Inhibition of histone deacetylation markedly induces myocardial differentiation of induced pluripotent stem cells in mice

Inhibition of histone deacetylation markedly induces myocardial differentiation of induced pluripotent stem cells in mice

Shinji Kaichi1, Koji Hasegawa2, Tomohide Takaya1, Tatsuya Morimoto2, Teruhisa Kawamura1, Koh Ono1, Noritaka Yokoo1, Takahiro Mima1, Shiro Baba1, Hiraku Doi1, Shinya Yamanaka3, Tatsutoshi Nakahata1, Toshio Heike1.

  1. Graduate School of Medicine, Kyoto University, Kyoto, Japan.
  2. Kyoto Medical Center, National Hospital Organization, Kyoto, Japan.
  3. Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

American Heart Association Scientific Sessions 2009 (Orlando, USA), 2009/11/18 (Poster).

Abstract

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.