Single cell qPCR reveals that additional HAND2 and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes

PLoS One. 2017 Aug 10;12(8):e0183000. doi: 10.1371/journal.pone.0183000. eCollection 2017.

Abstract

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.

MeSH terms

  • Basic Helix-Loop-Helix Transcription Factors / genetics*
  • Cell Differentiation
  • Cell Line
  • Cellular Reprogramming Techniques
  • Cellular Reprogramming*
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gene Expression Regulation
  • Human Embryonic Stem Cells / cytology*
  • Human Embryonic Stem Cells / metabolism
  • Humans
  • MicroRNAs / genetics*
  • Myocytes, Cardiac / cytology*
  • Myocytes, Cardiac / metabolism
  • Real-Time Polymerase Chain Reaction
  • Transcriptome

Substances

  • Basic Helix-Loop-Helix Transcription Factors
  • HAND2 protein, human
  • MIRN1 microRNA, human
  • MicroRNAs