Induction of human cardiomyocyte-like cells from fibroblasts by defined factors

Abstract

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca 2+ oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2′-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.cell fate conversion | regeneration | cardiogenesis C ardiovascular disease remains a leading cause of death worldwide, for which current therapeutic regimens remain limited. Given that adult human hearts have little regenerative capacity after injury, the demand is high for cardiac regenerative therapy. The recent discovery of induced pluripotent stem cells (iPSCs) allows the direct generation of specific cell types from differentiated somatic cells by overexpression of lineagespecific factors.Several previous studies have demonstrated that such direct lineage reprogramming can yield a diverse range of cell types, including pancreatic β cells, neurons, neural progenitors, blood progenitors, and hepatocyte-like cells (1-5). We previously reported that a minimum mixture of three cardiac-specific transcription factors-Gata4, Mef2c, and Tbx5 (GMT)-directly induced cardiomyocyte-like cells (iCMs) from mouse fibroblasts in vitro (6). Following our report, three other groups also reported generation of functional cardiomyocytes from mouse fibroblasts with various combinations of transcription factors, either with GMT plus Hand2 (GHMT) or Mef2c, Myocd, and Tbx5 or using microRNAs (7-9). Although full reprogramming into beating cardiomyocytes was not effic...

miR-322/-503 cluster is expressed in the earliest cardiac progenitor cells and drives cardiomyocyte specification

“…Reprogramming human somatic cells to cardiomyocytes often involves Mesp1 (30,36,37), justifying the identification of effectors downstream of Mesp1. The miRNAs identified here represent early regulators of cardiac fate, likely at the stages of cardiac mesoderm formation and cardiac program initiation.…”

Reprogramming Approaches to Cardiovascular Disease: From Developmental Biology to Regenerative Medicine

“…However, neither GMT nor GHMT, which reprogrammed iCMs from mouse fibroblasts, was able to reprogram human fibroblasts into iCMs in vitro [11][12][13]; however, inclusion of additional reprogramming factors resulted in successful reprogramming. Nam et al [12] found that GHT, without MEF2C, but with another transcription factor, myocardin, and two muscle-specific miRNAs, miR-1 and miR-133, could reprogram human fibroblasts into iCMs.…”

“…These reprogrammed iCMs expressed multiple cardiac genes, developed sarcomere-like structures, and generated Ca 2+ transients with a small subset of the cells exhibiting spontaneous contractility after 11 weeks in culture. Wada R. et al [13] reported that GMT with MESP1 and myocardin could activate cardiac gene expression in human neonatal and adult cardiac fibroblasts. In our study, pairing GMT with ESRRG and MESP1 induced global expression of cardiac genes and shifted the phenotype of human fibroblasts toward the CM-like state.…”

“…Since then, other labs around the world have reported success in reprogramming mouse fibroblasts into iCMs with similar cocktails of reprogramming factors [6][7][8][9][10]. We and others have also directly converted human cardiac and dermal fibroblasts into cardiac cells [11][12][13]. Several review papers published from different labs illustrate the potential and challenges of this new avenue for cardiac regenerative medicine [14][15][16][17][18][19].…”

Proceedings of the Uehara Memorial Foundation. Innovative Medicine: Basic Research and Development. Cardiac Reprogramming for Heart Repair

“…We next analyzed whether human fi broblasts could be directly converted to iCMs by defi ned factors [ 17 ]. We found that GMT was not suffi cient for cardiac induction in HCFs.…”