Here we demonstrated that the combination of three transcription factors, Gata4, Mef2c, and Tbx5, can rapidly and efficiently induce cardiomyocyte-like cells from post-natal cardiac and dermal fibroblasts. iCMs were similar to neonatal cardiomyocytes in global gene expression profile, electrophysiologically, and could contract spontaneously, demonstrating that functional cardiomyocytes can be generated from differentiated somatic cells by defined factors. Although much refinement and characterization of the reprogramming process will be necessary, the findings reported here raise the possibility of reprogramming the vast pool of endogenous fibroblasts that normally exists in the heart into functional cardiomyocytes for regenerative purposes.
The three reprogramming factors, Gata4, Mef2c, and Tbx5, are core transcription factors during early heart development (Olson, 2006
; Srivastava, 2006
; Zhao et al., 2008
). They interact with one another, coactivate cardiac gene expression (e.g., Nppa, Gja5
(Cx40) and Myh6
), and promote cardiomyocyte differentiation (Bruneau et al., 2001
; Garg et al., 2003
; Ghosh et al., 2009
; Lin et al., 1997
). Gata4 is considered a “pioneer” factor and might open chromatin structure in cardiac loci (Cirillo et al., 2002
), thus allowing binding of Mef2c and Tbx5 to their specific target sites and leading to full activation of the cardiac program. While the reprogramming event appears stable at the epigenetic level, as marked by histone methylation and DNA methylation, the global gene expression of iCMs is similar but not identical to neonatal cardiomyocytes. Whether they are more similar to adult ventricular cardiomyocytes or other sub-populations remains to be determined. Additional epigenetic regulators, microRNAs, or signaling proteins may be leveraged to increase the efficiency and robustness of the reprogramming event. Furthermore, other combinations of factors likely also induce cardiac reprogramming, much like the experience in the iPS field.
Several lines of evidence suggest that the iCMs we describe here originated from differentiated fibroblasts. We found that any potential rare cardiac “progenitor-like” cells, marked by c-kit or Isl1, were dispensable for cardiomyocyte induction (Beltrami et al., 2003
). Furthermore, the high efficiency of cardiac induction (up to 20%) does not favor the interpretation that rare stem or progenitor cells were the origin of induced cardiomyocytes. Most importantly, the ability to reprogram dermal fibroblasts into iCMs supports the conclusion that cardiac progenitors are not the target cells for the reprogramming factors. Remarkably, reprogramming of cardiac fibroblasts to myocytes occurred in a relatively short period, with the first GFP+
cells appearing at day 3, in contrast to iPS cell reprogramming, which typically takes 10–20 days and occurs with much lower efficiency (<0.1%) (Takahashi and Yamanaka, 2006
). Despite the early initiation of reprogramming, the process appears to continue for several weeks, with progressive changes in gene expression, contractile ability, and electrophysiologic maturation.
While many questions remain regarding the mechanisms of reprogramming, we were able to genetically test the “route” of cell fate alteration. Our findings suggest that cardiomyocytes were directly induced from cardiac fibroblasts without reverting to a cardiac progentior cell state, which may explain the rapid early reprogramming process. This conclusion was supported by the absence of Isl1-Cre-YFP or Mesp1-Cre-YFP activation during the process of reprogramming, which would have marked any cells that transiently expressed Isl1 or Mesp1 (Laugwitz et al., 2005
; Saga et al., 1999
The ability to reprogram endogenous cardiac fibroblasts into cardiomyocytes has many therapeutic implications. First, the avoidance of reprogramming to pluripotent cells before cardiac differentiation would greatly lower the risk of tumor formation in the setting of future cell-based therapies. Second, large amounts of an individual’s own fibroblasts can be grown from a cardiac biopsy or skin biopsy in vitro for transduction with the defined factors, followed by delivery of cells to damaged hearts. Third, and most promising, is the potential to introduce the defined factors, or factors that mimic their effects, directly into the heart to reprogram the endogenous fibroblast population, which represents more than 50% of the cells, into new cardiomyocytes that can contribute to the overall contractility of the heart. Our observation that injection of fibroblasts into the heart only 1 day after induction of Gata4/Mef2c/Tbx5 resulted in reprogramming of the transplanted cells suggests that this may be possible. Future studies in human cells and advances in safe delivery of defined factors will be necessary to advance this technology for potential regenerative therapies.