Bektik Emre, Dennis Adrienne, Prasanna Prateek, Madabhushi Anant, Fu Ji-Dong
Department of Medicine, Heart and Vascular Research Center, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio, United States of America.
School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Japan.
PLoS One. 2017 Aug 10;12(8):e0183000. doi: 10.1371/journal.pone.0183000. eCollection 2017.
The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional HAND2 or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more HAND2 expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, HAND2 and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells.
将心脏成纤维细胞直接重编程为诱导性心肌样细胞（iCMs）在恢复心脏功能方面具有巨大潜力。我们之前发现，人类成纤维细胞可通过7种重编程因子重编程为心肌样细胞；然而，人类成纤维细胞中的iCM重编程比小鼠细胞中的更困难且耗时更长。在本研究中，我们通过单细胞定量PCR研究了额外的重编程因子是否能在定量和/或定性方面改善7因子介导的人类iCM重编程。我们首先验证了46对TaqMan®引物/探针，它们具有足够的效率和灵敏度来检测个体H9人类胚胎干细胞（ESC）分化的心肌细胞（H9CMs）与人类成纤维细胞之间基因表达的显著差异。这46个基因的表达谱显示，与7因子重编程4周的iCMs相比，12周的iCMs重编程有所改善，表明人类iCM重编程过程中存在延长的随机阶段。尽管额外的一种重编程因子均未产生更多数量的iCMs，但我们的单细胞qPCR显示，额外的HAND2或microRNA-1可促进成纤维细胞基因的沉默，并在更多重编程的iCMs中产生更好的重编程程度。值得注意的是，HAND2表达越多，在7Fs + HAND2重编程的iCMs中激活的心脏基因水平越高。总之，HAND2和microRNA-1可帮助7种因子促进人类成纤维细胞iCM重编程的早期进程。我们的研究为进一步优化人类细胞中直接iCM重编程的方法提供了有价值的信息。
