Role of 5-azacytidine in differentiation of human mesenchymal stem cell sinto cardiomyocytes in vitro

Objective 5-azacytidine could induce the differentiation of stem cells into cardiomyocytes (CMs). The aim of this study was to screen the optimal condition for 5-azacytidine inducing differentiation of human mesenchumal stem cells (hMSCs) into CMs, and the effect of 5-azacytidine on adherence, cell vigor and chromosome karyotype of hMSCs. Methods hMSCs were isolated from human bone marrow and cultured in vitro. The phenotypes of hMSCs were identified by flow cytometric analyses. MTT test was used to investigate the effect of different concentrations of 5-azacytidine on proliferation of hMSCs. Four weeks after 5-azacytidine induction, semi-quantitative RT-PCR, transmission electron microscopy (TEM), single-cell action potentials, detection of cardio-enzyme AST and LDH, cell adherence, cell viability and chromosome karyotype test were performed. Results The typical morphological features of hMSCs were fibroblast-like in shape. hMSCs expressed CD44 and CD105,and did not express CD34, CD45 and CD31. The optimal concentration of 5-azacytidine was 10μmol/L. The shape of hMSCs treated with 5-Azacytidine changed from fusiform to polygon or astrocyte gradually, and passaged cells were evenly arranged as polarity structure. Induced-hMSCs connected with neighbouring cells, forming myotube-like structures 4 weeks later. It was confirmed that induced hMSCs shaped myotubule-like structure and had some of micro-histologic structures of CMs by TEM. RT-PCR showed that induced hMSCs expressed cardiac specific product BNNP and early cardio-myogenesis specific transcription factor NKX2.5mRNA. Besides, induced-MSCs led to the weak action potential and secreted cardio-enzyme AST and LDH. There was no significant difference in cell adherence and viability before and after induction. Both hMSCs and induced-hNSCs kept stable normal diploid nucleus. Conclusion The optimal condition for inducing effect of 5-azacytidine is 10μmol/L and 24-hour incubation; and under this condition, the adherence, vigor and chromosome karyotype of hMSCs would not be affected.