Date Published: March 29, 2017
Publisher: Public Library of Science
Author(s): Tareq Al-Maqtari, Kyung U. Hong, Bathri N. Vajravelu, Afsoon Moktar, Pengxiao Cao, Joseph B. Moore, Roberto Bolli, Maurizio Pesce.
Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy.
In contrast to the long-standing belief that the mammalian heart is a post-mitotic or terminally differentiated organ, previous reports have demonstrated that the adult mammalian heart possesses a capacity of cardiomyocyte renewal [1–5]. Beltrami and colleagues first described a unique resident cardiac cell population with characteristics of stem cells in the rat heart . This population of cells was found to be positive for c-kit (c-kit+), a receptor tyrosine kinase, and when isolated and grown in culture, they were self-renewing, clonogenic, and multipotent, being able to differentiate into cardiomyocytes, smooth muscle, and endothelial cells. Since then, c-kit+ CPCs have been described in multiple mammalian species, including human [7–11]. Also, discovery of specialized niches within the heart which contain clusters of undifferentiated c-kit+ CPCs and early-lineage committed cells (i.e., c-kit and GATA4, MEF2C, or Ets1 double-positive cells) strongly suggests that they not only reside stably in the heart but also are specifically “programmed” to give rise to multiple cardiac cell types . Moreover, when injected into an ischemic heart, they reconstitute differentiated myocardium with new vessels and myocytes . In a recent phase I clinical trial, c-kit+ CPCs isolated from patients with ischemic cardiomyopathy have been shown to significantly improve heart function and the quality of life when transplanted back into the patients via intracoronary injection [11, 12], clearly demonstrating the utility of these cells in developing stem cell therapies for the treatment of ischemic cardiomyopathy.
One of the limitations of the current regenerative CPC therapy for ischemic cardiomyopathy is the lack of robust de novo differentiation of the transplanted cells in the host myocardium [10, 14]. Although the cause of this is unknown, increasing the cardiogenic differentiation potential of CPCs may further enhance the efficacy of the CPC therapy. In support of this notion, Behfar and colleagues have shown that treatment of cells with ‘cardiogenic cocktail’ prior to transplantation augments the therapeutic benefit of the bone marrow mesenchymal stem cells in chronic ischemic cardiomyopathy . Although previous studies have shown that differentiation of CPCs can be induced by treating the cells with dexamethasone, 5-azacytidine followed by TGF-β1, or co-culturing with neonatal rat myocytes [6, 7, 31–33], the currently available differentiation protocols are often inefficient and result in variable results (Unpublished observations). Thus, in attempt to facilitate differentiation of CPCs, we tested the effects of ectopic expression of five TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) on ‘priming’ or ‘programming’ CPCs. Such method, often referred to as ‘forward programming,’ has been previously employed to drive cardiomyogenic differentiation of a variety of cell types [18–21, 24, 34], yet has never been tested in CPCs so far. These previous studies have mainly focused on inducing myogenic differentiation of cells of interest, and have not examined the ability of the TFs to induce differentiation of cells into other resident cell types in the heart. In our current study, we conducted a more comprehensive analysis of TF-induced differentiation of CPCs. We assessed the induction of genes associated not only with cardiomyocytes but also with other resident cardiac cell types upon introduction of the TFs. This was based on the observation that CPCs are multipotent and able to give rise to multiple cell types present in the heart, including myocytes, endothelial cells, and smooth muscle cells [6, 7, 9].