A Method for In Vitro Differentiation of Rabbit Adi-pose Derived-Mesenchymal Stromal/Stem Cells into Cardi-omyocyte-Like Cells

Objective: Cardiac diseases such as acute myocardial infarc-tion and ischemic cascade can rapidly develop and finally result in the loss of cardiomyocytes and congestive heart failure and are a reason for many deaths every year worldwide. Mesen-chymal stromal/stem cells (MSCs) can differentiate into mes-enchymal lineages, including cardiac cell types. This capacity is a promising therapeutic strategy for cell-based therapies to replace current invasive techniques.Materials and Methods: Different chemical substances, growth factors, and cytokines regulate the cardiomyocytes dif-ferentiation. We investigated the effect of new combination of differentiation materials on the third-passage of MSCs derived from adipose tissue. Firstly, these cells were pre-treated with ۵-Azacytidine which is an important chemical inducer for de-methylation of DNA, in concentration of ۵ and ۱۰ µM for two days respectively. Then cells were cultured with ۱۰ ng/ml basic fibroblast growth factor (bFGF) and ۵۰ µg/ml ascorbic acid for ۲۱ days to induce cardiomyocytes differentiation of MSCs. For characterization, quantitative real-time PCR (qRT-PCR) and Flow cytometery were performed.Results: Morphologically, cardiomyocytes are observed as rod-shaped cells that attach to adjacent cardiomyocytes via phase contrast light microscopy. Flow cytometery analysis displayed that increased expression cardiac troponin T and connexin ۴۳/ GJA۱ and quantitative real-time PCR analysis showed increased expression of cardiac markers such as GATA۴, NKX۲.۵, desmin and cardiac intracellular gap junction protein (connexin ۴۰ and ۴۳) when compared with negative control.Conclusion: Our results demonstrated that this differentia-tion protocol (۵-azacytidine combined with ascorbic acid and bFGF) improve cardiomyocyte-like cells differentiation from MSCs. Stem cell differentiation-based therapies have emerged as a promising therapeutic tool for improving cardiac regenera-tion and function.