SUMMARY The reprogramming of fibroblasts to induced pluripotent stem (iPS) cells raises the possibility that a somatic cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. A large pool of fibroblasts exists in the post-natal heart, yet no single “master regulator” of direct cardiac reprogramming has been identified. Here, we report that a combination of three developmental transcription factors (i.e., Gata4, Mef2c and Tbx5) rapidly and efficiently reprogrammed post-natal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells. Induced cardiomyocytes expressed cardiac-specific markers, had a global gene expression profile similar to cardiomyocytes, and contracted spontaneously. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells. These findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by defined factors. Reprogramming of endogenous or explanted fibroblasts might provide a source of cardiomyocytes for regenerative approaches.

Summary Growth and expansion of ventricular chambers is essential during heart development and is achieved by proliferation of cardiac progenitors. Adult cardiomyocytes, by contrast, achieve growth through hypertrophy rather than hyperplasia. Although epicardial-derived signals may contribute to the proliferative process in myocytes, the factors and cell types responsible for development of the ventricular myocardial thickness are unclear. Using a co-culture system, we found that embryonic cardiac fibroblasts induced proliferation of cardiomyocytes, in contrast to adult cardiac fibroblasts that promoted myocyte hypertrophy. We identified fibronectin, collagen and heparin-binding EGF-like growth factor as embryonic cardiac fibroblast-specific signals that collaboratively promoted cardiomyocyte proliferation in a paracrine fashion. β1-integrin was required for this proliferative response, and ventricular cardiomyocyte-specific deletion of β1-integrin in mice resulted in reduced myocardial proliferation and impaired ventricular compaction. These findings reveal a previously unrecognized paracrine function of embryonic cardiac fibroblasts in regulating cardiomyocyte proliferation.

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...