Progress has been made recently in inducing cardiac regeneration aimed at repairing the damaged cardiac tissue 
. BPCs and EPCs have been extensively used in these studies 
; however, the results have been variable and often improvements have been short-lived perhaps due to waning effects of the released humoral mediators from the transplanted cells and tissue and failure of effective engraftment 
. In addition, it is not always clear whether transplanted cells differentiate into cardiac or endothelial cells and thus whether they have the capability to regenerate injured tissue. Cells such as BPCs also consist of heterogeneous population of bone marrow cells containing mesenchymal cells, EPCs, and mature endothelial cells 
. These cells may not have trans-differentiation potential of cardiac progenitor cells 
. Here we demonstrate for the first time to our knowledge of the value of epigenetic modification of BPCs, which induces the BPCs to de-differentiate into multipotential cells, which we termed eiBPCs. Transplantation of cardiac progenitor cells derived from eiBPCs into mouse hearts induced significantly greater cardiac protection and myocyte regeneration in mice after myocardial infarction over that seen with BPCs alone. Based on these results, we posit that cardiac progenitor cells derived from epigenetic modification of autologous BPCs have promise for treating ischemic heart disease. These findings thus raise the possibility of converting BPCs into multipotent eiBPCs as a source of cardiac regenerating cells.
Epigenetic changes are defined as modifications of DNA or chromatin that do not involve alterations of the DNA sequence or genetic deletions 
. Recent studies into the mechanisms by which nuclear cloning and somatic-ESC fusion induce epigenetic changes in somatic cell nuclei suggest that the somatic cell chromatin is remodeled through chromatin condensation, DNA methylation/de-methylation, and histone modifications (acetylation/de-acetylation, phosphorylation, and methylation/de-methylation) 
. These histone and DNA modifications are functionally linked 
. DNA de-methylation/methylation is an essential epigenetic process required for gene activation or inactivation during development 
. A change in DNA methylation pattern is also a principal epigenetic event underlying de-differentiation of somatic cells 
. DNA methylation most often occurs on 5′-CpG-3′ dinucleotides 
. DNA methyl transferase enzymes (dnmt 1, dnmt3a, dnmt 3b) attach a methyl group to the 5th
carbon position of cytosine residues in the CG dinucleotide 
, the protein condenses into a protein complex consisting of methyl-binding proteins (MBD), HDACs, and repressor proteins at the methylated CpG sites 
, which leads to inhibition of gene expression 
. In addition to DNA methylation, post-translational modifications of histone proteins regulate gene activity by modulating chromatin structure 
. Acetylation of histone H3 and H4 generally correlates with gene activation whereas de-acetylation correlates with gene silencing 
Gene silencing is achieved by DNA methylation and histone deacetylation at the promoter region of genes 
whereas gene activation is mediated by DNA demethylation and histone acetylation 
. DNA demethylation and methylation are mediated and balanced by DNA demethyltransferases (DeMT) and DNA methyltransferases (Dnmts) respectively. In this study, we used Aza, an inhibitor for Dnmts, which blocks DNA methylation and thus promotes demethylation 
. Likewise histone acetylation and deacetylation is mediated and balanced by the enzymes histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. Here we also used TSA, a general inhibitor for HDACs that blocks deacetylation and promotes histone acetylation 
. Our data show that BPCs treated with Aza plus TSA in a dose-dependent manner activated the pluripotent genes Oct4, Nanog and Sox2 and resulted in expression of these proteins 
. However, we do not think that these cells became totipotent as defined earlier 
, since transplantation of these cells did not result in the formation of teratomas.
Transcriptional activation of pluripotent genes such as Oct4 requires acetylation of histone H3 at lysine 9 (aceH3K9), whereas histone deacetylation by HDACs 1, 2, 3, and 8 induces closed chromatin confirmation leading to repression of gene activity 
. Transcriptional activation is also modulated by histone demethylase activity of LSD1, a homolog of nuclear amine oxidase; however, depending on the cellular context, LSD1 can either repress transcription as a component of the CoREST transcriptional co-repressor complex 
or activate transcription by functioning as a co-activator 
. Our results show that treatment of BPCs with Aza plus TSA induced modifications resulting in expression of genes associated with a multipotent phenotype. We observed decreases in HDAC1 and LSD1 protein expression and increase in AceH3K9 protein expression indicative of epigenetic changes at the level of histones. We also observed increased aceH3K9 protein expression and its interaction with Oct4 promoter demonstrating that Oct4 promoter region was acetylated in the Aza- and TSA-treated BPCs. Thus, the chromatin modifying agents Aza and TSA induced epigenetic modification of BPCs at least in part by histone modifications and chromatin remodeling leading to the activation of dormant pluripotent genes.
We further transplantation of cardiac progenitor cells derived from the modified BPCs induced cardiomyogenesis and angiogenesis in infarcted hearts. As new vessel formation following cardiac progenitor cell transplantation was similar to that seen with BPC transplantation, it appears that angiogenesis is due to EPCs present in the transplanted cell population that were not fully committed to their myocyte fate.
Importantly, we did not observe teratoma formation following injection of cardiac progenitors. This is consistent with the use of cardiac progenitors derived from epigenetically multipotent BPCs rather fully de-differentiated iPSCs 
. We did not examine the in situ
electrophysiological properties of the regenerated CMCs; thus, although LV function was improved after cell transplantation, we do know whether the myocytes formed a proper syncytium. In addition, we observed only up to 5% of transplanted cells were retained in the myocardium at day 28 in absence of significant apoptosis, suggesting that the injected cells may have transitioned into myocytes during this period. Also it is possible that secretion of factors by transplanted cells or host tissue may contribute to the improvement in cardiac function. We observed the production of IL-10, an anti-inflammatory cytokine, in cardiac tissue after transplantation, which could improve LV function and remodeling via activation of STAT3 and suppression of p38 MAPKs 
Our observations are consistent with recent studies showing the feasibility of reprogramming of somatic cells into the CMC lineage 
. A recent study also demonstrated reprogramming of fibroblasts into multi-lineage hematopoietic progenitor cells and mature blood cells without establishing totipotency 
. They accomplished this by ectopic expression of Oct4-activated hematopoietic transcription factors and treatment with specific cytokines. We showed differentiation of BPCs into cardiac progenitor cells expressing the CMC markers Gata4, Nkx2.5, CTT, and α-SA is possible using the chromatin modifying agents Aza and TSA.
In summary, we showed that treatment of BPCs with chromatin modifying agents Aza and TSA induced expression of pluripotent genes Sox2, Oct4 and Nanog in BPCs, which can then be further programmed to generate cardiac progenitor cells that give rise to cardiac and endothelial cells in situ. Transplantation of these epigenetically reprogrammed cardiac progenitors cells induced cardiomyogenesis and also improved LV function of infarcted mouse hearts suggesting the utility of this approach. We used available chromatin modifying drugs to achieve multipotency of BPCs suggests clinical applicability and value of this cell transplantation approach.