Abstract 012: Directed Differentiation of c-kit+ Human Cardiac Stem Cells in vitro

Although transplantation of c-kit+ cardiac stem cells (CSCs) significantly alleviates post-myocardial infarction (MI) left ventricular dysfunction, the exogenous CSCs in the recipient heart do not generate cardiomyocytes robustly. Controlling the fate of transplanted CSCs and inducing their robust differentiation would be important in further improving the efficacy of cardiac stem cell therapies. We assessed the hypothesis that differentiation of human c-kit+ CSCs can be enhanced by programming them with cardiac transcription factors, including GATA4, MEF2C, Nkx2.5 and Tbx5. Transcription factors were introduced into CSCs in culture via lentivirus either individually or in combination. The transduced cells were then cultured for 1 or 2 weeks and analyzed for expression of 36 different markers of differentiated cardiac cell types by quantitative RT-PCR. Introduction of GATA4 led to marked increases in both early and late markers of cardiomyocyte differentiation, such as BNP and troponin T. By 2 weeks, GATA4 also significantly induced some of the smooth muscle cell markers, including calponin-1 and smooth muscle myosin heavy chain, as well as fibroblast markers. Interestingly, these gene expression changes induced by GATA4 were accompanied by dramatic morphological changes in CSCs. Overexpression of Tbx5 in CSCs resulted in induction of some of the cardiomyocyte markers specifically, although the number of genes differentially expressed was significantly less than those by GATA4. Interestingly, introduction of multiple transcription factors in combinations did not further increase the expression of cardiac marker genes. In conclusion, these findings suggest that cardiac gene programs can be activated in CSCs via introduction of defined transcription factors and that the pattern of expression of differentiated cardiac markers is unique to each transcription factor. However, none of the transcription factors alone was sufficient to induce differentiation of CSCs into mature cardiomyocyte-like cells, indicating that multiple and additional factors as well as temporal changes in their expression may be required for full differentiation and maturation of CSCs.