Ifkovits Jamie L, Addis Russell C, Epstein Jonathan A, Gearhart John D
Department of Cell and Developmental Biology, Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
PLoS One. 2014 Feb 26;9(2):e89678. doi: 10.1371/journal.pone.0089678. eCollection 2014.
Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ∼5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic.
最近的研究成功地利用转录因子的异位表达从成纤维细胞中诱导生成心肌细胞（iCMs），尽管在体外诱导频率较低。这项研究探讨了小分子的影响，这些小分子先前已被报道可改善向心肌细胞的分化以及与转录因子Hand2、Nkx2.5、Gata4、Mef2C和Tbx5的异位表达相结合重编程为诱导多能干细胞（iPSCs）对功能性iCMs转化的影响。我们使用了一种报告系统，其中钙指示剂GCaMP由心肌肌钙蛋白T启动子驱动，以量化iCM的产量。转化生长因子β（TGFβ）抑制剂SB431542（SB）被确定为一种小分子，能够将小鼠胚胎成纤维细胞和成年心脏成纤维细胞向iCMs的转化提高约5倍。进一步的表征表明，在重编程过程早期用SB抑制TGFβ以剂量反应方式导致成纤维细胞向iCMs转化的最大增加。在转录因子诱导后第3天进行的全基因组转录分析显示，与载体对照相比，在存在SB的情况下，与心肌发育相关的基因表达增加。在重编程过程中加入SB可提高iCM生成的效率，这是将iCMs用于基于发现的应用和临床的主要目标之一。
