Abstract 3871: Inhibition of Histone Deacetylation Markedly Induces Myocardial Differentiation of Induced Pluripotent Stem Cells in Mice

Background: Mouse and human fibroblasts can be directly reprogrammed to pluripotency by the ectopic expression of 4 transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) to yield induced pluripotent stem (iPS) cells. iPS cells can be generated even without the expression of c-Myc. The present study examined patterns in the differentiation of mouse iPS cells into cardiomyocytes within 3 different cell lines reprogrammed by 3 or 4 factors. Methods and Results: The level of SSEA-1, a stem cell marker, was similar at the undifferentiated stage in these lines. During the induction of differentiation on feeder-free gelatinized dishes, the expression of genes involved in cardiogenesis and myogenic contraction occurred in 2 iPS cell lines similarly to that in ES cells. However, in one iPS cell line (20D17) generated by 4 factors, the expression of cardiac-specific genes was extremely low, and contractile activity barely occurred. In contrast, patterns of VEGF and Flk-1 expression were similar among the 3 cell lines. The treatment of iPS cells with tricostatin A (TSA), an HDAC inhibitor, increased Nkx2.5 and ANF expression levels and contractile activities in all iPS cell lines. While basal Nkx2.5 expression was very low in 20D17, the TSA-induced increase was the most marked. Overall, Nkx2.5 mRNA levels in TSA-stimulated cells were similar among the 3 iPS cell lines. In the 20D17 iPS cell line, TSA increased mRNA and protein levels of cardiac myosin heavy chain, a contractile protein, clearly indicating TSA-induced myocardial differentiation in this line. The mRNA level of Oct3/4 and nuclear protein level of HDAC4 were higher in 20D17 with poor differentiation potency compared with the other iPS lines. DNA microarray analysis also identified genes up- and down-regulated in 20D17 versus the other cell line. One of the genes up-regulated in 20D17 was BMP4, whose expression in ES cells before differentiation induction reportedly disturbs myocardial differentiation. Conclusion: Thus, mouse iPS cells can differentiate into cardiomyocytes on feeder-free gelatinized dishes in a cell line-dependent manner. The results also suggest that TSA is useful to overcome cell line variation in differentiation efficiency, which possibly occurs in the clinical setting.