DNA methyltransferases inhibitors effectively induce gene expression changes suggestive of cardiomyogenic differentiation of human amniotic fluid‐derived mesenchymal stem cells via chromatin remodeling

Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) are a new potential stem cell source for cell therapy and regenerative medicine. These are fetal mesenchymal stem cells with multilineage differentiation potential found in amniotic fluid. The aim of the present study was to evaluate in vitro differentiation initiation of AF-MSCs into cardiac progenitors upon application of inhibitors of DNA methyltransferases (DNMT), such as Decitabine (DEC; 5-aza-2'-deoxycytidine) and Zebularine (ZEB). We assessed epigenetic changes and explored patterns of genes, enriched in association with hyperacetylated H4 after induced differentiation. Upregulation of cardiomyogenesis-related genes (TNNT2, MYH6, ACTN2, and DES) and cardiac ion channels genes, downregulation of pluripotency genes markers as well as increase in Connexin43 expression indicated cardiomyogenic commitment. Evaluation of global epigenetic changes showed that levels of chromatin modifying enzymes, such as Polycomb repressive complex 2 proteins (EZH2, SUZ12), DNMT1, histone deacetylases 1 and 2 were reduced to the similar extent by both differentiation agents. Levels of specific histone marks keeping active state of chromatin (H3K4me3, H3K9Ac, and H4hyperAc) increased and marks of repressed chromatin state (H3K27me3 and H3K9me3) decreased after DEC or ZEB treatment. Chip-Seq analysis after chromatin immunoprecipitation with H4hyperAc demonstrated enrichment of around 100 functionally annotated genes, related to chromatin reorganization and cardiomyogenesis and confirmed relation between H4 hyperacetylation and gene expression. Our results demonstrate that both DEC and ZEB can be potentially used as cardiomyogenic differentiation inducers in AF-MSCs, and they cause various genetic and epigenetic changes resulting in global chromatin remodeling.