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,1
* School of Life Science, Nanjing Normal University, Nanjing, Jiangsu, 210097, China; and School of Medical Technology, Jiangsu University, Zhenjiang, Jiangsu, 212001, China
To whom requests for reprints should be addressed at 1 School of Life Science, Nanjing Normal University, Nanjing, Jiangsu, 210097, China. E-mail: icls{at}ujs.edu.cn
A method for isolating adult human bone marrow mesenchymal stem cells (MSCs) was established, and the ability of human MSCs to differentiate into cells with characteristics of cardiomyocytes in vitro was investigated. Selected MSC surface antigens were analyzed by flow cytometry. The MSCs at Passage 2 were treated with 5-azacytidine to investigate their differentiation into cardiomyocytes. Characteristics of the putative myogenic cells were determined by immunohistochemistry and transmission electron and confocal microscopies. The expression of myogenic specific genes was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR, and DNA sequencing. The MSCs were spindle-shaped with irregular processes and were respectively positive for CD13, CD29, CD44, CD71 and negative for CD3, CD14, CD15, CD33, CD34, CD38, CD45, and HLA-DR. The myogenic cells differentiated from MSCs were positive for beta-myosin heavy chain (beta-MHC), desmin, and alpha-cardiac actin. When the myogenic cells were stimulated with low concentration of K+ (5.0 mM), an increase in intracellular calcium fluorescence was observed. Myofilament-like structures were observed in electron micrographs of the differentiated myogenic cells. The mRNAs of beta-MHC, desmin, alpha-cardiac actin, and cardiac troponin T were highly expressed in the myogenic cells. These results indicate that 5-azacytidine can induce human MSCs to differentiate in vitro into cells with characteristics commonly attributed to cardiomyocytes. Cardiomyocytes cultured from bone marrow sources are potentially valuable for repairing injured myocardium.
Key Words: mesenchymal stem cells • differentiation • cardiomyocytes
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