Bio

Honors & Awards


  • Postdoctoral Fellowship, American Heart Association (2013)

Professional Education


  • Doctor of Philosophy, Peking University (2009)

Stanford Advisors


Publications

Journal Articles


  • 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

  • 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

  • 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 Web of Science ID 000313839500014

    View details for PubMedID 23290139

  • Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique CIRCULATION 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. 2012; 126 (11): 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

  • 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 Web of Science ID 000288404400012

    View details for PubMedID 21362570

  • 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

  • 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 Web of Science ID 000314873000006

    View details for PubMedID 23136034

  • 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

  • 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

  • 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 Web of Science ID 000313573500014

    View details for PubMedID 22565608

  • 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 Web of Science ID 000296565500007

    View details for PubMedID 21898697

  • Induction of mutant p53-dependent apoptosis in human hepatocellular carcinoma by targeting stress protein mortalin INTERNATIONAL JOURNAL OF CANCER Lu, W., Lee, N. P., Kaul, S. C., Lan, F., Poon, R. T., Wadhwa, R., Luk, J. M. 2011; 129 (8): 1806-1814

    Abstract

    Stress protein mortalin (mtHSP70) is highly expressed in cancer cells. It was shown to contribute to carcinogenesis by sequestrating the wild type p53, a key tumor suppressor protein, in the cytoplasm resulting in an abrogation of its transcriptional activation function. We have found that the level of mortalin expression has significant correlation with human hepatocellular carcinoma (HCC) malignancy and therefore investigated whether it interacts with and influences the activities of mutant p53, frequently associated with HCC development. We have detected mortalin-p53 interactions in liver tumor and five HCC cell lines that harbored mutant p53. The data was in contrast to the normal liver and immortalized normal hepatocytes that lacked mortalin-p53 interaction. Furthermore, we have found that the shRNA-mediated mortalin silencing could induce mutant p53-mediated tumor-specific apoptosis in HCC. Such allotment of apoptotic function to mutant p53 by targeting mortalin-p53 interaction in cancer cells is a promising strategy for HCC therapy.

    View details for DOI 10.1002/ijc.25857

    View details for Web of Science ID 000294224300002

    View details for PubMedID 21165951

  • 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

  • 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 Web of Science ID 000294726800078

    View details for PubMedID 21719696

  • Mortalin-p53 interaction in cancer cells is stress dependent and constitutes a selective target for cancer therapy CELL DEATH AND DIFFERENTIATION Lu, W., Lee, N. P., Kaul, S. C., Lan, F., Poon, R. T., Wadhwa, R., Luk, J. M. 2011; 18 (6): 1046-1056

    Abstract

    Stress protein mortalin is a multifunctional protein and is highly expressed in cancers. It has been shown to interact with tumor suppressor protein-p53 (both wild and mutant types) and inactivates its transcriptional activation and apoptotic functions in cancer cells. In the present study, we found that, unlike most of the cancer cells, HepG2 hepatoma lacked mortalin-p53 interaction. We demonstrate that the mortalin-p53 interaction exists in cancer cells that are either physiologically stressed (frequently associated with p53 mutations) or treated with stress-inducing chemicals. Targeting mortalin-p53 interaction with either mortalin small hairpin RNA or a chemical or peptide inhibitor could induce p53-mediated tumor cell-specific apoptosis in hepatocellular carcinoma; p53-null hepatoma or normal hepatocytes remain unaffected.

    View details for DOI 10.1038/cdd.2010.177

    View details for Web of Science ID 000290379300013

    View details for PubMedID 21233847

  • Inducible expression of stem cell associated intermediate filament nestin reveals an important role in glioblastoma carcinogenesis INTERNATIONAL JOURNAL OF CANCER Lu, W. J., Lan, F., He, Q., Lee, A., Tang, C. Z., Dong, L., Lan, B., Ma, X., Wu, J. C., Shen, L. 2011; 128 (2): 343-351

    Abstract

    The intermediate filament nestin is transiently expressed in neural stem/progenitor cells during the development of central nervous system. Recently, increasing evidence has shown that upregulation of nestin is related to malignancy of several cancers, especially glioblastoma. However, the function of nestin in carcinogenesis remains unclear. In this study, we investigated the role of nestin in glioblastoma carcinogenesis by comparing subclones of rat C6 glioblastoma cells that were either high or low for nestin expression. We found that while nestin expression did not influence the in vitro proliferation of glioblastoma cells, subclones characterized by high levels of nestin formed tumors in vivo at significantly faster rates than subclones with low expression. Importantly, C6 subclones that expressed nestin at low levels in vitro were also found to give rise to tumors highly positive for the protein, suggesting that induction of nestin plays an important role in glioblastoma carcinogenesis. Derivation of nestin positive tumors from nestin negative human U87 glioblastoma cells in immunodeficient mice further confirmed that a switch to positive expression of nestin is fundamental to the course of glioblastoma development. Blocking the expression of nestin in glioblastoma tumors via intratumor injection of shRNA significantly slowed tumor growth and volume. These results demonstrated that nestin plays a crucial role in development of glioblastoma and may potentially be targeted for treatment of the disease.

    View details for DOI 10.1002/ijc.25586

    View details for Web of Science ID 000285263100010

    View details for PubMedID 20669222

  • Overexpression of CD133 promotes the phosphorylation of Erk in U87MG human glioblastoma cells NEUROSCIENCE LETTERS Dong, L., Qi, N., Ge, R., Cao, C., Lan, F., Shen, L. 2010; 484 (3): 210-214

    Abstract

    An increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. However, the biological function of CD133 protein remains unknown. In this study, we observed no significant effects on cell proliferation and migration in CD133 overexpressed U87MG human glioblastoma cells. It is reported that MAPK/Erk was constitutively activated in CD133 positive liver cancer stem cell. To find out possible mechanism between CD133 and Erk phosphorylation, we performed this study to evaluate the level of Erk phosphorylation in CD133 overexpressed U87MG cells. We found that CD133 overexpression significantly activated Erk, which suggested CD133 involved in activation of MAPK/Erk pathway.

    View details for DOI 10.1016/j.neulet.2010.08.057

    View details for Web of Science ID 000282998400011

    View details for PubMedID 20800650

  • Generation of Human-Induced Pluripotent Stem Cells from Gut Mesentery-Derived Cells by Ectopic Expression of OCT4/SOX2/NANOG CELLULAR REPROGRAMMING Li, Y., Zhao, H., Lan, F., Lee, A., Chen, L., Lin, C., Yao, Y., Li, L. 2010; 12 (3): 237-247

    Abstract

    Induced pluripotent stem (iPS) cells have been generated from human somatic cells by ectopic expression of defined transcription factors. Application of this approach in human cells may have enormous potential to generate patient-specific pluripotent stem cells. However, traditional methods of reprogramming in human somatic cells involve the use of oncogenes c-MYC and KLF4, which are not applicable to clinical translation. In the present study, we investigated whether human fetal gut mesentery-derived cells (hGMDCs) could be successfully reprogrammed into induced pluripotent stem (iPS) cells by OCT4, SOX2, and NANOG alone. We used lentiviruses to express OCT4, SOX2, NANOG, in hGMDCs, then generated iPS cells that were identified by morphology, presence of pluripotency markers, global gene expression profile, DNA methylation status, capacity to form embryoid bodies (EBs), and terotoma formation. iPS cells resulting from hGMDCs were similar to human embryonic stem (ES) cells in morphology, proliferation, surface markers, gene expression, and epigenetic status of pluripotent cell-specific genes. Furthermore, these cells were able to differentiate into cell types of all three germ layers both in vitro and in vivo, as shown by EB and teratoma formation assays. DNA fingerprinting showed that the human iPS cells were derived from the donor cells, and are not a result of contamination. Our results provide proof that hGMDCs can be reprogrammed into pluripotent cells by ectopic expression of three factors (OCT4, SOX2, and NANOG) without the use of oncogenes c-MYC and KLF4.

    View details for DOI 10.1089/cell.2009.0103

    View details for Web of Science ID 000279402900002

    View details for PubMedID 20698766

  • Adenovirus-Mediated CTLA41g Gene Transfer Improves the Survival of Grafted Human Hepatic Progenitors in Mouse Liver TRANSPLANTATION PROCEEDINGS Lan, F., Ma, X., Liu, Y., Shen, I. 2009; 41 (5): 1862-1864

    Abstract

    The invasive nature of surgery and limited numbers of donor livers for end-stage patients has prompted the search for alternative cell therapies for intractable hepatic disease. Hepatocyte transplantations have been performed for a variety of indications, but sustained benefits have not been observed in most cases. Rat fetal liver epithelial cells (liver stem cells) have demonstrated self-renewal in vivo and functional repopulation of the liver. We have previously isolated and expanded epithelial progenitor cells (EPC) from the human fetal liver to investigate their differentiation potential. In this study, we applied suppression of immunorejection by adenoviral CTLA4Ig gene delivery mediated to examine the survival and differentiation of human fetal EPC transplanted into normal mouse liver. The grafted EPC showed extensive proliferation at both 1 and 2 months after transplantation compared with controls. Moreover, most EPC differentiated into hepatocytes, while a small fraction became bile ductular cells. This finding suggested that human fetal EPC may be a ideal source of cell-based therapy for various liver diseases.

    View details for DOI 10.1016/j.transproceed.2009.01.103

    View details for Web of Science ID 000267449100094

    View details for PubMedID 19545745

  • In vivo gamma imaging of the secondary tumors of transplanted human fetal striatum neural stem cells-derived primary tumor cells NEUROREPORT He, Q., Liu, Z., Jia, B., Li, X., Shi, J., Zhang, J., Lan, F., Yang, Z., Liu, Y., Shen, L., Wang, F. 2008; 19 (10): 1009-1014

    Abstract

    This study describes gamma-imaging of the secondary tumors from the transplanted human fetal striatum neural stem cells-derived primary tumor cells in nude mice. The subcutaneous primary tumors were detected to express integrin alphavbeta3, and the corresponding cells were isolated and enriched in vitro, then transplanted to the nude mice. The technetium-99m-labeled Arg-Gly-Asp peptide, with high affinity to integrin alphavbeta3, was prepared for biodistribution and gamma-imaging. The secondary tumors were readily visualized at 1-h postinjection, and the tumor uptake of radiotracer was similar to that of positive control animals transplanted with U87MG human glioma cells. The tumor specificity of radiotracer was demonstrated by blocking experiment. We concluded that gamma-imaging is a promising approach in imaging the tumorigenesis of transplanted stem cells in vivo.

    View details for Web of Science ID 000257488500003

    View details for PubMedID 18580570

  • Hepatocyte growth factor promotes proliferation and migration in immortalized progenitor cells NEUROREPORT Lan, F., Xu, J., Zhang, X., Wong, V. W., Li, X., Lu, A., Lu, W., Shen, L., Li, L. 2008; 19 (7): 765-769

    Abstract

    Hepatocyte growth factor (HGF) and its receptor c-Met are widely expressed in the developing and adult brain. However, little is known about the role of HGF during the development of the human dopaminergic neuronal system. We have established telomerase-immortalized dopaminergic progenitor cells isolated from the fetal striatum that express markers for neural progenitor cells and tyrosine hydroxylase. We show that the cells were able to differentiate into dopaminergic neurons and release dopamine. Exogenous HGF-induced proliferation was inhibited by U0126, whereas migration was completely blocked by LY294002. Study demonstrates that HGF regulates the proliferation and migration of dopaminergic progenitor cells. Modulating dopaminergic progenitor cells in the striatum may prove to be a new approach for treating Parkinson's disease.

    View details for Web of Science ID 000255530600012

    View details for PubMedID 18418254

Conference Proceedings


  • Novel MicroRNA Prosurvival Cocktail for Improving Engraftment and Function of Cardiac Progenitor Cell Transplantation 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 Web of Science ID 000294782800004

    View details for PubMedID 21911815

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