Cardiomyocyte-like differentiation of human bone marrow mesenchymal stem cells after exposure to 5-azacytidine in vitro

Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073 g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnl), GATA4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34-CD45' CD31" CDlla'. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14 days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin, GATA4, cTnl and connexin-43 in 5,10 /.imol/L 5-aza groups, but no cardiac specific protein was found in neither 3 jumol/L 5-aza group nor in the control group. The ratio of cTnl positively stained cells in 10 jumol/L group was higher than that in 5 jumol/L group (65.3±4.7% vs48.2±5.4%, P < 0. 05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain ( MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration.