Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Cardiovasc Res. 2010 Nov 1;88(2):314-23. doi: 10.1093/cvr/cvq189. Epub 2010 Jun 14.
Mouse and human fibroblasts can be directly reprogrammed to pluripotency by the ectopic expression of four 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 of differentiation of mouse iPS cells into cardiomyocytes in three different cell lines reprogrammed by three or four factors.
During the induction of differentiation on feeder-free gelatinized dishes, genes involved in cardiogenesis were expressed as in embryonic stem cells and myogenic contraction occurred in two iPS cell lines. However, in one iPS cell line (20D17) generated by four factors, the expression of cardiac-specific genes and the beating activity were extremely low. Treating iPS cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased Nkx2.5 expression in all iPS cell lines. While the basal Nkx2.5 expression was very low in 20D17, the TSA-induced increase was the greatest. TSA also induced the expression of contractile proteins in 20D17. Furthermore, we demonstrated the increased mRNA level of Oct3/4 and nuclear protein level of HDAC4 in 20D17 compared with the other two iPS cell lines. DNA microarray analysis identified genes whose expression is up- or down-regulated in 20D17.
Mouse iPS cells differentiate into cardiomyocytes in a cell line-dependent manner. TSA induces myocardial differentiation in mouse iPS cells and might be useful to overcome cell line variation in the differentiation efficiency.
通过异位表达四个转录因子（Oct3/4、Sox2、Klf4 和 c-Myc），将小鼠和人成纤维细胞直接重编程为多能性，从而产生诱导多能干细胞（iPS）细胞。即使不表达 c-Myc，也可以产生 iPS 细胞。本研究检查了在由三种或四种因子重编程的三种不同细胞系中，小鼠 iPS 细胞向心肌细胞分化的分化模式。
在无饲养层的明胶化培养皿上诱导分化期间，参与心脏发生的基因如胚胎干细胞中那样表达，并且在两种 iPS 细胞系中发生肌原性收缩。然而，在由四种因子产生的一个 iPS 细胞系（20D17）中，心脏特异性基因的表达和搏动活性极低。用组蛋白去乙酰化酶（HDAC）抑制剂曲古抑菌素 A（TSA）处理 iPS 细胞，可增加所有 iPS 细胞系中 Nkx2.5 的表达。虽然 20D17 中的基础 Nkx2.5 表达水平很低，但 TSA 诱导的增加幅度最大。TSA 还诱导了 20D17 中的收缩蛋白表达。此外，我们证明了与其他两种 iPS 细胞系相比，20D17 中 Oct3/4 的 mRNA 水平和 HDAC4 的核蛋白水平增加。DNA 微阵列分析鉴定了表达上调或下调的基因在 20D17 中。
小鼠 iPS 细胞以细胞系依赖性方式分化为心肌细胞。TSA 诱导小鼠 iPS 细胞的心肌分化，可能有助于克服分化效率中的细胞系差异。
