attributed to sudden cardiac death. Total mortality was lower in the SC group (14%) than in controls (35%; P=0.01). The same was true of pump failure (5% vs. 18%; P=0.03), but not of sudden cardiac death (9% vs. 16%; P=0.39).Intracoronary stem cell transplantation may be associated with improved ventricular function, exercise tolerance, and long-term survival in patients with dilated cardiomyopathy. Higher intramyocardial homing is associated with better stem cell therapy response.

  View details for DOI 10.1161/CIRCRESAHA.112.276519

  View details for Web of Science ID 000313053000021

  View details for PubMedID 23065358

• Abnormal Calcium Handling Properties Underlie Familial Hypertrophic Cardiomyopathy Pathology in Patient-Specific Induced Pluripotent Stem Cells CELL STEM CELL Lan, F., Lee, A. S., Liang, P., Sanchez-Freire, V., Nguyen, P. K., Wang, L., Han, L., Yen, M., Wang, Y., Sun, N., Abilez, O. J., Hu, S., Ebert, A. D., Navarrete, E. G., Simmons, C. S., Wheeler, M., Pruitt, B., Lewis, R., Yamaguchi, Y., Ashley, E. A., Bers, D. M., Robbins, R. C., Longaker, M. T., Wu, J. C. 2013; 12 (1): 101-113

  Abstract

  Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca(2+)) imaging indicated dysregulation of Ca(2+) cycling and elevation in intracellular Ca(2+) ([Ca(2+)](i)) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca(2+) homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.

  View details for DOI 10.1016/j.stem.2012.10.010

  View details for PubMedID 23290139

• Enhanced Aß(1-40) production in endothelial cells stimulated with fibrillar Aß(1-42). PloS one Rajadas, J., Sun, W., Li, H., Inayathullah, M., Cereghetti, D., Tan, A., de Mello Coelho, V., Chrest, F. J., Kusiak, J. W., Smith, W. W., Taub, D., Wu, J. C., Rifkind, J. M. 2013; 8 (3)

  Abstract

  Amyloid accumulation in the brain of Alzheimer's patients results from altered processing of the 39- to 43-amino acid amyloid β protein (Aβ). The mechanisms for the elevated amyloid (Aβ(1-42)) are considered to be over-expression of the amyloid precursor protein (APP), enhanced cleavage of APP to Aβ, and decreased clearance of Aβ from the central nervous system (CNS). We report herein studies of Aβ stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving Aβ(1-42) fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of Aβ(1-40) by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that Aβ(1-40) accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar Aβ(1-42) treatment involving eNOS upregulation and increased autophagy are also reported.

  View details for DOI 10.1371/journal.pone.0058194

  View details for PubMedID 23505467

• Loss of CDKN2B Promotes p53-Dependent Smooth Muscle Cell Apoptosis and Aneurysm Formation ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Leeper, N. J., Raiesdana, A., Kojima, Y., Kundu, R. K., Cheng, H., Maegdefessel, L., Toh, R., Ahn, G., Ali, Z. A., Anderson, D. R., Miller, C. L., Roberts, S. C., Spin, J. M., de Almeida, P. E., Wu, J. C., Xu, B., Cheng, K., Quertermous, M., Kundu, S., Kortekaas, K. E., Berzin, E., Downing, K. P., Dalman, R. L., Tsao, P. S., Schadt, E. E., Owens, G. K., Quertermous, T. 2013; 33 (1): E1-?

  Abstract

  Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear.Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow-derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model.These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

  View details for DOI 10.1161/ATVBAHA.112.300399

  View details for Web of Science ID 000312392500001

  View details for PubMedID 23162013

  View details for PubMedCentralID PMC3569043

• Cardiovascular molecular imaging as a tool to study biology. Theranostics Rodriguez-Porcel, M., Wu, J. C. 2013; 3 (11): 914-915

  View details for DOI 10.7150/thno.6333

  View details for PubMedID 24396501

• American Journal of Nuclear Medicine and Molecular Imaging: Editorial Board (2014) e-Century Publishing Corporation. American journal of nuclear medicine and molecular imaging Seto, B., Jacobs, P. M., Mach, R. H., Bulte, J. W., Cai, W., Alavi, A., Divgi, C. R., Wu, J. C. 2013; 4 (1): 89-95

  View details for PubMedID 24380049

  View details for PubMedCentralID PMC3867733

• Imaging Neural Stem Cell Graft-Induced Structural Repair in Stroke CELL TRANSPLANTATION Daadi, M. M., Hu, S., Klausner, J., Li, Z., Sofilos, M., Sun, G., Wu, J. C., Steinberg, G. K. 2013; 22 (5): 881-892

  Abstract

  Stem cell therapy ameliorates motor deficits in experimental stroke model. Multimodal molecular imaging enables real time longitudinal monitoring of infarct location, size and transplant survival. In the present study, we used magnetic resonance imaging (MRI) and positron emission tomography (PET) to track the infarct evolution, tissue repair and the fate of grafted cells. We genetically engineered embryonic stem cell -derived neural stem cells (NSCs) with a triple fusion reporter gene to express monomeric red fluorescence protein and herpes simplex virus truncated thymidine kinase for multimodal molecular imaging and SPIO labeled for MRI. The infarct size, as well as fate and function of grafted cells were tracked in real time for 3 months using MRI and PET. We report that grafted NSCs reduced the infarct size in animals with less then 1 cm³ initial infarct in a dose-dependent manner, while larger stroke was not amenable to such beneficial effects. PET imaging revealed increased metabolic activity in grafted animals and visualized functioning grafted cells in vivo. Immunohistopathological analysis demonstrated that, after 3-month survival period grafted NSCs dispersed in the stroke-lesioned parenchyma and differentiated into neurons, astrocytes and oligodendrocytes. Longitudinal multimodal imaging provides insights into time course dose-dependant interactions between NSC grafts and structural changes in infracted tissue.

  View details for DOI 10.3727/096368912X656144

  View details for Web of Science ID 000318585300010

• Generation of Human iPSCs from Human Peripheral Blood Mononuclear Cells Using Non-integrative Sendai Virus in Chemically Defined Conditions. Methods in molecular biology (Clifton, N.J.) Churko, J. M., Burridge, P. W., Wu, J. C. 2013; 1036: 81-88

  Abstract

  Human-induced pluripotent stem cells (hiPSCs) have received enormous attention because of their ability to differentiate into multiple cell types that demonstrate the patient's original phenotype. The use of hiPSCs is particularly valuable to the study of cardiac biology, as human cardiomyocytes are difficult to isolate and culture and have a limited proliferative potential. By deriving iPSCs from patients with heart disease and subsequently differentiating these hiPSCs to cardiomyocytes, it is feasible to study cardiac biology in vitro and model cardiac diseases. While there are many different methods for deriving hiPSCs, clinical use of these hiPSCs will require derivation by methods that do not involve modification of the original genome (non-integrative) or incorporate xeno-derived products (such as bovine serum albumin) which may contain xeno-agents. Ideally, this derivation would be carried out under chemically defined conditions to prevent lot-to-lot variability and enhance reproducibility. Additionally, derivation from cell types such as fibroblasts requires extended culture (4-6 weeks), greatly increasing the time required to progress from biopsy to hiPSC. Herein, we outline a method of culturing peripheral blood mononuclear cells (PBMCs) and reprogramming PBMCs into hiPSCs using a non-integrative Sendai virus.

  View details for DOI 10.1007/978-1-62703-511-8_7

  View details for PubMedID 23807788

• A transgenic tri-modality reporter mouse. PloS one Yan, X., Ray, P., Paulmurugan, R., Tong, R., Gong, Y., Sathirachinda, A., Wu, J. C., Gambhir, S. S. 2013; 8 (8)

  Abstract

  Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This "Tri-Modality Reporter Mouse" system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent (tdTomato), and positron emission tomography (PET) (ttk) modalities. Transgenic colonies with different levels of tri-fusion reporter gene expression showed a linear correlation between all three-reporter proteins (R(2)=0.89 for TdTomato vs Fluc, R(2)=0.94 for Fluc vs TTK, R(2)=0.89 for TdTomato vs TTK) in vitro from tissue lysates and in vivo by optical and PET imaging. Mesenchymal stem cells (MSCs) isolated from this transgenics showed high level of reporter gene expression, which linearly correlated with the cell numbers (R(2)=0.99 for bioluminescence imaging (BLI)). Both BLI (R(2)=0.93) and micro-PET (R(2)=0.94) imaging of the subcutaneous implants of Tri-Modality Reporter Mouse derived MSCs in nude mice showed linear correlation with the cell numbers and across different imaging modalities (R(2)=0.97). Serial imaging of MSCs transplanted to mice with acute myocardial infarction (MI) by intramyocardial injection exhibited significantly higher signals in MI heart at days 2, 3, 4, and 7 (p<0.01). MSCs transplanted to the ischemic hindlimb of nude mice showed significantly higher BLI and PET signals in the first 2 weeks that dropped by 4(th) week due to poor cell survival. However, laser Doppler perfusion imaging revealed that blood circulation in the ischemic limb was significantly improved in the MSCs transplantation group compared with the control group. In summary, this mouse can be used as a source of donor cells and organs in various research areas such as stem cell research, tissue engineering research, and organ transplantation.

  View details for DOI 10.1371/journal.pone.0073580

  View details for PubMedID 23951359

• Induced pluripotency of human prostatic epithelial cells. PloS one Zhao, H., Sun, N., Young, S. R., Nolley, R., Santos, J., Wu, J. C., Peehl, D. M. 2013; 8 (5)

  Abstract

  Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore, provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification.

  View details for DOI 10.1371/journal.pone.0064503

  View details for PubMedID 23717621

• MicroRNA expression profiling of human-induced pluripotent and embryonic stem cells. Methods in molecular biology (Clifton, N.J.) Sharma, A., Wu, J. C. 2013; 936: 247-256

  Abstract

  Clinical implications of induced pluripotent stem (iPS) cell technology are enormous for personalized medicine. However, extensive use of viral approach for ectopic expression of reprogramming factors is a major hurdle in realization of its true potential. Non-viral methods for making iPS cells, although plausible, are impractical because of high cost. MicroRNAs are important cellular modulators that have been shown to rival transcription factors and are important players in embryonic development. We have generated distinct "microRNA-omes" signature of iPS cells that remain in a near embryonic stem (ES) cell state and distinct from differentiated cells. Recent advances in the microRNA field and experimentally validated microRNAs warrant a review in experimental protocols for microRNA expression profile.

  View details for PubMedID 23007513

• Pluripotent Stem Cells: Immune to the Immune System? SCIENCE TRANSLATIONAL MEDICINE Pearl, J. I., Kean, L. S., Davis, M. M., Wu, J. C. 2012; 4 (164)

  Abstract

  Human embryonic stem cells (hESCs), initially thought to be immune privileged cells, are now known to be susceptible to immune recognition. Human induced pluripotent stem cells (iPSCs) have been proposed as a potential source of autologous stem cells for therapy, but even these autologous stem cells may be targets of immune rejection. With clinical trials on the horizon, it is imperative that the immunogenicity of hESCs and iPSCs be definitively understood.

  View details for DOI 10.1126/scitranslmed.3005090

  View details for PubMedID 23241742

• In vivo directed differentiation of pluripotent stem cells for skeletal regeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Levi, B., Hyun, J. S., Montoro, D. T., Lo, D. D., Chan, C. K., Hu, S., Sun, N., Lee, M., Grova, M., Connolly, A. J., Wu, J. C., Gurtner, G. C., Weissman, I. L., Wan, D. C., Longaker, M. T. 2012; 109 (50): 20379-20384

  Abstract

  Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

  View details for DOI 10.1073/pnas.1218052109

  View details for PubMedID 23169671

• Genome Editing of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells With Zinc Finger Nucleases for Cellular Imaging CIRCULATION RESEARCH Wang, Y., Zhang, W. Y., Hu, S., Lan, F., Lee, A. S., Huber, B., Lisowski, L., Liang, P., Huang, M., de Almeida, P. E., Won, J. H., Sun, N., Robbins, R. C., Kay, M. A., Urnov, F. D., Wu, J. C. 2012; 111 (12): 1494-?

  Abstract

  Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied on random integration, a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression.To address this barrier, we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging.We used ZFN technology to integrate a construct containing monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging, bioluminescence imaging, and positron emission tomography imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine.Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo.

  View details for DOI 10.1161/CIRCRESAHA.112.274969

  View details for Web of Science ID 000311994700042

  View details for PubMedID 22967807

  View details for PubMedCentralID PMC3518748

• Short-Term Costimulatory Molecule Blockade Promotes Long-Term Engraftment of Transplanted Human Embryonic Stem Derivatives and Improves Cardiac Recovery Huber, B. C., Ransohoff, J. D., Ransohoff, K. J., de Almeida, P. E., Gold, J. D., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2012

  View details for Web of Science ID 000208885004413

• Transient, Inducible, Placenta-Specific Gene Expression in Mice ENDOCRINOLOGY Fan, X., Petitt, M., Gamboa, M., Huang, M., Dhal, S., Druzin, M. L., Wu, J. C., Chen-Tsai, Y., Nayak, N. R. 2012; 153 (11): 5637-5644

  Abstract

  Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues.

  View details for DOI 10.1210/en.2012-1556

  View details for PubMedID 23011919

• Returns of the Living Dead Therapeutic Action of Irradiated and Mitotically Inactivated Embryonic Stem Cells CIRCULATION RESEARCH Gold, J. D., Wu, J. C. 2012; 111 (10): 1250-1252

  View details for DOI 10.1161/CIRCRESAHA.112.279398

  View details for Web of Science ID 000310501300004

  View details for PubMedID 23104873

  View details for PubMedCentralID PMC3556461

• Human Cardiac Progenitor Cells Engineered With Pim-I Kinase Enhance Myocardial Repair JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Mohsin, S., Khan, M., Toko, H., Bailey, B., Cottage, C. T., Wallach, K., Nag, D., Lee, A., Siddiqi, S., Lan, F., Fischer, K. M., Gude, N., Quijada, P., Avitabile, D., Truffa, S., Collins, B., Dembitsky, W., Wu, J. C., Sussman, M. A. 2012; 60 (14): 1278-1287

  Abstract

  The goal of this study was to demonstrate the enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for the treatment of myocardial infarction.Regenerative potential of stem cells to repair acute infarction is limited. Improved hCPC survival, proliferation, and differentiation into functional myocardium will increase efficacy and advance translational implementation of cardiac regeneration.hCPCs isolated from the myocardium of heart failure patients undergoing left ventricular assist device implantation were engineered to express green fluorescent protein (hCPCe) or Pim-1-GFP (hCPCeP). Functional tests of hCPC regenerative potential were performed with immunocompromised mice by using intramyocardial adoptive transfer injection after infarction. Myocardial structure and function were monitored by echocardiographic and hemodynamic assessment for 20 weeks after delivery. hCPCe and hCPCeP expressing luciferase were observed by using bioluminescence imaging to noninvasively track persistence.hCPCeP exhibited augmentation of reparative potential relative to hCPCe control cells, as shown by significantly increased proliferation coupled with amelioration of infarction injury and increased hemodynamic performance at 20 weeks post-transplantation. Concurrent with enhanced cardiac structure and function, hCPCeP demonstrated increased cellular engraftment and differentiation with improved vasculature and reduced infarct size. Enhanced persistence of hCPCeP versus hCPCe was revealed by bioluminescence imaging at up to 8 weeks post-delivery.Genetic engineering of hCPCs with Pim-1 enhanced repair of damaged myocardium. Ex vivo gene delivery to modify stem cells has emerged as a viable option addressing current limitations in the field. This study demonstrates that efficacy of hCPCs from the failing myocardium can be safely and significantly enhanced through expression of Pim-1 kinase, setting the stage for use of engineered cells in pre-clinical settings.

  View details for DOI 10.1016/j.jacc.2012.04.047

  View details for Web of Science ID 000309508400012

  View details for PubMedID 22841153

  View details for PubMedCentralID PMC3461098

• Induced Pluripotent Stem Cells as a Disease Modeling and Drug Screening Platform JOURNAL OF CARDIOVASCULAR PHARMACOLOGY Ebert, A. D., Liang, P., Wu, J. C. 2012; 60 (4): 408-416

  Abstract

  Induced pluripotent stem cells (iPSCs) hold great hopes for therapeutic application in various diseases. Although ongoing research is dedicated to achieving clinical translation of iPSCs, further understanding of the mechanisms that underlie complex pathogenic conditions is required. Compared with other classical models for studying diseases, iPSCs provide considerable advantages. A newly emerging application of iPSCs is in vitro disease modeling, which can significantly improve the never-ending search for new pharmacological cures. Here, we will discuss current efforts to create iPSC-dependent patient-specific disease models. Furthermore, we will review the use of iPSCs for development and testing of new therapeutic agents and the implications for high-throughput drug screening.

  View details for DOI 10.1097/FJC.0b013e318247f642

  View details for PubMedID 22240913

• Pushing the Reset Button: Chemical-Induced Conversion of Amniotic Fluid Stem Cells Into a Pluripotent State MOLECULAR THERAPY Diecke, S., Wu, J. C. 2012; 20 (10): 1839-1841

  View details for DOI 10.1038/mt.2012.192

  View details for PubMedID 23023056

• Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation CIRCULATION RESEARCH Gu, M., Nguyen, P. K., Lee, A. S., Xu, D., Hu, S., Plews, J. R., Han, L., Huber, B. C., Lee, W. H., Gong, Y., de Almeida, P. E., Lyons, J., Ikeno, F., Pacharinsak, C., Connolly, A. J., Gambhir, S. S., Robbins, R. C., Longaker, M. T., Wu, J. C. 2012; 111 (7): 882-893

  Abstract

  Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models.To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia.Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery.In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

  View details for DOI 10.1161/CIRCRESAHA.112.269001

  View details for PubMedID 22821929

• Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique Meeting of the American-Heart-Association Lan, F., Liu, J., Narsinh, K. H., Hu, S., Han, L., Lee, A. S., Karow, M., Nguyen, P. K., Nag, D., Calos, M. P., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2012: S20-?

  Abstract

  Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts.We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging.The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

  View details for DOI 10.1161/CIRCULATIONAHA.111.084913

  View details for Web of Science ID 000314150200003

  View details for PubMedID 22965984

  View details for PubMedCentralID PMC3481839

• Modeling Pathogenesis in Familial Hypertrophic Cardiomyopathy Using Patient-Specific Induced Pluripotent Stem Cells Basic Cardiovascular Sciences Scientific Session Lan, F., Lee, A., Liang, P., Navarrete, E., Wang, L., Leng, H., Sanchez, V., Yen, M., Wang, Y., Nguyen, P., Sun, N., Abilez, O., Lewis, R., Yamaguchi, Y., Ashley, E., Bers, D., Robbins, R., Longaker, M., Wu, J. LIPPINCOTT WILLIAMS & WILKINS. 2012

  View details for Web of Science ID 000312506400056

• Biomaterial applications in cardiovascular tissue repair and regeneration. Expert review of cardiovascular therapy Lam, M. T., Wu, J. C. 2012; 10 (8): 1039-1049

  Abstract

  Cardiovascular disease physically damages the heart, resulting in loss of cardiac function. Medications can help alleviate symptoms, but it is more beneficial to treat the root cause by repairing injured tissues, which gives patients better outcomes. Besides heart transplants, cardiac surgeons use a variety of methods for repairing different areas of the heart such as the ventricular septal wall and valves. A multitude of biomaterials are used in the repair and replacement of impaired heart tissues. These biomaterials fall into two main categories: synthetic and natural. Synthetic materials used in cardiovascular applications include polymers and metals. Natural materials are derived from biological sources such as human donor or harvested animal tissues. A new class of composite materials has emerged to take advantage of the benefits of the strengths and minimize the weaknesses of both synthetic and natural materials. This article reviews the current and prospective applications of biomaterials in cardiovascular therapies.

  View details for DOI 10.1586/erc.12.99

  View details for PubMedID 23030293

• The manipulation of natural killer cells to target tumor sites using magnetic nanoparticles BIOMATERIALS Jang, E., Shin, J., Ren, G., Park, M., Cheng, K., Chen, X., Wu, J. C., Sunwoo, J. B., Cheng, Z. 2012; 33 (22): 5584-5592

  Abstract

  The present work demonstrates that Cy5.5 conjugated Fe(3)O(4)/SiO(2) core/shell nanoparticles could allow us to control movement of human natural killer cells (NK-92MI) by an external magnetic field. Required concentration of the nanoparticles for the cell manipulation is as low as ~20 μg Fe/mL. However, the relative ratio of the nanoparticles loaded NK-92MI cells infiltrated into the target tumor site is enhanced by 17-fold by applying magnetic field and their killing activity is still maintained as same as the NK-92MI cells without the nanoparticles. This approach allows us to open alternative clinical treatment with reduced toxicity of the nanoparticles and enhanced infiltration of immunology to the target site.

  View details for DOI 10.1016/j.biomaterials.2012.04.041

  View details for PubMedID 22575830

• Early Stem Cell Engraftment Predicts Late Cardiac Functional Recovery Preclinical Insights From Molecular Imaging CIRCULATION-CARDIOVASCULAR IMAGING Liu, J., Narsinh, K. H., Lan, F., Wang, L., Nguyen, P. K., Hu, S., Lee, A., Han, L., Gong, Y., Huang, M., Nag, D., Rosenberg, J., Chouldechova, A., Robbins, R. C., Wu, J. C. 2012; 5 (4): 481-490

  Abstract

  Human cardiac progenitor cells have demonstrated great potential for myocardial repair in small and large animals, but robust methods for longitudinal assessment of their engraftment in humans is not yet readily available. In this study, we sought to optimize and evaluate the use of positron emission tomography (PET) reporter gene imaging for monitoring human cardiac progenitor cell (hCPC) transplantation in a mouse model of myocardial infarction.hCPCs were isolated and expanded from human myocardial samples and stably transduced with herpes simplex virus thymidine kinase (TK) PET reporter gene. Thymidine kinase-expressing hCPCs were characterized in vitro and transplanted into murine myocardial infarction models (n=57). Cardiac echocardiographic, magnetic resonance imaging and pressure-volume loop analyses revealed improvement in left ventricular contractile function 2 weeks after transplant (hCPC versus phosphate-buffered saline, P<0.03). Noninvasive PET imaging was used to track hCPC fate over a 4-week time period, demonstrating a substantial decline in surviving cells. Importantly, early cell engraftment as assessed by PET was found to predict subsequent functional improvement, implying a "dose-effect" relationship. We isolated the transplanted cells from recipient myocardium by laser capture microdissection for in vivo transcriptome analysis. Our results provide direct evidence that hCPCs augment cardiac function after their transplantation into ischemic myocardium through paracrine secretion of growth factors.PET reporter gene imaging can provide important diagnostic and prognostic information regarding the ultimate success of hCPC treatment for myocardial infarction.

  View details for DOI 10.1161/CIRCIMAGING.111.969329

  View details for PubMedID 22565608

• Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5 CIRCULATION RESEARCH Chen, J. X., Krane, M., Deutsch, M., Wang, L., Rav-Acha, M., Gregoire, S., Engels, M. C., Rajarajan, K., Karra, R., Abel, E. D., Wu, J. C., Milan, D., Wu, S. M. 2012; 111 (1): 50-55

  Abstract

  Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown.To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT).We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes.Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.

  View details for DOI 10.1161/CIRCRESAHA.112.270264

  View details for PubMedID 22581928

• Large animal induced pluripotent stem cells as pre-clinical models for studying human disease JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Plews, J. R., Gu, M., Longaker, M. T., Wu, J. C. 2012; 16 (6): 1196-1202

  Abstract

  The path to induced pluripotency Discovery of a pan-species pluripotency network Animal iPSCs and disease modelling Issues with large animal iPSCs Conclusions The derivation of human embryonic stem cells and subsequently human induced pluripotent stem cells (iPSCs) has energized regenerative medicine research and enabled seemingly limitless applications. Although small animal models, such as mouse models, have played an important role in the progression of the field, typically, they are poor representations of the human disease phenotype. As an alternative, large animal models should be explored as a potentially better approach for clinical translation of cellular therapies. However, only fragmented information regarding the derivation, characterization and clinical usefulness of pluripotent large animal cells is currently available. Here, we briefly review the latest advances regarding the derivation and use of large animal iPSCs.

  View details for DOI 10.1111/j.1582-4934.2012.01521.x

  View details for PubMedID 22212700

• Atomic Force Mechanobiology of Pluripotent Stem Cell-Derived Cardiomyocytes PLOS ONE Liu, J., Sun, N., Bruce, M. A., Wu, J. C., Butte, M. J. 2012; 7 (5)

  Abstract

  We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent, stem cell-derived cardiomyocytes, including contraction force, rate, duration, and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy, and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive, inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents, or as a platform for studying cardiomyocyte biology.

  View details for DOI 10.1371/journal.pone.0037559

  View details for PubMedID 22624048

• Dynamic microRNA expression during the transition from right ventricular hypertrophy to failure PHYSIOLOGICAL GENOMICS Reddy, S., Zhao, M., Hu, D., Fajardo, G., Hu, S., Ghosh, Z., Rajagopalan, V., Wu, J. C., Bernstein, D. 2012; 44 (10): 562-575

  Abstract

  MicroRNAs (miRs) are small, noncoding RNAs that are emerging as crucial regulators of cardiac remodeling in left ventricular hypertrophy (LVH) and failure (LVF). However, there are no data on their role in right ventricular hypertrophy (RVH) and failure (RVF). This is a critical question given that the RV is uniquely at risk in patients with congenital right-sided obstructive lesions and in those with systemic RVs. We have developed a murine model of RVH and RVF using pulmonary artery constriction (PAC). miR microarray analysis of RV from PAC vs. control demonstrates altered miR expression with gene targets associated with cardiomyocyte survival and growth during hypertrophy (miR 199a-3p) and reactivation of the fetal gene program during heart failure (miR-208b). The transition from hypertrophy to heart failure is characterized by apoptosis and fibrosis (miRs-34, 21, 1). Most are similar to LVH/LVF. However, there are several key differences between RV and LV: four miRs (34a, 28, 148a, and 93) were upregulated in RVH/RVF that are downregulated or unchanged in LVH/LVF. Furthermore, there is a corresponding downregulation of their putative target genes involving cell survival, proliferation, metabolism, extracellular matrix turnover, and impaired proteosomal function. The current study demonstrates, for the first time, alterations in miRs during the process of RV remodeling and the gene regulatory pathways leading to RVH and RVF. Many of these alterations are similar to those in the afterload-stressed LV. miRs differentially regulated between the RV and LV may contribute to the RVs increased susceptibility to heart failure.

  View details for DOI 10.1152/physiolgenomics.00163.2011

  View details for PubMedID 22454450

• Imaging Stem Cell Therapy for the Treatment of Peripheral Arterial Disease CURRENT VASCULAR PHARMACOLOGY Ransohoff, J. D., Wu, J. C. 2012; 10 (3): 361-373

  Abstract

  Arteriosclerotic cardiovascular diseases are among the leading causes of morbidity and mortality worldwide. Therapeutic angiogenesis aims to treat ischemic myocardial and peripheral tissues by delivery of recombinant proteins, genes, or cells to promote neoangiogenesis. Concerns regarding the safety, side effects, and efficacy of protein and gene transfer studies have led to the development of cell-based therapies as alternative approaches to induce vascular regeneration and to improve function of damaged tissue. Cell-based therapies may be improved by the application of imaging technologies that allow investigators to track the location, engraftment, and survival of the administered cell population. The past decade of investigations has produced promising clinical data regarding cell therapy, but design of trials and evaluation of treatments stand to be improved by emerging insight from imaging studies. Here, we provide an overview of pre-clinical and clinical experience using cell-based therapies to promote vascular regeneration in the treatment of peripheral arterial disease. We also review four major imaging modalities and underscore the importance of in vivo analysis of cell fate for a full understanding of functional outcomes.

  View details for PubMedID 22239638

• Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy SCIENCE TRANSLATIONAL MEDICINE Sun, N., Yazawa, M., Liu, J., Han, L., Sanchez-Freire, V., Abilez, O. J., Navarrete, E. G., Hu, S., Wang, L., Lee, A., Pavlovic, A., Lin, S., Chen, R., Hajjar, R. J., Snyder, M. P., Dolmetsch, R. E., Butte, M. J., Ashley, E. A., Longaker, M. T., Robbins, R. C., Wu, J. C. 2012; 4 (130)

  Abstract

  Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca(2+)), decreased contractility, and abnormal distribution of sarcomeric α-actinin. When stimulated with a β-adrenergic agonist, DCM iPSC-derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric α-actinin distribution. Treatment with β-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.

  View details for DOI 10.1126/scitranslmed.3003552

  View details for PubMedID 22517884

• Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns NATURE PROTOCOLS Sanchez-Freire, V., Ebert, A. D., Kalisky, T., Quake, S. R., Wu, J. C. 2012; 7 (5): 829-838

  Abstract

  Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation.

  View details for DOI 10.1038/nprot.2012.021

  View details for PubMedID 22481529

• Production of De Novo Cardiomyocytes: Human Pluripotent Stem Cell Differentiation and Direct Reprogramming CELL STEM CELL Burridge, P. W., Keller, G., Gold, J. D., Wu, J. C. 2012; 10 (1): 16-28

  Abstract

  Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes, both in vitro and in vivo. Beyond uses in cell replacement therapy, patient-specific cardiomyocytes may find applications in drug testing, drug discovery, and disease modeling. Recently, approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs), adult heart-derived cardiac progenitor cells (CPCs), and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts, highlighting potential applications and future challenges.

  View details for DOI 10.1016/j.stem.2011.12.013

  View details for PubMedID 22226352

• In Vivo Functional and Transcriptional Profiling of Bone Marrow Stem Cells After Transplantation Into Ischemic Myocardium ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Sheikh, A. Y., Huber, B. C., Narsinh, K. H., Spin, J. M., van der Bogt, K., de Almeida, P. E., Ransohoff, K. J., Kraft, D. L., Fajardo, G., Ardigo, D., Ransohoff, J., Bernstein, D., Fischbein, M. P., Robbins, R. C., Wu, J. C. 2012; 32 (1): 92-102

  Abstract

  Clinical trials of bone marrow-derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlies their potential efficacy remains unknown. In the present study, we evaluated the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in a murine myocardial infarction model.We used bioluminescence imaging and high-throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks posttransplantation. Moreover, transcriptomic analysis of BMMCs revealed nonspecific upregulation of various cell regulatory genes, with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography. Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis.Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy.

  View details for DOI 10.1161/ATVBAHA.111.238618

  View details for PubMedID 22034515

• Multimodality Imaging Evaluation of Functional and Clinical Benefits of Percutaneous Coronary Intervention in Patients with Chronic Total Occlusion Lesion THERANOSTICS Sun, D., Wang, J., Tian, Y., Narsinh, K., Wang, H., Li, C., Ma, X., Wang, Y., Wang, D., Li, C., Wu, J. C., Tian, J., Cao, F. 2012; 2 (8): 788-800

  Abstract

  To determine the effects of percutaneous coronary intervention (PCI) on cardiac perfusion, cardiac function, and quality of life in patients with chronic total occlusion (CTO) lesion in left anterior descending (LAD) coronary artery.Patients (n=99) with CTO lesion in the LAD coronary artery who had successfully undergone PCI were divided into three groups based on the SPECT/CTCA fusion imaging: (a) no severe cardiac perfusion defects (n=9); (b) reversible cardiac perfusion defects (n=40); or (c) fixed cardiac perfusion defects (n=50). No statistical difference of perfusion abnormality was observed at 6 months and 1 year after PCI in group (a). In group (b), SPECT/CTCA fusion imaging demonstrated that cardiac perfusion abnormality was significantly decreased 6 month and 1 year after PCI. Left ventricular ejection fraction (LVEF) increased significantly at 6 months and 1 year follow up. Quality of life improved at 6 months and 1 year after PCI procedure. Moreover, patients in group (c) also benefited from PCI therapy: a decrease in cardiac perfusion abnormality, an increase in LVEF, and an improvement in quality of life. PCI of coronary arteries in addition to LAD did not significantly affect cardiac function and quality of life improvement in each group.PCI exerts functional and clinical benefits in patients with CTO lesion in LAD coronary artery, particularly in patients with reversible cardiac perfusion defects. SPECT/CTCA fusion imaging may serve as a useful tool to evaluate the outcomes of patients with CTO lesion in LAD coronary artery.

  View details for DOI 10.7150/thno.4717

  View details for Web of Science ID 000307648500005

  View details for PubMedID 22916078

  View details for PubMedCentralID PMC3425125

• Epigenetic Modulations of Induced Pluripotent Stem Cells: Novel Therapies and Disease Models. Drug discovery today. Disease models Huang, C., Wu, J. C. 2012; 9 (4): e153-e160

  Abstract

  Recent breakthroughs in induced pluripotent stem cell (iPSC) technology hold promise for novel cell-based therapies as well as for effective drug development. The therapeutic potential of iPSCs makes it important to understand the reprogramming mechanisms and iPSC differentiation process. Epigenetic states that mediate exogenous stimulations on cell-intrinsic transcriptional features play a key role in iPSCs. This review focuses on epigenetic mechanisms that control iPSC pluripotency and differentiation. We discuss the potential application of epigenetic modulations in development of iPSC-based therapies and disease models.

  View details for PubMedID 23646061

• Molecular Imaging of Stem Cells: Tracking Survival, Biodistribution, Tumorigenicity, and Immunogenicity THERANOSTICS Gu, E., Chen, W., Gu, J., Burridge, P., Wu, J. C. 2012; 2 (4): 335-345

  Abstract

  Being able to self-renew and differentiate into virtually all cell types, both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have exciting therapeutic implications for myocardial infarction, neurodegenerative disease, diabetes, and other disorders involving irreversible cell loss. However, stem cell biology remains incompletely understood despite significant advances in the field. Inefficient stem cell differentiation, difficulty in verifying successful delivery to the target organ, and problems with engraftment all hamper the transition from laboratory animal studies to human clinical trials. Although traditional histopathological techniques have been the primary approach for ex vivo analysis of stem cell behavior, these postmortem examinations are unable to further elucidate the underlying mechanisms in real time and in vivo. Fortunately, the advent of molecular imaging has led to unprecedented progress in understanding the fundamental behavior of stem cells, including their survival, biodistribution, immunogenicity, and tumorigenicity in the targeted tissues of interest. This review summarizes various molecular imaging technologies and how they have advanced the current understanding of stem cell survival, biodistribution, immunogenicity, and tumorigenicity.

  View details for DOI 10.7150/thno.3666

  View details for Web of Science ID 000304031200002

  View details for PubMedID 22509197

  View details for PubMedCentralID PMC3326720

• Enhancement of Human Adipose-Derived Stromal Cell Angiogenesis through Knockdown of a BMP-2 Inhibitor PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., Nelson, E. R., Hyun, J. S., Glotzbach, J. P., Li, S., Nauta, A., Montoro, D. T., Lee, M., Commons, G. C., Hu, S., Wu, J. C., Gurtner, G. C., Longaker, M. T. 2012; 129 (1): 53-66

  Abstract

  Previous studies have demonstrated the role of noggin, a bone morphogenetic protein-2 inhibitor, in vascular development and angiogenesis. The authors hypothesized that noggin suppression in human adipose-derived stromal cells would enhance vascular endothelial growth factor secretion and angiogenesis in vitro and in vivo to a greater extent than bone morphogenetic protein-2 alone.Human adipose-derived stromal cells were isolated from human lipoaspirate (n = 6) noggin was knocked down using lentiviral techniques. Knockdown was confirmed and angiogenesis was assessed by tubule formation and quantitative real-time polymerase chain reaction. Cells were seeded onto scaffolds and implanted into a 4-mm critical size calvarial defect. In vivo angiogenic signaling was assessed by immunofluorescence and immunohistochemistry.Human adipose-derived stromal cells with noggin suppression secreted significantly higher amounts of angiogenic proteins, expressed higher levels of angiogenic genes, and formed more tubules in vitro. In vivo, calvarial defects seeded with noggin shRNA human adipose-derived stromal cells exhibited a significantly higher number of vessels in the defect site than controls by immunohistochemistry (p < 0.05). In addition, bone morphogenetic protein-2-releasing scaffolds significantly enhanced vascular signaling in the defect site.Human adipose-derived stromal cells demonstrate significant increases in angiogenesis in vitro and in vivo with both noggin suppression and BMP-2 supplementation. By creating a cell with noggin suppressed and by using a scaffold with increased bone morphogenetic protein-2 signaling, a more angiogenic niche can be created.

  View details for DOI 10.1097/PRS.0b013e3182361ff5

  View details for PubMedID 21915082

• American Journal of Nuclear Medicine and Molecular Imaging: Editorial Board (2012) e-Century Publishing Corporation. American journal of nuclear medicine and molecular imaging Seto, B., Jacobs, P. M., Mach, R. H., Bulte, J. W., Cai, W., Alavi, A., Divgi, C. R., Wu, J. C. 2012; 2 (4): 508-513

  View details for PubMedID 23145366

• Molecular Imaging of Bone Marrow Mononuclear Cell Survival and Homing in Murine Peripheral Artery Disease JACC-CARDIOVASCULAR IMAGING van der Bogt, K. E., Hellingman, A. A., Lijkwan, M. A., Bos, E., de Vries, M. R., van Rappard, J. R., Fischbein, M. P., Quax, P. H., Robbins, R. C., Hamming, J. F., Wu, J. C. 2012; 5 (1): 46-55

  Abstract

  This study aims to provide insight into cellular kinetics using molecular imaging after different transplantation methods of bone marrow-derived mononuclear cells (MNCs) in a mouse model of peripheral artery disease (PAD).MNC therapy is a promising treatment for PAD. Although clinical translation has already been established, there is a lack of knowledge about cell behavior after transplantation and about the mechanism whereby MNC therapy might ameliorate complaints of PAD.MNCs were isolated from F6 transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). Male FVB and C57Bl6 mice (n = 50) underwent femoral artery ligation and were randomized into 4 groups receiving the following: 1) single intramuscular (IM) injection of 2 × 10(6) MNCs; 2) 4 weekly IM injections of 5 × 10(5) MNCs; 3) 2 × 10(6) MNCs intravenously; and 4) phosphate-buffered saline as control. Cells were characterized by flow cytometry and in vitro bioluminescence imaging (BLI). Cell survival, proliferation, and migration were monitored by in vivo BLI, which was validated by ex vivo BLI, post-mortem immunohistochemistry, and flow cytometry. Paw perfusion and neovascularization was measured with laser Doppler perfusion imaging (LDPI) and histology, respectively.In vivo BLI revealed near-complete donor cell death 4 weeks after IM transplantation. After intravenous transplantation, BLI revealed that cells migrated to the injured area in the limb, as well as to the liver, spleen, and bone marrow. Ex vivo BLI showed presence of MNCs in the scar tissue and adductor muscle. However, no significant effects on neovascularization were observed, as monitored by LDPI and histology.This is one of the first studies to assess kinetics of transplanted MNCs in PAD using in vivo molecular imaging. MNC survival is short-lived, MNCs do not preferentially home to injured areas, and MNCs do not significantly stimulate perfusion in this particular model.

  View details for DOI 10.1016/j.jcmg.2011.07.011

  View details for PubMedID 22239892

• Endothelial progenitor cells in cardiovascular disease and chronic inflammation: from biomarker to therapeutic agent BIOMARKERS IN MEDICINE Grisar, J. C., Haddad, F., Gomari, F. A., Wu, J. C. 2011; 5 (6): 731-744

  Abstract

  The discovery of endothelial progenitor cells in the 1990s challenged the paradigm of angiogenesis by showing that cells derived from hematopoietic stem cells are capable of forming new blood vessels even in the absence of a pre-existing vessel network, a process termed vasculogenesis. Since then, the majority of studies in the field have found a strong association between circulating endothelial progenitor cells and cardiovascular risk. Several studies have also reported that inflammation influences the mobilization and differentiation of endothelial progenitor cells. In this review, we discuss the emerging role of endothelial progenitor cells as biomarkers of cardiovascular disease as well as the interplay between inflammation and endothelial progenitor cell biology. We will also review the challenges in the field of endothelial progenitor cell-based therapy.

  View details for DOI 10.2217/BMM.11.92

  View details for Web of Science ID 000298488200005

  View details for PubMedID 22103609

  View details for PubMedCentralID PMC3285378

• Non-invasive Bioluminescence Imaging of Myoblast-Mediated Hypoxia-Inducible Factor-1 Alpha Gene Transfer MOLECULAR IMAGING AND BIOLOGY Gheysens, O., Chen, I. Y., Rodriguez-Porcel, M., Chan, C., Rasooly, J., Vaerenberg, C., Paulmurugan, R., Willmann, J. K., Deroose, C., Wu, J., Gambhir, S. S. 2011; 13 (6): 1124-1132

  Abstract

  We tested a novel imaging strategy, in which both the survival of transplanted myoblasts and their therapeutic transgene expression, a recombinant hypoxia-inducible factor-1α (HIF-1α-VP2), can be monitored using firefly luciferase (fluc) and Renilla luciferase (hrl) bioluminescence reporter genes, respectively.The plasmid pUbi-hrl-pUbi-HIF-1α-VP2, which expresses both hrl and HIF-1α-VP2 using two ubiquitin promoters, was characterized in vitro. C2c12 myoblasts stably expressing fluc and transiently transfected with pUbi-hrl-pUbi-HIF-1α-VP2 were injected into the mouse hindlimb. Both hrl and fluc expression were monitored using bioluminescence imaging (BLI).Strong correlations existed between the expression of hRL and each of HIF-1α-VP2, VEGF, and PlGF (r(2) > 0.83, r(2) > 0.82, and r(2) > 0.97, respectively). In vivo, both transplanted cells and HIF-1α-VP2 transgene expression were successfully imaged using BLI.An objective evaluation of myoblast-mediated gene transfer in living mice can be performed by monitoring both the survival and the transgene expression of transplanted myoblasts using the techniques developed herein.

  View details for DOI 10.1007/s11307-011-0471-9

  View details for PubMedID 21267661

• Nonintegrating Knockdown and Customized Scaffold Design Enhances Human Adipose-Derived Stem Cells in Skeletal Repair STEM CELLS Levi, B., Hyun, J. S., Nelson, E. R., Li, S., Montoro, D. T., Wan, D. C., Jia, F. J., Glotzbach, J. C., James, A. W., Lee, M., Huang, M., Quarto, N., Gurtner, G. C., Wu, J. C., Longaker, M. T. 2011; 29 (12): 2018-2029

  Abstract

  An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose-derived stem cells (hASCs) represent a readily available, autogenous cell source with well-documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP-releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine.

  View details for DOI 10.1002/stem.757

  View details for PubMedID 21997852

• Patient-Specific Induced Pluripotent Stem Cell as a Model for Familial Dilated Cardiomyopathy Scientific Sessions of the American-Heart-Association/Resuscitation Science Symposium Sun, N., Yazawa, M., Liu, J., Han, L., Sanchez-Freire, V., Hu, S., Ricardo, D., Butte, M., Ashley, E., Longaker, M., Robbins, R., Wu, J. LIPPINCOTT WILLIAMS & WILKINS. 2011

  View details for Web of Science ID 000299738705089

• Site-Specific Reporter Gene Imaging of Stem Cell Transplantation: Early Engraftment Predicts Late Improvement in Cardiac Function Narsinh, K. H., Lan, F., Liu, J., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2011

  View details for Web of Science ID 000299738701337

• Site-Specific Recombinase Strategy to Create Induced Pluripotent Stem Cells Efficiently with Plasmid DNA STEM CELLS Karow, M., Chavez, C. L., Farruggio, A. P., Geisinger, J. M., Keravala, A., Jung, W. E., Lan, F., Wu, J. C., Chen-Tsai, Y., Calos, M. P. 2011; 29 (11): 1696-1704

  Abstract

  Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.

  View details for DOI 10.1002/stem.730

  View details for PubMedID 21898697

• Functional Characterization and Expression Profiling of Human Induced Pluripotent Stem Cell- and Embryonic Stem Cell-Derived Endothelial Cells STEM CELLS AND DEVELOPMENT Li, Z., Hu, S., Ghosh, Z., Han, Z., Wu, J. C. 2011; 20 (10): 1701-1710

  Abstract

  With regard to human induced pluripotent stem cells (hiPSCs), in which adult cells are reprogrammed into embryonic-like cells using defined factors, their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study, hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method, and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the "distances" among hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and HUVECs. We further conclude that hiPSCs can differentiate into functional endothelial cells, but with limited expansion potential compared with hESC-ECs; thus, extensive studies should be performed to explore the cause and extent of such differences before clinical application of hiPSC-ECs can begin.

  View details for DOI 10.1089/scd.2010.0426

  View details for PubMedID 21235328

• Imaging Guiding the Clinical Translation of Cardiac Stem Cell Therapy CIRCULATION RESEARCH Nguyen, P. K., Lan, F., Wang, Y., Wu, J. C. 2011; 109 (8): 962-979

  Abstract

  Stem cells have been touted as the holy grail of medical therapy, with promises to regenerate cardiac tissue, but it appears the jury is still out on this novel therapy. Using advanced imaging technology, scientists have discovered that these cells do not survive nor engraft long-term. In addition, only marginal benefit has been observed in large-animal studies and human trials. However, all is not lost. Further application of advanced imaging technology will help scientists unravel the mysteries of stem cell therapy and address the clinical hurdles facing its routine implementation. In this review, we will discuss how advanced imaging technology will help investigators better define the optimal delivery method, improve survival and engraftment, and evaluate efficacy and safety. Insights gained from this review may direct the development of future preclinical investigations and clinical trials.

  View details for DOI 10.1161/CIRCRESAHA.111.242909

  View details for Web of Science ID 000295368300015

  View details for PubMedID 21960727

  View details for PubMedCentralID PMC3206967

• Preclinical Derivation and Imaging of Autologously Transplanted Canine Induced Pluripotent Stem Cells JOURNAL OF BIOLOGICAL CHEMISTRY Lee, A. S., Xu, D., Plews, J. R., Nguyen, P. K., Nag, D., Lyons, J. K., Han, L., Hu, S., Lan, F., Liu, J., Huang, M., Narsinh, K. H., Long, C. T., de Almeida, P. E., Levi, B., Kooreman, N., Bangs, C., Pacharinsak, C., Ikeno, F., Yeung, A. C., Gambhir, S. S., Robbins, R. C., Longaker, M. T., Wu, J. C. 2011; 286 (37): 32697-32704

  Abstract

  Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.

  View details for DOI 10.1074/jbc.M111.235739

  View details for PubMedID 21719696

• Novel MicroRNA Prosurvival Cocktail for Improving Engraftment and Function of Cardiac Progenitor Cell Transplantation Annual Meeting of the American-Heart-Association Hu, S., Huang, M., Nguyen, P. K., Gong, Y., Li, Z., Jia, F., Lan, F., Liu, J., Nag, D., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2011: S27–S34

  Abstract

  Although stem cell therapy has provided a promising treatment for myocardial infarction, the low survival of the transplanted cells in the infarcted myocardium is possibly a primary reason for failure of long-term improvement. Therefore, the development of novel prosurvival strategies to boost stem cell survival will be of significant benefit to this field.Cardiac progenitor cells (CPCs) were isolated from transgenic mice, which constitutively express firefly luciferase and green fluorescent protein. The CPCs were transduced with individual lentivirus carrying the precursor of miR-21, miR-24, and miR-221, a cocktail of these 3 microRNA precursors, or green fluorescent protein as a control. After challenge in serum free medium, CPCs treated with the 3 microRNA cocktail showed significantly higher viability compared with untreated CPCs. After intramuscular and intramyocardial injections, in vivo bioluminescence imaging showed that microRNA cocktail-treated CPCs survived significantly longer after transplantation. After left anterior descending artery ligation, microRNA cocktail-treated CPCs boost the therapeutic efficacy in terms of functional recovery. Histological analysis confirmed increased myocardial wall thickness and CPC engraftment in the myocardium with the microRNA cocktail. Finally, we used bioinformatics analysis and experimental validation assays to show that Bim, a critical apoptotic activator, is an important target gene of the microRNA cocktail, which collectively can bind to the 3'UTR region of Bim and suppress its expression.We have demonstrated that a microRNA prosurvival cocktail (miR-21, miR-24, and miR-221) can improve the engraftment of transplanted cardiac progenitor cells and therapeutic efficacy for treatment of ischemic heart disease.

  View details for DOI 10.1161/CIRCULATIONAHA.111.017954

  View details for PubMedID 21911815

• Double Knockdown of Prolyl Hydroxylase and Factor-Inhibiting Hypoxia-Inducible Factor With Nonviral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction Annual Meeting of the American-Heart-Association Huang, M., Nguyen, P., Jia, F., Hu, S., Gong, Y., de Almeida, P. E., Wang, L., Nag, D., Kay, M. A., Giaccia, A. J., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2011: S46–S54

  Abstract

  Under normoxic conditions, hypoxia-inducible factor (HIF)-1α is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure.PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1α protein and angiogenesis genes, respectively.We demonstrated that HIF-1α upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.

  View details for DOI 10.1161/CIRCULATIONAHA.110.014019

  View details for PubMedID 21911818

• An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells NATURE BIOTECHNOLOGY Tang, C., Lee, A. S., Volkmer, J., Sahoo, D., Nag, D., Mosley, A. R., Inlay, M. A., Ardehali, R., Chavez, S. L., Pera, R. R., Behr, B., Wu, J. C., Weissman, I. L., Drukker, M. 2011; 29 (9): 829-U86

  Abstract

  An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.

  View details for DOI 10.1038/nbt.1947

  View details for PubMedID 21841799

• In vivo bioluminescence for tracking cell fate and function AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY de Almeida, P. E., van Rappard, J. R., Wu, J. C. 2011; 301 (3): H663-H671

  Abstract

  Tracking the fate and function of cells in vivo is paramount for the development of rational therapies for cardiac injury. Bioluminescence imaging (BLI) provides a means for monitoring physiological processes in real time, ranging from cell survival to gene expression to complex molecular processes. In mice and rats, BLI provides unmatched sensitivity because of the absence of endogenous luciferase expression in mammalian cells and the low background luminescence emanating from animals. In the field of stem cell therapy, BLI provides an unprecedented means to monitor the biology of these cells in vivo, giving researchers a greater understanding of their survival, migration, immunogenicity, and potential tumorigenicity in a living animal. In addition to longitudinal monitoring of cell survival, BLI is a useful tool for semiquantitative measurements of gene expression in vivo, allowing a better optimization of drug and gene therapies. Overall, this technology not only enables rapid, reproducible, and quantitative monitoring of physiological processes in vivo but also can measure the influences of therapeutic interventions on the outcome of cardiac injuries.

  View details for DOI 10.1152/ajpheart.00337.2011

  View details for PubMedID 21666118

• Imaging Atherosclerosis With F18-Fluorodeoxyglucose Positron Emission Tomography What Are We Actually Seeing? JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Wu, J. C., Nguyen, P. K. 2011; 58 (6): 615-617

  View details for DOI 10.1016/j.jacc.2011.04.021

  View details for Web of Science ID 000293167200010

  View details for PubMedID 21798424

• Pretreatment with angiotensin-converting enzyme inhibitor improves doxorubicin-induced cardiomyopathy via preservation of mitochondrial function JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Hiona, A., Lee, A. S., Nagendran, J., Xie, X., Connolly, A. J., Robbins, R. C., Wu, J. C. 2011; 142 (2): 396-U529

  Abstract

  Doxorubicin is a widely used chemotherapy drug, but its application is associated with cardiotoxicity. Free radical generation and mitochondrial dysfunction are thought to contribute to doxorubicin-induced cardiac failure. Angiotensin-converting enzyme inhibitors are commonly used as cardioprotective agents and have recently been shown in clinical studies to be efficacious in the prevention of anthracycline-induced heart failure. This study evaluated a mechanism for these protective effects by testing the ability of the angiotensin-converting enzyme inhibitor enalapril to preserve mitochondrial function in a model of chronic doxorubicin treatment in rats.Sprague Dawley rats were divided into 3 groups and followed for a total of 10 weeks: (1) control-untreated, (2) doxorubicin treated, and (3) doxorubicin + enalapril treated. Doxorubicin was administered via intraperitoneal injection at weekly intervals from weeks 2 to 7. Enalapril was administered in the drinking water of the doxorubicin + enalapril group for the study duration.Doxorubicin treatment produced a significant loss in left ventricular contractility (P < .05), decrease in mitochondrial function via impairment of state-3 respiration, decrease in the cytosolic fraction of adenosine triphosphate, and up-regulation of free radical production. Enalapril significantly attenuated the decrease in percent fractional shortening (P < .05) and prevented the doxorubicin-associated reduction in respiratory efficiency and cytosolic adenosine triphosphate content (P < .05). Enalapril also abolished the robust doxorubicin-induced increase in free radical formation.Administration of enalapril attenuates doxorubicin-induced cardiac dysfunction via preservation of mitochondrial respiratory efficiency and reduction in doxorubicin-associated free radical generation.

  View details for DOI 10.1016/j.jtcvs.2010.07.097

  View details for PubMedID 21094500

• Differences in Osteogenic Differentiation of Adipose-Derived Stromal Cells from Murine, Canine, and Human Sources In Vitro and In Vivo PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., Nelson, E. R., Brown, K., James, A. W., Xu, D., Dunlevie, R., Wu, J. C., Lee, M., Wu, B., Commons, G. W., Vistnes, D., Longaker, M. T. 2011; 128 (2): 373-386

  Abstract

  Given the diversity of species from which adipose-derived stromal cells are derived and studied, the authors set out to delineate the differences in the basic cell biology that may exist across species. Briefly, the authors found that significant differences exist with regard to proliferation and osteogenic potentials of adipose-derived stromal cells across species.Adipose-derived stromal cells were derived from human, mouse, and canine sources as previously described. Retinoic acid, insulin-like growth factor-1, and bone morphogenetic protein-2 were added to culture medium; proliferation and osteogenic differentiation were assessed by standardized assays. In vivo methods included seeding 150,000 adipose-derived stromal cells on a biomimetic scaffold and analyzing healing by micro-computed tomography and histology.Adipose-derived stromal cells from all species had the capability to undergo osteogenic differentiation. Canine adipose-derived stromal cells were the most proliferative, whereas human adipose-derived stromal cells were the most osteogenic (p < 0.05). Human cells, however, had the most significant osteogenic response to osteogenic media. Retinoic acid stimulated osteogenesis in mouse and canine cells but not in human adipose-derived stromal cells. Insulin-like growth factor-1 enhanced osteogenesis across all species, most notably in human- and canine-derived cells.Adipose-derived stromal cells derived from human, mouse, and canine all have the capacity to undergo osteogenic differentiation. Canine adipose-derived stromal cells appear to be the most proliferative, whereas human adipose-derived stromal cells appear to be the most osteogenic. Different cytokines and chemicals can be used to modulate this osteogenic response. These results are promising as attempts are made to optimize tissue-engineered bone using adipose-derived stromal cells.

  View details for DOI 10.1097/PRS.0b013e31821e6e49

  View details for PubMedID 21788829

• Imaging Cardiac Stem Cell Therapy: Translations to Human Clinical Studies JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH Zhang, W. Y., Ebert, A. D., Narula, J., Wu, J. C. 2011; 4 (4): 514-522

  Abstract

  Stem cell therapy promises to open exciting new options in the treatment of cardiovascular diseases. Although feasible and clinically safe, the in vivo behavior and integration of stem cell transplants still remain largely unknown. Thus, the development of innovative non-invasive imaging techniques capable of effectively tracking such therapy in vivo is vital for a more in-depth investigation into future clinical applications. Such imaging modalities will not only generate further insight into the mechanisms behind stem cell-based therapy, but also address some major concerns associated with translational cardiovascular stem cell therapy. In the present review, we summarize the principles underlying three major stem cell tracking methods: (1) radioactive labeling for positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging, (2) iron particle labeling for magnetic resonance imaging (MRI), and (3) reporter gene labeling for bioluminescence, fluorescence, MRI, SPECT, and PET imaging. We then discuss recent clinical studies that have utilized these modalities to gain biological insights into stem cell fate.

  View details for DOI 10.1007/s12265-011-9281-3

  View details for PubMedID 21538182

• Dissecting the Oncogenic and Tumorigenic Potential of Differentiated Human Induced Pluripotent Stem Cells and Human Embryonic Stem Cells CANCER RESEARCH Ghosh, Z., Huang, M., Hu, S., Wilson, K. D., Dey, D., Wu, J. C. 2011; 71 (14): 5030-5039

  Abstract

  Pluripotent stem cells, both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However, the tumorigenic potential of these cells remains a great concern, as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice, most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal, cardiac, or endothelial cells prior to human transplantation, drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study, we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells, and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer, whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall, our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation, and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy.

  View details for DOI 10.1158/0008-5472.CAN-10-4402

  View details for PubMedID 21646469

• Efficient gene delivery of primary human cells using peptide linked polyethylenimine polymer hybrid BIOMATERIALS Dey, D., Inayathullah, M., Lee, A. S., LeMieux, M. C., Zhang, X., Wu, Y., Nag, D., De Almeida, P. E., Han, L., Rajadas, J., Wu, J. C. 2011; 32 (20): 4647-4658

  Abstract

  Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nanogels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly, when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of 'reactive oxygen species' (ROS) in the presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress.

  View details for DOI 10.1016/j.biomaterials.2011.03.016

  View details for PubMedID 21477858

• A Novel Method of Local Gene Delivery and Noninvasive Imaging of Transgene Expression in the Mouse Endometrium 44th Annual Meeting of the Society-for-the-Study-of-Reproduction (SSR) Fan, X., Dhal, S., Wu, J. C., Kuo, C. J., Druzin, M. L., Nayak, N. R. SOC STUDY REPRODUCTION. 2011

  View details for Web of Science ID 000310746200585

• Report of the National Heart, Lung, and Blood Institute Working Group on the Translation of Cardiovascular Molecular Imaging CIRCULATION Buxton, D. B., Antman, M., Danthi, N., Dilsizian, V., Fayad, Z. A., Garcia, M. J., Jaff, M. R., Klimas, M., Libby, P., Nahrendorf, M., Sinusas, A. J., Wickline, S. A., Wu, J. C., Bonow, R. O., Weissleder, R. 2011; 123 (19): 2157-2163

  View details for DOI 10.1161/CIRCULATIONAHA.110.000943

  View details for Web of Science ID 000290616600017

  View details for PubMedID 21576680

  View details for PubMedCentralID PMC3119488

• Derivation of Human Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling CIRCULATION RESEARCH Narsinh, K., Narsinh, K. H., Wu, J. C. 2011; 108 (9): 1146-1156

  Abstract

  The successful derivation of human induced pluripotent stem cells (hiPSCs) by dedifferentiation of somatic cells offers significant potential to overcome obstacles in the field of cardiovascular disease. hiPSC derivatives offer incredible potential for new disease models and regenerative medicine therapies. However, many questions remain regarding the optimal starting materials and methods to enable safe, efficient derivation of hiPSCs suitable for clinical applications. Initial reprogramming experiments were carried out using lentiviral or retroviral gene delivery methods. More recently, various nonviral methods that avoid permanent and random transgene insertion have emerged as alternatives. These include transient DNA transfection using plasmids or minicircles, protein transduction, or RNA transfection. In addition, several small molecules have been found to significantly augment hiPSC derivation efficiency, allowing the use of a fewer number of genes during pluripotency induction. We review these various methods for the derivation of hiPSCs, focusing on their ultimate clinical applicability, with an emphasis on their potential for use as cardiovascular therapies and disease-modeling platforms.

  View details for DOI 10.1161/CIRCRESAHA.111.240374

  View details for PubMedID 21527744

• Comparison of Human Induced Pluripotent and Embryonic Stem Cells: Fraternal or Identical Twins? MOLECULAR THERAPY Narsinh, K. H., Plews, J., Wu, J. C. 2011; 19 (4): 635-638

  View details for DOI 10.1038/mt.2011.41

  View details for PubMedID 21455209

• Short-Term Immunosuppression Promotes Engraftment of Embryonic and Induced Pluripotent Stem Cells CELL STEM CELL Pearl, J. I., Lee, A. S., Leveson-Gower, D. B., Sun, N., Ghosh, Z., Lan, F., Ransohoff, J., Negrin, R. S., Davis, M. M., Wu, J. C. 2011; 8 (3): 309-317

  Abstract

  Embryonic stem cells (ESCs) are an attractive source for tissue regeneration and repair therapies because they can be differentiated into virtually any cell type in the adult body. However, for this approach to succeed, the transplanted ESCs must survive long enough to generate a therapeutic benefit. A major obstacle facing the engraftment of ESCs is transplant rejection by the immune system. Here we show that blocking leukocyte costimulatory molecules permits ESC engraftment. We demonstrate the success of this immunosuppressive therapy for mouse ESCs, human ESCs, mouse induced pluripotent stem cells (iPSCs), human induced pluripotent stem cells, and more differentiated ESC/(iPSCs) derivatives. Additionally, we provide evidence describing the mechanism by which inhibition of costimulatory molecules suppresses T cell activation. This report describes a short-term immunosuppressive approach capable of inducing engraftment of transplanted ESCs and iPSCs, providing a significant improvement in our mechanistic understanding of the critical role costimulatory molecules play in leukocyte activation.

  View details for DOI 10.1016/j.stem.2011.01.012

  View details for PubMedID 21362570

• Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells JOURNAL OF CLINICAL INVESTIGATION Narsinh, K. H., Sun, N., Sanchez-Freire, V., Lee, A. S., Almeida, P., Hu, S., Jan, T., Wilson, K. D., Leong, D., Rosenberg, J., Yao, M., Robbins, R. C., Wu, J. C. 2011; 121 (3): 1217-1221

  Abstract

  Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.

  View details for DOI 10.1172/JCI44635

  View details for PubMedID 21317531

• Bioluminescence Reporter Gene Imaging Characterize Human Embryonic Stem Cell-Derived Teratoma Formation JOURNAL OF CELLULAR BIOCHEMISTRY Su, W., Zhou, M., Zheng, Y., Fan, Y., Wang, L., Han, Z., Kong, D., Zhao, R., Wu, J. C., Xiang, R., Li, Z. 2011; 112 (3): 840–48

  Abstract

  Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However, teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore, tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study, hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology, we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover, FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4, and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency, retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers.

  View details for DOI 10.1002/jcb.22982

  View details for Web of Science ID 000287910200012

  View details for PubMedID 21328457

  View details for PubMedCentralID PMC3138474

• Studies in Adipose-Derived Stromal Cells: Migration and Participation in Repair of Cranial Injury after Systemic Injection PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Nelson, E. R., Hu, S., Sun, N., Peng, M., Wu, J., Longaker, M. T. 2011; 127 (3): 1130-1140

  Abstract

  Adipose-derived stromal cells are a multipotent cell type with the ability to undergo osteogenic differentiation. The authors sought to examine whether systemically administered adipose-derived stromal cells would migrate to and heal surgically created defects of the mouse cranial skeleton.Mouse adipose-derived stromal cells were harvested from luciferase-positive transgenic mice; human adipose-derived stromal cells were harvested from human lipoaspirate and labeled with luciferase and green fluorescent protein. A 4-mm calvarial defect (critical sized) was made in the mouse parietal bone; skin incisions alone were used as a control (n = 5 per group). Adipose-derived stromal cells were injected intravenously (200,000 cells per animal) and compared with saline injection only. Methods of analyses included micro-computed tomographic scanning, in vivo imaging system detection of luciferase activity, and standard histology.Migration of adipose-derived stromal cells to calvarial defect sites was confirmed by accumulation of luciferase activity and green fluorescent protein stain as early as 4 days and persisting up to 4 weeks. Little activity was observed among control groups. Intravenous administration of either mouse or human adipose-derived stromal cells resulted in histologic evidence of bone formation within the defect site, in comparison with an absence of bone among control defects. By micro-computed tomographic analysis, human but not mouse adipose-derived stromal cells stimulated significant osseous healing.Intravenously administered adipose-derived stromal cells migrate to sites of calvarial injury. Thereafter, intravenous human adipose-derived stromal cells contribute to bony calvarial repair. Intravenous administration of adipose-derived stromal cells may be an effective delivery method for future efforts in skeletal regeneration.

  View details for DOI 10.1097/PRS.0b013e3182043712

  View details for PubMedID 21364416

• Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells HUMAN MOLECULAR GENETICS Panula, S., Medrano, J. V., Kee, K., Bergstrom, R., Ha Nam Nguyen, N. N., Byers, B., Wilson, K. D., Wu, J. C., Simon, C., Hovatta, O., Pera, R. A. 2011; 20 (4): 752-762

  Abstract

  Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications.

  View details for DOI 10.1093/hmg/ddq520

  View details for PubMedID 21131292

• Sex differences in the diagnostic evaluation of coronary artery disease JOURNAL OF NUCLEAR CARDIOLOGY Nguyen, P. K., Nag, D., Wu, J. C. 2011; 18 (1): 144-152

  View details for DOI 10.1007/s12350-010-9315-2

  View details for PubMedID 21136229

• Response to Letter Regarding Article, "Dynamic MicroRNA Expression Programs During Cardiac Differentiation of Human Embryonic Stem Cells: Role for miR-499" CIRCULATION-CARDIOVASCULAR GENETICS Wilson, K. D., Wu, J. C. 2011; 4 (1): E4

  View details for DOI 10.1161/CIRCGENETICS.110.958942

  View details for Web of Science ID 000287353200004

• Radiation exposure from imaging tests: is there an increased cancer risk? Expert review of cardiovascular therapy Nguyen, P. K., Wu, J. C. 2011; 9 (2): 177-183

  Abstract

  The increasing exposure to low-dose radiation from diagnostic testing has prompted renewed interest in evaluating its carcinogenic risk, but quantifying health risk from low-dose radiation exposure remains controversial. The current approach is to adopt the linear non-threshold model, which is commonly applied to high-dose exposure, and apply it to assess risk from low-dose exposure. However, existing data are conflicting and limited to epidemiological studies and/or in vitro analyses. In this article, we will discuss the potential cancer risk from low- and high-dose radiation, their effects on DNA repair response pathways, and the best course of action for patients and providers to minimize risk.

  View details for DOI 10.1586/erc.10.184

  View details for PubMedID 21453214

• Cardiovascular Molecular Imaging Focus on Clinical Translation CIRCULATION Chen, I. Y., Wu, J. C. 2011; 123 (4): 425-443

  View details for DOI 10.1161/CIRCULATIONAHA.109.916338

  View details for PubMedID 21282520

• Elastic Properties of Induced Pluripotent Stem Cells TISSUE ENGINEERING PART A Hammerick, K. E., Huang, Z., Sun, N., Lam, M. T., Prinz, F. B., Wu, J. C., Commons, G. W., Longaker, M. T. 2011; 17 (3-4): 495-502

  Abstract

  The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell, we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was as follows: hASC-iPSC, human embryonic stem cell, fibroblast-iPSC, fibroblasts, and, lastly, as the stiffest cell, hASC. In comparing hASC-iPSCs to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence, material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state.

  View details for DOI 10.1089/ten.tea.2010.0211

  View details for PubMedID 20807017

• Molecular imaging of RNA interference therapy targeting PHD2 for treatment of myocardial ischemia. Methods in molecular biology (Clifton, N.J.) Huang, M., Wu, J. C. 2011; 709: 211-221

  Abstract

  Coronary artery disease is the number one cause of morbidity and mortality in the Western world. It typically occurs when heart muscle receives inadequate blood supply due to rupture of atherosclerotic plaques. During ischemia, up-regulation of hypoxia inducible factor-1 alpha (HIF-1α) transcriptional factor can activate several downstream angiogenic genes. However, HIF-1α is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Recently, we cloned the mouse PHD2 gene by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted behind H1 promoter, followed by a separate hypoxia response element (HRE)-incorporated promoter driving a firefly luciferase (Fluc) reporter gene. This construct allowed us to monitor gene expression noninvasively and was used to test the hypothesis that inhibition of PHD2 by short hairpin RNA interference (shRNA) can lead to significant improvement in angiogenesis and contractility as revealed by in vitro and in vivo experiments.

  View details for DOI 10.1007/978-1-61737-982-6_13

  View details for PubMedID 21194030

• Enhanced Healing of Diabetic Wounds by Topical Administration of Adipose Tissue-Derived Stromal Cells Overexpressing Stromal-Derived Factor-1: Biodistribution and Engraftment Analysis by Bioluminescent Imaging STEM CELLS INTERNATIONAL Di Rocco, G., Gentile, A., Antonini, A., Ceradini, F., Wu, J. C., Capogrossi, M. C., Toietta, G. 2011

  View details for DOI 10.4061/2011/304562

  View details for Web of Science ID 000208760300013

• Launch of the american journal of nuclear medicine and molecular imaging. American journal of nuclear medicine and molecular imaging Seto, B., Jacobs, P. M., Alavi, A., Mach, R. H., Divgi, C. R., Bulte, J. W., Wu, J. C., Cai, W. 2011; 1 (1): 1-2

  View details for PubMedID 23133791

  View details for PubMedCentralID PMC3477714

• American Journal of Nuclear Medicine and Molecular Imaging: Editorial Board (2011) e-Century Publishing Corporation. American journal of nuclear medicine and molecular imaging Seto, B., Jacobs, P. M., Mach, R. H., Bulte, J. W., Cai, W., Alavi, A., Divgi, C. R., Wu, J. C. 2011; 1 (1): 80-83

  View details for PubMedID 23133798

• Effects of Long-Term Culture on Human Embryonic Stem Cell Aging STEM CELLS AND DEVELOPMENT Xie, X., Hiona, A., Lee, A. S., Cao, F., Huang, M., Li, Z., Cherry, A., Pei, X., Wu, J. C. 2011; 20 (1): 127-138

  Abstract

  In recent years, human embryonic stem (hES) cells have become a promising cell source for regenerative medicine. Although hES cells have the ability for unlimited self-renewal, potential adverse effects of long-term cell culture upon hES cells must be investigated before therapeutic applications of hES cells can be realized. Here we investigated changes in molecular profiles associated with young (<60 passages) and old (>120 passages) cells of the H9 hES cell line as well as young (<85 passages) and old (>120 passages) cells of the PKU1 hES cell line. Our results show that morphology, stem cell markers, and telomerase activity do not differ significantly between young and old passage cells. Cells from both age groups were also shown to differentiate into derivatives of all 3 germ layers upon spontaneous differentiation in vitro. Interestingly, mitochondrial dysfunction was found to occur with prolonged culture. Old passage cells of both the H9 and PKU1 lines were characterized by higher mitochondrial membrane potential, larger mitochondrial morphology, and higher reactive oxygen species content than their younger counterparts. Teratomas derived from higher passage cells were also found to have an uneven preference for differentiation compared with tumors derived from younger cells. These findings suggest that prolonged culture of hES cells may negatively impact mitochondrial function and possibly affect long-term pluripotency.

  View details for DOI 10.1089/scd.2009.0475

  View details for PubMedID 20629482

• Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors NATURE PROTOCOLS Narsinh, K. H., Jia, F., Robbins, R. C., Kay, M. A., Longaker, M. T., Wu, J. C. 2011; 6 (1): 78-88

  Abstract

  Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼4 weeks.

  View details for DOI 10.1038/nprot.2010.173

  View details for PubMedID 21212777

• Imaging of embryonic stem cell migration in vivo. Methods in molecular biology (Clifton, N.J.) Lee, A. S., Wu, J. C. 2011; 750: 101-114

  Abstract

  Conventional reporter gene technology and histological methods cannot routinely be used to track the in vivo behavior of embryonic stem (ES) cells longitudinally after cellular transplantation. Here we describe a protocol for monitoring the in vivo survival, proliferation, and migration of ES cells without necessitating animal sacrifice. Stable ES cell lines containing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase) reporter genes can be established within 4-6 weeks by lentiviral transduction followed by fluorescence-activated cell sorting. The cell fate and behavior of these DF or TF ES cells can subsequently be tracked noninvasively by bioluminescence and microPET imaging for a prolonged period of time.

  View details for DOI 10.1007/978-1-61779-145-1_7

  View details for PubMedID 21618086

• Tumorigenicity of pluripotent stem cells: biological insights from molecular imaging JOURNAL OF THE ROYAL SOCIETY INTERFACE Kooreman, N. G., Wu, J. C. 2010; 7: S753-S763

  Abstract

  Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the ability (i) to duplicate indefinitely while maintaining pluripotency and (ii) to differentiate into cell types of all three embryonic germ layers. These two properties of ESCs and iPSCs make them potentially suitable for tissue engineering and cell replacement therapy for many different diseases, including Parkinson's disease, diabetes and heart disease. However, one critical obstacle in the clinical application of ESCs or iPSCs is the risk of teratoma formation. The emerging field of molecular imaging is allowing researchers to track transplanted ESCs or iPSCs in vivo, enabling early detection of teratomas.

  View details for DOI 10.1098/rsif.2010.0353.focus

  View details for PubMedID 20880852

• Dynamic MicroRNA Expression Programs During Cardiac Differentiation of Human Embryonic Stem Cells Role for miR-499 CIRCULATION-CARDIOVASCULAR GENETICS Wilson, K. D., Hu, S., Venkatasubrahmanyam, S., Fu, J., Sun, N., Abilez, O. J., Baugh, J. J., Jia, F., Ghosh, Z., Li, R. A., Butte, A. J., Wu, J. C. 2010; 3 (5): 426-U97

  Abstract

  MicroRNAs (miRNAs) are a newly discovered endogenous class of small, noncoding RNAs that play important posttranscriptional regulatory roles by targeting messenger RNAs for cleavage or translational repression. Human embryonic stem cells are known to express miRNAs that are often undetectable in adult organs, and a growing body of evidence has implicated miRNAs as important arbiters of heart development and disease.To better understand the transition between the human embryonic and cardiac "miRNA-omes," we report here the first miRNA profiling study of cardiomyocytes derived from human embryonic stem cells. Analyzing 711 unique miRNAs, we have identified several interesting miRNAs, including miR-1, -133, and -208, that have been previously reported to be involved in cardiac development and disease and that show surprising patterns of expression across our samples. We also identified novel miRNAs, such as miR-499, that are strongly associated with cardiac differentiation and that share many predicted targets with miR-208. Overexpression of miR-499 and -1 resulted in upregulation of important cardiac myosin heavy-chain genes in embryoid bodies; miR-499 overexpression also caused upregulation of the cardiac transcription factor MEF2C.Taken together, our data give significant insight into the regulatory networks that govern human embryonic stem cell differentiation and highlight the ability of miRNAs to perturb, and even control, the genes that are involved in cardiac specification of human embryonic stem cells.

  View details for DOI 10.1161/CIRCGENETICS.109.934281

  View details for PubMedID 20733065

• Methods to assess stem cell lineage, fate and function ADVANCED DRUG DELIVERY REVIEWS Nguyen, P. K., Nag, D., Wu, J. C. 2010; 62 (12): 1175-1186

  Abstract

  Stem cell therapy has the potential to regenerate injured tissue. For stem cells to achieve their full therapeutic potential, stem cells must differentiate into the target cell, reach the site of injury, survive, and engraft. To fully characterize these cells, evaluation of cell morphology, lineage specific markers, cell specific function, and gene expression must be performed. To monitor survival and engraftment, cell fate imaging is vital. Only then can organ specific function be evaluated to determine the effectiveness of therapy. In this review, we will discuss methods for evaluating the function of transplanted cells for restoring the heart, nervous system, and pancreas. We will also highlight the specific challenges facing these potential therapeutic areas.

  View details for DOI 10.1016/j.addr.2010.08.008

  View details for PubMedID 20816906

• MicroRNA-210 as a Novel Therapy for Treatment of Ischemic Heart Disease 82nd National Conference and Exhibitions and Scientific Sessions of the American-Heart-Association Hu, S., Huang, M., Li, Z., Jia, F., Ghosh, Z., Lijkwan, M. A., Fasanaro, P., Sun, N., Wang, X., Li, F. M., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2010: S124–S131

  Abstract

  MicroRNAs are involved in various critical functions, including the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. We hypothesize that microRNA-210 can rescue cardiac function after myocardial infarction by upregulation of angiogenesis and inhibition of cellular apoptosis in the heart.Using microRNA microarrays, we first showed that microRNA-210 was highly expressed in live mouse HL-1 cardiomyocytes compared with apoptotic cells after 48 hours of hypoxia exposure. We confirmed by polymerase chain reaction that microRNA-210 was robustly induced in these cells. Gain-of-function and loss-of-function approaches were used to investigate microRNA-210 therapeutic potential in vitro. After transduction, microRNA-210 can upregulate several angiogenic factors, inhibit caspase activity, and prevent cell apoptosis compared with control. Afterward, adult FVB mice underwent intramyocardial injections with minicircle vector carrying microRNA-210 precursor, minicircle carrying microRNA-scramble, or sham surgery. At 8 weeks, echocardiography showed a significant improvement of left ventricular fractional shortening in the minicircle vector carrying microRNA-210 precursor group compared with the minicircle carrying microRNA-scramble control. Histological analysis confirmed decreased cellular apoptosis and increased neovascularization. Finally, 2 potential targets of microRNA-210, Efna3 and Ptp1b, involved in angiogenesis and apoptosis were confirmed through additional experimental validation.MicroRNA-210 can improve angiogenesis, inhibit apoptosis, and improve cardiac function in a murine model of myocardial infarction. It represents a potential novel therapeutic approach for treatment of ischemic heart disease.

  View details for DOI 10.1161/CIRCULATIONAHA.109.928424

  View details for PubMedID 20837903

• The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat BIOMATERIALS Yu, J., Du, K. T., fang, q., Gu, Y., Mihardja, S. S., Sievers, R. E., Wu, J. C., Lee, R. J. 2010; 31 (27): 7012-7020

  Abstract

  The combination of scaffold material and cell transplantation therapy has been extensively investigated in cardiac tissue engineering. However, many polymers are difficult to administer or lack the structural integrity to restore LV function. Additionally, polymers need to be biological friendly, favorably influence the microenvironment and increase stem cell retention and survival. This study determined whether human mesenchymal stem cells (hMSCs) encapsulated in RGD modified alginate microspheres are capable of facilitating myocardial repair. The in vitro study of hMSCs demonstrated that the RGD modified alginate can improve cell attachment, growth and increase angiogenic growth factor expression. Alginate microbeads and hMSCs encapsulated in microbeads successfully maintained LV shape and prevented negative LV remodeling after an MI. Cell survival was significantly increased in the encapsulated hMSC group compared with PBS control or cells alone. Microspheres, hMSCs, and hMSCs in microspheres groups reduced infarct area and enhanced arteriole formation. In summary, surface modification and microencapsulation techniques can be combined with cell transplantation leading to the maintenance of LV geometry, preservation of LV function, increase of angiogenesis and improvement of cell survival.

  View details for DOI 10.1016/j.biomaterials.2010.05.078

  View details for Web of Science ID 000280616300013

  View details for PubMedID 20566215

• Role of Molecular Imaging in Stem Cell Therapy for Myocardial Restoration TRENDS IN CARDIOVASCULAR MEDICINE Lijkwan, M. A., Bos, E. J., Wu, J. C., Robbins, R. C. 2010; 20 (6): 183-188

  Abstract

  During the past two decades, stem cells have created enthusiasm as a regenerative therapy for ischemic heart disease. Transplantation of bone marrow stem cells, skeletal myoblasts, and endothelial progenitor cells has shown to improve myocardial function after infarction. Recently, attention has focused on the potential use of embryonic stem cells and induced pluripotent stem cells because they possess the capacity to differentiate into various cell types, including cardiac and endothelial cells. Clinical trials have shown positive effects on the functional recovery of heart after myocardial infarction and have answered questions on timing, dosage, and cell delivery route of stem cells such as those derived from bone marrow. Despite the current advances in stem cell research, one main hurdle remains the lack of reliable information about the fate of cell engraftment, survival, and proliferation after transplantation. This review discusses the different cell types used in cardiac cell therapy as well as molecular imaging modalities relevant to survival issues.

  View details for PubMedID 22137639

• Effects of Ionizing Radiation on Self-Renewal and Pluripotency of Human Embryonic Stem Cells CANCER RESEARCH Wilson, K. D., Sun, N., Huang, M., Zhang, W. Y., Lee, A. S., Li, Z., Wang, S. X., Wu, J. C. 2010; 70 (13): 5539-5548

  Abstract

  Human embryonic stem cells (hESC) present a novel platform for in vitro investigation of the early embryonic cellular response to ionizing radiation. Thus far, no study has analyzed the genome-wide transcriptional response to ionizing radiation in hESCs, nor has any study assessed their ability to form teratomas, the definitive test of pluripotency. In this study, we use microarrays to analyze the global gene expression changes in hESCs after low-dose (0.4 Gy), medium-dose (2 Gy), and high-dose (4 Gy) irradiation. We identify genes and pathways at each radiation dose that are involved in cell death, p53 signaling, cell cycling, cancer, embryonic and organ development, and others. Using Gene Set Enrichment Analysis, we also show that the expression of a comprehensive set of core embryonic transcription factors is not altered by radiation at any dose. Transplantation of irradiated hESCs to immune-deficient mice results in teratoma formation from hESCs irradiated at all doses, definitive proof of pluripotency. Further, using a bioluminescence imaging technique, we have found that irradiation causes hESCs to initially die after transplantation, but the surviving cells quickly recover by 2 weeks to levels similar to control. To conclude, we show that similar to somatic cells, irradiated hESCs suffer significant death and apoptosis after irradiation. However, they continue to remain pluripotent and are able to form all three embryonic germ layers. Studies such as this will help define the limits for radiation exposure for pregnant women and also radiotracer reporter probes for tracking cellular regenerative therapies.

  View details for DOI 10.1158/0008-5472.CAN-09-4238

  View details for PubMedID 20530673

• Advances in cardiovascular molecular imaging for tracking stem cell therapy THROMBOSIS AND HAEMOSTASIS Ransohoff, K. J., Wu, J. C. 2010; 104 (1): 13-22

  Abstract

  The high mortality rate associated with cardiovascular disease is partially due to the lack of proliferative cells in the heart. Without adequate repair following myocardial infarction, progressive dilation can lead to heart failure. Stem cell therapies present one promising option for treating cardiovascular disease, though the specific mechanisms by which they benefit the heart remain unclear. Before stem cell therapies can be used safely in human populations, their biology must be investigated using innovative technologies such as multi-modality molecular imaging. The present review will discuss the basic principles, labelling techniques, clinical applications, and drawbacks associated with four major modalities: radionuclide imaging, magnetic resonance imaging, bioluminescence imaging, and fluorescence imaging.

  View details for DOI 10.1160/TH09-08-0530

  View details for PubMedID 20458434

• Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy GENE THERAPY Chen, I. Y., Gheysens, O., Ray, S., Wang, Q., Padmanabhan, P., Paulmurugan, R., Loening, A. M., Rodriguez-Porcel, M., Willmann, J. K., Sheikh, A. Y., Nielsen, C. H., Hoyt, G., Contag, C. H., Robbins, R. C., Biswal, S., Wu, J. C., Gambhir, S. S. 2010; 17 (7): 827-838

  Abstract

  Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

  View details for DOI 10.1038/gt.2010.30

  View details for PubMedID 20237511

• Micro-CT for Characterization of Murine CV Disease Models JACC-CARDIOVASCULAR IMAGING Sheikh, A. Y., van der Bogt, K. E., Doyle, T. C., Sheikh, M. K., Ransohoff, K. J., Ali, Z. A., Palmer, O. P., Robbins, R. C., Fischbein, M. P., Wu, J. C. 2010; 3 (7): 783-785

  View details for DOI 10.1016/j.jcmg.2010.01.012

  View details for Web of Science ID 000281626700015

  View details for PubMedID 20633858

  View details for PubMedCentralID PMC2952324

• Gene Correction in Human Embryonic and Induced Pluripotent Stem Cells: Promises and Challenges Ahead MOLECULAR THERAPY Narsinh, K. H., Wu, J. C. 2010; 18 (6): 1061-1063

  View details for DOI 10.1038/mt.2010.92

  View details for PubMedID 20514030

• In vivo visualization of transplanted bone marrow mononuclear cell behavior in murine peripheral artery occlusive disease 45th Congress of the European-Society-for-Surgical-Research van der Bogt, K. E., Hellingman, A. A., Lijkwan, M. A., Bos, E. J., de Vries, M. R., FISCHBEIN, M., QUAX, P. H., Robbins, R. C., Wu, J. C., Hamming, J. F. WILEY-BLACKWELL. 2010: S9–S9

  View details for Web of Science ID 000281136400032

• Antioxidants Improve Early Survival of Cardiomyoblasts After Transplantation to the Myocardium MOLECULAR IMAGING AND BIOLOGY Rodriguez-Porcel, M., Gheysens, O., Paulmurugan, R., Chen, I. Y., Peterson, K. M., Willmann, J. K., Wu, J. C., Zhu, X., Lerman, L. O., Gambhir, S. S. 2010; 12 (3): 325-334

  Abstract

  We tested the hypothesis that modulation of the microenvironment (using antioxidants) will increase stem cell survival in hypoxia and after transplantation to the myocardium.Rat cardiomyoblasts were stably transfected with a reporter gene (firefly luciferase) for bioluminescence imaging (BLI). First, we examined the role of oxidative stress in cells under hypoxic conditions. Subsequently, stem cells were transplanted to the myocardium of rats using high-resolution ultrasound, and their survival was monitored daily using BLI.Under hypoxia, oxidative stress was increased together with decreased cell survival compared to control cells, both of which were preserved by antioxidants. In living subjects, oxidative stress blockade increased early cell survival after transplantation to the myocardium, compared to untreated cells/animals.Modulation of the local microenvironment (with antioxidants) improves stem cell survival. Increased understanding of the interaction between stem cells and their microenvironment will be critical to advance the field of regenerative medicine.

  View details for DOI 10.1007/s11307-009-0274-4

  View details for Web of Science ID 000277375300011

  View details for PubMedID 20013064

  View details for PubMedCentralID PMC2865580

• Current Perspectives on Imaging Cardiac Stem Cell Therapy JOURNAL OF NUCLEAR MEDICINE Wu, J. C., Abraham, M. R., Kraitchman, D. L. 2010; 51: 128S-136S

  Abstract

  Molecular imaging is a new discipline that makes possible the noninvasive visualization of cellular and molecular processes in living subjects. In the field of cardiovascular regenerative therapy, imaging cell fate after transplantation is a high priority in both basic research and clinical translation. For cell-based therapy to truly succeed, we must be able to track the locations of delivered cells, the duration of cell survival, and any potential adverse effects. The insights gathered from basic research imaging studies will yield valuable insights into better designs for clinical trials. This review highlights the different types of stem cells used for cardiovascular repair, the development of various imaging modalities to track their fate in vivo, and the challenges of clinical translation of cardiac stem cell imaging in the future.

  View details for DOI 10.2967/jnumed.109.068239

  View details for PubMedID 20395348

• Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Huang, N. F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M. D., Wu, J. C., Gambhir, S. S., Cooke, J. P. 2010; 30 (5): 984-U224

  Abstract

  We examined the effect of delivery modality on the survival, localization, and functional effects of exogenously administered embryonic stem cells (ESCs) or endothelial cells derived from them (ESC-ECs) in the ischemic hindlimb.Murine ESCs or ESC-ECs were stably transduced with a construct for bioluminescence imaging (BLI) and fluorescent detection. In a syngeneic murine model of limb ischemia, ESCs or ESC-ECs were delivered by intramuscular (IM), intrafemoral artery (IA), or intrafemoral vein injections (n=5 in each group). For 2 weeks, cell survival and localization were tracked by BLI and confirmed by immunohistochemistry, and functional improvement was assessed by laser Doppler perfusion. BLI showed that ESCs localized to the ischemic limb after IM or IA, but not after intrafemoral vein administration. Regardless of the route of administration, ESCs were detected outside the hindlimb circulation in the spleen or lungs. ESCs did not improve limb perfusion and generated teratomas. In contrast, ESC-ECs delivered by all 3 modalities localized to the ischemic limb, as assessed by BLI. Most surprisingly, ESC-EC injected intrafemoral vein eventually localized to the ischemic limb after initially lodging in the pulmonary circulation. Immunohistochemical studies confirmed the engraftment of ESC-ECs into the limb vasculature after 2 weeks. Notably, ESC-ECs were not detected in the spleen or lungs after 2 weeks, regardless of route of administration. Furthermore, ESC-ECs significantly improved limb perfusion and neovascularization compared with the parental ESCs or the vehicle control group.In contrast to parental ESCs, ESC-ECs preferentially localized in the ischemic hindlimb by IA, IM, and intrafemoral vein delivery. ESC-ECs engrafted into the ischemic microvasculature, enhanced neovascularization, and improved limb perfusion.

  View details for DOI 10.1161/ATVBAHA.110.202796

  View details for PubMedID 20167654

• Rosiglitazone Increases Myocardial Glucose Metabolism in Insulin-Resistant Cardiomyopathy JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Kao, D. P., Witteles, R. M., Quon, A., Wu, J. C., Gambhir, S. S., Fowler, M. B. 2010; 55 (9): 926-927

  View details for DOI 10.1016/j.jacc.2009.08.085

  View details for Web of Science ID 000274865100015

  View details for PubMedID 20185047

• Human iPS cell-based therapy Considerations before clinical applications CELL CYCLE Sun, N., Longaker, M. T., Wu, J. C. 2010; 9 (5): 880-885

  Abstract

  Generation of induced pluripotent stem (iPS) cells has revolutionized the field of regenerative medicine. With the exponential increase in iPS cell research in the past three years, human iPS cells have been derived with different technologies and from various cell types. From a translational perspective, however, a number of issues must be addressed before safe and high quality patient-specific iPS cells can be derived for clinical applications. In addition, iPS cell-based therapies also need to be thoroughly evaluated in pre-clinical animal models before they can be applied to human subjects.

  View details for PubMedID 20160515

• A nonviral minicircle vector for deriving human iPS cells NATURE METHODS Jia, F., Wilson, K. D., Sun, N., Gupta, D. M., Huang, M., Li, Z., Panetta, N. J., Chen, Z. Y., Robbins, R. C., Kay, M. A., Longaker, M. T., Wu, J. C. 2010; 7 (3): 197-U46

  Abstract

  Owing to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem cells (iPSCs). We report the use of 'minicircle' DNA, a vector type that is free of bacterial DNA and capable of high expression in cells, for this purpose. Here we use a single minicircle vector to generate transgene-free iPSCs from adult human adipose stem cells.

  View details for DOI 10.1038/NMETH.1426

  View details for PubMedID 20139967

• Human Neural Stem Cell Grafts Modify Microglial Response and Enhance Axonal Sprouting in Neonatal Hypoxic-Ischemic Brain Injury STROKE Daadi, M. M., Davis, A. S., Arac, A., Li, Z., Maag, A., Bhatnagar, R., Jiang, K., Sun, G., Wu, J. C., Steinberg, G. K. 2010; 41 (3): 516-523

  Abstract

  Hypoxic-ischemic (HI) brain injury in newborn infants represents a major cause of cerebral palsy, development delay, and epilepsy. Stem cell-based therapy has the potential to rescue and replace the ischemic tissue caused by HI and to restore function. However, the mechanisms by which stem cell transplants induce functional recovery are yet to be elucidated. In the present study, we sought to investigate the efficacy of human neural stem cells derived from human embryonic stem cells in a rat model of neonatal HI and the mechanisms enhancing brain repair.The human neural stem cells were genetically engineered for in vivo molecular imaging and for postmortem histological tracking. Twenty-four hours after the induction of HI, animals were grafted with human neural stem cells into the forebrain. Motor behavioral tests were performed the fourth week after transplantation. We used immunocytochemistry and neuroanatomical tracing to analyze neural differentiation, axonal sprouting, and microglia response. Treatment-induced changes in gene expression were investigated by microarray and quantitative polymerase chain reaction.Bioluminescence imaging permitted real time longitudinal tracking of grafted human neural stem cells. HI transplanted animals significantly improved in their use of the contralateral impeded forelimb and in the Rotorod test. The grafts showed good survival, dispersion, and differentiation. We observed an increase of uniformly distributed microglia cells in the grafted side. Anterograde neuroanatomical tracing demonstrated significant contralesional sprouting. Microarray analysis revealed upregulation of genes involved in neurogenesis, gliogenesis, and neurotrophic support.These results suggest that human neural stem cell transplants enhance endogenous brain repair through multiple modalities in response to HI.

  View details for DOI 10.1161/STROKEAHA.109.573691

  View details for PubMedID 20075340

• Comparison of Gene-Transfer Efficiency in Human Embryonic Stem Cells MOLECULAR IMAGING AND BIOLOGY Cao, F., Xie, X., Gollan, T., Zhao, L., Narsinh, K., Lee, R. J., Wu, J. C. 2010; 12 (1): 15-24

  Abstract

  Technologies designed to allow manipulation and modification of human embryonic stem (hES) cells are numerous and vary in the complexity of their methods, efficiency, reliability, and safety. The most commonly studied and practiced of these methods include electroporation, lipofection, nucleofection, and lentiviral transduction. However, at present, it is unclear which protocol offers the most efficient and reliable method of gene transfer to hES cells. In this study, a bi-fusion construct with ubiquitin promoter driving enhanced green fluorescent protein reporter and the firefly luciferase (pUb-eGFP-Fluc) along with neomycin selection marker was used for in vitro and in vivo studies. In vitro studies examined the transfection efficiency and viability of each technique using two hES cell lines (male H1 and female H9 cells). Lentiviral transduction demonstrated the highest efficiency (H1: 25.3 +/- 4.8%; H9: 22.4 +/- 6.5%) with >95% cell viability. Nucleofection demonstrated transfection efficiency of 16.1 +/- 3.6% (H1) and 5.8 +/- 3.2% (H9). However, minimal transfection efficiency was observed with electroporation (2.1 +/- 0.4% (H1) and 1.9 +/- 0.3% (H9)) and lipofection (1.5 +/- 0.5% (H1) and 1.3 +/- 0.2% (H9); P < 0.05 vs. lentiviral transduction). Electroporation also demonstrated the highest cell death (62 +/- 11% (H1) and 42 +/- 10% (H9)) followed by nucleofection (25 +/- 9% (H1) and 30 +/- 15 (H9)). Importantly, lentiviral transduction generated a greater number of hES cell lines stably expressing the double-fusion reporter gene (hES-DF) compared to other transfection techniques. Finally, following subcutaneous transplantation into immunodeficient nude mice, the hES-eGFP-Fluc cells showed robust proliferation as determined by longitudinal bioluminescence imaging. In summary, this study demonstrates that lentiviral transduction and nucleofection are efficient, simple, and safe techniques for reliable gene transfer in hES cells. The double-fusion construct provides an attractive approach for generating stable hES cell lines and monitoring engraftment and proliferation in vitro and in vivo.

  View details for DOI 10.1007/s11307-009-0236-x

  View details for PubMedID 19551446

• Persistent Donor Cell Gene Expression among Human Induced Pluripotent Stem Cells Contributes to Differences with Human Embryonic Stem Cells PLOS ONE Ghosh, Z., Wilson, K. D., Wu, Y., Hu, S., Quertermous, T., Wu, J. C. 2010; 5 (2)

  Abstract

  Human induced pluripotent stem cells (hiPSCs) generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs), as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.

  View details for DOI 10.1371/journal.pone.0008975

  View details for PubMedID 20126639

• Timing of Bone Marrow Cell Delivery Has Minimal Effects on Cell Viability and Cardiac Recovery After Myocardial Infarction CIRCULATION-CARDIOVASCULAR IMAGING Swijnenburg, R., Govaert, J. A., van der Bogt, K. E., Pearl, J. I., Huang, M., Stein, W., Hoyt, G., Vogel, H., Contag, C. H., Robbins, R. C., Wu, J. C. 2010; 3 (1): 77-U109

  Abstract

  Despite ongoing clinical trials, the optimal time for delivery of bone marrow mononuclear cells (BMCs) after myocardial infarction is unclear. We compared the viability and effects of transplanted BMCs on cardiac function in the acute and subacute inflammatory phases of myocardial infarction.The time course of acute inflammatory cell infiltration was quantified by FACS analysis of enzymatically digested hearts of FVB mice (n=12) after left anterior descending artery ligation. Mac-1(+)Gr-1(high) neutrophil infiltration peaked at day 4. BMCs were harvested from transgenic FVB mice expressing firefly luciferase (Fluc) and green fluorescent protein (GFP). Afterward, 2.5x10(6) BMCs were injected into the left ventricle of wild-type FVB mice either immediately (acute BMC) or 7 days (subacute BMC) after myocardial infarction, or after a sham procedure (n=8 per group). In vivo bioluminescence imaging showed an early signal increase in both BMC groups at day 7, followed by a nonsignificant trend (P=0.203) toward improved BMC survival in the subacute BMC group that persisted until the bioluminescence imaging signal reached<0.01) and 6 weeks (both BMC groups versus saline; P<0.05) but no significant differences between the 2 BMC groups. FACS analysis of BMC-injected hearts at day 7 revealed that GFP(+) BMCs expressed hematopoietic (CD45, Mac-1, Gr-1), minimal progenitor (Sca-1, c-kit), and no endothelial (CD133, Flk-1) or cardiac (Trop-T) cell markers.Timing of BMC delivery has minimal effects on intramyocardial retention and preservation of cardiac function. In general, there is poor long-term engraftment and BMCs tend to adopt inflammatory cell phenotypes.

  View details for DOI 10.1161/CIRCIMAGING.109.872085

  View details for PubMedID 19920031

• Embryonic stem cell biology: insights from molecular imaging. Methods in molecular biology (Clifton, N.J.) Sallam, K., Wu, J. C. 2010; 660: 185-199

  Abstract

  Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.

  View details for DOI 10.1007/978-1-60761-705-1_12

  View details for PubMedID 20680820

• Functional and Transcriptional Characterization of Human Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Myocardial Infarction PLOS ONE Li, Z., Wilson, K. D., Smith, B., Kraft, D. L., Jia, F., Huang, M., Xie, X., Robbins, R. C., Gambhir, S. S., Weissman, I. L., Wu, J. C. 2009; 4 (12)

  Abstract

  Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However, the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product, both of which can limit the future clinical application of hESC-ECs. Moreover, to fully understand the beneficial effects of stem cell therapy, investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time.In this study, we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray, and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover, our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods.Taken together, we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes, form functional vessels in vivo, and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future.

  View details for DOI 10.1371/journal.pone.0008443

  View details for PubMedID 20046878

• Current-Controlled Electrical Point-Source Stimulation of Embryonic Stem Cells CELLULAR AND MOLECULAR BIOENGINEERING Chen, M. Q., Xie, X., Wilson, K. D., Sun, N., Wu, J. C., Giovangrandi, L., Kovacs, G. T. 2009; 2 (4): 625-635

  Abstract

  Stem cell therapy is emerging as a promising clinical approach for myocardial repair. However, the interactions between the graft and host, resulting in inconsistent levels of integration, remain largely unknown. In particular, the influence of electrical activity of the surrounding host tissue on graft differentiation and integration is poorly understood. In order to study this influence under controlled conditions, an in vitro system was developed. Electrical pacing of differentiating murine embryonic stem (ES) cells was performed at physiologically relevant levels through direct contact with microelectrodes, simulating the local activation resulting from contact with surrounding electroactive tissue. Cells stimulated with a charged balanced voltage-controlled current source for up to 4 days were analyzed for cardiac and ES cell gene expression using real-time PCR, immunofluorescent imaging, and genome microarray analysis. Results varied between ES cells from three progressive differentiation stages and stimulation amplitudes (nine conditions), indicating a high sensitivity to electrical pacing. Conditions that maximally encouraged cardiomyocyte differentiation were found with Day 7 EBs stimulated at 30 microA. The resulting gene expression included a sixfold increase in troponin-T and a twofold increase in beta-MHCwithout increasing ES cell proliferation marker Nanog. Subsequent genome microarray analysis revealed broad transcriptome changes after pacing. Concurrent to upregulation of mature gene programs including cardiovascular, neurological, and musculoskeletal systems is the apparent downregulation of important self-renewal and pluripotency genes. Overall, a robust system capable of long-term stimulation of ES cells is demonstrated, and specific conditions are outlined that most encourage cardiomyocyte differentiation.

  View details for DOI 10.1007/s12195-009-0096-0

  View details for Web of Science ID 000272671600015

  View details for PubMedCentralID PMC2905819

• Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion 82nd National Conference and Exhibitions and Scientific Sessions of the American-Heart-Association Huang, N. F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M. D., Wu, J. C., Gambhir, S. S., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 2009: S1152–S1152

  View details for Web of Science ID 000271831504251

• Enhancing HIF-1a by Double shRNA Knockdown in Murine Myocardial Infarction 82nd National Conference and Exhibitions and Scientific Sessions of the American-Heart-Association Huang, M., Jia, F., Hu, S., Wang, X., Kay, M. A., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2009: S827–S827

  View details for Web of Science ID 000271831502527

• MicroRNA-210 as a Novel Therapy for Treatment of Ischemic Heart Disease 82nd National Conference and Exhibitions and Scientific Sessions of the American-Heart-Association Hu, S., Huang, M., Wang, X., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2009: S899–S899

  View details for Web of Science ID 000271831503012

• Poor Functional Recovery After Transplantation of Diabetic Bone Marrow Stem Cells in Ischemic Myocardium JOURNAL OF HEART AND LUNG TRANSPLANTATION Govaert, J. A., Swijnenburg, R., Schrepfer, S., Xie, X., van der Bogt, K. E., Hoyt, G., Stein, W., Ransohoff, K. J., Robbins, R. C., Wu, J. C. 2009; 28 (11): 1158-1165

  Abstract

  Autologous bone marrow mononuclear cell (BMMC) therapy has shown promise for improving cardiac function after myocardial infarction. The efficiency of such therapy for diabetic patients remains unknown.BMMCs were harvested from type 2 diabetic male BKS.Cg-m+/+Lepr(db)/J mice or C57BLKS/J (non-diabetic control) mice and were isolated using Ficoll-based separation. Cell characterization was performed by flow cytometry. Cell viability was determined by apoptosis and proliferation assays. Female BKS.Cg-m+/+Lepr(db)/J mice underwent left anterior descending artery ligation and were randomized into 3 groups receiving 2.5 x 10(6) diabetic BMMCs (n = 8), 2.5 x 10(6) control BMMCs (n = 8), or phosphate-buffered saline (n = 6). At Week 5, cardiac function was assessed with echocardiography and invasive hemodynamic measurements. Post-mortem cell survival was quantified by TaqMan real-time transcription polymerase chain reaction (RT-PCR) for the male Sry gene.BKS.Cg-m+/+Lepr(db)/J BMMCs showed a significantly lower mononuclear fraction and a significantly lower proliferation rate compared with C57BLKS/J BMMCs. Fractional shorting (40.1% +/- 1.2% vs 30.3% +/- 1.9%; p = 0.001) and cardiac output (4,166 +/- 393 vs 2,246 +/- 462 microl/min; p = 0.016) significantly improved for mice treated with control BMMCs injection compared with those treated with diabetic BMMCs, respectively. This difference could not be attributed to difference in cell engraftment because TaqMan RT-PCR showed no significant difference in cell survival in infarcted hearts between the 2 groups.Diabetic BMMCs are significantly impaired in their ability to improve cardiac function after myocardial infarction compared with control BMMCs. These findings could have significant clinical implication regarding autologous BMMC therapy in diabetic patients.

  View details for DOI 10.1016/j.healun.2009.06.018

  View details for Web of Science ID 000271795900007

  View details for PubMedID 19782602

  View details for PubMedCentralID PMC3163601

• Hepatocyte Growth Factor or Vascular Endothelial Growth Factor Gene Transfer Maximizes Mesenchymal Stem Cell-Based Myocardial Salvage After Acute Myocardial Infarction 81st Annual Scientific Session of the American-Heart-Association Deuse, T., Peter, C., Fedak, P. W., Doyle, T., Reichenspurner, H., Zimmermann, W. H., Eschenhagen, T., Stein, W., Wu, J. C., Robbins, R. C., Schrepfer, S. LIPPINCOTT WILLIAMS & WILKINS. 2009: S247–S254

  Abstract

  Mesenchymal stem cell (MSC)-based regenerative strategies were investigated to treat acute myocardial infarction and improve left ventricular function.Murine AMI was induced by coronary ligation with subsequent injection of MSCs, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), or MSCs +HGF/VEGF into the border zone. Left ventricular ejection fraction was calculated using micro-computed tomography imaging after 6 months. HGF and VEGF protein injection (with or without concomitant MSC injection) significantly and similarly improved the left ventricular ejection fraction and reduced scar size compared with the MSC group, suggesting that myocardial recovery was due to the cytokines rather than myocardial regeneration. To provide sustained paracrine effects, HGF or VEGF overexpressing MSCs were generated (MSC-HGF, MSC-VEGF). MSC-HGF and MSC-VEGF showed significantly increased in vitro proliferation and increased in vivo proliferation within the border zone. Cytokine production correlated with MSC survival. MSC-HGF- and MSC-VEGF-treated animals showed smaller scar sizes, increased peri-infarct vessel densities, and better preserved left ventricular function when compared with MSCs transfected with empty vector. Murine cardiomyocytes were exposed to hypoxic in vitro conditions. The LDH release was reduced, fewer cardiomyocytes were apoptotic, and Akt activity was increased if cardiomyocytes were maintained in conditioned medium obtained from MSC-HGF or MSC-VEGF cultures.This study showed that (1) elevating the tissue levels of HGF and VEGF after acute myocardial infarction seems to be a promising reparative therapeutic approach, (2) HGF and VEGF are cardioprotective by increasing the tolerance of cardiomyocytes to ischemia, reducing cardiomyocyte apoptosis and increasing prosurvival Akt activation, and (3) MSC-HGF and MSC-VEGF are a valuable source for increased cytokine production and maximize the beneficial effect of MSC-based repair strategies.

  View details for DOI 10.1161/CIRCULATIONAHA.108.843680

  View details for PubMedID 19752375

• nAChRs Mediate Human Embryonic Stem Cell-Derived Endothelial Cells: Proliferation, Apoptosis, and Angiogenesis PLOS ONE Yu, J., Huang, N. F., Wilson, K. D., Velotta, J. B., Huang, M., Li, Z., Lee, A., Robbins, R. C., Cooke, J. P., Wu, J. C. 2009; 4 (9)

  Abstract

  Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).To induce endothelial cell differentiation, undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days, CD31(+) cells (13.7+/-2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation, these hESC-ECs expressed endothelial specific markers such as vWF (96.3+/-1.4%), CD31 (97.2+/-2.5%), and VE-cadherin (93.7+/-2.8%), form vascular-like channels, and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward, 5x10(6) hESC-ECs treated for 24 hours with nicotine (10(-8) M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 microg/ml) in the drinking water. Surprisingly, bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally, in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs, and enhance their angiogenic effects in vivo. Furthermore, activation of nAChRs has anti-apoptotic, angiogenic, and proliferative effects through MAPK and Akt signaling pathways.

  View details for DOI 10.1371/journal.pone.0007040

  View details for Web of Science ID 000269796500020

  View details for PubMedID 19753305

  View details for PubMedCentralID PMC2737633

• Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sun, N., Panetta, N. J., Gupta, D. M., Wilson, K. D., Lee, A., Jia, F., Hu, S., Cherry, A. M., Robbins, R. C., Longaker, M. T., Wu, J. C. 2009; 106 (37): 15720-15725

  Abstract

  Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.

  View details for DOI 10.1073/pnas.0908450106

  View details for PubMedID 19805220

• Novel Minicircle Vector for Gene Therapy in Murine Myocardial Infarction 81st Annual Scientific Session of the American-Heart-Association Huang, M., Chen, Z., Hu, S., Jia, F., Li, Z., Hoyt, G., Robbins, R. C., Kay, M. A., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2009: S230–S237

  Abstract

  Conventional plasmids for gene therapy produce low-level and short-term gene expression. In this study, we develop a novel nonviral vector that robustly and persistently expresses the hypoxia-inducible factor-1 alpha (HIF-1alpha) therapeutic gene in the heart, leading to functional benefits after myocardial infarction.We first created minicircles (MC) carrying double-fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) for noninvasive measurement of transfection efficiency. Mouse C2C12 myoblasts and normal FVB/N mice were used for in vitro and in vivo confirmation, respectively. Bioluminescence imaging showed stable MC gene expression in the heart for >12 weeks and the activity level was 5.6+/-1.2-fold stronger than regular plasmid at day 4 (P<0.01). Next, we created MC carrying HIF-1alpha (MC-HIF-1alpha) therapeutic gene for treatment of myocardial infarction. Adult FVB/N mice underwent left anterior descending ligation and were injected intramyocardially with: (1) MC-HIF-1alpha; (2) regular plasmid carrying HIF-1alpha (PL-HIF-1alpha) as positive control; and (3) PBS as negative control (n=10/group). Echocardiographic study showed a significantly greater improvement of left ventricular ejection fraction in the MC group (51.3%+/-3.6%) compared to regular plasmid group (42.3%+/-4.1%) and saline group (30.5%+/-2.8%) at week 4 (P<0.05 for both). Histology demonstrated increased neoangiogenesis in both treatment groups. Finally, Western blot showed MC express >50% higher HIF-1alpha level than regular plasmid.Taken together, this is the first study to our knowledge to demonstrate that MC can significantly improve transfection efficiency, duration of transgene expression, and cardiac contractility. Given the serious drawbacks associated with most viral vectors, we believe this novel nonviral vector can be of great value for cardiac gene therapy protocols.

  View details for DOI 10.1161/CIRCULATIONAHA.108.841155

  View details for PubMedID 19752373

• Rapid and efficient feeder-free generation of human adipose stromal cell-derived induced pluripotent stem cells (hASC-iPSCs) 95th Annual Clinical Congress of the American-College-of-Surgeons/64th Annual Sessions of the Owen H Wangensteen Forum on Fundamental Surgical Problems Gupta, D. M., Panetta, N. J., Sun, N., Wilson, K. D., Lee, A., Jia, F., Robbins, R. C., Longaker, M. T., Wu, J. C. ELSEVIER SCIENCE INC. 2009: S87–S87

  View details for Web of Science ID 000269755300193

• Effects of cell number on teratoma formation by human embryonic stem cells CELL CYCLE Lee, A. S., Tang, C., Cao, F., Xie, X., van der Bogt, K., Hwang, A., Connolly, A. J., Robbins, R. C., Wu, J. C. 2009; 8 (16): 2608-2612

  Abstract

  Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source, potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n = 35) or skeletal muscle (n = 35) injection of 1 x 10(2), 1 x 10(3), 1 x 10(4), 1 x 10(5) or 1 x 10(6) DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after 8 weeks were followed out to day 365 to confirm tumor absence. Significantly, in this study, a minimum of 1 x 10(5) ES cells in the myocardium and 1 x 10(4) cells in the skeletal muscle was observed to be requisite for teratoma development, suggesting that human ES cell number may be a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic.

  View details for PubMedID 19597339

• Imaging Gene Expression in Human Mesenchymal Stem Cells: From Small to Large Animals RADIOLOGY Willmann, J. K., Paulmurugan, R., Rodriguez-Porcel, M., Stein, W., Brinton, T. J., Connolly, A. J., Nielsen, C. H., Lutz, A. M., Lyons, J., Ikeno, F., Suzuki, Y., Rosenberg, J., Chen, I. Y., Wu, J. C., Yeung, A. C., Yock, P., Robbins, R. C., Gambhir, S. S. 2009; 252 (1): 117-127

  Abstract

  To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning.Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Transduction of human MSCs by using different doses of adenovirus that contained a cytomegalovirus (CMV) promoter driving the mutant herpes simplex virus type 1 thymidine kinase reporter gene (Ad-CMV-HSV1-sr39tk) was characterized in a cell culture. A total of 2.25 x 10(6) transduced (n = 5) and control nontransduced (n = 5) human MSCs were injected into the myocardium of 10 rats, and reporter gene expression in human MSCs was visualized with micro-PET by using the radiotracer 9-(4-[fluorine 18]-fluoro-3-hydroxymethylbutyl)-guanine (FHBG). Different numbers of transduced human MSCs suspended in either phosphate-buffered saline (PBS) (n = 4) or matrigel (n = 5) were injected into the myocardium of nine swine, and gene expression was visualized with a clinical PET-CT. For analysis of cell culture experiments, linear regression analyses combined with a t test were performed. To test differences in radiotracer uptake between injected and remote myocardium in both rats and swine, one-sided paired Wilcoxon tests were performed. In swine experiments, a linear regression of radiotracer uptake ratio on the number of injected transduced human MSCs was performed.In cell culture, there was a viral dose-dependent increase of gene expression and FHBG accumulation in human MSCs. Human MSC viability was 96.7% (multiplicity of infection, 250). Cardiac FHBG uptake in rats was significantly elevated (P < .0001) after human MSC injection (0.0054% injected dose [ID]/g +/- 0.0007 [standard deviation]) compared with that in the remote myocardium (0.0003% ID/g +/- 0.0001). In swine, myocardial radiotracer uptake was not elevated after injection of up to 100 x 10(6) human MSCs (PBS group). In the matrigel group, signal-to-background ratio increased to 1.87 after injection of 100 x 10(6) human MSCs and positively correlated (R(2) = 0.97, P < .001) with the number of administered human MSCs.Reporter gene imaging in human MSCs can be translated to large animals. The study highlights the importance of co-administering a "scaffold" for increasing intramyocardial retention of human MSCs.

  View details for DOI 10.1148/radiol.2513081616

  View details for PubMedID 19366903

• Molecular and Magnetic Resonance Imaging of Human Embryonic Stem Cell-Derived Neural Stem Cell Grafts in Ischemic Rat Brain MOLECULAR THERAPY Daadi, M. M., Li, Z., Arac, A., Grueter, B. A., Sofilos, M., Malenka, R. C., Wu, J. C., Steinberg, G. K. 2009; 17 (7): 1282-1291

  Abstract

  Real-time imaging of transplanted stem cells is essential for understanding their interactions in vivo with host environments, for tracking cell fate and function and for successful delivery and safety monitoring in the clinical setting. In this study, we used bioluminescence (BLI) and magnetic resonance imaging (MRI) to visualize the fate of grafted human embryonic stem cell (hESC)-derived human neural stem cells (hNSCs) in stroke-damaged rat brain. The hNSCs were genetically engineered with a lentiviral vector carrying a double fusion (DF) reporter gene that stably expressed enhanced green fluorescence protein (eGFP) and firefly luciferase (fLuc) reporter genes. The hNSCs were self-renewable, multipotent, and expressed markers for neural stem cells. Cell survival was tracked noninvasively by MRI and BLI for 2 months after transplantation and confirmed histologically. Electrophysiological recording from grafted GFP(+) cells and immuno-electronmicroscopy demonstrated connectivity. Grafted hNSCs differentiated into neurons, into oligodendrocytes in stroke regions undergoing remyelination and into astrocytes extending processes toward stroke-damaged vasculatures. Our data suggest that the combination of BLI and MRI modalities provides reliable real-time monitoring of cell fate.

  View details for DOI 10.1038/mt.2009.104

  View details for PubMedID 19436269

• MicroRNA Profiling of Human-Induced Pluripotent Stem Cells STEM CELLS AND DEVELOPMENT Wilson, K. D., Venkatasubrahmanyam, S., Jia, F., Sun, N., Butte, A. J., Wu, J. C. 2009; 18 (5): 749-757

  Abstract

  MicroRNAs (miRNAs) are a newly discovered endogenous class of small noncoding RNAs that play important posttranscriptional regulatory roles by targeting mRNAs for cleavage or translational repression. Accumulating evidence now supports the importance of miRNAs for human embryonic stem cell (hESC) self-renewal, pluripotency, and differentiation. However, with respect to induced pluripotent stem cells (iPSC), in which embryonic-like cells are reprogrammed from adult cells using defined factors, the role of miRNAs during reprogramming has not been well-characterized. Determining the miRNAs that are associated with reprogramming should yield significant insight into the specific miRNA expression patterns that are required for pluripotency. To address this lack of knowledge, we use miRNA microarrays to compare the "microRNA-omes" of human iPSCs, hESCs, and fetal fibroblasts. We confirm the presence of a signature group of miRNAs that is up-regulated in both iPSCs and hESCs, such as the miR-302 and 17-92 clusters. We also highlight differences between the two pluripotent cell types, as in expression of the miR-371/372/373 cluster. In addition to histone modifications, promoter methylation, transcription factors, and other regulatory control elements, we believe these miRNA signatures of pluripotent cells likely represent another layer of regulatory control over cell fate decisions, and should prove important for the cellular reprogramming field.

  View details for DOI 10.1089/scd.2008.0247

  View details for PubMedID 19284351

• Comparison of Optical Bioluminescence Reporter Gene and Superparamagnetic Iron Oxide MR Contrast Agent as Cell Markers for Noninvasive Imaging of Cardiac Cell Transplantation MOLECULAR IMAGING AND BIOLOGY Chen, I. Y., Greve, J. M., Gheysens, O., Willmann, J. K., Rodriguez-Porcel, M., Chu, P., Sheikh, A. Y., Faranesh, A. Z., Paulmurugan, R., Yang, P. C., Wu, J. C., Gambhir, S. S. 2009; 11 (3): 178-187

  Abstract

  In this study, we compared firefly luciferase (Fluc) reporter gene and superparamagnetic iron oxide (Feridex) as cell markers for longitudinal monitoring of cardiomyoblast graft survival using optical bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), respectively.Rats (n = 31) underwent an intramyocardial injection of cardiomyoblasts (2 x 10(6)) labeled with Fluc, Feridex, or no marker (control) or an injection of Feridex alone (75 microg). Afterward, rats were serially imaged with BLI or MRI and killed at different time points for histological analysis.BLI revealed a drastically different cell survival kinetics (half-life = 2.65 days over 6 days) than that revealed by MRI (half-life = 16.8 days over 80 days). Injection of Feridex alone led to prolonged tissue retention of Feridex (> or =16 days) and persistent MR signal (> or =42 days).Fluc BLI reporter gene imaging is a more accurate gauge of transplanted cell survival as compared to MRI of Feridex-labeled cells.

  View details for DOI 10.1007/s11307-008-0182-z

  View details for PubMedID 19034584

• Tracking cardiac engraftment and distribution of implanted bone marrow cells: Comparing intra-aortic, intravenous, and intramyocardial delivery JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Li, S., Lai, T. Y., Sun, Z., Han, M., Moriyama, E., Wilson, B., Fazel, S., Weisel, R. D., Yau, T., Wu, J. C., Li, R. 2009; 137 (5): 1225-U216

  Abstract

  Cell therapy improved cardiac function after a myocardial infarction in several preclinical studies; however, the functional benefits were limited in the initial clinical trials, perhaps because of inadequate cell engraftment. We used noninvasive molecular imaging to compare the distribution and myocardial retention of cells implanted by using clinical delivery routes.Bone marrow stromal cells isolated from male rats and transfected with a firefly luciferase reporter gene were injected by using 3 increasingly invasive techniques (ie, intravenous, intra-aortic, and intramyocardial) into female rats 3 or 28 days after coronary ligation. Whole-body bioluminescence imaging was performed 2, 24, and 48 hours later; implanted cells were quantified at 48 hours in explanted organs by means of bioluminescence and real-time polymerase chain reaction.Variations in cell distribution among groups were profound, with nearly complete trapping of the injected cells in the lungs after intravenous delivery. Cell delivery into the aortic root (with the distal aorta occluded) produced minimal cell retention in the heart. Direct intramyocardial injection facilitated the best early targeting of the cells (P < .05 vs intravenous and intra-aortic injection). Rapid signal loss over 48 hours indicated very poor cell survival in all 3 groups, although implanted cell retention was greater in mature compared with acute infarcts.This is the first study to correlate live cell imaging with quantitative genetic and histologic techniques. Noninvasive molecular imaging tracked delivered cells and will permit the evaluation of new and improved delivery platforms designed to increase cell homing, retention, and engraftment.

  View details for DOI 10.1016/j.jtcvs.2008.11.001

  View details for Web of Science ID 000265299000029

  View details for PubMedID 19379996

• Imaging Survival and Function of Transplanted Cardiac Resident Stem Cells JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Li, Z., Lee, A., Huang, M., Chun, H., Chung, J., Chu, P., Hoyt, G., Yang, P., Rosenberg, J., Robbins, R. C., Wu, J. C. 2009; 53 (14): 1229-1240

  Abstract

  The goal of this study is to characterize resident cardiac stem cells (CSCs) and investigate their therapeutic efficacy in myocardial infarction by molecular imaging methods.CSCs have been isolated and characterized in vitro. These cells offer a provocative method to regenerate the damaged myocardium. However, the survival kinetics and function of transplanted CSCs have not been fully elucidated.CSCs were isolated from L2G85 transgenic mice (FVB strain background) that constitutively express both firefly luciferase and enhanced green fluorescence protein reporter gene. CSCs were characterized in vitro and transplanted in vivo into murine infarction models. Multimodality noninvasive imaging techniques were used to assess CSC survival and therapeutic efficacy for restoration of cardiac function.CSCs can be isolated from L2G85 mice, and fluorescence-activated cell sorting analysis showed expression of resident CSC markers (Sca-1, c-Kit) and mesenchymal stem cell markers (CD90, CD106). Afterwards, 5 x 10(5) CSCs (n = 30) or phosphate-buffered saline control (n = 15) was injected into the hearts of syngeneic FVB mice undergoing left anterior descending artery ligation. Bioluminescence imaging showed poor donor cell survival by week 8. Echocardiogram, invasive hemodynamic pressure-volume analysis, positron emission tomography imaging with fluorine-18-fluorodeoxyglucose, and cardiac magnetic resonance imaging demonstrated no significant difference in cardiac contractility and viability between the CSC and control group. Finally, postmortem analysis confirmed transplanted CSCs integrated with host cardiomyocytes by immunohistology.In a mouse myocardial infarction model, Sca-1-positive CSCs provide no long-term engraftment and benefit to cardiac function as determined by multimodality imaging.

  View details for DOI 10.1016/j.jacc.2008.12.036

  View details for PubMedID 19341866

• Noninvasive De novo Imaging of Human Embryonic Stem Cell-Derived Teratoma Formation CANCER RESEARCH Cao, F., Li, Z., Lee, A., Liu, Z., Chen, K., Wang, H., Cai, W., Chen, X., Wu, J. C. 2009; 69 (7): 2709-2713

  Abstract

  Teratoma formation can be a serious drawback after the therapeutic transplantation of human embryonic stem (hES) cells. Therefore, noninvasive imaging of teratomas could be a valuable tool for monitoring patients undergoing hES cell treatment. Here, we investigated the angiogenic process within teratomas derived from hES cells and now report the first example of using (64)Cu-labeled RGD tetramer ((64)Cu-DOTA-RGD4) for positron emission tomography imaging of teratoma formation by targeting alpha(v)beta(3) integrin. H9 hES cells (2 x 10(6)), stably expressing firefly luciferase, and enhanced green fluorescence protein (Fluc-eGFP) were injected into adult nude mice (n=12) s.c. Eight weeks after transplantation, these hES cell grafts evolved into teratomas as confirmed by longitudinal bioluminescence imaging. Under micropositron emission tomography imaging, 2-deoxy-2-[(18)F]fluoro-D-glucose and 3'-deoxy-3'-[(18)F]-fluorothymidine both failed to detect hES cell-derived teratomas (0.8+/-0.5 versus 1.1+/-0.4 %ID/g, respectively; P=not significant versus background signals). By contrast, (64)Cu-DOTA-RGD4 revealed specific and prominent uptake in vascularized teratoma and significantly lower uptake in control tumors (human ovarian carcinoma 2008 cell line), which had low integrin expression (10.1+/-3.4 versus 1.4+/-1.2 %ID/g; P<0.01). Immunofluorescence staining of CD31 and beta(3) integrin also supported our in vivo imaging results (P<0.05). Moreover, we found that the cells dissociated from teratomas showed higher alpha(v)beta(3) integrin expression than the 2008 cells. In conclusion, by targeting alpha(v)beta(3) integrin, we successfully showed the ability of (64)Cu-DOTA-RGD4 to noninvasively visualize teratoma formation in vivo for the first time.

  View details for DOI 10.1158/0008-5472.CAN-08-4122

  View details for Web of Science ID 000264908100004

  View details for PubMedID 19318556

  View details for PubMedCentralID PMC2866177

• Comparison of Transplantation of Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stem Cells in the Infarcted Heart TRANSPLANTATION van der Bogt, K. E., Schrepfer, S., Yu, J., Sheikh, A. Y., Hoyt, G., Govaert, J. A., Velotta, J. B., Contag, C. H., Robbins, R. C., Wu, J. C. 2009; 87 (5): 642-652

  Abstract

  Mesenchymal stem cells hold promise for cardiovascular regenerative therapy. Derivation of these cells from the adipose tissue might be easier compared with bone marrow. However, the in vivo fate and function of adipose stromal cells (ASC) in the infarcted heart has never been compared directly to bone marrow-derived mesenchymal cells (MSC).ASC and MSC were isolated from transgenic FVB mice with a beta-actin promoter driving firefly luciferase and green fluorescent protein double fusion reporter gene, and they were characterized using flow cytometry, microscopy, bioluminescence imaging and luminometry. FVB mice (n=8 per group) underwent myocardial infarction followed by intramyocardial injection of 5x10(5) ASC, MSC, fibroblasts (Fibro, positive control), or saline (negative control). Cell survival was measured using bioluminescence imaging for 6 weeks and cardiac function was monitored by echocardiography and pressure-volume analysis. Ventricular morphology was assessed using histology.ASC and MSC were CD34(-), CD45(-), c-Kit(-), CD90(+), Sca-1(+), shared similar morphology and had a population doubling time of approximately 2 days. Cells expressed Fluc reporter genes in a number-dependent fashion as confirmed by luminometry. After cardiac transplantation, both cell types showed drastic donor cell death within 4 to 5 weeks. Furthermore, transplantation of either cell type was not capable of preserving ventricular function and dimensions, as confirmed by pressure-volume-loops and histology.This is the first study comparing the in vivo behavior of both cell types in the infarcted heart. ASC and MSC do not tolerate well in the cardiac environment, resulting in acute donor cell death and a subsequent loss of cardiac function similar to control groups.

  View details for DOI 10.1097/TP.0b013e31819609d9

  View details for PubMedID 19295307

• Imaging of STAT3 Signaling Pathway During Mouse Embryonic Stem Cell Differentiation STEM CELLS AND DEVELOPMENT Xie, X., Chan, K. S., Cao, F., Huang, M., Li, Z., Lee, A., Weissman, I. L., Wu, J. C. 2009; 18 (2): 205-214

  Abstract

  Signal transducers and activators of transcription 3 (STAT3) is a pleiotropic transcription factor involved in a variety of physiological processes. STAT3 acts as a key transcriptional determinant of mouse embryonic stem (ES) cell self-renewal and plays a pivotal function in early mammalian embryogenesis because the development of many organs requires STAT3 activation. However, little is known about the role of STAT3 function during ES cell differentiation. To answer this question, we built a lentiviral construct with 7-repeat STAT3-binding sequence (enhancer) and minimal TA (promoter) driving renilla luciferase and monomeric red fluorescence protein (Rluc-mRFP), followed by a constitutive cytomegalovirus promoter driving green fluorescent protein as a selection marker. The specificity of our custom-designed 7-repeat STAT3 reporter construct was first confirmed by cotransfection with constitutively active version of STAT3 (STAT3C) into human embryonic kidney 293T cells. Next, a mouse ES cell line stably transduced with STAT3 reporter construct was isolated. This ES cell line showed a tight response in reporter gene expression with leukemia inhibitory factor (LIF) induction and was chosen as a developmental model for the STAT3 functional study. Using serial noninvasive bioluminescence imaging, we showed that the onset of embryoid body (EB) formation involved inhibition of STAT3 activity. However, during differentiation, STAT3 activity steadily increased from day 5 to 14 and was reduced by day 21. STAT3 activity was also confirmed separately by Western blots. Finally, phosphorylation of STAT3 was also found to correspond with cardiomyocyte differentiation. In summary, this is the first study to monitor real-time STAT3 activity during ES cell differentiation. This genetically modified line can be used to study the biological role of STAT3 during ES cell differentiation into different derivatives.

  View details for DOI 10.1089/scd.2008.0152

  View details for PubMedID 18576943

• Transplantation of Human Embryonic Stem Cell-Derived Endothelial Cells for Vascular Diseases JOURNAL OF CELLULAR BIOCHEMISTRY Li, Z., Han, Z., Wui, J. C. 2009; 106 (2): 194-199

  Abstract

  Using endothelial cells for therapeutic angiogenesis/vasculogenesis of ischemia diseases has led to exploring human embryonic stem cells (hESCs) as a potentially unlimited source for endothelial progenitor cells. With their capacity for self-renewal and pluripotency, hESCs and their derived endothelial cells (hESC-ECs) may be more advantageous than other endothelial cells obtained from diseased populations. However, hESC-ECs' poor differentiation efficiency and poorly characterized in vivo function after transplantation present significant challenges for their future clinical application. This review will focus on the differentiation pathways of hESCs and their therapeutic potential for vascular diseases, as well as the monitoring of transplanted cells' fate via molecular imaging. Finally, cell enhancement strategies to improve the engraftment efficiency of hESC-ECs will be discussed.

  View details for DOI 10.1002/jcb.22003

  View details for PubMedID 19097085

• Bioluminescence reporter gene imaging of human embryonic stem cell survival, proliferation, and fate. Methods in molecular biology (Clifton, N.J.) Wilson, K. D., Huang, M., Wu, J. C. 2009; 574: 87-103

  Abstract

  The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging, and specifically bioluminescent reporter gene imaging, has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatiotemporal kinetics of hESC engraftment and proliferation in living subjects. In this chapter, the specific materials and methods needed for tracking stem cell proliferation with bioluminescence imaging will be described.

  View details for DOI 10.1007/978-1-60327-321-3_8

  View details for PubMedID 19685302

• Rapid and efficient feeder-free generation of human adipose stromal cell-derived induced pluripotent stem cells 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Panetta, N. J., Gupta, D. M., Sun, N., Wilson, K. D., Lee, A., Jia, F., Robbins, R. C., Longaker, M. T., Wu, J. C. MEDIMOND S R L. 2009: 159–161

  View details for Web of Science ID 000303383400030

• Multimodality Cardiovascular Molecular Imaging, Part II CIRCULATION-CARDIOVASCULAR IMAGING Nahrendorf, M., Sosnovik, D. E., French, B. A., Swirski, F. K., Bengel, F., Sadeghi, M. M., Lindner, J. R., Wu, J. C., Kraitchman, D. L., Fayad, Z. A., Sinusas, A. J. 2009; 2 (1): 56-70

  View details for DOI 10.1161/CIRCIMAGING.108.839092

  View details for Web of Science ID 000266129000010

  View details for PubMedID 19808565

  View details for PubMedCentralID PMC2760054

• Molecular imaging of human embryonic stem cells. Methods in molecular biology (Clifton, N.J.) Narsinh, K. H., Cao, F., Wu, J. C. 2009; 515: 13-32

  Abstract

  Human embryonic stem cells (hESCs) are a renewable source of differentiated cell types that may be employed in various tissue regeneration strategies. However, clinical implementation of cell transplantation therapy is hindered by legitimate concerns regarding the in vivo teratoma formation of undifferentiated hESCs and host immune reactions to allogenic cells. Investigating in vivo hESC behaviour and the ultimate feasibility of cell transplantation therapy necessitates the development of novel molecular imaging techniques to longitudinally monitor hESC localization, proliferation, and viability in living subjects. An innovative approach to harness the respective strengths of various imaging platforms is the creation and use of a fusion reporter construct composed of red fluorescent protein (RFP), firefly luciferase (fluc), and herpes simplex virus thymidine kinase (HSV-tk). The imaging modalities made available by use of this construct, including optical fluorescence, bioluminescence, and positron emission tomography (PET), mat be adapted to investigate a variety of physiological phenomena, including the spatio-temporal kinetics of hESC engraftment and proliferation in living subjects. This chapter describes the applications of reporter gene imaging to accelerate basic science research and clinical studies involving hESCs through (1) isolation of a homogenous hESC population, (2) noninvasive, longitudinal tracking of the location and proliferation of hESCs administered to a living subject, and (3) ablation of the hESC graft in the event of cellular misbehavior.

  View details for PubMedID 19405224

• Modeling Conduction in Host-Graft Interactions Between Stem Cell Grafts and Cardiomyocytes Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society Chen, M. Q., Yu, J., Whittington, R. H., Wu, J. C., Kovacs, G. T., Giovangrandi, L. IEEE. 2009: 6014–6017

  Abstract

  Cell therapy has recently made great strides towards aiding heart failure. However, while transplanted cells may electromechanically integrate into host tissue, there may not be a uniform propagation of a depolarization wave between the heterogeneous tissue boundaries. A model using microelectrode array technology that maps the electrical interactions between host and graft tissues in co-culture is presented and sheds light on the effects of having a mismatch of conduction properties at the boundary. Skeletal myoblasts co-cultured with cardiomyocytes demonstrated that conduction velocity significantly decreases at the boundary despite electromechanical coupling. In an attempt to improve the uniformity of conduction with host cells, differentiating human embryonic stem cells (hESC) were used in co-culture. Over the course of four to seven days, synchronous electrical activity was observed at the hESC boundary, implying differentiation and integration. Activity did not extend far past the boundary, and conduction velocity was significantly greater than that of the host tissue, implying the need for other external measures to properly match the conduction properties between host and graft tissue.

  View details for PubMedID 19964687

• Trafficking Mesenchymal Stem Cell Engraftment and Differentiation in Tumor-Bearing Mice by Bioluminescence Imaging STEM CELLS Wang, H., Cao, F., De, A., Cao, Y., Contag, C., Gambhir, S. S., Wu, J. C., Chen, X. 2009; 27 (7): 1548-1558

  Abstract

  The objective of the study was to track the distribution and differentiation of mesenchymal stem cells (MSCs) in tumor-bearing mice. The 4T1 murine breast cancer cells were labeled with renilla luciferase-monomeric red fluorescence protein (rLuc-mRFP) reporter gene. The MSCs labeled with firefly luciferase-enhanced green fluorescence protein (fLuc-eGFP) reporter gene (MSCs-R) were isolated from L2G85 transgenic mice that constitutively express fLuc-eGFP reporter gene. To study the tumor tropism of MSCs, we established both subcutaneous and lung metastasis models. In lung metastasis tumor mice, we injected MSCs-R intravenously either on the same day or 4 days after 4T1 tumor cell injection. In subcutaneous tumor mice, we injected MSCs-R intravenously 7 days after subcutaneous 4T1 tumor inoculation. The tumor growth was monitored by rLuc bioluminescence imaging (BLI). The fate of MSCs-R was monitored by fLuc BLI. The localization of MSCs-R in tumors was examined histologically. The osteogenic and adipogenic differentiation of MSCs-R was investigated by alizarin red S and oil red O staining, respectively. The mechanism of the dissimilar differentiation potential of MSCs-R under different tumor microenvironments was investigated. We found that the 4T1 cells were successfully labeled with rLuc-mRFP. The MSCs-R isolated from L2G85 transgenic mice constitutively express fLuc-eGFP reporter gene. When injected intravenously, MSCs-R survived, proliferated, and differentiated in tumor sites but not elsewhere. The localization of GFP(+) MSCs-R in tumor lesions was confirmed ex vivo. In conclusion, the MSCs-R can selectively localize, survive, and proliferate in both subcutaneous tumor and lung metastasis as evidenced by noninvasive bioluminescence imaging and ex vivo validation. The MSCs-R migrated to lung tumor differentiated into osteoblasts, whereas the MSCs-R targeting subcutaneous tumor differentiated into adipocytes.

  View details for DOI 10.1002/stem.81

  View details for PubMedID 19544460

• Long term non-invasive imaging of embryonic stem cells using reporter genes NATURE PROTOCOLS Sun, N., Lee, A., Wu, J. C. 2009; 4 (8): 1192-1201

  Abstract

  Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol, we describe the in vivo monitoring of stem cell survival, proliferation and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase and herpes simplex virus thymidine kinase (HSVtk)) reporter genes using lentiviral transduction. We used fluorescence-activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography (PET) imaging to track them in vivo and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking, such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny.

  View details for DOI 10.1038/nprot.2009.100

  View details for PubMedID 19617890

• In Vivo Imaging of Embryonic Stem Cells Reveals Patterns of Survival and Immune Rejection Following Transplantation STEM CELLS AND DEVELOPMENT Swijnenburg, R., Schrepfer, S., Cao, F., Pearl, J. I., Xie, X., Connolly, A. J., Robbins, R. C., Wu, J. C. 2008; 17 (6): 1023-1029

  Abstract

  Embryonic stem cell (ESC)-based transplantation is considered a promising novel therapy for a variety of diseases. This is bolstered by the suggested immune-privileged properties of ESCs. In this study, we used in vivo bioluminescent imaging (BLI) to non-invasively track the fate of transplanted murine ESCs (mESCs), which are stably transduced with a double fusion reporter gene consisting of firefly luciferase (FLuc) and enhanced green fluorescent protein (eGFP). Following syngeneic intramuscular transplantation of 1 x 10(6) mESCs, the cells survived and differentiated into teratomas. In contrast, allogeneic mESC transplants were infiltrated by a variety of inflammatory cells, leading to rejection within 28 days. Acceleration of rejection was observed when mESCs were allotransplanted following prior sensitization of the host. Finally, we demonstrate that the mESC derivatives were more rapidly rejected compared to undifferentiated mESCs. These data show that mESCs do not retain immune-privileged properties in vivo and are subject to immunological rejection as assessed by novel molecular imaging approaches.

  View details for DOI 10.1089/scd.2008.0091

  View details for PubMedID 18491958

• Molecular Imaging Antidote to Cardiac Stem Cell Controversy JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Wu, J. C. 2008; 52 (20): 1661-1664

  View details for DOI 10.1016/j.jacc.2008.08.020

  View details for Web of Science ID 000260904000009

  View details for PubMedID 18992657

• Hemodynamic forces regulate embryonic stem cell commitment to vascular progenitors. Current cardiology reviews Hsiai, T. K., Wu, J. C. 2008; 4 (4): 269-274

  Abstract

  Pluripotent embryonic stem can (ES) cells can differentiate into all cell lineages. During the process of embryonic development, ES cells are exposed to fluid flow or blood flow generated by the contracting heart. Absence of fluid flow results in the formation of abnormal cardiac chambers and valve formation. Thus, hemodynamic forces and ES cell differentiation to vascular progenitor cells (VPCs) are of emerging interests for restoring endothelial dysfunction, inducing angiogenesis, and forming blood vessel networks. Hemodynamic forces such as fluid shear stress increase the percentage of cells in the S and G(2)-M phases, and induce decondensation of chromatin for gene transcription. Fluid shear stress further accelerates ES commitment to CD31(+) VPC vascular progenitor cells. These ES-derived CD31(+) cells express endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). They are also capable of LDL uptake and tubular network formation. In this context, understanding hemodynamic forces and ES cell kinetics of differentiation towards endothelial lineage has potential therapeutic applications for repairing vascular damage and engineering vascular graft. Multidisciplinary team approach will likely garner momentum and synergize expertise to address the current road blocks in basic stem cell research for engraftable, restorative, low immunogenic, and non-tumorigenic endothelial progenitors in high purity and stability.

  View details for DOI 10.2174/157340308786349471

  View details for PubMedID 20066134

  View details for PubMedCentralID PMC2801858

• Multimodality Cardiovascular Molecular Imaging, Part I CIRCULATION-CARDIOVASCULAR IMAGING Sinusas, A. J., Bengel, F., Nahrendorf, M., Epstein, F. H., Wu, J. C., Villanueva, F. S., Fayad, Z. A., Gropler, R. J. 2008; 1 (3): 244-256

  Abstract

  In Part I of this consensus article, the imaging methodology, evolving imaging technology, and development of novel targeted molecular probes relevant to the developing field of cardiovascular molecular imaging were reviewed. Novel reporter gene and reporter probe imaging approaches for tracking of cardiac transgene expression were also discussed and have important future implications for evaluation of gene- and cell-based therapies for the failing heart. The current role of metabolic and receptor imaging was also briefly reviewed, as these represent the beginning of our clinical application of molecular imaging within the cardiovascular system. Part II will summarize the available targeted imaging probes as well as specific future applications of molecular imaging for identification and evaluation of critical pathophysiological processes of the cardiovascular system.

  View details for DOI 10.1161/CIRCIMAGING.108.824359

  View details for Web of Science ID 000266128800011

  View details for PubMedID 19808549

• Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes PLOS ONE Cao, F., Wagner, R. A., Wilson, K. D., Xie, X., Fu, J., Drukker, M., Lee, A., Li, R. A., Gambhir, S. S., Weissman, I. L., Robbins, R. C., Wu, J. C. 2008; 3 (10)

  Abstract

  Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

  View details for DOI 10.1371/journal.pone.0003474

  View details for PubMedID 18941512

• Multimodal evaluation of in vivo magnetic resonance imaging of myocardial restoration by mouse embryonic stem cells JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Hendry, S. L., van der Bogt, K. E., Sheikh, A. Y., Arai, T., Dylla, S. J., Drukker, M., McConnell, M. V., Kutschka, I., Hoyt, G., Cao, F., Weissman, I. L., Connolly, A. J., Pelletier, M. P., Wu, J. C., Robbins, R. C., Yang, P. C. 2008; 136 (4): 1028-U14

  Abstract

  Mouse embryonic stem cells have demonstrated potential to restore infarcted myocardium after acute myocardial infarction. Although the underlying mechanism remains controversial, magnetic resonance imaging has provided reliable in vivo assessment of functional recovery after cellular transplants. Multimodal comparison of the restorative effects of mouse embryonic stem cells and mouse embryonic fibroblasts was performed to validate magnetic resonance imaging data and provide mechanistic insight.SCID-beige mice (n = 55) underwent coronary artery ligation followed by injection of 2.5 x 10(5) mouse embryonic stem cells, 2.5 x 10(5) mouse embryonic fibroblasts, or normal saline solution. In vivo magnetic resonance imaging of myocardial restoration by mouse embryonic stem cells was evaluated by (1) in vivo pressure-volume loops, (2) in vivo bioluminescence imaging, and (3) ex vivo TaqMan (Roche Molecular Diagnostics, Pleasanton, Calif) polymerase chain reaction and immunohistologic examination.In vivo magnetic resonance imaging demonstrated significant improvement in left ventricular ejection fraction at 1 week in the mouse embryonic stem cell group. This finding was validated with (1) pressure-volume loop analysis demonstrating significantly improved systolic and diastolic functions, (2) bioluminescence imaging and polymerase chain reaction showing superior posttransplant survival of mouse embryonic stem cells, (3) immunohistologic identification of cardiac phenotype within engrafted mouse embryonic stem cells, and (4) polymerase chain reaction measuring increased expressions of angiogenic and antiapoptotic genes and decreased expressions of antifibrotic genes.This study validates in vivo magnetic resonance imaging as an effective means of evaluating the restorative potential of mouse embryonic stem cells.

  View details for DOI 10.1016/j.jtcvs.2007.12.053

  View details for PubMedID 18954646

• Short hairpin RNA interference therapy for ischemic heart disease 80th Annual Scientific Session of the American-Heart-Association (AHA) Huang, M., Chan, D. A., Jia, F., Xie, X., Li, Z., Hoyt, G., Robbins, R. C., Chen, X., Giaccia, A. J., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2008: S226–U289

  Abstract

  During hypoxia, upregulation of hypoxia inducible factor-1 alpha transcriptional factor can activate several downstream angiogenic genes. However, hypoxia inducible factor-1 alpha is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Here we hypothesize that short hairpin RNA (shRNA) interference therapy targeting PHD2 can be used for treatment of myocardial ischemia and this process can be followed noninvasively by molecular imaging.PHD2 was cloned from mouse embryonic stem cells by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted into the pSuper vector driven by the H1 promoter followed by a separate hypoxia response element-incorporated promoter driving a firefly luciferase reporter gene. This construct was used to transfect mouse C2C12 myoblast cell line for in vitro confirmation. Compared with the control short hairpin scramble (shScramble) as control, inhibition of PHD2 increased levels of hypoxia inducible factor-1 alpha protein and several downstream angiogenic genes by >30% (P<0.01). Afterward, shRNA targeting PHD2 (shPHD2) plasmid was injected intramyocardially following ligation of left anterior descending artery in mice. Animals were randomized into shPHD2 experimental group (n=25) versus shScramble control group (n=20). Bioluminescence imaging detected plasmid-mediated transgene expression for 4 to 5 weeks. Echocardiography showed the shPHD2 group had improved fractional shortening compared with the shScramble group at Week 4 (33.7%+/-1.9% versus 28.4%+/-2.8%; P<0.05). Postmortem analysis showed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. Finally, Western blot analysis of explanted hearts also confirmed that animals treated with shPHD2 had significantly higher levels of hypoxia inducible factor-1 alpha protein.This is the first study to image the biological role of shRNA therapy for improving cardiac function. Inhibition of PHD2 by shRNA led to significant improvement in angiogenesis and contractility by in vitro and in vivo experiments. With further validation, the combination of shRNA therapy and molecular imaging can be used to track novel cardiovascular gene therapy applications in the future.

  View details for DOI 10.1161/CIRCULATIONAHA.107.760785

  View details for PubMedID 18824759

• Comparison of different adult stem cell types for treatment of myocardial ischemia 80th Annual Scientific Session of the American-Heart-Association (AHA) van der Bogt, K. E., Sheikh, A. Y., Schrepfer, S., Hoyt, G., Cao, F., Ransohoff, K. J., Swijnenburg, R., Pearl, J., Lee, A., Fischbein, M., Contag, C. H., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2008: S121–U166

  Abstract

  A comparative analysis of the efficacy of different cell candidates for the treatment of heart disease remains to be described. This study is designed to evaluate the therapeutic efficacy of 4 cell types in a murine model of myocardial infarction.Bone marrow mononuclear cells (MN), mesenchymal stem cells (MSC), skeletal myoblasts (SkMb), and fibroblasts (Fibro) expressing firefly luciferase (Fluc) and green fluorescence protein (GFP) were characterized by flow cytometry, bioluminescence imaging (BLI), and luminometry. Female FVB mice (n=70) underwent LAD ligation and intramyocardially received one cell type (5x10(5)) or PBS. Cell survival was measured by BLI and by TaqMan PCR. Cardiac function was assessed by echocardiography and invasive hemodynamic measurements. Fluc expression correlated with cell number in all groups (r(2)>0.93). In vivo BLI revealed acute donor cell death of MSC, SkMb, and Fibro within 3 weeks after transplantation. By contrast, cardiac signals were still present after 6 weeks in the MN group, as confirmed by TaqMan PCR (P<0.01). Echocardiography showed significant preservation of fractional shortening in the MN group compared to controls (P<0.05). Measurements of left ventricular end-systolic/diastolic volumes revealed that the least amount of ventricular dilatation occurred in the MN group (P<0.05). Histology confirmed the presence of MN, although there was no evidence of transdifferentiation by donor MN into cardiomyocytes.This is the first study to show that compared to MSC, SkMB, and Fibro, MN exhibit a more favorable survival pattern, which translates into a more robust preservation of cardiac function.

  View details for DOI 10.1161/CIRCULATIONAHA.107.759480

  View details for PubMedID 18824743

• Immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Swijnenburg, R., Schrepfer, S., Govaert, J. A., Cao, F., Ransohoff, K., Sheikh, A. Y., Haddad, M., Connolly, A. J., Davis, M. M., Robbins, R. C., Wu, J. C. 2008; 105 (35): 12991-12996

  Abstract

  Given their self-renewing and pluripotent capabilities, human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However, the host immune response against transplanted hESCs is not well characterized. In fact, controversy remains as to whether hESCs have immune-privileged properties. To address this issue, we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death, suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses, resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover, we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally, we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together, these data suggest that hESCs are immunogenic, trigger both cellular and humoral-mediated pathways, and, as a result, are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches.

  View details for DOI 10.1073/pnas.0805802105

  View details for PubMedID 18728188

• Surgically palliated double-inlet left ventricle with transposition of the great arteries mistaken for aortic aneurysm with dissection INTERNATIONAL JOURNAL OF CARDIOLOGY Gupta, A., Fleischmann, D., Murphy, D. J., Wu, J. C. 2008; 128 (2): E82-E84

  Abstract

  We report a 23-year-old male with history of double-inlet single ventricle with transposition of the great arteries who is s/p pulmonary artery banding, a Damus-Kaye-Stanzel anastomosis, and Fontan procedure during infancy and childhood who now presents with chest pain. A chest CTA at an outside hospital was thought concerning for the presence of a thoracic aortic aneurysm with dissection, prompting immediate transfer to our institution. However, repeat chest CTA at our institution revealed the predicted anastomoses based on his surgical procedures, which was misinterpreted as aortic aneurysm and dissection. An understanding of the physiology of his surgically repaired congenital heart disease is critical in interpreting his subsequent chest CTA and arriving at the appropriate clinical conclusion.

  View details for DOI 10.1016/j.ijcard.2007.04.165

  View details for Web of Science ID 000257950500043

  View details for PubMedID 17689761

• Noninvasive radionuclide imaging of cardiac gene therapy: progress and potential NATURE CLINICAL PRACTICE CARDIOVASCULAR MEDICINE Hiona, A., Wu, J. C. 2008; 5: S87-S95

  Abstract

  Over the past decade, several clinical trials have evaluated the efficacy of cardiac-specific gene therapy. Despite encouraging results in basic research and preclinical studies, most of the recent large, randomized, placebo-controlled cardiac gene therapy trials have failed to provide convincing evidence of improvements in clinical outcomes. Because many of these problems are due to the lack of appropriate monitoring techniques, there is a critical need to develop noninvasive imaging techniques that can verify vector delivery and gene expression in target and nontarget tissues. The field of molecular imaging of cardiac gene expression is rapidly advancing because it offers distinct advantages over conventional methods, including the ability to noninvasively measure the location, time course, and magnitude of gene expression. We aim to give readers a clear understanding of how molecular imaging can enable noninvasive tracking of cardiac gene transfer and expression. We discuss limitations of current methods for analyzing gene transfer and describe how reporter gene imaging works.

  View details for DOI 10.1038/ncpcardio1113

  View details for PubMedID 18641612

• Intracellular and extracellular targets of molecular imaging in the myocardium NATURE CLINICAL PRACTICE CARDIOVASCULAR MEDICINE Chun, H. J., Narula, J., Hofstra, L., Wu, J. C. 2008; 5: S33-S41

  Abstract

  Utilization of molecular imaging has significantly advanced the field of cardiovascular medicine. In addition to the targets currently in use, novel targets are being developed, including those involved in the processes of myocardial metabolism, myocardial injury, cardiac neurotransmission, and interstitial dysregulation. Further development of these imaging targets may lead to improved characterization of disease processes and guide provision of individualized therapies.

  View details for DOI 10.1038/ncpcardio1161

  View details for PubMedID 18641605

• Transcriptome Alteration in the Diabetic Heart by Rosiglitazone: Implications for Cardiovascular Mortality PLOS ONE Wilson, K. D., Li, Z., Wagner, R., Yue, P., Tsao, P., Nestorova, G., Huang, M., Hirschberg, D. L., Yock, P. G., Quertermous, T., Wu, J. C. 2008; 3 (7)

  Abstract

  Recently, the type 2 diabetes medication, rosiglitazone, has come under scrutiny for possibly increasing the risk of cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiac transcriptional profiling and imaging studies of a murine model of type 2 diabetes, the C57BL/KLS-lepr(db)/lepr(db) (db/db) mouse.We compared cardiac gene expression profiles from three groups: untreated db/db mice, db/db mice after rosiglitazone treatment, and non-diabetic db/+ mice. Prior to sacrifice, we also performed cardiac magnetic resonance (CMR) and echocardiography. As expected, overall the db/db gene expression signature was markedly different from control, but to our surprise was not significantly reversed with rosiglitazone. In particular, we have uncovered a number of rosiglitazone modulated genes and pathways that may play a role in the pathophysiology of the increase in cardiac mortality as seen in several recent meta-analyses. Specifically, the cumulative upregulation of (1) a matrix metalloproteinase gene that has previously been implicated in plaque rupture, (2) potassium channel genes involved in membrane potential maintenance and action potential generation, and (3) sphingolipid and ceramide metabolism-related genes, together give cause for concern over rosiglitazone's safety. Lastly, in vivo imaging studies revealed minimal differences between rosiglitazone-treated and untreated db/db mouse hearts, indicating that rosiglitazone's effects on gene expression in the heart do not immediately turn into detectable gross functional changes.This study maps the genomic expression patterns in the hearts of the db/db murine model of diabetes and illustrates the impact of rosiglitazone on these patterns. The db/db gene expression signature was markedly different from control, and was not reversed with rosiglitazone. A smaller number of unique and interesting changes in gene expression were noted with rosiglitazone treatment. Further study of these genes and molecular pathways will provide important insights into the cardiac decompensation associated with both diabetes and rosiglitazone treatment.

  View details for DOI 10.1371/journal.pone.0002609

  View details for PubMedID 18648539

• Seeing is believing: tracking cells to determine the effects of cell transplantation. Seminars in thoracic and cardiovascular surgery Pearl, J., Wu, J. C. 2008; 20 (2): 102-109

  Abstract

  Stem cell therapy holds promise as a therapeutic option for cardiovascular disease. As the field of cellular transplantation matures, novel methodologies are needed to longitudinally track and evaluate the functional effects of transplanted cells. At present, available techniques that hold the greatest promise for tracking cell fate include radionuclide labeling, ferromagnetic particle labeling, and genetic modification with reporter genes. This review describes the benefits and limitations of each technique and provides a summary of critical issues regarding stem cell transplantation that have been addressed by each imaging modality.

  View details for DOI 10.1053/j.semtcvs.2008.04.002

  View details for PubMedID 18707641

• Shear stress influences spatial variations in vascular Mn-SOD expression: implication for LDL nitration AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Ai, L., Rouhanizadeh, M., Wu, J. C., Takabe, W., Yu, H., Alavi, M., Li, R., Chu, Y., Miller, J., Heistad, D. D., Hsiai, T. K. 2008; 294 (6): C1576-C1585

  Abstract

  Fluid shear stress modulates vascular production of endothelial superoxide anion (O2*-) and nitric oxide (*NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress (tau ave) of 23 dyn/cm2 upregulated Mn-SOD mRNA expression at a higher level than did OSS at tau ave = 0.02 dyn/cm2 +/- 3.0 dyn.cm(-2).s(-1) and at 1 Hz (PSS by 11.3 +/- 0.4-fold vs. OSS by 5.0 +/- 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration (P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration.

  View details for DOI 10.1152/ajpcell.00518.2007

  View details for Web of Science ID 000256574900031

  View details for PubMedID 18434620

  View details for PubMedCentralID PMC3008554

• Can radionuclide imaging predict future response to stem cell therapy? JOURNAL OF NUCLEAR CARDIOLOGY Wu, J. C. 2008; 15 (3): 308-310

  View details for DOI 10.1016/j.nuclcard.2008.03.005

  View details for Web of Science ID 000256389300004

  View details for PubMedID 18513636

• Imaging of VEGF receptor in a rat myocardial infarction model using PET JOURNAL OF NUCLEAR MEDICINE Rodriguez-Porcel, M., Cai, W., Gheysens, O., Willmann, J. K., Chen, K., Wang, H., Chen, I. Y., He, L., Wu, J. C., Li, Z., Mohamedali, K. A., Kim, S., Rosenblum, M. G., Chen, X., Gambhir, S. S. 2008; 49 (4): 667-673

  Abstract

  Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factor's receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject.In Sprague-Dawley rats, MI was induced by ligation of the left coronary artery and confirmed by ultrasound (n = 8). To image and study the kinetics of VEGFRs, 64Cu-DOTA-VEGF121 PET scans were performed before MI induction (baseline) and on days 3, 10, 17, and 24 after MI. Sham-operated animals served as controls (n = 3).Myocardial origin of the 64Cu-DOTA-VEGF121 signal was confirmed by CT coregistration and autoradiography. VEGFR specificity of the 64Cu-DOTA-VEGF121 probe was confirmed by in vivo use of a 64Cu-DOTA-VEGFmutant. Baseline myocardial uptake of 64Cu-DOTA-VEGF121 was minimal (0.30 +/- 0.07 %ID/g [percentage injected dose per gram of tissue]); it increased significantly after MI (day 3, 0.97 +/- 0.05 %ID/g; P < 0.05 vs. baseline) and remained elevated for 2 wk (up to day 17 after MI), after which time it returned to baseline levels.We demonstrate the feasibility of imaging VEGFRs in the myocardium. In summary, we imaged and described the kinetics of 64Cu-DOTA-VEGF121 uptake in a rat model of MI. Studies such as the one presented here will likely play a major role when studying pathophysiology and assessing therapies in different animal models of disease and, potentially, in patients.

  View details for DOI 10.2967/jnumed.107.040576

  View details for PubMedID 18375924

• Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects STEM CELLS Li, Z., Suzuki, Y., Huang, M., Cao, F., Xie, X., Connolly, A. J., Yang, P. C., Wu, J. C. 2008; 26 (4): 864-873

  Abstract

  Human embryonic stem (hES) cells are pluripotent stem cells capable of self-renewal and differentiation into virtually all cell types. Thus, they hold tremendous potential as cell sources for regenerative therapies. The concurrent development of accurate, sensitive, and noninvasive technologies capable of monitoring hES cells engraftment in vivo can greatly expedite basic research prior to future clinical translation. In this study, hES cells were stably transduced with a lentiviral vector carrying a novel double-fusion reporter gene that consists of firefly luciferase and enhanced green fluorescence protein. Reporter gene expression had no adverse effects on cell viability, proliferation, or differentiation to endothelial cells (human embryonic stem cell-derived endothelial cells [hESC-ECs]). To compare the two popular imaging modalities, hES cells and hESC-ECs were then colabeled with superparamagnetic iron oxide particles before transplantation into murine hind limbs. Longitudinal magnetic resonance (MR) imaging showed persistent MR signals in both cell populations that lasted up to 4 weeks. By contrast, bioluminescence imaging indicated divergent signal patterns for hES cells and hESC-ECs. In particular, hESC-ECs showed significant bioluminescence signals at day 2, which decreased progressively over the following 4 weeks, whereas bioluminescence signals from undifferentiated hES cells increased dramatically during the same period. Post-mortem histology and immunohistochemistry confirmed teratoma formation after injection of undifferentiated hES cells but not hESC-ECs. From these data taken together, we concluded that reporter gene is a better marker for monitoring cell viability, whereas iron particle labeling is a better marker for high-resolution detection of cell location by MR. Furthermore, transplantation of predifferentiated rather than undifferentiated hES cells would be more suited for avoiding teratoma formation.

  View details for DOI 10.1634/stemcells.2007-0843

  View details for PubMedID 18218820

• Cardiac resident stem cells exhibit mesenchymal features and provide no long term benefit to cardiac function in mouse ischemic model of heart failure 57th Annual Scientific Session of the American-College-of-Cardiology Li, Z., Chun, H., Xie, X., Huang, M., Hoyt, G., Chu, P., Contag, C. H., Robbins, R. C., Wu, J. C. ELSEVIER SCIENCE INC. 2008: A375–A375

  View details for Web of Science ID 000253997102511

• Reporter gene imaging following percutaneous delivery in swine - Moving toward clinical applications JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Rodriguez-Porcel, M., Brinton, T. J., Chen, I. Y., Gheysens, O., Lyons, J., Ikeno, F., Willmann, J. K., Wu, L., Wu, J. C., Yeung, A. C., Yock, P., Gambhir, S. S. 2008; 51 (5): 595-597

  View details for DOI 10.1016/j.jacc.2007.08.063

  View details for Web of Science ID 000252908600013

  View details for PubMedID 18237691

  View details for PubMedCentralID PMC2853907

• In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells. Journal of visualized experiments : JoVE Wilson, K., Yu, J., Lee, A., Wu, J. C. 2008

  Abstract

  The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects. A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin. To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

  View details for DOI 10.3791/740

  View details for PubMedID 19066577

• Cardiac Differentiation of Embryonic Stem Cells with Point-Source Electrical Stimulation 30th Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society Chen, M. Q., Xie, X., Whittington, R. H., Kovacs, G. T., Wu, J. C., Giovangrandi, L. IEEE. 2008: 1729–1732

  Abstract

  The use of pluripotent stem cells as a means to repair damaged heart tissue has recently emerged as a promising, yet controversial therapy. Despite the different approaches and the variety of cell types used, many of these procedures have been met with mixed success. The lack of understanding of the differentiation and integration process, notably with respect to electrical signaling, significantly hampers the development of these therapies. A system was thus developed allowing the use of point source electrical stimulation on embryonic stem (ES) cells to study the effect of physiologically-relevant electrical stimulus. When modulating the amplitude of the stimulus over various differentiation stages of embryonic stem cells, differences in the proportions of cardiomyocytes to embryonic stem cells were observed through quantitative PCR. The use of this technique might have larger applications in understanding molecular pathways towards the regeneration process.

  View details for PubMedID 19163013

• A novel platform device for rodent echocardiography. ILAR journal Kutschka, I., Sheikh, A. Y., Sista, R., Hendry, S. L., Chun, H. J., Hoyt, G., Kutschka, W., Pelletier, M. P., Quertermous, T., Wu, J. C., Robbins, R. C. 2008; 49: E1-7

  Abstract

  Acquisition of echocardiographic data from rodents is subject to wide variability due to variations in technique. We hypothesize that a dedicated imaging platform can aid in standardization of technique and improve the quality of images obtained. We constructed a device consisting of a boom-mounted steel platform frame (25 x 35 x 3 cm) on which a transparent polyethylene membrane is mounted. The animal is placed onto the membrane and receives continual inhaled anesthesia via an integrated port. The membrane allows for probe positioning from beneath the animal to obtain standard echo-views in left lateral decubitus or prone positions. The frame can be set at any desired angle ranging from 0 to 360 degrees along either the long or short axis. Adult male Sprague-Dawley rats (n = 5) underwent echocardiography (General Electric, Vivid 7, 14 MHz) using the platform. The device allowed for optimal positioning of animals for a variety of standard echocardiographic measurements. Evaluations among all animals showed minimal variability between two different operators and time points. We tested the feasibility of the device for supporting the assessment of cardiac function in a disease model by evaluating a separate cohort of adult male spontaneously hypertensive rats (n = 5) that underwent left anterior descending coronary artery ligation. Serial echocardiography demonstrated statistically significant decreases of fractional shortening and ejection fraction (p < 0.01) 240 days after surgery. Our novel imaging platform allowed for consistent collection of high-quality echocardiographic data from rats. Future studies will focus on improving this technology to allow for standardized high-throughput echocardiographic analysis in small animal models of disease.

  View details for PubMedID 18506056

• Comparison of imaging techniques for tracking cardiac stem cell therapy JOURNAL OF NUCLEAR MEDICINE Wu, J. C. 2007; 48 (12): 1916-1919

  View details for DOI 10.2967/jnumed.107.043299

  View details for PubMedID 18056330

• Spatial and temporal kinetics, of teratoma formation from murine embryonic stem cell transplantation STEM CELLS AND DEVELOPMENT Cao, F., van der Bogt, K. E., Sadrzadeh, A., Xie, X., Sheikh, A. Y., Wang, H., Connolly, A. J., Robbins, R. C., Wu, J. C. 2007; 16 (6): 883-891

  Abstract

  Pluripotent embryonic stem (ES) cells have the potential to form teratomas composed of derivatives from all three germ layers in animal models. This tumorigenic potential prevents clinical translation of ES cell research. In order to understand the biology and physiology of teratoma formation, we investigated the influence of undifferentiated ES cell number, migration, and long-term follow up after transplantation. Murine ES cells were stably transduced with a self-inactivating (SIN) lentiviral vector with a constitutive ubiquitin promoter driving a double-fusion (DF) reporter gene that consists of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP). To assess effects of cell numbers, varying numbers of ES-DF cells (1, 10, 100, 1,000, and 10,000) were injected subcutaneously into the dorsal regions of adult nude mice. To assess cell migration, 1 x 10(6) ES-DF cells were injected intramyocardially into adult Sv129 mice, and leakage to other extracardiac sites was monitored. To assess effects of long-term engraftment, 1 x 10(4) ES-DF cells were injected intramyocardially into adult nude rats, and cell survival response was monitored for 10 months. Our results show that ES-DF cells caused extracardiac teratoma in both immunocompetent and immunodeficient hosts; the lowest number of undifferentiated ES cells capable of causing teratoma was 500-1,000; and long-term engraftment could be shown for >300 days. Collectively, these results illustrate the potent tumorigenic potential of ES cells, which presents an enormous obstacle for future clinical studies.

  View details for DOI 10.1089/scd.2007.0160

  View details for PubMedID 17896868

• In vivo imaging and evaluation of different biomatrices for improvement of stem cell survival JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE Cao, F., Rafie, A. H., Abilez, O. J., Wang, H., Blundo, J. T., Pruitt, B., Zarins, C., Wu, J. C. 2007; 1 (6): 465-468

  Abstract

  Therapeutic effects from injection of stem cells are often hampered by acute donor cell death as well as migration away from damaged areas. This is likely due to the fact that injected cells do not have the physical and biochemical cues for ordered engrafment. Here we evaluate 3 common biomatrices (Matrigel, Collagen I, Purmatrix) that has the potential of providing suitable scaffolds needed to enhance stem cell survival. The longitudinal fate of transplanted stem cells was monitored by reporter imaging techniques.

  View details for DOI 10.1002/term.55

  View details for Web of Science ID 000256520300008

  View details for PubMedID 18163533

  View details for PubMedCentralID PMC3657504

• Cardiac engraftment and distribution of bone marrow stem cells implanted by intravenous, intracoronary and intramyocardial injections using molecular Imaging 80th Annual Scientific Session of the American-Heart-Association (AHA) Li, S., Lai, T. Y., Sun, Z., Han, M., Moriyama, E., Wilson, B., Fazel, S., Weisel, R. D., Yau, T., Wu, J. C., Li, R. LIPPINCOTT WILLIAMS & WILKINS. 2007: 397–97

  View details for Web of Science ID 000250394301778

• Transplantation of bone-marrow mononuclear cells into the infarcted heart has favorable engraftment compared to mesenchymal stem cells, skeletal myoblasts and fibroblasts van der Bogt, K. E., Sheikh, A. Y., Schrepfer, S., Hoyt, G., Cao, F., Ransohoff, K., Contag, C. H., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2007: 397

  View details for Web of Science ID 000250394301776

• Genomic profiling of bone marrow cells transplanted into the heart 80th Annual Scientific Session of the American-Heart-Association (AHA) Sheikh, A. Y., Cao, F., Spin, J. M., Fajardo, G., Gambhir, S. S., Weissman, I. L., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2007: 167–67

  View details for Web of Science ID 000250394300738

• Short hairpin RNA interference therapy in murine myocardial infarction 80th Annual Scientific Session of the American-Heart-Association (AHA) Huang, M., Chan, D., Jia, F., Xie, X., Li, Z., Giaccia, A., Wu, J. LIPPINCOTT WILLIAMS & WILKINS. 2007: 590–90

  View details for Web of Science ID 000250394302665

• Quantum dot imaging for embryonic stem cells BMC BIOTECHNOLOGY Lin, S., Xie, X., Patel, M. R., Yang, Y., Li, Z., Cao, F., Gheysens, O., Zhang, Y., Gambhir, S. S., Rao, J. H., Wu, J. C. 2007; 7

  Abstract

  Semiconductor quantum dots (QDs) hold increasing potential for cellular imaging both in vitro and in vivo. In this report, we aimed to evaluate in vivo multiplex imaging of mouse embryonic stem (ES) cells labeled with Qtracker delivered quantum dots (QDs).Murine embryonic stem (ES) cells were labeled with six different QDs using Qtracker. ES cell viability, proliferation, and differentiation were not adversely affected by QDs compared with non-labeled control cells (P = NS). Afterward, labeled ES cells were injected subcutaneously onto the backs of athymic nude mice. These labeled ES cells could be imaged with good contrast with one single excitation wavelength. With the same excitation wavelength, the signal intensity, defined as (total signal-background)/exposure time in millisecond was 11 +/- 2 for cells labeled with QD 525, 12 +/- 9 for QD 565, 176 +/- 81 for QD 605, 176 +/- 136 for QD 655, 167 +/- 104 for QD 705, and 1,713 +/- 482 for QD 800. Finally, we have shown that QD 800 offers greater fluorescent intensity than the other QDs tested.In summary, this is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements, QDs will have a greater potential for tracking stem cells within deep tissues. These results provide a promising tool for imaging stem cell therapy non-invasively in vivo.

  View details for DOI 10.1186/1472-6750-7-67

  View details for PubMedID 17925032

• Differentiation, survival, and function of embryonic stem cell-derived endothelial cells for ischemic heart disease 79th Annual Scientific Session of the American-Heart-Association Li, Z., Wu, J. C., Sheikh, A. Y., Kraft, D., Cao, F., Xie, X., Patel, M., Gambhir, S. S., Robbins, R. C., Cooke, J. P., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2007: I46–I54

  Abstract

  Embryonic stem (ES) cells are distinguished by their capacity for self-renewal and pluripotency. Here we characterize the differentiation of ES cell-derived endothelial cells (ESC-ECs), use molecular imaging techniques to examine their survival in vivo, and determine the therapeutic efficacy of ESC-ECs for restoration of cardiac function after ischemic injury.Murine ES cells were transfected with a construct composed of a vascular endothelial cadherin promoter driving enhanced green fluorescence protein (pVE-cadherin-eGFP). Differentiation of ES cells to ECs was detected by FACS analysis using Flk-1 (early EC marker at day 4) and VE-cadherin (late EC marker at day 8). After isolation, these ESC-ECs express endothelial cell markers similar to adult mouse lung endothelial cells, form vascular-like channels, and incorporate DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). For in vivo imaging, ES cells were transduced with an ubiquitin promoter driving firefly luciferase and monomeric red fluorescence protein (pUb-Fluc-mRFP). A robust correlation exists between Fluc signals and cell numbers by ex vivo imaging analysis (R2=0.98) and by in vitro enzyme assay (R2=0.94). Afterward, 5x10(5) ESC-ECs or PBS (as control) was injected into the hearts of mice undergoing LAD ligation (n=15 per group). Bioluminescence imaging showed longitudinal survival of transplanted ESC-ECs for approximately 8 weeks. Echocardiogram demonstrated significant functional improvement in the ESC-EC group compared with control (P=0.04). Finally, postmortem analysis confirmed increased presence of small capillaries and venules in the infarcted zones by CD31 staining.This is the first study to track the fate and function of transplanted ESC-ECs in the heart. With further validation, these ESC-ECs could become a valuable source of cell therapy for induction of angiogenesis in the treatment of myocardial ischemia.

  View details for DOI 10.1161/CIRCULATIONAHA.106.680561

  View details for PubMedID 17846325

• Cardiovascular molecular imaging RADIOLOGY Wu, J. C., Bengel, F. M., Gambhir, S. S. 2007; 244 (2): 337-355

  Abstract

  The goal of this review is to highlight how molecular imaging will impact the management and improved understanding of the major cardiovascular diseases that have substantial clinical impact and research interest. These topics include atherosclerosis, myocardial ischemia, myocardial viability, heart failure, gene therapy, and stem cell transplantation. Traditional methods of evaluation for these diseases will be presented first, followed by methods that incorporate conventional and molecular imaging approaches.

  View details for PubMedID 17592037

• Positron emission tomography imaging of conditional gene activation in the heart JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Chang, G. Y., Cao, F., Krishnan, M., Huang, M., Li, Z., Xie, X., Sheikh, A. Y., Hoyt, G., Robbins, R. C., Hsial, T., Schneider, M. D., Wu, J. C. 2007; 43 (1): 18-26

  Abstract

  The Cre-loxP system has been routinely used for conditional activation and deletion of gene expression. However, the spatiotemporal manner of these events in the heart has not yet been defined by in vivo imaging. Adenovirus (1 x 10(9 )pfu) carrying the silent positron emission tomography (PET) reporter gene, herpes simplex virus type 1 thymidine kinase (HSV1-tk), was injected into the left ventricular wall of male transgenic mice (n=15) or FVB controls (n=8). Transgenic mice expressed Cre recombinase driven by a cardiac-specific alpha-myosin heavy chain (alpha-MHC) promoter. Following injection of the 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([18F]-FHBG; 137+/-25 microCi) reporter probe, microPET imaging was used to assess the expression of HSV1-tk reporter gene in the myocardium. Two days following adenoviral injection, cardiac HSV1-tk gene activation resulted in tracer uptake of 3.20+/-0.51% ID/g for alpha-MHC-Cre and 0.05+/-0.02%ID/g for control mice (P<0.01). The in vivo results were confirmed by RT-PCR and Western blot analysis. Similar transfections were evaluated in both cardiac-specific and non-cardiac-specific cell lines. Enzyme activity showed a robust correlation (r2=0.82) between in vivo molecular imaging technique and traditional in vitro enzyme assays. With further development and validation, PET imaging will likely play an important role in the noninvasive, repetitive, and quantitative measurement of conditional gene activation in the future.

  View details for DOI 10.1016/j.yjmcc.2007.03.809

  View details for PubMedID 17467733

• Integration of genomics, proteomics, and imaging for cardiac stem cell therapy EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Chun, H. J., Wilson, K. O., Huang, M., Wu, J. C. 2007; 34: S20-S26

  Abstract

  Cardiac stem cell therapy is beginning to mature as a valid treatment for heart disease. As more clinical trials utilizing stem cells emerge, it is imperative to establish the mechanisms by which stem cells confer benefit in cardiac diseases. In this paper, we review three methods - molecular cellular imaging, gene expression profiling, and proteomic analysis - that can be integrated to provide further insights into the role of this emerging therapy.

  View details for DOI 10.1007/s00259-007-0437-y

  View details for PubMedID 17464506

• Magnetic resonance imaging of progressive cardiomyopathic changes in the db/db mouse AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Yue, P., Arai, T., Terashima, M., Sheikh, A. Y., Cao, F., Charo, D., Hoyt, G., Robbins, R. C., Ashley, E. A., Wu, J., Yang, P. C., Tsao, P. S. 2007; 292 (5): H2106-H2118

  Abstract

  The db/db mouse is a well-established model of diabetes. Previous reports have documented contractile dysfunction (i.e., cardiomyopathy) in these animals, although the extant literature provides limited insights into cardiac structure and function as they change over time. To better elucidate the natural history of cardiomyopathy in db/db mice, we performed cardiac magnetic resonance (CMR) scans on these animals. CMR imaging was conducted with a 4.7-T magnet on female db/db mice and control db/+ littermates at 5, 9, 13, 17, and 22 wk of age. Gated gradient echo sequences were used to obtain cineographic short-axis slices from apex to base. From these images left ventricular (LV) mass (LVM), wall thickness, end-diastolic volume (LVEDV), and ejection fraction (LVEF) were determined. Additionally, cardiac [(18)F]fluorodeoxyglucose ([(18)F]FDG) PET scanning, pressure-volume loops, and real-time quantitative PCR on db/db myocardium were performed. Relative to control, db/db mice developed significant increases in LVM and wall thickness as early as 9 wk of age. LVEDV diverged slightly later, at 13 wk. Interestingly, compared with the baseline level, LVEF in the db/db group did not decrease significantly until 22 wk. Additionally, [(18)F]FDG metabolic imaging showed a 40% decrease in glucose uptake in db/db mice. Furthermore, contractile dysfunction was observed in 15-wk db/db mice undergoing pressure-volume loops. Finally, real-time quantitative PCR revealed an age-dependent recapitulation of the fetal gene program, consistent with a myopathic process. In summary, as assessed by CMR, db/db mice develop characteristic structural and functional changes consistent with cardiomyopathy.

  View details for DOI 10.1152/ajpheart.00856.2006

  View details for PubMedID 17122193

• An unusual cause of stroke from a left atrial mass. Journal of the American Society of Echocardiography Wu, J. C., Fishbein, M. C., Child, J. S. 2007; 20 (5): 537 e1-2

  Abstract

  A 59-year-old woman was admitted to the hospital after having severe headache for 1 day. Her medical history was significant for coronary artery disease, diabetes, atrial fibrillation, and rheumatic heart disease. The patient was in remission from stage II left breast cancer (T2 N0 M0) in 1997, treated with lumpectomy, 6 cycles of chemotherapy (cyclophosphamide, methotrexate, and 5-fluorouracil), and local radiation therapy (total 6000 cGy). Head magnetic resonance imaging scan showed left occipital hemorrhage. Subsequent angiogram showed a possible mycotic aneurysm of distal parietal occipital branch of the posterior cerebral artery. Cardiology was consulted to evaluate for a cardioembolic source.

  View details for PubMedID 17484995

• Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation CLONING AND STEM CELLS Cao, F., Drukker, M., Lin, S., Sheikh, A. Y., Xie, X., Li, Z., Connolly, A. J., Weissman, I. L., Wu, J. C. 2007; 9 (1): 107-117

  Abstract

  Numerous studies have demonstrated the potential use of stem cells for the repair and regeneration of injured tissues. However, tracking transplanted stem cell fate and function in vivo remains problematic. To address these issues, murine embryonic stem (ES) cells were stably transduced with self-inactivating lentiviral vectors carrying either a triple fusion (TF) or double fusion (DF) reporter gene construct. The TF consisted of monomeric red fluorescence protein (mrfp), firefly luciferase (Fluc), and herpes simplex virus truncated thymidine kinase (HSV-ttk) reporter genes. The DF consisted of enhanced green fluorescence protein (egfp) and Fluc reporter genes but lacked HSV-ttk. Stably transduced ES-TF or ES-DF cells were selected by fluorescence activated cell sorting based on either mrfp (TF) or egfp (DF) expression. Afterwards, cells were injected subcutaneously into the right (ES-TF cells) and left (ES-DF cells) shoulders of adult female nude mice. Cell survival was tracked noninvasively by bioluminescence and positron emission tomography imaging of Fluc and HSV-ttk reporter genes, respectively. Imaging signals progressively increased from day 2 to day 14, consistent with ES cell survival and proliferation in vivo. However, teratoma formation occurred in all nude mice after 5 weeks. Administration of ganciclovir (GCV), targeting the HSV-ttk gene, resulted in selective ablation of teratomas arising from the ES-TF cells but not ES-DF cells. These data demonstrate the novel use of multimodality imaging techniques to (1) monitor transplanted ES cell survival and proliferation in vivo and (2) assess the efficacy of suicide gene therapy as a backup safety measure against teratoma formation.

  View details for DOI 10.1089/clo.2006.0016

  View details for PubMedID 17386018

• Clinical hurdles for the transplantation of cardiomyocytes derived from human embryonic stem cells: role of molecular imaging CURRENT OPINION IN BIOTECHNOLOGY Swijnenburg, R., van der Bogt, K. E., Sheikh, A. Y., Cao, F., Wu, J. C. 2007; 18 (1): 38-45

  Abstract

  Over the past few years, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, there remain significant hurdles before safe and successful translation of hESC-based treatment into clinical therapy, including the ability to assess cells following transplant. To this end, molecular imaging has proven a reliable methodology for tracking the long-term fate of transplanted cells. Imaging reporter genes that are introduced into the cells before transplantation enable non-invasive and longitudinal studies of cell viability, location and behaviour in vivo. Therefore, molecular imaging is expected to play an increasing role in characterizing the biology and physiology of hESC-derived cardiac cells in living subjects.

  View details for DOI 10.1016/j.copbio.2006.12.003

  View details for PubMedID 17196814

• Clinical dilemmas in treating left ventricular thrombus. International journal of cardiology Leeper, N. J., Gupta, A., Schnittger, I., Wu, J. C. 2007; 114 (3): e118-9

  View details for PubMedID 17049652

• Genetic modification of embryonic stem cells with VEGF enhances cell survival and improves cardiac function CLONING AND STEM CELLS Xie, X., Cao, F., Sheikh, A. Y., Li, Z., Connolly, A. J., Pei, X., Li, R., Robbins, R. C., Wui, J. C. 2007; 9 (4): 549-563

  Abstract

  Cardiac stem cell therapy remains hampered by acute donor cell death posttransplantation and the lack of reliable methods for tracking cell survival in vivo. We hypothesize that cells transfected with inducible vascular endothelial growth factor 165 (VEGF(165)) can improve their survival as monitored by novel molecular imaging techniques. Mouse embryonic stem (ES) cells were transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF(165) and renilla luciferase (Rluc). Addition of doxycycline induced Bi-Tet expression of VEGF(165) and Rluc significantly compared to baseline (p<0.05). Expression of VEGF(165) enhanced ES cell proliferation and inhibited apoptosis as determined by Annexin-V staining. For noninvasive imaging, ES cells were transduced with a double fusion (DF) reporter gene consisting of firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP). There was a robust correlation between cell number and Fluc activity (R(2)=0.99). Analysis by immunostaining, histology, and RT-PCR confirmed that expression of Bi-Tet and DF systems did not affect ES cell self-renewal or pluripotency. ES cells were differentiated into beating embryoid bodies expressing cardiac markers such as troponin, Nkx2.5, and beta-MHC. Afterward, 5 x 10(5) cells obtained from these beating embryoid bodies or saline were injected into the myocardium of SV129 mice (n=36) following ligation of the left anterior descending (LAD) artery. Bioluminescence imaging (BLI) and echocardiography showed that VEGF(165) induction led to significant improvements in both transplanted cell survival and cardiac function (p<0.05). This is the first study to demonstrate imaging of embryonic stem cell-mediated gene therapy targeting cardiovascular disease. With further validation, this platform may have broad applications for current basic research and further clinical studies.

  View details for DOI 10.1089/clo.2007.0032

  View details for PubMedID 18154515

• Endothelial progenitor cells from murine embryonic stem cells: isolation and transplantation for myocardial infarction Li, Z., Wu, J., Sheikh, A., Kraft, D., Cao, F., Xie, X., Patel, M., Gambhir, S., Robbins, R., Cooke, J., Wu, J. SAGE PUBLICATIONS LTD. 2007: 152–52

  View details for Web of Science ID 000247470400044

• Molecular Imaging of bone marrow mononuclear cell homing and engraftment in ischemic myocardium STEM CELLS Sheikh, A. Y., Lin, S., Cao, F., Cao, Y., van der Bogt, K. E., Chu, P., Chang, C., Contag, C. H., Robbins, R. C., Wu, J. C. 2007; 25 (10): 2677-2684

  Abstract

  Bone marrow mononuclear cell (BMMC) therapy shows promise as a treatment for ischemic heart disease. However, the ability to monitor long-term cell fate remains limited. We hypothesized that molecular imaging could be used to track stem cell homing and survival after myocardial ischemia-reperfusion (I/R) injury. We first harvested donor BMMCs from adult male L2G85 transgenic mice constitutively expressing both firefly luciferase (Fluc) and enhanced green fluorescence protein reporter gene. Fluorescence-activated cell sorting analysis revealed approximately 0.07% of the population to consist of classic hematopoietic stem cells (lin-, thy-int, c-kit+, Sca-1+). Afterward, adult female FVB recipients (n = 38) were randomized to sham surgery or acute I/R injury. Animals in the sham (n = 16) and I/R (n = 22) groups received 5 x 10(6) of the L2G85-derived BMMCs via tail vein injection. Bioluminescence imaging (BLI) was used to track cell migration and survival in vivo for 4 weeks. BLI showed preferential homing of BMMCs to hearts with I/R injury compared with sham hearts within the first week following cell injection. Ex vivo analysis of explanted hearts by histology confirmed BLI imaging results, and quantitative real-time polymerase chain reaction (for the male Sry gene) further demonstrated a greater number of BMMCs in hearts with I/R injury compared with the sham group. Functional evaluation by echocardiography demonstrated a trend toward improved left ventricular fractional shortening in animals receiving BMMCs. Taken together, these data demonstrate that molecular imaging can be used to successfully track BMMC therapy in murine models of heart disease. Specifically, we have demonstrated that systemically delivered BMMCs preferentially home to and are retained by injured myocardium. Disclosure of potential conflicts of interest is found at the end of this article.

  View details for DOI 10.1634/stemcells.2007-0041

  View details for PubMedID 17628019

• Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells PROTEOMICS Wu, J. C., Cao, F., Dutta, S., Xie, X., Kim, E., Chungfat, N., Gambhir, S., Mathewson, S., Connolly, A. J., Brown, M., Wang, E. W. 2006; 6 (23): 6234-6249

  Abstract

  Study of stem cells may reveal promising treatment for diseases. The fate and function of transplanted stem cells remain poorly defined. Recent studies demonstrate that reporter genes can monitor real-time survival of transplanted stem cells in living subjects. We examined the effects of a novel and versatile triple fusion (TF) reporter gene construction on embryonic stem (ES) cell function by proteomic analysis. Murine ES cells were stably transduced with a self-inactivating lentiviral vector containing fluorescence (firefly luciferase; Fluc), bioluminescence (monomeric red fluorescence protein; mRFP), and positron emission tomography (herpes simplex virus type 1 truncated thymidine kinase; tTK) reporter genes. Fluorescence-activated cell sorting (FACS) analysis isolated stably transduced populations. TF reporter gene effects on cellular function were evaluated by quantitative proteomic profiling of control ES cells versus ES cells stably expressing the TF construct (ES-TF). Overall, no significant changes in protein quantity were observed. TF reporter gene expression had no effect on ES cell viability, proliferation, and differentiation capability. Molecular imaging studies tracked ES-TF cell survival and proliferation in living animals. In summary, this is the first proteomic study, demonstrating the unique potential of reporter gene imaging for tracking ES cell transplantation non-invasively, repetitively, and quantitatively.

  View details for DOI 10.1002/pmic.200600150

  View details for PubMedID 17080479

• Molecular imaging of human embryonic stem cells - Keeping an eye on differentiation, tumorigenicity and immunogenicity CELL CYCLE van der Bogt, K. E., Swijnenburg, R., Cao, F., Wu, J. C. 2006; 5 (23): 2748-2752

  Abstract

  Human embryonic stem cells (hESCs) are capable of differentiation into every cell type of the human being. They are under extensive investigation for their regenerative potential in a variety of debilitating diseases. However, the field of hESC research is still in its infancy, as there are several critical issues that need to be resolved before clinical translation. Two major concerns are the ability of undifferentiated hESCs to form teratomas and the possibility of a provoked immune reaction after transplantation of hESCs into a new host. Therefore, it is imperative to develop noninvasive imaging modalities that allow for longitudinal, repetitive, and quantitative assessment of transplanted cell survival, proliferation, and migration in vivo. Reporter gene-based molecular imaging offers these characteristics and has great potential in the field of stem cell therapy. Moreover, it has recently been shown that reporter gene imaging can be combined with therapeutic strategies. Here, we provide an outline of the current status of hESC research and discuss the concerns of tumorigenicity and immunogenicity. Furthermore, we describe how molecular imaging can be utilized to follow and resolve these issues.

  View details for PubMedID 17172859

• Molecular imaging of cardiac stem cell mediated angiogenic gene therapy 79th Annual Scientific Session of the American-Heart-Association Xie, X., Cao, F., Sheikh, A. Y., Li, Z., Pei, X., Gambhir, S. S., Li, R., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2006: 566–66

  View details for Web of Science ID 000241792803549

• Noninvasive imaging of bone marrow stem cell homing, survival, and proliferation in. ischemic myocardium 79th Annual Scientific Session of the American-Heart-Association Lin, S., Sheikh, A. Y., Cao, F., Gambhir, S. S., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2006: 660–60

  View details for Web of Science ID 000241792804305

• Right coronary cameral fistula resulting from surgery of double chamber right ventricle. Journal of the American Society of Echocardiography Leeper, N. J., Gupta, A., Murphy, D. J., Wu, J. C. 2006; 19 (9): 1191 e9-11

  View details for PubMedID 16950481

• Murine model of ischemia reperfusion for myocardial restoration with cellular transplantation 3rd Annual Symposium of the American-Heart-Association-Council-on-Basic-Cardiovascular-Sciences Hendry, S., Sheikh, A., Schrepfer, S., Pelletier, M., Wu, J., Yang, P., Robbins, R. LIPPINCOTT WILLIAMS & WILKINS. 2006: E19–E19

  View details for Web of Science ID 000240209800022

• Overview of stem cells and imaging modalities for cardiovascular diseases JOURNAL OF NUCLEAR CARDIOLOGY Chang, G. Y., Xie, X., Wu, J. C. 2006; 13 (4): 554-569

  Abstract

  Stem cell therapy is emerging as a promising approach to treat heart diseases. Considerable evidence from experimental studies and initial clinical trials suggests that stem cell transplantation promotes systolic function and prevent ventricular remodeling. However, the specific mechanisms by which stem cells improve heart function remain largely unknown. In addition, interpreting the long-term effects of stem cell therapy is difficult because of the limitations of conventional techniques. The recent development of molecular imaging techniques offers great potential to address these critical issues by noninvasively tracking the fate of the transplanted cells. This review offers a focused discussion on the use of stem cell therapy and imaging in the context of cardiology.

  View details for DOI 10.1016/j.nuclcard.2006.05.012

  View details for PubMedID 16919579

• Noninvasive evaluation of immunosuppressive drug efficacy on acute donor cell survival MOLECULAR IMAGING AND BIOLOGY Gheysens, O., Lin, S., Cao, F., Wang, D., Chen, I. Y., Rodriguez-Porcel, M., Min, J. J., Gambhir, S. S., Wu, J. C. 2006; 8 (3): 163-170

  Abstract

  The therapeutic benefits of cell transplantation may depend on the survival of sufficient numbers of grafted cells. We evaluate four potent immunosuppressive medications aimed at preventing acute donor cell death.Embryonic rat H9c2 myoblasts were stably transduced to express firefly luciferase reporter gene (H9c2-Fluc). H9c2-Fluc cells (3x10(6)) were injected into thigh muscles of Sprague-Dawley rats (N=30) treated with cyclosporine, dexamethasone, mycophenolate mofetil, tacrolimus, or saline from day -3 to day +14. Longitudinal optical bioluminescence imaging was performed over two weeks. Fluc activity was 40.0+/-12.1% (dexamethasone), 30.5+/-12.5% (tacrolimus), and 21.5+/-3.5% (mycophenolate) vs. 12.0+/-5.0% (control) and 8.3+/-5.0% (cyclosporine) at day 4 (P<0.05). However, by day 14, cell signals had decreased drastically to <10% for all groups despite drug therapy.This study demonstrates the ability of optical molecular imaging for tracking cell survival noninvasively and raises important questions with regard to the overall efficacy of immunosuppressives for prolonging transplanted cell survival.

  View details for DOI 10.1007/s11307-006-0038-3

  View details for PubMedID 16555032

• In vivo bioluminescence imaging of cord blood derived mesenchymal stem cell transplantation into rat myocardium ANNALS OF NUCLEAR MEDICINE Min, J. J., Ahn, Y., Moon, S., Kim, Y. S., Park, J. E., Mn, J. J., Le, U. N., Wu, J. C., Joo, S. Y., Hong, M. H., Yang, D. H., Jeong, M. H., Song, C. H., Jeong, Y. H., Yoo, K. Y., Kang, K. S., Bom, H. S. 2006; 20 (3): 165-170

  Abstract

  The conventional method for the analysis of myocardial cell transplantation depends on postmortem histology. Here, we have sought to demonstrate the feasibility of a longitudinal monitoring of transplanted cell survival in living animals, accomplished with optical imaging techniques and pharmacological interventions.Human cord blood (50 ml) was donated with parental consent. After getting cord blood derived mesenchymal stem cells (CBMSCs), cells were transfected (MOI = 100) overnight with adenovirus encoding firefly luciferase gene (Ad-CMV-Fluc). Our experimental Sprague-Dawley rats (n = 12) were given intramyocardial injections containing 1 x 10(6) CBMSCs, which had been made to express the firefly luciferase (Fluc) reporter gene. Optical bioluminescence imaging was then conducted using a cooled charged-coupled device (CCD) camera (Xenogen), beginning on the day after the transplantation (day 1). Groups of mice were intraperitoneally injected with cyclosporine (5 mg/kg) or tacrolimus (1 mg/kg), in an attempt to determine the degree to which cell survival had been prolonged, and these values were then compared with the cell survival values of the negative control group. The presence of transplanted CBMSCs on in vivo images confirmed by in situ hybridization for human specific Alu in the myocardium.Cardiac bioluminescence signals were determined to be present for 6 days after transplantation: day 1 (97000 +/- 9100 x 10(5) p/s/cm2/sr), day 3 (9600 +/- 1110 p/s/cm2/sr), and day 5 (3200 +/- 550 p/s/cm2/sr). The six mice that received either cyclosporine or tacrolimus displayed cardiac bioluminescence signals for a period of 8 days after transplantation. We observed significant differences between the treated group and the non-treated group, beginning on day 3 (tacrolimus; 26500 +/- 4340 p/s/cm2/sr, cyclosporine; 27200 +/- 3340 p/s/cm2/sr, non-treated; 9630 +/- 1180 p/s/cm2/sr, p < 0.01), and persisting until day 7 (tacrolimus; 12500 +/- 2946 p/s/cm2/sr, cyclosporine; 7310 +/- 1258 p/s/cm2/sr, non-treated; 2460 +/- 160 p/s/cm2/sr, p < 0.01). The human-derived CBMSCs were detected in the myocardium 3 days after transplantation by in situ hybridization.The locations, magnitude, and survival duration of the CBMSCs were noninvasively monitored with a bioluminescence optical imaging system. We determined that optical molecular imaging expedites the fast throughput screening of pharmaceutical agents, allowing for the noninvasive tracking of cell survival within animals. In rat cardiac CBMSC transplant models, transient immunosuppressive treatment with tacrolimus or cyclosporine was shown to improve donor cell survival.

  View details for Web of Science ID 000237375100001

  View details for PubMedID 16715945

• Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation PHYSIOLOGICAL GENOMICS Wu, J. C., Spin, J. M., Cao, F., Lin, S. A., Xie, X. Y., Gheysens, O., Chen, I. Y., Sheikh, A. Y., Robbins, R. C., Tsalenko, A., Gambhir, S. S., Quertermous, T. 2006; 25 (1): 29-38

  Abstract

  Stem cell therapy offers exciting promise for treatment of ischemic heart disease. Recent advances in molecular imaging techniques now allow investigators to monitor cell fate noninvasively and repetitively. Here we examine the effects of a triple-fusion reporter gene on embryonic stem (ES) cell transcriptional profiles. Murine ES cells were stably transfected with a self-inactivating lentiviral vector carrying a triple-fusion (TF) construct consisting of fluorescence, bioluminescence, and positron emission tomography (PET) reporter genes. Fluorescence-activated cell sorting (FACS) analysis allowed isolation of stably transfected populations. Microarray studies comparing gene expression in nontransduced control ES cells vs. stably transduced ES cells expressing triple fusion (ES-TF) revealed some increases in transcriptional variability. Annotation analysis showed that ES-TF cells downregulated cell cycling, cell death, and protein and nucleic acid metabolism genes while upregulating homeostatic and anti-apoptosis genes. Despite these transcriptional changes, expression of the TF reporter gene had no significant effects on ES cell viability, proliferation, and differentiation capability. Importantly, transplantation studies in murine myocardium demonstrated the feasibility of tracking ES-TF cells in living subjects using bioluminescence and PET imaging. Taken together, this is the first study to analyze in detail the effects of reporter genes on molecular imaging of ES cells.

  View details for DOI 10.1152/physiolgenomics.00254.2005

  View details for PubMedID 16390873

• Molecular imaging of cardiac stem cell transplantation. Current cardiology reports Sheikh, A. Y., Wu, J. C. 2006; 8 (2): 147-154

  Abstract

  In recent years, stem cell therapy for the treatment of heart disease has translated from the imagination of investigators to the bedside of patients. The initial results from trials evaluating cell therapy for the heart are encouraging. As this new field of cellular transplantation matures, it is imperative that novel methodologies for evaluating cell therapy are developed and applied to guide therapy. Molecular imaging is a discipline that is evolving to address these needs and is expected to play an increasing role in the characterization and assessment of cell therapy. This article provides a focused overview of clinical stem cell therapy for the heart, followed by a discussion of how novel molecular imaging techniques are presently being applied to monitor cell therapy.

  View details for PubMedID 16524542

• F-18-labeled bombesin analogs for targeting GRP receptor-expressing prostate cancer JOURNAL OF NUCLEAR MEDICINE Zhang, X., Cai, W., Cao, F., Schreibmann, E., Wu, Y., Wu, J. C., Xing, L., Chen, X. 2006; 47 (3): 492-501

  Abstract

  The gastrin-releasing peptide receptor (GRPR) is found to be overexpressed in a variety of human tumors. The aim of this study was to develop 18F-labeled bombesin analogs for PET of GRPR expression in prostate cancer xenograft models.[Lys3]Bombesin ([Lys3]BBN) and aminocaproic acid-bombesin(7-14) (Aca-BBN(7-14)) were labeled with 18F by coupling the Lys3 amino group and Aca amino group, respectively, with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) under slightly basic condition (pH 8.5). Receptor-binding affinity of FB-[Lys3]BBN and FB-Aca-BBN(7-14) was tested in PC-3 human prostate carcinoma cells. Internalization and efflux of both radiotracers were also evaluated. Tumor-targeting efficacy and in vivo kinetics of both radiotracers were examined in male athymic nude mice bearing subcutaneous PC-3 tumors by means of biodistribution and dynamic microPET imaging studies. 18F-FB-[Lys3]BBN was also tested for orthotopic PC-3 tumor delineation. Metabolic stability of 18F-FB-[Lys3]BBN was determined in mouse blood, urine, liver, kidney, and tumor homogenates at 1 h after injection.The typical decay-corrected radiochemical yield was about 30%-40% for both tracers, with a total reaction time of 150 +/- 20 min starting from 18F-. 18F-FB-[Lys3]BBN had moderate stability in the blood and PC-3 tumor, whereas it was degraded rapidly in the liver, kidneys, and urine. Both radiotracers exhibited rapid blood clearance. 18F-FB-[Lys3]BBN had predominant renal excretion. 18F-FB-Aca-BBN(7-14) exhibited both hepatobiliary and renal clearance. Dynamic microPET imaging studies revealed that the PC-3 tumor uptake of 18F-FB-[Lys3]BBN in PC-3 tumor was much higher than that of 18F-FB-Aca-BBN(7-14) at all time points examined (P < 0.01). The receptor specificity of 18F-FB-[Lys3]BBN in vivo was demonstrated by effective blocking of tumor uptake in the presence of [Tyr4]BBN. No obvious blockade was found in PC-3 tumor when 18F-FB-Aca-BBN(7-14) was used as radiotracer under the same condition. 18F-FB-[Lys3]BBN was also able to visualize orthotopic PC-3 tumor at early time points after tracer administration, at which time minimal urinary bladder activity was present to interfere with the receptor-mediated tumor uptake.This study demonstrates that 18F-FB-[Lys3]BBN and PET are suitable for detecting GRPR-positive prostate cancer in vivo.

  View details for PubMedID 16513619

• In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery CIRCULATION Cao, F., Lin, S., Xie, X. Y., Ray, P., Patel, M., Zhang, X. Z., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X. Y., Weissman, I. L., Gambhir, S. S., Wu, J. C. 2006; 113 (7): 1005-1014

  Abstract

  Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities.Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (P=NS). Afterward, 1x10(7) of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (n=20). Control animals received nontransduced ES cells (n=6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7x10(7)+/-5.8x10(6) photons.s(-1).cm(-2) per steradian (sr) and 0.08+/-0.03% injected dose/g, respectively (P<0.05 versus control). Both signals increased progressively from week 1 to week 4, which indicated ES cell survival and proliferation in the host. Histological analysis demonstrated the formation of intracardiac and extracardiac teratomas. Finally, animals (n=4) that were treated with intraperitoneal injection of ganciclovir (50 mg/kg) did not develop teratomas when compared with control animals (n=4) treated with saline (1 mL/kg).This is the first study to characterize ES cells that stably express fluorescence, bioluminescence, and positron emission tomography reporter genes and monitor the kinetics of ES cell survival, proliferation, and migration. This versatile imaging platform should have broad applications for basic research and clinical studies on stem cell therapy.

  View details for DOI 10.1161/CIRCULATIONHA.105.588954

  View details for PubMedID 16476845

• Quantitative evaluation of sex mismatch cell transplantation using real-time polymerase chain reaction is efficacious for determining myocardial cell delivery efficiency i2 Summit 2006 on Innovation in Intervention Brinton, T. J., Wilson, K. D., Price, E. T., Zhang, S. X., Krishnan, M., Wu, J. C., Yeung, A. C., Yock, P. G. ELSEVIER SCIENCE INC. 2006: 42B–42B

  View details for Web of Science ID 000235530500178

• Imaging chemically modified adenovirus for targeting tumors expressing integrin alpha(v)beta(3) in living mice with mutant herpes simplex virus type 1 thymidine kinase PET reporter gene JOURNAL OF NUCLEAR MEDICINE Xiong, Z. M., Cheng, Z., Zhang, X. Z., Patel, M., Wu, J. C., Gambhir, S. S., Chen, X. Y. 2006; 47 (1): 130-139

  Abstract

  The aim of this study was to change adenovirus tropism by chemical modification of the fiber knobs with PEGylated RGD peptide for targeting integrin alpha(v)beta(3) that is uniquely or highly expressed in tumor cells and neovasculature of tumors of various origins.The first generation Ad (Ad) vector, which expresses the herpes simplex virus type 1 mutant thymidine kinase (HSV1-sr39tk) gene under the control of cytomegalovirus (CMV) promoter was conjugated with poly(ethylene glycol) (PEG) or RGD-PEG. The transduction efficiency of Ads (Adtk, PEG-Adtk, and RGD-PEG-Adtk) into different types of cells (293T, MCF7, MDA-MB-435, and U87MG) was analyzed and quantified by thymidine kinase (TK) assay using 8-(3)H-penciclovir (8-(3)H-PCV) as substrate. The in vivo infectivity of the Ad vectors after intravenous administration into integrin alpha(v)beta(3)-positive U87MG and MDA-MB-435 tumor-bearing athymic nude mice was measured by both noninvasive microPET using 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) as a reporter probe and ex vivo TK assay of the tumor and tissue homogenates.PEGylation completely abrogated coxsackievirus and adenovirus receptor (CAR)-knob interaction and the infectivity of PEG-Adtk is significantly lower than that of unmodified Adtk in CAR-positive cells. RGD-PEG-modified virus (RGD-PEG-Adtk) had significantly higher infectivity than PEG-Adtk and the extent of increase is related to both CAR and integrin alpha(v)beta(3) expression levels. (18)F-FHBG had minimal nonspecific uptake in the liver and tumors that are void of sr39tk. Mice preinjected intravenously with unmodified Adtk resulted in high hepatic uptake and moderate tumor accumulation of the tracer. In contrast, RGD-PEG-Adtk administration resulted in significantly lower liver uptake without compromising the tumor accumulation of (18)F-FHBG. Expression of TK in the liver and tumor homogenates corroborated with the magnitude of (18)F-FHBG uptake quantified by noninvasive microPET. Analysis of liver and tumor tissue integrin level confirmed that RGD-integrin interaction is responsible for the enhanced tumor infectivity of RGD-PEG-Adtk.The results of this study suggest that RGD-PEG conjugation is an effective way to modify Ad vector tropism for improved systemic gene delivery. Noninvasive PET and (18)F-FHBG are able to monitor in vivo transfectivity of both Adtk and RGD-PEG-Adtk vectors in the liver and tumors after intravenous injection.

  View details for PubMedID 16391197

• Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging. FASEB journal Krishnan, M., Park, J. M., Cao, F., Wang, D., Paulmurugan, R., Tseng, J. R., Gonzalgo, M. L., Gambhir, S. S., Wu, J. C. 2006; 20 (1): 106-108

  Abstract

  Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.

  View details for PubMedID 16246867

• Image-guided cardiac cell delivery using high-resolution small-animal ultrasound MOLECULAR THERAPY Rodriguez-Porcel, M., Gheysens, O., Chen, I. Y., Wu, J. C., Gambhir, S. S. 2005; 12 (6): 1142-1147

  Abstract

  Open-chest cardiac injection is the preferred delivery method for cardiac gene and stem cell therapy in small animals, but it is invasive and the operator is unable to see the actual delivery site. High-resolution ultrasound has recently been developed for small-animal imaging. We tested the hypothesis that image-guided cardiac cell delivery using high-resolution ultrasound guidance is feasible and reproducible. Sprague-Dawley rats (n = 11) were imaged using high-resolution ultrasound, and stably transfected cardiomyoblasts (plasmid-CMV-firefly luciferase) were injected into the anterior cardiac wall under ultrasound guidance (parasternal long-axis view), using a 28-gauge needle. After injection, bioluminescence imaging was performed using a cooled charged-coupled camera. Injection was successful in all animals and was associated with no mortality. The signal detected was positively correlated with the amount of cells transplanted (R(2) = 0.94, P = 0.03) and highly correlated with ex vivo assays (R(2) = 0.82). In addition, the optical signal could be followed longitudinally using bioluminescence imaging. Ultrasound image-guided cardiac cell delivery is an effective, safe, and reproducible way to perform cell delivery to a specific myocardial region and can be combined with assessment of cardiac function. We are confident that the use of these technologies will play a significant role in the future of gene and cell therapy.

  View details for DOI 10.1016/j.ymthe.2005.07.532

  View details for Web of Science ID 000233864700017

  View details for PubMedID 16111921

• Human gene therapy and imaging: cardiology EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Wu, J. C., Yla-Herttuala, S. 2005; 32: S346-S357

  Abstract

  This review discusses the basics of cardiovascular gene therapy, the results of recent human clinical trials, and the rapid progress in imaging techniques in cardiology. Improved understanding of the molecular and genetic basis of coronary heart disease has made gene therapy a potential new alternative for the treatment of cardiovascular diseases. Experimental studies have established the proof-of-principle that gene transfer to the cardiovascular system can achieve therapeutic effects. First human clinical trials provided initial evidence of feasibility and safety of cardiovascular gene therapy. However, phase II/III clinical trials have so far been rather disappointing and one of the major problems in cardiovascular gene therapy has been the inability to verify gene expression in the target tissue. New imaging techniques could significantly contribute to the development of better gene therapeutic approaches. Although the exact choice of imaging modality will depend on the biological question asked, further improvement in image resolution and detection sensitivity will be needed for all modalities as we move from imaging of organs and tissues to imaging of cells and genes.

  View details for DOI 10.1007/s00259-005-1897-6

  View details for PubMedID 16096829

• Myocardial restoration with embryonic stem cell transplantation in a murine myocardial infarction model 78th Annual Scientific Session of the American-Heart-Association Hendry, S. L., Arai, T., Dylla, S. J., Drukker, M., Sheikh, A. Y., Kutschka, I., Hoyt, G., Connolly, A., Pelletier, M., Wu, J. C., Yang, P. C., Robbins, R. C. LIPPINCOTT WILLIAMS & WILKINS. 2005: U37–U38

  View details for Web of Science ID 000232956400031

• Tracking embryonic stem cell transplant in the heart using a novel triple fusion reporter gene 78th Annual Scientific Session of the American-Heart-Association Cao, F., Lin, S., Krishnan, M., Drukker, M. E., Patel, M. R., Ray, P., Zhang, X. Z., Chen, X. Y., Gambhir, S. S., Weissman, I., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2005: U144–U145

  View details for Web of Science ID 000232956400464

• Myocardial restoration with embryonic stem cell transplantation in a murine myocardial infarction model 78th Annual Scientific Session of the American-Heart-Association Hendry, S. L., Arai, T., Dylla, S. J., Drukker, M., Sheikh, A. Y., Kutschka, I., Hoyt, G., Connolly, A., Pelletier, M., Wu, J. C., Yang, P. C., Robbins, R. C. LIPPINCOTT WILLIAMS & WILKINS. 2005: U432–U432

  View details for Web of Science ID 000232956402439

• Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging FASEB JOURNAL Krishnan, M., Park, J. M., Cao, F., Wang, D. X., Paulmurugan, R., Tseng, J. R., Gonzalgo, M. L., Gambhir, S. S., Wu, J. C. 2005; 19 (12): 106-?

  Abstract

  Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.

  View details for DOI 10.1096/fj.05-4551fje

  View details for Web of Science ID 000232991100011

  View details for PubMedCentralID PMC3625424

• Common congenital heart disorders in adults CURRENT PROBLEMS IN CARDIOLOGY Wu, J. C., Child, J. S. 2004; 29 (11): 641-700

  View details for DOI 10.1016/j.cpcardiol.2004.04.003

  View details for Web of Science ID 000225317800002

  View details for PubMedID 15536453

• Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium CIRCULATION Wu, J. C., Chen, I. Y., Wang, Y. L., Tseng, J. R., Chhabra, A., Salek, M., Min, J. J., Fishbein, M. C., Crystal, R., Gambhir, S. S. 2004; 110 (6): 685-691

  Abstract

  Angiogenic gene therapy is a promising treatment paradigm for patients with ischemic heart disease. In this study, we used micro-positron emission tomography (microPET) to monitor the transgene expression, function, and effects in a whole-body system.Adenovirus with cytomegalovirus promoter driving an angiogenic gene (vascular endothelial growth factor [VEGF]) linked to a PET reporter gene (herpes simplex virus type 1 mutant thymidine kinase; Ad-CMV-VEGF121-CMV-HSV1-sr39tk) was used to transfect rat embryonic cardiomyoblasts in vitro. Expression of both genes correlated strongly (r=0.98; P<0.001). Afterward, rats underwent ligation of the left anterior descending artery followed by injection of 1x10(10) pfu of Ad-CMV-VEGF121-CMV-HSV1-sr39tk (study; n=35) or Ad-null (control; n=15) at the peri-infarct region. Noninvasive microPET imaging was used to assess the uptake of 9-(4-[18F]-fluoro-hydroxymethylbutyl)guanine ([18F]-FHBG) PET reporter probe by cells expressing the HSV1-sr39tk PET reporter gene. Cardiac transgene expression peaked at day 1 and declined over the next 2 weeks. Repeat adenoviral injections at day 60 yielded no detectable signal. The in vivo reporter gene expression (% injected dose/g of [18F]-FHBG) correlated well with ex vivo gamma counting (r=0.92), myocardial tissue HSV1-sr39TK enzyme activity (r=0.95), and myocardial tissue VEGF level (r=0.94; P<0.001 for all). The VEGF121 isoform induced significant increases in capillaries and small blood vessels. However, the level of neovasculature did not translate into significant improvements in functional parameters such as myocardial contractility by echocardiography, perfusion by nitrogen-13 ammonia imaging, and metabolism by [18F]-fluorodeoxyglucose imaging.Taken together, these findings establish the feasibility of molecular imaging for monitoring angiogenic gene expression with a PET reporter gene and probe noninvasively, quantitatively, and repetitively. The principles demonstrated here can be used to evaluate other therapeutic genes of interest in animal models before future clinical trials are initiated.

  View details for DOI 10.1161/01.CIR.0000138153.02213.22

  View details for Web of Science ID 000223194700008

  View details for PubMedID 15302807

• Molecular imaging of cardiovascular gene products JOURNAL OF NUCLEAR CARDIOLOGY Wu, J. C., Tseng, J. R., Gambhir, S. S. 2004; 11 (4): 491-505

  View details for DOI 10.1016/j.nuclcard.2004.04.004

  View details for PubMedID 15295418

• Cardiac-Specific Gene Expression Facilitated by an Enhanced Myosin Light Chain Promoter MOLECULAR IMAGING Boecker, W., Bernecker, O. Y., Wu, J. C., Zhu, X., Sawa, T., Grazette, L., Rosenzweig, A., del Monte, F., Schmidt, U., Hajjar, R. J. 2004; 3 (2): 69-75

  Abstract

  Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression.To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc) reporter gene (Ad.4 x MLC250.Luc). Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 x MLC.Luc or control vectors. For in vivo testing, Ad.4 x MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera.In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells) was 20.4 versus 0.9 (Ad.4 x MLC250.Luc vs. Ad.CMV). In vivo: Ad.4 x MLC250.Luc significantly reduced luc activity in liver (38.4-fold), lung (16.1-fold), and kidney (21.8-fold) versus Ad.CMV (p =.01); whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 x MLC.Luc) versus 1:1.4 (Ad.CMV.Luc).This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.

  View details for Web of Science ID 000208288100001

  View details for PubMedID 15296671

• Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery CIRCULATION Chen, I. Y., Wu, J. C., Min, J. J., Sundaresan, G., Lewis, X., Liang, Q. W., Herschman, H. R., Gambhir, S. S. 2004; 109 (11): 1415-1420

  Abstract

  We have previously validated the use of micro-positron emission tomography (microPET) for monitoring the expression of a single PET reporter gene in rat myocardium. We now report the use of a bicistronic adenoviral vector (Ad-CMV-D2R80a-IRES-HSV1-sr39tk) for linking the expression of 2 PET reporter genes, a mutant rat dopamine type 2 receptor (D2R80a) and a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk), with the aid of an internal ribosomal entry site (IRES).Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5x10(8) pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer (r2=0.96, P<0.001 for D2R80A; r2=0.98, P<0.001 for HSV1-sr39TK) and each other (r2=0.97; P<0.001). Experimental (n=8) and control (n=6) athymic rats underwent intramyocardial injection of up to 2x10(9) pfu of Ad-CMV-D2R80a-IRES-HSV1-sr39tk and saline, respectively. Forty-eight hours later and weekly thereafter, rats were assessed for D2R80a-dependent myocardial accumulation of 3-(2-[18F]fluoroethyl)spiperone ([18F]-FESP) and HSV1-sr39tk-dependent sequestration of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]-FHBG) using microPET. Longitudinal [18F]-FESP and [18F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes (r2=0.73; P<0.001). The location of adenovirus-mediated transgene expression, as inferred from microPET images, was confirmed by ex vivo gamma counting of explanted heart.The IRES-based bicistronic adenoviral vector can potentially be used in conjunction with PET for indirect imaging of therapeutic gene expression by replacing 1 of the 2 PET reporter genes with a therapeutic gene of choice.

  View details for DOI 10.1161/01.CIR.0000121727.59564.5B

  View details for Web of Science ID 000220364700015

  View details for PubMedID 15007006

• Molecular imaging of cardiac cell transplantation in living animals using optical bioluminescence and positron emission tomography CIRCULATION Wu, J. C., Chen, I. Y., Sundaresan, G., Min, J. J., De, A., Qiao, J. H., Fishbein, M. C., Gambhir, S. S. 2003; 108 (11): 1302-1305

  Abstract

  The current method of analyzing myocardial cell transplantation relies on postmortem histology. We sought to demonstrate the feasibility of monitoring transplanted cell survival in living animals using molecular imaging techniques.For optical bioluminescence charged-coupled device imaging, rats (n=20) underwent intramyocardial injection of embryonic rat H9c2 cardiomyoblasts (3x10(6) to 5x10(5)) expressing firefly luciferase (Fluc) reporter gene. Cardiac bioluminescence signals were present for more than 2 weeks with 3x10(6) cells: day 1 (627 000+/-15%), day 2 (346 100+/-21%), day 4 (112 800+/-20%), day 8 (78 860+/-24%), day 12 (67 780+/-12%), and day 16 (62 200+/-5% p x s(-1) x cm(2-1) x sr(-1)). For micro-positron emission tomography imaging, rats (n=20) received cardiomyoblasts (3x10(6)) expressing mutant herpes simplex type 1 thymidine kinase (HSV1-sr39tk) reporter gene. Detailed tomography of transplanted cells is shown by 9-(4-[18F]-fluoro-3hydroxymethylbutyl)guanine ([18F]-FHBG) reporter probe and nitrogen-13 ammonia ([13N]-NH3) perfusion images. Within the transplanted region, there was a 4.48+/-0.71-fold increase of in vivo [18F]-FHBG activity and a 4.01+/-0.51-fold increase of ex vivo gamma counting compared with control animals. Finally, the in vivo images of cell survival were confirmed by ex vivo autoradiography, histology, immunohistochemistry, and reporter protein assays.The location(s), magnitude, and survival duration of embryonic cardiomyoblasts were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation biology.

  View details for DOI 10.1161/01.CIR.0000091252.20010.6E

  View details for Web of Science ID 000185328800006

  View details for PubMedID 12963637

• Positron-emission tomography reporter gene expression imaging in rat myocardium CIRCULATION Inubushi, M., Wu, J. C., Gambhir, S. S., Sundaresan, G., Satyamurthy, N., Namavari, M., Yee, S., Barrio, J. R., Stout, D., Chatziioannou, A. F., Wu, L. L., Schelbert, H. R. 2003; 107 (2): 326-332

  Abstract

  This study examines the quantitative accuracy, detection sensitivity, and time course of imaging the expression of a mutant herpes simplex type-1 virus thymidine kinase (HSV1-sr39tk) PET reporter gene in rat myocardium by using the PET reporter probe 9-(4-[18F]-Fluoro-3-Hydroxymethylbutyl)-Guanine ([18F]-FHBG) and a small-animal PET (microPET).In 40 rats, adenovirus expressing HSV1-sr39tk driven by a cytomegalovirus promoter (Ad-CMV-HSV1-sr39tk, 1x10(6) to 1x10(9) pfu) was injected through a thoracotomy directly into the left ventricular myocardium. After 3 days, myocardial perfusion was imaged with [13N]-ammonia for delineating the left ventricular myocardium, followed by imaging the expression of the reporter gene with intravenous [18F]-FHBG. The total myocardial [18F]-FHBG accumulation was quantified in percent of injected dose (%ID). Immunohistochemistry and autoradiography demonstrated HSV1-sr39tk enzyme (HSV1-sr39TK) and accumulation of [18F]-FHBG in the inoculated myocardium in 3 rats each. In 24 rats with various viral titers, the %ID was correlated with ex vivo well counting (r2=0.981, P<0.0001) and myocardial HSV1-sr39TK activity by tissue enzyme activity assay (r2=0.790, P<0.0001). Myocardial [18F]-FHBG accumulation was identified at viral titers down to 1x10(7) pfu. In 6 rats serially imaged up to day 17, myocardial [18F]-FHBG accumulation on microPET peaked on days 3 to 5 and was no longer identified on days 10 to 17.HSV1-sr39tk reporter gene expression can be monitored with [18F]-FHBG and microPET in rat myocardium quantitatively and serially with high detection sensitivity. Cardiac PET reporter gene imaging offers the potential of monitoring the expression of therapeutic genes in cardiac gene therapy.

  View details for DOI 10.1161/01.CIR.0000044385.60972.AE

  View details for Web of Science ID 000180785700022

  View details for PubMedID 12538436

• Positron emission tomography imaging of cardiac reporter gene expression in living rats CIRCULATION Wu, J. C., Inubushi, M., Sundaresan, G., Schelbert, H. R., Gambhir, S. S. 2002; 106 (2): 180-183

  Abstract

  Imaging reporter gene expression is useful for noninvasive monitoring of gene therapy. In this study, we imaged cardiac reporter gene expression in living rats using micro positron emission tomography (microPET).Rats (n=10) underwent intramyocardial injection with 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving herpes simplex virus type 1 mutant thymidine kinase (Ad-CMV-HSV1-sr39tk) as PET reporter gene. Control rats (n=4) received 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving firefly luciferase (Ad-CMV-Fluc). On days 2 to 4, microPET images were obtained after a tail vein injection of nitrogen-13 ammonia ([13N]-NH3) as myocardial perfusion tracer, followed by 9-(4-[18F]-fluoro-3 hydroxymethylbutyl) guanine ([18F]-FHBG) to assess HSV1-sr39tk expression. After imaging, hearts were removed for ex vivo [18F] gamma counting and thymidine kinase enzyme assay. Results show homogenous myocardial distribution of [13N]-NH3 on all microPET images. Rats injected with Ad-CMV-HSV1-sr39tk have significant [18F]-FHBG uptake in the anterolateral wall compared with background signal in controls. Gamma counting shows 20.0+/-4.4-fold increase of radioactivity, whereas enzyme assay shows 22.1+/-6.1-fold increase of thymidine kinase activity in Ad-CMV-HSV1-sr39tk injected rats (P<0.05).Successful imaging of cardiac HSV1-sr39tk expression was performed in living rats with microPET. The presence of [18F]-FHBG uptake is confirmed by gamma counting and the presence of HSV1-sr39TK protein by thymidine kinase enzyme assay. Cardiac reporter gene imaging by PET may eventually be applied toward human gene therapy studies.

  View details for DOI 10.1161/01.CIR.0000023620.59633.53

  View details for Web of Science ID 000176820500016

  View details for PubMedID 12105155

• Optical imaging of cardiac reporter gene expression in living rats CIRCULATION Wu, J. C., Inubushi, M., Sundaresan, G., Schelbert, H. R., Gambhir, S. S. 2002; 105 (14): 1631-1634

  Abstract

  Studies of cardiac gene transfer rely on postmortem analysis using histologic staining or enzyme assays. Noninvasive imaging of the temporal and spatial characteristics of cardiac gene expression in the same subject offers significant advantages.Rats underwent direct myocardial injection via left thoracotomy with adenovirus-expressing firefly luciferase (Ad-CMV-Fluc; n=30). The reporter substrate D-luciferin was injected intraperitoneally. Serial images were acquired by use of a cooled charged couple detector (CCD) camera. Results are expressed as relative light unit per minute (RLU/min). Rats transduced with 1x10(9) plaque-forming units show decremental cardiac luciferase activity over time: 152 070+/-21 170 (day 2), 195 806+/-62 630 (day 5), 7250+/-2941 (day 8), and 2040+/-971 RLU/min (day 14). To assess the detection sensitivity, serially diluted titers of Ad-CMV-Fluc were injected: 1x10(9) (195 393+/-14 896), 1x10(8) (33 777+/-18 179), 1x10(7) (417+/-91), 1x10(6) (185+/-64), 1x10(5) (53+/-1), and control (54+/-1) (P<0.05 for 1x10(9), 1x10(8), and 1x10(7) plaque-forming units versus control adenovirus-expressing mutant thymidine kinase [Ad-CMV-HSV1-sr39tk]; n=3). Finally, rats were euthanized, and in vitro luciferase activity correlated with in vivo CCD signals (r2=0.92).This study demonstrates for the first time the feasibility of imaging the location, magnitude, and time course of cardiac reporter gene expression in living rats. Cardiac gene therapy studies could be aided with wider application of this approach.

  View details for DOI 10.1161/01.CIR.0000014984.95520.AD

  View details for Web of Science ID 000174987300185

  View details for PubMedID 11940538

• Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice MOLECULAR THERAPY Wu, J. C., Sundaresan, G., Iyer, M., Gambhir, S. S. 2001; 4 (4): 297-306

  Abstract

  The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/-8% standard deviation from mean values, and a detection sensitivity (range tested: 1 x 10(4) - 1 x 10(9) plaque form-ing units (pfu)) of 1 x 10(6) pfu of E1-deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-FL). The duration and magnitude of adenoviral mediated (1 x 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for >150 days. In contrast, FL activity in nude mice remains elevated for >110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2)=0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.

  View details for Web of Science ID 000171403200006

  View details for PubMedID 11592831

• Severe regional ischemia alters coronary flow reserve in the remote perfusion area JOURNAL OF NUCLEAR CARDIOLOGY Wu, J. C., Yun, J. J., Dione, D. P., Heller, E. N., Deckelbaum, L. I., Sinusas, A. J. 2000; 7 (1): 43-52

  Abstract

  Clinical and experimental studies suggest that coronary flow reserve (CFR) may be abnormal in regions remote from myocardial infarction. We sought to determine the possible relation among stenosis severity, ischemic dysfunction, and impairment of CFR in remote regions.In 7 open-chest dogs, acute graded left circumflex (LCX) ischemia was created and maintained based on measurement of the transstenotic (aortic-distal LCX) pressure gradient (measured in millimeters of mercury). Regional thickening was assessed with sonomicrometers. Regional myocardial flow was assessed at rest with radiolabeled microspheres. Doppler flow probes were placed on proximal LCX and left anterior descending (LAD) arteries to measure resting flow and CFR in response to intracoronary injection of adenosine (36 microg). These parameters were assessed under baseline conditions and during transstenotic gradients of 10, 20, 30, and 40 mm Hg. Increasing LCX stenosis severity caused progressive impairment of LCX CFR: baseline (2.22+/-0.10), stenosis 10 (1.80+/-0.06), stenosis 20 (1.56+/-0.08), stenosis 30 (1.30+/-0.04), and stenosis 40 (1.17+/-0.06) (P<.01 vs. baseline). Remote LAD CFR was not altered by mild to moderate LCX stenosis (baseline [2.33+/-0.19]; stenosis 10 [2.30+/-0.25]; stenosis 20 [2.15+/-0.26]). However, critical LCX stenosis producing mild to moderate reduction in thickening in the ischemic region was associated with a significant impairment of LAD CFR: stenosis 30 (1.90+/-0.26) and stenosis 40 (1.80+/-0.22) (P<.01 vs. baseline). These changes in remote CFR persisted after correction for changes in the rate-pressure product.In an acute canine model of progressive LCX coronary stenosis, CFR was impaired in both ischemic and remote nonischemic regions in association with mild to moderate ischemic-induced regional myocardial dysfunction. Thus pharmacologic vasodilation provoked only mild heterogeneity in CFR in the presence of a critical LCX stenosis as a result of concurrent reduction of LAD CFR. This phenomenon warrants further clinical and experimental investigation because it may affect detection of flow heterogeneity during acute ischemia (which induced myocardial dysfunction).

  View details for Web of Science ID 000085428600007

  View details for PubMedID 10698234

• Limitations of dobutamine for enhancing flow heterogeneity in the presence of single coronary stenosis: Implications for technetium-99m-sestamibi imaging 44th Annual Scientific Session of the American-College-of-Cardiology Wu, J. C., Yun, J. J., Heller, E. N., Dione, D. P., DeMan, P., Liu, Y. H., Zaret, B. L., Wackers, F. J., Sinusas, A. J. SOC NUCLEAR MEDICINE INC. 1998: 417–25

  Abstract

  Dobutamine is used as an alternative to exercise in conjunction with 99mTc-sestamibi SPECT perfusion imaging for detection of coronary artery disease. However, the use of quantitative dobutamine 99mTc-sestamibi SPECT imaging for enhanced detection of coronary stenosis has not been established. The goal of this study is to examine the effects of dobutamine stress on regional myocardial blood flow and relative myocardial 99mTc-sestamibi activity in the presence of a single-vessel stenosis.In six open-chest dogs with left circumflex artery stenosis, radiolabeled microspheres were injected during baseline, severe stenosis and peak dobutamine stress (10 microg/kg/min). Technetium-99m-sestamibi was injected intravenously at peak dobutamine. Hearts were excised 20 min after 99mTc-sestamibi injection for SPECT imaging and post-mortem gamma-well counting.Dobutamine significantly increased heart rate, rate-pressure product and the first derivative of left ventricular pressure. Ischemic zone (left circumflex) myocardial blood flows (in ml/min/g) were: baseline, 0.92 +/- 0.15; stenosis, 0.65 +/- 0.16; and dobutamine, 1.19 +/- 0.38. Nonischemic zone myocardial blood flows were: baseline, 0.99 +/- 0.18; stenosis, 1.01 +/- 0.12; and dobutamine, 1.94 +/- 0.32 (p < 0.01 versus stenosis). Ischemic flows, expressed as percentages of nonischemic flows, were: baseline, 94% +/- 2%; stenosis, 63% +/- 11% (p < 0.05 versus baseline) and dobutamine, 60% +/- 12% (p was not significant versus stenosis). Technetium-99m-sestamibi activity in the ischemic zone (75% +/- 6% nonischemic) underestimated the relative flow deficit produced during dobutamine stress (p = 0.056). Myocardial 99mTc-sestamibi activity correlated with flow when flow was less than 1.0 ml/min/g. At higher flow ranges (1.0 ml/min/g-3.5 ml/min/g), 99mTc-sestamibi did not track flow.In a canine model of flow-limiting, single-vessel stenosis, dobutamine (10 microg/kg/min) did not augment flow heterogeneity. In addition, relative myocardial 99mTc-sestamibi activity underestimated microsphere flow at higher flows induced by dobutamine, leading to underestimation of ischemia. These findings suggest that dobutamine stress 99mTc-sestamibi scintigraphy may underestimate the relative flow deficit.

  View details for Web of Science ID 000072421600012

  View details for PubMedID 9529285