Sean M. Wu, MD, PhD, FACC

All Publications

• 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 Web of Science ID 000377443800001

  View details for PubMedID 27280684

• Endocardium Minimally Contributes to Coronary Endothelium in the Embryonic Ventricular Free Walls. Circulation research Zhang, H., Pu, W., Li, G., Huang, X., He, L., Tian, X., Liu, Q., Zhang, L., Wu, S. M., Sucov, H. M., Zhou, B. 2016

  Abstract

  There is persistent uncertainty regarding the developmental origins of coronary vessels, with two principal sources suggested as ventricular endocardium or sinus venosus (SV). These two proposed origins implicate fundamentally distinct mechanisms of vessel formation. Resolution of this controversy is critical for deciphering the programs that result in the formation of coronary vessels, and has implications for research on therapeutic angiogenesis.To resolve the controversy over the developmental origin of coronary vessels.We first generatedNfatc1-CreandNfatc1-Drelineage tracers for endocardium labeling. We found that Nfatc1 recombinases also label a significant portion of SV endothelial cells in addition to endocardium. Therefore, restricted endocardial lineage tracing requires a specific marker that distinguishes endocardium from SV. By single cell gene expression analysis, we identified a novel endocardial gene natriuretic peptide receptor 3 (Npr3). Npr3 is expressed in the entirety of the endocardium but not in the SV. Genetic lineage tracing based onNpr3-CreERshowed that endocardium contributes to a minority of coronary vessels in the free walls of embryonic heart. Intersectional genetic lineage tracing experiments demonstrated that endocardium minimally contributes to coronary endothelium in the embryonic ventricular free walls.Our study suggested that SV, but not endocardium, is the major origin for coronary endothelium in the embryonic ventricular free walls. This work thus resolves the recent controversy over the developmental origin of coronary endothelium, providing the basis for studying coronary vessel formation and regeneration after injury.

  View details for DOI 10.1161/CIRCRESAHA.116.308749

  View details for PubMedID 27056912

• Members Only: Hypoxia-Induced Cell-Cycle Activation in Cardiomyocytes. Cell metabolism Sharma, A., Wu, S. M. 2015; 22 (3): 365-366

  Abstract

  A low level of cardiomyocyte turnover occurs in the adult mammalian heart, but the source of these new cells remains unknown. Kimura et al., 2015 utilized a novel hypoxia-induced fate mapping system to identify a rare population of adult cardiomyocytes undergoing cell-cycle entry and expansion in healthy adult hearts and following ischemic injury.

  View details for DOI 10.1016/j.cmet.2015.08.004

  View details for PubMedID 26331604

• Fetal Mammalian Heart Generates a Robust Compensatory Response to Cell Loss. Circulation Sturzu, A. C., Rajarajan, K., Passer, D., Plonowska, K., Riley, A., Tan, T. C., Sharma, A., Xu, A. F., Engels, M. C., Feistritzer, R., Li, G., Selig, M. K., Geissler, R., Robertson, K. D., Scherrer-Crosbie, M., Domian, I. J., Wu, S. M. 2015; 132 (2): 109-121

  Abstract

  -Heart development is tightly regulated by signaling events acting upon a defined number of progenitor and differentiated cardiac cells. While loss-of-function of these signaling pathways leads to congenital malformation, the consequences of cardiac progenitor cell (CPC) or embryonic cardiomyocyte loss are less clear. In this study, we tested the hypothesis that embryonic mouse hearts exhibit a robust mechanism for regeneration following extensive cell loss.-By combining a conditional cell ablation approach with a novel blastocyst complementation strategy, we generated murine embryos that exhibit a full spectrum of CPC or cardiomyocyte ablation. Remarkably, ablation of up to 60% of CPCs at embryonic day 7.5 was well-tolerated and permitted embryo survival. Ablation of embryonic cardiomyocytes to a similar degree (50-60%) at embryonic day 9.0 could be fully rescued by residual myocytes with no obvious adult cardiac functional deficit. In both ablation models, an increase in cardiomyocyte proliferation rate was detected and accounted for at least some of the rapid recovery of myocardial cellularity and heart size.-Our study defines the threshold for cell loss in the embryonic mammalian heart and reveals a robust cardiomyocyte compensatory response that sustains normal fetal development.

  View details for DOI 10.1161/CIRCULATIONAHA.114.011490

  View details for PubMedID 25995316

• Identification of cardiovascular lineage descendants at single-cell resolution DEVELOPMENT Li, G., Plonowska, K., Kuppusamy, R., Sturzu, A., Wu, S. M. 2015; 142 (5): 846-857

  Abstract

  The transcriptional profiles of cardiac cells derived from murine embryos and from mouse embryonic stem cells (mESCs) have primarily been studied within a cell population. However, the characterization of gene expression in these cells at a single-cell level might demonstrate unique variations that cannot be appreciated within a cell pool. In this study, we aimed to establish a single-cell quantitative PCR platform and perform side-by-side comparison between cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) derived from mESCs and mouse embryos. We first generated a reference map for cardiovascular single cells through quantifying lineage-defining genes for CPCs, CMs, smooth muscle cells (SMCs), endothelial cells (EDCs), fibroblasts and mESCs. This panel was then applied against single embryonic day 10.5 heart cells to demonstrate its ability to identify each endocardial cell and chamber-specific CM. In addition, we compared the gene expression profile of embryo- and mESC-derived CPCs and CMs at different developmental stages and showed that mESC-derived CMs are phenotypically similar to embryo-derived CMs up to the neonatal stage. Furthermore, we showed that single-cell expression assays coupled with time-lapse microscopy can resolve the identity and the lineage relationships between progenies of single cultured CPCs. With this approach, we found that mESC-derived Nkx2-5(+) CPCs preferentially become SMCs or CMs, whereas single embryo-derived Nkx2-5(+) CPCs represent two phenotypically distinct subpopulations that can become either EDCs or CMs. These results demonstrate that multiplex gene expression analysis in single cells is a powerful tool for examining the unique behaviors of individual embryo- or mESC-derived cardiac cells.

  View details for DOI 10.1242/dev.116897

  View details for Web of Science ID 000351697400005

  View details for PubMedID 25633351

• 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 DOI 10.1126/science.350.6261.640-a

  View details for PubMedID 26542560

• Patching up broken hearts: cardiac cell therapy gets a bioengineered boost. Cell stem cell Serpooshan, V., Wu, S. M. 2014; 15 (6): 671-673

  Abstract

  Preclinical and clinical studies for cardiac cell therapy have only seen moderate success due to poor engraftment and survival of transplanted cells. In this issue of Cell Stem Cell, Ye et al. (2014) employ a growth-factor-loaded fibrin patch and show improved cardiovascular cell survival after cell transplantation into a porcine model of ischemia reperfusion.

  View details for DOI 10.1016/j.stem.2014.11.008

  View details for PubMedID 25479741

• Harnessing the potential of induced pluripotent stem cells for regenerative medicine NATURE CELL BIOLOGY Wu, S. M., Hothedlinger, K. 2011; 13 (5): 497-505

  Abstract

  The discovery of methods to convert somatic cells into induced pluripotent stem cells (iPSCs) through expression of a small combination of transcription factors has raised the possibility of producing custom-tailored cells for the study and treatment of numerous diseases. Indeed, iPSCs have already been derived from patients suffering from a large variety of disorders. Here we review recent progress that has been made in establishing iPSC-based disease models, discuss associated technical and biological challenges, and highlight possible solutions to overcome these barriers. We believe that a better understanding of the molecular basis of pluripotency, cellular reprogramming and lineage-specific differentiation of iPSCs is necessary for progress in regenerative medicine.

  View details for DOI 10.1038/ncb0511-497

  View details for Web of Science ID 000290148700004

  View details for PubMedID 21540845

• Generation of Functional Ventricular Heart Muscle from Mouse Ventricular Progenitor Cells SCIENCE Domian, I. J., Chiravuri, M., van der Meer, P., Feinberg, A. W., Shi, X., Shao, Y., Wu, S. M., Parker, K. K., Chien, K. R. 2009; 326 (5951): 426-429

  Abstract

  The mammalian heart is formed from distinct sets of first and second heart field (FHF and SHF, respectively) progenitors. Although multipotent progenitors have previously been shown to give rise to cardiomyocytes, smooth muscle, and endothelial cells, the mechanism governing the generation of large numbers of differentiated progeny remains poorly understood. We have employed a two-colored fluorescent reporter system to isolate FHF and SHF progenitors from developing mouse embryos and embryonic stem cells. Genome-wide profiling of coding and noncoding transcripts revealed distinct molecular signatures of these progenitor populations. We further identify a committed ventricular progenitor cell in the Islet 1 lineage that is capable of limited in vitro expansion, differentiation, and assembly into functional ventricular muscle tissue, representing a combination of tissue engineering and stem cell biology.

  View details for DOI 10.1126/science.1177350

  View details for Web of Science ID 000270818600053

  View details for PubMedID 19833966

• Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart NATURE Zhou, B., Ma, Q., Rajagopal, S., Wu, S. M., Domian, I., Rivera-Feliciano, J., Jiang, D., von Gise, A., Ikeda, S., Chien, K. R., Pu, W. T. 2008; 454 (7200): 109-U5

  Abstract

  The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 (refs 1 and 2). These multipotent progenitors give rise to cardiomyocyte, smooth muscle and endothelial cells, the major lineages of the mature heart. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium-an epithelial sheet overlying the heart. During normal murine heart development, a subset of these Wt1(+) precursors differentiated into fully functional cardiomyocytes. Wt1(+) proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5(+) and Isl1(+) progenitors. These results identify Wt1(+) epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.

  View details for DOI 10.1038/nature07060

  View details for Web of Science ID 000257308300047

  View details for PubMedID 18568026

• Origins and fates of cardiovascular progenitor cells CELL Wu, S. M., Chien, K. R., Mummery, C. 2008; 132 (4): 537-543

  Abstract

  Multipotent cardiac progenitor cells are found in the fetal and adult heart of many mammalian species including humans and form as intermediates during the differentiation of embryonic stem cells. Despite similar biological properties, the molecular identities of these different cardiac progenitor cell populations appear to be distinct. Elucidating the origins and lineage relationships of these cell populations will accelerate clinical applications such as drug screening and cell therapy as well as shedding light on the pathogenic mechanisms underlying cardiac diseases.

  View details for DOI 10.1016/j.cell.2008.02.002

  View details for Web of Science ID 000253817900012

  View details for PubMedID 18295570

• Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart CELL Wu, S. M., Fujiwara, Y., Cibulsky, S. M., Clapham, D. E., Lien, C., Schultheiss, T. M., Orkin, S. H. 2006; 127 (6): 1137-1150

  Abstract

  Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood. To explore the relationship between developmental fate and potential, we isolated a cardiac-specific Nkx2.5(+) cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5(+) cells from in vitro differentiated murine embryonic stem cells and found approximately 28% of these cells expressed c-kit. These c-kit(+) cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell. We confirmed these findings by isolating c-kit(+)Nkx2.5(+) cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.

  View details for DOI 10.1016/j.cell.2006.10.028

  View details for Web of Science ID 000242991000013

  View details for PubMedID 17123591

• Regenerative Medicine: Potential Mechanisms of Cardiac Recovery in Takotsubo Cardiomyopathy. Current treatment options in cardiovascular medicine Chang, A. Y., Kittle, J. T., Wu, S. M. 2016; 18 (3): 20-?

  Abstract

  Takotsubo cardiomyopathy is an increasingly reported cause of acute chest pain and acute heart failure and is often associated with significant hemodynamic compromise. The illness is remarkable for the reversibility in systolic dysfunction seen in the disease course. While the pathophysiology of takotsubo syndrome is not completely elucidated, research suggests the presence of a cytoprotective process that allows the myocardium to recover following the inciting insult. Here, we summarize molecular and histologic studies exploring the response to injury in takotsubo disease and provide some discussion on how they may contribute to further investigations in cardiac recovery and regeneration.

  View details for DOI 10.1007/s11936-016-0443-0

  View details for PubMedID 26874708

• Cardioprotective Actions of TGF beta RI Inhibition Through Stimulating Autocrine/Paracrine of Survivin and Inhibiting Wnt in Cardiac Progenitors STEM CELLS Ho, Y., Tsai, W., Lin, F., Huang, W., Lin, L., Wu, S. M., Liu, Y., Chen, W. 2016; 34 (2): 445-455

  Abstract

  Heart failure due to myocardial infarction (MI) is a major cause of morbidity and mortality in the world. We found previously that A83-01, a TGFβRI inhibitor, could facilitate cardiac repair in post-MI mice and induce the expansion of a Nkx2.5+ cardiomyoblast population. The present study aimed to investigate the key autocrine/paracrine factors regulated by A83-01 in the injured heart and the mechanism of cardioprotection by this molecule. Using a previously described transgenic Nkx2.5 enhancer-GFP reporter mice, we isolated cardiac progenitor cells (CPC) including Nkx2.5-GFP+ (Nkx2.5+), sca1+ and Nkx2.5+/sca1+ cells. A83-01 was found to induce proliferation of these three subpopulations mainly through increasing Birc5 expression in the MEK/ERK-dependent pathway. Survivin, encoded by Birc5, could also directly proliferate Nkx2.5+ cells and enhance cultured cardiomyocytes viability. A83-01 could also reverse the down-regulation of Birc5 in post-injured mice hearts (n=6) to expand CPCs. Moreover, the increased Wnt3a in post-injured hearts could decrease CPCs, which could be reversed by A83-01 via inhibiting Fzd6 and WISP1 expressions in CPCs. Next, we used inducible αMHC-cre/mTmG mice to label cardiomyocytes with GFP and non-myocytes with RFP. We found A83-01 preserved more GFP+ myocytes (68.6±3.1% vs 80.9±3.0%; P<0.05, n=6) and fewer renewed RFP+ myocytes (0.026±0.005 vs 0.062±0.008%; P<0.05, n=6) in parallel with less cardiac fibrosis in isoprenaline-injected mice treated with A83-01. TGFβRI inhibition in an injured adult heart could both stimulate the autocrine/paracrine activity of survivin and inhibit Wnt in CPCs to mediate cardioprotection and improve cardiac function. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/stem.2216

  View details for Web of Science ID 000370353200017

  View details for PubMedID 26418219

• Harnessing the Induction of Cardiomyocyte Proliferation for Cardiac Regenerative Medicine. Current treatment options in cardiovascular medicine Sharma, A., Zhang, Y., Wu, S. M. 2015; 17 (10): 404-?

  Abstract

  Adult human cardiomyocytes are terminally differentiated and have limited capacity for cell division. Hence, they are not naturally replaced following ischemic injury to the heart. As such, cardiac function is often permanently compromised after an event such as myocardial infarction. In recent years, investigators have focused intensively on ways to reactivate cardiomyocyte mitotic activity in both in vitro cell culture systems and in vivo animal models. In parallel, advances in stem cell biology have allowed for the mass production of patient-specific human cardiomyocytes from human-induced pluripotent stem cells. These cells can be produced via chemically defined differentiation of human pluripotent stem cells in a matter of weeks and could theoretically be utilized directly for therapeutic purposes to replace damaged myocardium. However, stem cell-derived cardiomyocytes, like their adult counterparts, are post-mitotic and incapable of large-scale expansion after reaching a certain stage of in vitro differentiation. Due to this shared characteristic, these stem cell-derived cardiomyocytes may provide a platform for studying genes, pathways, and small molecules that induce cell cycle reentry and proliferation of human cardiomyocytes. Ultimately, the discovery of novel mechanisms or pathways to induce human cardiomyocyte proliferation should improve our ability to regenerate adult cardiomyocytes and help restore cardiac function following injury.

  View details for DOI 10.1007/s11936-015-0404-z

  View details for PubMedID 26324824

• Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes PLOS ONE Tarnawski, L., Xian, X., Monnerat, G., Macaulay, I. C., Malan, D., Borgman, A., Wu, S. M., Fleischmann, B. K., Jovinge, S. 2015; 10 (8)

  View details for DOI 10.1371/journal.pone.0135880

  View details for Web of Science ID 000360435500010

• Small RNAs make big impact in cardiac repair. Circulation research Krane, M., Deutsch, M., Doppler, S., Lange, R., Wu, S. M. 2015; 116 (3): 393-395

  View details for DOI 10.1161/CIRCRESAHA.114.305676

  View details for PubMedID 25634967

• Molecular Regulation of Cardiomyocyte Differentiation CIRCULATION RESEARCH Paige, S. L., Plonowska, K., Xu, A., Wu, S. M. 2015; 116 (2): 341-353

  View details for DOI 10.1161/CIRCRESAHA.116.302752

  View details for Web of Science ID 000347939000019

• Pharmacological inhibition of TGF beta receptor improves Nkx2.5 cardiomyoblast-mediated regeneration CARDIOVASCULAR RESEARCH Chen, W., Liu, Y., Ho, Y., Wu, S. M. 2015; 105 (1): 44-54

  Abstract

  Our previous study found that A83-01, a small molecule type 1 TGFβ receptor inhibitor, could induce proliferation of postnatal Nkx2.5(+) cardiomyoblasts in vitro and enhance their cardiomyogenic differentiation. The present study addresses whether A83-01 treatment in vivo could increase cardiomyogenesis and improve cardiac function after myocardial infarction through an Nkx2.5(+) cardiomyoblast-dependent process.To determine the effect of A83-01 on the number of Nkx2.5(+) cardiomyoblasts in the heart after myocardial injury, we treated transgenic Nkx2.5 enhancer-GFP reporter mice for 7 days with either A83-01 or DMSO and measured the number of GFP(+) cardiomyoblasts in the heart at 1 week after injury by flow cytometry. To determine the degree of new cardiomyocyte formation after myocardial injury and the effect of A83-01 in this process, we employed inducible Nkx2.5 enhancer-Cre transgenic mice to lineage label postnatal Nkx2.5(+) cardiomyoblasts and their differentiated progenies after myocardial injury. We also examined the cardiac function of each animal by intracardiac haemodynamic measurements. We found that A83-01 treatment significantly increased the number of Nkx2.5(+) cardiomyoblasts at baseline and after myocardial injury, resulting in an increase in newly formed cardiomyocytes. Finally, we showed that A83-01 treatment significantly improved ventricular elastance and stroke work, leading to improved contractility after injury.Pharmacological inhibition of TGFβ signalling improved cardiac function in injured mice and promoted the expansion and cardiomyogenic differentiation of Nkx2.5(+) cardiomyoblasts. Direct modulation of resident cardiomyoblasts in vivo may be a promising strategy to enhance therapeutic cardiac regeneration.

  View details for DOI 10.1093/cvr/cvu229

  View details for Web of Science ID 000350217200007

  View details for PubMedID 25362681

• Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation. Journal of visualized experiments : JoVE Sharma, A., Li, G., Rajarajan, K., Hamaguchi, R., Burridge, P. W., Wu, S. M. 2015

  Abstract

  Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

  View details for DOI 10.3791/52628

  View details for PubMedID 25867738

• Comparing mouse and human pluripotentstem cell derived cardiac cells: both systemshave advantages for pharmacological and toxicological screening. Journal of pharmacological and toxicological methods Lagerqvist, E. L., Finnin, B. A., Elliott, D., Anderson, D., Wu, S. M., Pouton, C. W., Haynes, J. M. 2015

  Abstract

  Pluripotent stem cells offer an unparalleled opportunity to investigate cardiac physiology, pharmacology, toxicology and pathophysiology. In this paper we describe the use of both mouse (Nkx2-5(eGFP/w)) and human (NKX2-5(eGFP/w)) pluripotent stem cell reporter lines, differentiated toward cardiac lineage, for live single cell high acquisition rate calcium imaging. We also assess the potential of NKX2-5(eGFP/w) cardiac lineage cells for use toxicological screening as well as establish their sensitivity to a shift between low and high oxygen environments. Differentiated mouse Nkx2-5(eGFP/w) cells demonstrated a wide range of spontaneous oscillation rates that could be reduced by ryanodine (10 μM), thapsigargin (1 μM) and ZD7288 (10μM). In contrast human NKX2-5(eGFP/w) cell activity was only reduced by thapsigargin (1 μM). Human cells were also sensitive to the addition of trastuzumab and doxorubicin as well as a low oxygen environment. We suggest that the human NKX2-5(eGFP/w) cells are less suitable for studies of compounds affecting cardiac pacemaker activity than mouse Nkx2-5(eGFP/w) cells, but are very suitable for cardiac toxicity studies.

  View details for DOI 10.1016/j.vascn.2015.04.009

  View details for PubMedID 25957031

• Myeloid Zinc Finger 1 (Mzf1) Differentially Modulates Murine Cardiogenesis by Interacting with an Nkx2.5 Cardiac Enhancer PLOS ONE Doppler, S. A., Werner, A., Barz, M., Lahm, H., Deutsch, M., Dressen, M., Schiemann, M., Voss, B., Gregoire, S., Kuppusamy, R., Wu, S. M., Lange, R., Krane, M. 2014; 9 (12)

  View details for DOI 10.1371/journal.pone.0113775

  View details for Web of Science ID 000347114900078

• 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

• Somatic Cell Reprogramming into Cardiovascular Lineages JOURNAL OF CARDIOVASCULAR PHARMACOLOGY AND THERAPEUTICS Chen, J. X., Plonowska, K., Wu, S. M. 2014; 19 (4): 340-349

  View details for DOI 10.1177/1074248414527641

  View details for Web of Science ID 000338394200003

• Insulin-like growth factor promotes cardiac lineage induction in vitro by selective expansion of early mesoderm. Stem cells Engels, M. C., Rajarajan, K., Feistritzer, R., Sharma, A., Nielsen, U. B., Schalij, M. J., de Vries, A. A., Pijnappels, D. A., Wu, S. M. 2014; 32 (6): 1493-1502

  Abstract

  A thorough understanding of the developmental signals that direct pluripotent stem cells (PSCs) towards a cardiac fate is essential for translational applications in disease modeling and therapy. We screened a panel of 44 cytokines/signaling molecules for their ability to enhance Nkx2.5(+) cardiac progenitor cell (CPC) formation during in vitro embryonic stem cell (ESC) differentiation. Treatment of murine ESCs with insulin or insulin-like growth factors (IGF1/2) during early differentiation increased mesodermal cell proliferation and, consequently, CPC formation. Furthermore, we show that downstream mediators of IGF signaling (e.g. phospho-Akt and mTOR) are required for this effect. These data support a novel role for IGF family ligands to expand the developing mesoderm and promote cardiac differentiation. Insulin or IGF treatment could provide an effective strategy to increase the PSC-based generation of CPCs and cardiomyocytes for applications in regenerative medicine. Stem Cells 2014.

  View details for DOI 10.1002/stem.1660

  View details for PubMedID 24496962

• Telocytes in human heart valves JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Yang, Y., Sun, W., Wu, S. M., Xiao, J., Kong, X. 2014; 18 (5): 759-765

  Abstract

  Valve interstitial cells (VICs) are responsible for maintaining the structural integrity and dynamic behaviour of the valve. Telocytes (TCs), a peculiar type of interstitial cells, have been recently identified by Popescu's group in epicardium, myocardium and endocardium (visit www.telocytes.com). The presence of TCs has been identified in atria, ventricles and many other tissues and organ, but not yet in heart valves. We used transmission electron microscopy and immunofluorescence methods (double labelling for CD34 and c-kit, or vimentin, or PDGF Receptor-β) to provide evidence for the existence of TCs in human heart valves, including mitral valve, tricuspid valve and aortic valve. TCs are found in both apex and base of heart valves, with a similar density of 27-28 cells/mm(2) in mitral valve, tricuspid valve and aortic valve. Since TCs are known for the participation in regeneration or repair biological processes, it remains to be determined how TCs contributes to the valve attempts to re-establish normal structure and function following injury, especially a complex junction was found between TCs and a putative stem (progenitor) cell.

  View details for DOI 10.1111/jcmm.12285

  View details for Web of Science ID 000335861500002

  View details for PubMedID 24674389

• 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

• 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

• Meta-Analysis of Stem Cell Therapy in Chronic Ischemic Cardiomyopathy AMERICAN JOURNAL OF CARDIOLOGY Kandala, J., Upadhyay, G. A., Pokushalov, E., Wu, S., Drachman, D. E., Singh, J. P. 2013; 112 (2): 217-225

  Abstract

  Studies investigating bone marrow stem cell therapy (BMSCT) in patients with chronic ischemic cardiomyopathy have yielded mixed results. A meta-analysis of randomized controlled trials of BMSCT in patients with chronic ischemic cardiomyopathy was undertaken to assess its efficacy and the best route of administration. The MEDLINE, Embase, Cumulative Index to Nursing & Allied Health Literature, and Cochrane Library databases were searched through April 2012 for randomized controlled trials that investigated the impact of BMSCT and its route of administration on left ventricular (LV) function in patients with chronic ischemic cardiomyopathy and systolic dysfunction. Of the 226 reports identified, 10 randomized controlled trials including 519 patients (average LV ejection fraction [LVEF] at baseline 32 ± 7%) were included in the analysis. On the basis of a random-effects model, BMSCT improved the LVEF at 6 months by 4.48% (95% confidence interval [CI] 2.43% to 6.53%, p = 0.0001). A greater improvement in the LVEF was seen with intramyocardial injection compared with intracoronary infusion (5.13% [95% CI 3.17% to 7.10%], p <0.00001, vs 2.32% [95% CI -2.06% to 6.70%], p = 0.30). Overall, there were reductions in LV end-systolic volume of -20.64 ml (95% CI -33.21 to -8.07, p = 0.001) and LV end-diastolic volume of -16.71 ml (95% CI -31.36 to -2.06, p = 0.03). In conclusion, stem cell therapy may improve LVEF and favorably remodel the heart in patients with chronic ischemic cardiomyopathy. On the basis of current limited data, intramyocardial injection may be superior to intracoronary infusion in patients with LV systolic dysfunction.

  View details for DOI 10.1016/j.amjcard.2013.03.021

  View details for Web of Science ID 000322206500013

  View details for PubMedID 23623290

• A83-01, a TGF beta RI inhibitor, can proliferate adult cardiac progenitor cells and improve cardiac contractility of myocardial infarcted mice ACTA PHARMACOLOGICA SINICA Chen, W., Liu, Y., Ho, Y., Wu, S. M. 2013; 34: 57-57

  View details for Web of Science ID 000322051500249

• Autophagy - the friendly fire in endothelial cell regeneration. Focus on "Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions" AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Sharma, A., Wu, S. M. 2013; 304 (7): C614-C616

  View details for DOI 10.1152/ajpcell.00046.2013

  View details for Web of Science ID 000316995700003

  View details for PubMedID 23407881

• At a Crossroad Cell Therapy for Cardiac Repair CIRCULATION RESEARCH Deutsch, M., Sturzu, A., Wu, S. M. 2013; 112 (6): 884-890

  View details for DOI 10.1161/CIRCRESAHA.112.275974

  View details for Web of Science ID 000316189900005

  View details for PubMedID 23493304

• Essential and Unexpected Role of Yin Yang 1 to Promote Mesodermal Cardiac Differentiation CIRCULATION RESEARCH Gregoire, S., Karra, R., Passer, D., Deutsch, M., Krane, M., Feistritzer, R., Sturzu, A., Domian, I., Saga, Y., Wu, S. M. 2013; 112 (6): 900-U104

  Abstract

  Cardiogenesis is regulated by a complex interplay between transcription factors. However, little is known about how these interactions regulate the transition from mesodermal precursors to cardiac progenitor cells (CPCs). Objective: To identify novel regulators of mesodermal cardiac lineage commitment.We performed a bioinformatic-based transcription factor binding site analysis on upstream promoter regions of genes that are enriched in embryonic stem cell-derived CPCs. From 32 candidate transcription factors screened, we found that Yin Yang 1 (YY1), a repressor of sarcomeric gene expression, is present in CPCs in vivo. Interestingly, we uncovered the ability of YY1 to transcriptionally activate Nkx2.5, a key marker of early cardiogenic commitment. YY1 regulates Nkx2.5 expression via a 2.1-kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays, in vivo chromatin immunoprecipitation, and genome-wide sequencing analysis. Furthermore, the ability of YY1 to activate Nkx2.5 expression depends on its cooperative interaction with Gata4 at a nearby chromatin. Cardiac mesoderm-specific loss-of-function of YY1 resulted in early embryonic lethality. This was corroborated in vitro by embryonic stem cell-based assays in which we showed that the overexpression of YY1 enhanced the cardiogenic differentiation of embryonic stem cells into CPCs.These results demonstrate an essential and unexpected role for YY1 to promote cardiogenesis as a transcriptional activator of Nkx2.5 and other CPC-enriched genes.

  View details for DOI 10.1161/CIRCRESAHA.113.259259

  View details for Web of Science ID 000316189900007

  View details for PubMedID 23307821

• Of fish and men: clonal lineage analysis identifies divergence in myocardial development. Circulation research Sharma, A., Wu, S. M. 2013; 112 (4): 583-585

  View details for DOI 10.1161/CIRCRESAHA.113.300964

  View details for PubMedID 23410876

• iPS Cell Modeling of Cardiometabolic Diseases JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH Nakamura, K., Hirano, K., Wu, S. M. 2013; 6 (1): 46-53

  Abstract

  Cardiometabolic diseases encompass simple monogenic enzyme deficiencies with well-established pathogenesis and clinical outcomes to complex polygenic diseases such as the cardiometabolic syndrome. The limited availability of relevant human cell types such as cardiomyocytes has hampered our ability to adequately model and study pathways or drugs relevant to these diseases in the heart. The recent discovery of induced pluripotent stem (iPS) cell technology now offers a powerful opportunity to establish translational platforms for cardiac disease modeling, drug discovery, and pre-clinical testing. In this article, we discuss the excitement and challenges of modeling cardiometabolic diseases using iPS cell and their potential to revolutionize translational research.

  View details for DOI 10.1007/s12265-012-9413-4

  View details for Web of Science ID 000313657700006

  View details for PubMedID 23070616

• Early cardiac development: a view from stem cells to embryos CARDIOVASCULAR RESEARCH van Vliet, P., Wu, S. M., Zaffran, S., Puceat, M. 2012; 96 (3): 352-362

  Abstract

  From the 1920s, early cardiac development has been studied in chick and, later, in mouse embryos in order to understand the first cell fate decisions that drive specification and determination of the endocardium, myocardium, and epicardium. More recently, mouse and human embryonic stem cells (ESCs) have demonstrated faithful recapitulation of early cardiogenesis and have contributed significantly to this research over the past few decades. Derived almost 15 years ago, human ESCs have provided a unique developmental model for understanding the genetic and epigenetic regulation of early human cardiogenesis. Here, we review the biological concepts underlying cell fate decisions during early cardiogenesis in model organisms and ESCs. We draw upon both pioneering and recent studies and highlight the continued role for in vitro stem cells in cardiac developmental biology.

  View details for DOI 10.1093/cvr/cvs270

  View details for Web of Science ID 000311306800006

  View details for PubMedID 22893679

• Reprogramming the Beat Kicking It Up a Notch CIRCULATION Wu, S. M., Milan, D. J. 2012; 126 (9): 1009-1011

  View details for DOI 10.1161/CIRCULATIONAHA.112.126482

  View details for Web of Science ID 000308355100008

  View details for PubMedID 22837161

• 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 Web of Science ID 000306061700012

  View details for PubMedID 22581928

• Small molecule regulators of postnatal Nkx2.5 cardiomyoblast proliferation and differentiation JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Chen, W., Wu, S. M. 2012; 16 (5): 961-965

  Abstract

  While recent data have supported the capacity for a neonatal heart to undergo cardiomyogenesis, it is unclear whether these new cardiomyocytes arise from an immature cardiomyoblast population or from the division of mature cardiomyocytes. By following the expression of enhanced Green Fluorescent Protein (eGFP) in an Nkx2.5 enhancer-eGFP transgenic mice, we have identified a population of immature cells that can undergo cardiomyogenic as well as smooth muscle cell differentiation in the neonatal heart. Here, we examined growth factors and small molecule regulators that potentially regulate the proliferation and cardiomyogenic versus smooth muscle cell differentiation of neonatal Nkx2.5-GFP (+) cells in vitro. We found that A83-01 (A83), an inhibitor of TGF-βRI, was able to induce an expansion of neonatal Nkx2.5-eGFP (+) cells. In addition, the ability of A83 to expand eGFP (+) cells in culture was dependent on signalling from the mitogen-activated protein kinase kinase (MEK) as treatment with a MEK inhibitor, PD0325901, abolished this effect. On the other hand, activation of neonatal Nkx2.5-eGFP (+) cells with TGF-β1, but not activin A nor BMP2, led to smooth muscle cell differentiation, an effect that can be reversed by treatment with A83. In summary, small molecule inhibition of TGF-β signalling may be a promising strategy to induce the expansion of a rare population of postnatal cardiomyoblasts.

  View details for DOI 10.1111/j.1582-4934.2011.01513.x

  View details for Web of Science ID 000303239500002

  View details for PubMedID 22212626

• Putting the Pieces Together: Stem Cells and The Quest to Heal A Broken Heart CardioSource World News Wu, S. M., Singh, J. P. 2012; 12: 22-27
• Induced pluripotent stem cell modeling of complex genetic diseases. Drug discovery today. Disease models Hinson, J. T., Nakamura, K., Wu, S. M. 2012; 9 (4): e147-e152

  Abstract

  The study of complex disease genetics by genome-wide association studies (GWAS) has led to hundreds of genomic loci associated with disease traits in humans. However, the functional consequences of most loci are largely undefined. We discuss here the potential for human induced pluripotent stem (iPS) cells to bridge the gap between genetic variant and mechanisms of complex disease. We also highlight specific diseases and the roadblocks that must be overcome before iPS cell technology can be widely adopted for complex disease modeling.

  View details for DOI 10.1016/j.ddmod.2012.04.002

  View details for PubMedID 23690830

• A Brief Primer on the Development of the Heart Heart Failure, 2nd Ed. Gregoire S, Wu SM 2012: Chapter 1
• Epigenetic mechanisms in cardiac development and disease ACTA BIOCHIMICA ET BIOPHYSICA SINICA Vallaster, M., Vallaster, C. D., Wu, S. M. 2012; 44 (1): 92-102

  Abstract

  During mammalian development, cardiac specification and ultimately lineage commitment to a specific cardiac cell type is accomplished by the action of specific transcription factors (TFs) and their meticulous control on an epigenetic level. In this review, we detail how cardiac-specific TFs function in concert with nucleosome remodeling and histone-modifying enzymes to regulate a diverse network of genes required for processes such as cell growth and proliferation, or epithelial to mesenchymal transition (EMT), for instance. We provide examples of how several cardiac TFs, such as Nkx2.5, WHSC1, Tbx5, and Tbx1, which are associated with developmental and congenital heart defects, are required for the recruitment of histone modifiers, such as Jarid2, p300, and Ash2l, and components of ATP-dependent remodeling enzymes like Brg1, Baf60c, and Baf180. Binding of these TFs to their respective sites at cardiac genes coincides with a distinct pattern of histone marks, indicating that the precise regulation of cardiac gene networks is orchestrated by interactions between TFs and epigenetic modifiers. Furthermore, we speculate that an epigenetic signature, comprised of TF occupancy, histone modifications, and overall chromatin organization, is an underlying mechanism that governs cardiac morphogenesis and disease.

  View details for DOI 10.1093/abbs/gmr090

  View details for Web of Science ID 000298386700010

  View details for PubMedID 22194017

• Reprogramming of mouse, rat, pig, and human fibroblasts into iPS cells. Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] Rajarajan, K., Engels, M. C., Wu, S. M. 2012; Chapter 23: Unit 23 15-?

  Abstract

  The induction of pluripotency in somatic cells by transcription-factor overexpression has been widely regarded as one of the major breakthroughs in stem cell biology within this decade. The generation of these induced pluripotent stem cells (iPSCs) has enabled investigators to develop in vitro disease models for biological discovery and drug screening, and in the future, patient-specific therapy for tissue or organ regeneration. While new technologies for reprogramming are continually being discovered, the availability of iPSCs from different species is also increasing rapidly. Comparison of iPSCs across species may provide new insights into key aspects of pluripotency and early embryonic development. iPSCs from large animals may enable the generation of genetically modified large animal models or potentially transplantable donor tissues or organs. This unit describes the procedure for the generation of iPSCs from mouse, rat, pig and human fibroblasts.

  View details for DOI 10.1002/0471142727.mb2315s97

  View details for PubMedID 22237859

• Developmental and Regenerative Biology of Multipotent Cardiovascular Progenitor Cells CIRCULATION RESEARCH Sturzu, A. C., Wu, S. M. 2011; 108 (3): 353-364

  Abstract

  Our limited ability to improve the survival of patients with heart failure is attributable, in part, to the inability of the mammalian heart to meaningfully regenerate itself. The recent identification of distinct families of multipotent cardiovascular progenitor cells from endogenous, as well as exogenous, sources, such as embryonic and induced pluripotent stem cells, has raised much hope that therapeutic manipulation of these cells may lead to regression of many forms of cardiovascular disease. Although the exact source and cell type remains to be clarified, our greater understanding of the scientific underpinning behind developmental cardiovascular progenitor cell biology has helped to clarify the origin and properties of diverse cells with putative cardiogenic potential. In this review, we highlight recent advances in the understanding of cardiovascular progenitor cell biology from embryogenesis to adulthood and their implications for therapeutic cardiac regeneration. We believe that a detailed understanding of cardiogenesis will inform future applications of cardiovascular progenitor cells in heart failure therapy and regenerative medicine.

  View details for DOI 10.1161/CIRCRESAHA.110.227066

  View details for Web of Science ID 000286930500012

  View details for PubMedID 21293007

• Regenerative strategies for cardiac disease In: Stem Cells and Regenerative Medicine. Humana Press. Huang X, Oh JB, Wu SM 2011; 1: 579-593
• Origin of Cardiac Progenitor Cells in the Developing and Postnatal Heart JOURNAL OF CELLULAR PHYSIOLOGY Kuhn, E. N., Wu, S. M. 2010; 225 (2): 321-325

  Abstract

  The mammalian heart lacks the capacity to replace the large numbers of cardiomyocytes lost due to cardiac injury. Several different cell-based routes to myocardial regeneration have been explored, including transplantation of cardiac progenitors and cardiomyocytes into injured myocardium. As seen with cell-based therapies in other solid organ systems, inherent limitations, such as host immune response, cell death and long-term graft instability have hampered meaningful cardiac regeneration. An understanding of the cell biology of cardiac progenitors, including their developmental origin, lineage markers, renewal pathways, differentiation triggers, microenvironmental niche, and mechanisms of homing and migration to the site of injury, will enable further refinement of therapeutic strategies to enhance clinically meaningful cardiac repair.

  View details for DOI 10.1002/jcp.22281

  View details for Web of Science ID 000283003400007

  View details for PubMedID 20568226

• Isolation and functional characterization of pluripotent stem cell-derived cardiac progenitor cells. Current protocols in stem cell biology Huang, X., Wu, S. M. 2010; Chapter 1: Unit 1F 10-?

  Abstract

  The use of transgenic markers in pluripotent stem cells allows the facile isolation of transient cell populations that appear at certain phases of embryonic development. Here, we describe a procedure for deriving cardiac progenitors from mouse pluripotent stem cells carrying a GFP reporter under the control of an Nkx2.5 enhancer sequence. The cells are propagated under standard conditions and are differentiated using the hanging-droplet method with medium optimized for commitment to the cardiac lineage. Cardiac progenitors are isolated from the differentiation culture using fluorescence-activated cell sorting (FACS) and can be cultured further for functional characterization and experimentation. The protocols described here can be applied to both embryonic and induced pluripotent stem cells and can easily be adapted to transgenic lines carrying other cardiac cell lineage reporters.

  View details for DOI 10.1002/9780470151808.sc01f10s14

  View details for PubMedID 20814937

• Promises and pitfalls in cell replacement therapy for heart failure. Drug discovery today. Disease mechanisms Krane, M., Wernet, O., Wu, S. M. 2010; 7 (2): e109-e115

  Abstract

  Symptomatic heart failure is a complex clinical syndrome with a poor prognosis. Many efforts have been made to develop new therapeutic strategies to improve prognosis associated with heart failure. In this context, different stem cell populations for cardiac regenerative therapy have been examined recently. Here we discuss the potential strategies for using stem cells in cardiac regenerative therapy and the barriers that remain before an effective cell-based cardiac regenerative therapy can be employed clinically.

  View details for PubMedID 21180399

• The integrative aspects of cardiac physiology and their implications for cell-based therapy ANALYSIS OF CARDIAC DEVELOPMENT: FROM EMBRYO TO OLD AGE Pijnappels, D. A., Gregoire, S., Wu, S. M. 2010; 1188: 7-14

  Abstract

  Cardiac development is characterized by a complex interplay of chemical, mechanical, and electrical forces, which together contribute to the proper formation of the heart muscle. In adult myocardium, cardiomyocytes are elongated, well-coupled by gap junctions, and organized in spatially well-defined muscle fibers. This specific tissue architecture affects electromechanical activation and global cardiac function. Since the adult heart has only limited capacity for repair after injury, a significant loss of myocardial tissue often leads to impaired cardiac function. Recent efforts to transplant autologous cells to counteract this cardiomyocyte loss have resulted in marginal functional improvement and no evidence of myocyte regeneration. In order to achieve durable therapeutic efficiency, the transplanted cells will need to not only be cardiomyogenic, but also functionally integrate with host myocardial tissue and thereby contribute to both structural and functional restoration.

  View details for DOI 10.1111/j.1749-6632.2009.05077.x

  View details for Web of Science ID 000277731600002

  View details for PubMedID 20201880

• Cardiac progenitor cells: from embryonic to the aging heart. Aging Health Liu, Y-H., Kuhn, E.B., Wu, S.M. 2010; 6 (6): 679-686
• Myocardial Injury Induces the Expansion and Cardiomyogenic Differentiation of Postnatal Nkx2.5 Progenitor Cells via Inflammatory Signals CIRCULATION Liu, Y., Rawnsley, D., Zeng, M., Yu, E., Pijnappels, D., Thibault, H., Scherrer-Crosbie, M., Wu, S. M. 2009; 120 (18): S756-S756

  View details for Web of Science ID 000271831502235

• VISIONS: the art of science. Molecular reproduction and development Wu, S. M. 2009; 76 (6): 525-?

  View details for DOI 10.1002/mrd.21022

  View details for PubMedID 19358263

• Committed Ventricular Progenitors in the Islet-1 Lineage Expand and Assemble Into Functional Ventricular Heart Muscle JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY van der Meer, P., Domian, I. J., Chiravuri, M., Feinberg, A. F., Wu, S. M., Parker, K. K., Chien, K. R. 2009; 53 (10): A468-A468

  View details for Web of Science ID 000263864201940

• Platypnea-orthodeoxia syndrome in two previously healthy adults: a case-based review Clinical Medicine Insights: Cardiology Ptaszek LM, Saldana F, Palacios IF, Wu SM 2009; 3: 37-43
• Derivation and Functional Characterization of Nkx2.5+Cardiac Progenitor Cells from Mouse Induced Pluripotent Stem Cells CIRCULATION Pijnappels, D. A., Stadtfeld, M., Zeng, M., Yu, E., Fujiwara, Y., Wang, G., Orkin, S. H., Jackson-Grusby, L., Hochedlinger, K., Wu, S. M. 2008; 118 (18): S428-S428

  View details for Web of Science ID 000262104500714

• Mesp1 at the heart of mesoderm lineage specification CELL STEM CELL Wu, S. M. 2008; 3 (1): 1-2

  Abstract

  Stem cell-based cardiac regeneration requires a detailed understanding of the factors that induce cardiac lineage commitment. In this issue of Cell Stem Cell, Lindsley et al. (2008) and Bondue et al. (2008) use embryonic stem cells to identify a key role for Mesp1 in this process.

  View details for DOI 10.1016/j.stem.2008.06.017

  View details for Web of Science ID 000257622300001

  View details for PubMedID 18593549

• Multipotent stem cells in cardiac regenerative therapy REGENERATIVE MEDICINE Karra, R., Wu, S. M. 2008; 3 (2): 189-198

  Abstract

  The potential for stem cells to ameliorate or cure heart diseases has galvanized a cadre of cardiovascular translational and clinical scientists to take a 'first-in-man' approach using autologous stem cells from a variety of tissues. However, recent clinical trial data show that when these cells are given by intracoronary infusion or direct myocardial injection, limited improvement in heart function occurs with no evidence of cardiomyogenesis. These studies illustrate the great need to understand the logic of cell-lineage commitment and the principles of cardiac differentiation. Recent identification of stem/progenitor cells of embryological origin with intrinsic competence to differentiate into multiple lineages within the heart offers new possibilities for cardiac regeneration. When combined with developments in nuclear reprogramming and provided that tumor risks and other challenges of embryonic cell transplantation can be overcome, the prospect of achieving autologous, cardiomyogenic, stem cell-based therapy may be within reach.

  View details for DOI 10.2217/17460751.3.2.189

  View details for Web of Science ID 000257995000013

  View details for PubMedID 18307403

• Cardiovascular Stem Cells in Regenerative Medicine: Ready for Prime Time? Drug discovery today. Therapeutic strategies Liu, Y. H., Karra, R., Wu, S. M. 2008; 5 (4): 201-207

  Abstract

  Restoration of cardiovascular function is the ultimate goal of stem cell-based therapy. In principle, cardiovascular stem cells can improve cardiac function via de novo cardiomyogenesis, enhanced myocardial neovascularization, and prevention of post-infarct remodeling. Stem cell transplantation to improve cardiac function has received mixed results in human clinical trials. These early data suggest that a critical reassessment of the scientific basis to stem cell-based therapy is needed in order to bring this highly promising treatment modality to mainstream clinical care.

  View details for PubMedID 20054428

• alpha(2)-Macraglobulin from rheumatoid arthritis synovial fluid: Functional analysis defines a role for oxidation in inflammation ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Wu, S. M., Pizzo, S. V. 2001; 391 (1): 119-126

  Abstract

  A hallmark of inflammation is the release of oxidants, proteinases, and cytokines, all important mediators of the inflammatory cascade. alpha(2)-Macroglobulin (alpha(2)M) is a high-affinity, broad-specificity proteinase inhibitor that also binds and regulates the biological activities of a number of cytokines. We demonstrated recently that hypochlorite-oxidized alpha(2)M has decreased ability to inhibit proteinases and regulate cytokines in vitro. The role of oxidation in regulating alpha(2)M functions in vivo is largely unknown. To determine the extent and biological consequence of in vivo alpha(2)M oxidation, we measured the degree of oxidative alpha(2)M modification from rheumatoid arthritis (RA) synovial fluid and compared this with osteoarthritis (OA) as noninflammatory controls. We found that RA synovial fluid alpha(2)M is significantly more oxidized than that from OA. RA synovial fluid also contains a twofold higher median alpha(2)M level than OA, while having only half the alpha(2)M-proteinase inhibitory activity. Detailed biochemical analysis demonstrates proteolytically degraded alpha(2)M in RA greater than in OA synovial fluid. Additionally, the hypochlorite-mediated oxidation product, chlorotyrosine, is present in RA more than in OA or plasma alpha(2)M samples. Taken together, these findings confirm a role for oxidative regulation of inflammation by altering the functions of extracellular mediators such as alpha(2)M.

  View details for Web of Science ID 000169700200015

  View details for PubMedID 11414692

• Differential regulation of the fibroblast growth factor (FGF) family by alpha(2)-macroglobulin: evidence for selective modulation of FGF-2-induced angiogenesis BLOOD Asplin, I. R., Wu, S. M., Mathew, S., Bhattacharjee, G., Pizzo, S. V. 2001; 97 (11): 3450-3457

  Abstract

  The fibroblast growth factor (FGF) family has an important role in processes such as angiogenesis, wound healing, and development in which precise control of proteinase activity is important. The human plasma proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M) regulates cellular growth by binding and modulating the activity of many cytokines and growth factors. These studies investigate the ability of native and activated alpha(2)M (alpha(2)M*) to bind to members of the FGF family. Both alpha(2)M and alpha(2)M* bind specifically and saturably to FGF-1, -2, -4, and -6, although the binding to alpha(2)M* is of significantly higher affinity. Neither alpha(2)M nor alpha(2)M* bind to FGF-5, -7, -9, or -10. FGF-2 was chosen for more extensive study in view of its important role in angiogenesis. It was demonstrated that FGF-2 binds to the previously identified TGF-beta binding site. The alpha(2)M* inhibits FGF-2-dependent fetal bovine heart endothelial cell proliferation in a dose-dependent manner. Unexpectedly, alpha(2)M* does not affect FGF-2-induced vascular tubule formation on Matrigel basement membrane matrix or collagen gels. Further studies demonstrate that FGF-2 partitions between fluid-phase alpha(2)M* and solid-phase Matrigel or collagen. These studies suggest that the ability of alpha(2)M* to modulate the activity of FGF-2 is dependent on an interplay with extracellular matrix components. (Blood. 2001;97:3450-3457)

  View details for Web of Science ID 000168927900019

  View details for PubMedID 11369636

• The conformation-dependent interaction of alpha(2)-macroglobulin with vascular endothelial growth factor - A novel mechanism of alpha(2)-macroglobulin/growth factor binding JOURNAL OF BIOLOGICAL CHEMISTRY Bhattacharjee, G., Asplin, I. R., Wu, S. M., Gawdi, G., Pizzo, S. V. 2000; 275 (35): 26806-26811

  Abstract

  alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these factors is poorly understood. We examined in detail the interaction between alpha(2)M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to alpha(2)M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of alpha(2)M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated alpha(2)M and to the exterior of native and proteinase-converted alpha(2)M; and 3) VEGF (molecular mass approximately 40 kDa) can access the interior of receptor-recognized alpha(2)M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of alpha(2)M is internalized and degraded by macrophages via the alpha(2)M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized alpha(2)M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of alpha(2)M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca(2+) levels. We demonstrate that under physiological conditions, alpha(2)M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca(2+).

  View details for Web of Science ID 000089144800021

  View details for PubMedID 10862607

• a-Macroglobulins/Kunins In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 4th Ed. Pizzo SV, Wu SM 2000: 367-379
• Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha(2)-Macroglobulin BIOCHEMISTRY Wu, S. M., Pizzo, S. V. 1999; 38 (42): 13983-13990

  Abstract

  The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.

  View details for Web of Science ID 000083288400025

  View details for PubMedID 10529245

• Oxidized alpha(2)-macroglobulin (alpha(2)M) differentially regulates receptor binding by cytokines growth factors: Implications for tissue injury and repair mechanisms in inflammation JOURNAL OF IMMUNOLOGY Wu, S. M., Patel, D. D., Pizzo, S. V. 1998; 161 (8): 4356-4365

  Abstract

  Alpha2M binds specifically to TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), beta-nerve growth factor (beta-NGF), platelet-derived growth factor (PDGF), and TGF-beta. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of alpha2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized alpha2M exhibits increased binding to TNF-alpha, IL-2, and IL-6 and decreased binding to beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Hypochlorite oxidation of methylamine-treated alpha2M (alpha2M*), an analogue of the proteinase/alpha2M complex, also results in decreased binding to bFGF, beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Concomitantly, we observed decreased ability to inhibit TGF-beta binding and regulation of cells by oxidized alpha2M and alpha2M*. We then isolated alpha2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-beta compared with alpha2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.

  View details for Web of Science ID 000076343300073

  View details for PubMedID 9780213

• The binding of receptor-recognized alpha(2)-macroglobulin to the low density lipoprotein receptor-related protein and the alpha(2)M signaling receptor is decoupled by oxidation JOURNAL OF BIOLOGICAL CHEMISTRY Wu, S. M., BOYER, C. M., Pizzo, S. V. 1997; 272 (33): 20627-20635

  Abstract

  Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by alpha2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of alpha2M* using 200 microM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.

  View details for Web of Science ID A1997XR22100048

  View details for PubMedID 9252378

• Crashing the Boards: A User Friendly Study Guide for the USMLE Step 1 Lippincott-Raven Yeh B, Paydafar JA, Flynn M, Biswas SS, Bulsara KR, Liao L, Wu SM 1997; 1
• Low-density lipoprotein receptor-related protein alpha(2)-macroglobulin receptor on murine peritoneal macrophages mediates the binding and catabolism of low-density lipoprotein ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Wu, S. M., Pizzo, S. V. 1996; 326 (1): 39-47

  Abstract

  Low-density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor is a member of the low-density lipoprotein receptor family. It is known to bind a wide variety of unrelated ligands including alpha 2-macroglobulin-proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, and Pseudomonas exotoxin A. Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP. Recent studies have shown that some ligands can bind to more than one receptor in this family. However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently. In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4 degrees C (Kd = 1.8 nM) and competes for the binding of a receptor-recognized form of alpha 2-macroglobulin (alpha 2M*) (Ki = 3 nM). alpha 2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase. Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd = 5 nM). LDL can also completely inhibit the binding of alpha 2M* to purified LRP. These results indicate that LDL binds directly to LRP. The ability of LDL to cross-compete with alpha 2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as alpha 2M*. In addition, the ability of alpha 2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.

  View details for Web of Science ID A1996TT88500006

  View details for PubMedID 8579370