Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (40): 7597-7600.doi: 10.3969/j.issn.1673-8225.2010.40.043

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Myogenic differentiation factors and 5-azacytidine induce the differentiation of rat bone marrow mesenchymal stem cells into skeletal muscle cells in vitro　

Chen Zhen-qiang1, Sun Zhan-sheng2, Zhi Wei3   

  1. 1 Department of Orthopedics, Liaocheng People’s Hospital, Liaocheng   252000, Shandong Province, China; 2 Department of Orthopedic Trauma, Shandong Provincial Hospital, Jinan   250021, Shandong Province, China; 3 Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu   610000, Sichuan Province, China
  • Online:2010-10-01 Published:2010-10-01
  • Contact: Sun Zhan-sheng, Chief physician, Master’s supervisor, Department of Orthopedic Trauma, Shandong Provincial Hospital, Jinan 250021, Shandong Province, China
  • About author:Chen Zhen-qiang★, Master, Associate chief physician, Department of Orthopedics, Liaocheng People’s Hospital, Liaocheng 252000, Shandong Province, China
  • Supported by:

    the Natural Science Foundation of Shandong Provice, No.Y2003C35*

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Abstract:

BACKGROUND: Muscle transposition is a conventional method to treat muscle tissue defects, but it results in damage to another piece of muscle. For this reason, we designed this study to search for a method to in situ repair muscle tissue defects.
OBJECTIVE: To investigate the conditions for in vitro induced differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into skeletal muscle cells.
METHODS: Following isolation and culture, passage 3 BMSCs were induced to differentiate in vitro by a combination of 5-azacytidine, myogenic differentiation factor, transforming growth factor β1, and insulin like growth factor. At 9 days after induction, cells were harvested and identified by immunohistochemistry.
RESULTS AND CONCLUSION: Primary cultured BMSCs exhibited an adherent, colony-like growth. After 5-7 days, multi-synaptic cells, thin and flat polygonal cells, polygonal cells, and triangle-shaped cells were observed. After 12 days, cells confluenced and covered the whole bottom of culture flask, with slightly altered morphology of BMSCs. After 5-azacytidine induction, some cells died and grew slowly. After 7 days, cells markedly grew and soma was gradually enlarged, presenting with an oval, spindle-shaped, or irregular appearance. After 14 days, spindle-shaped cells become more. After 18-22 days, myotubes were increased in number and enlarged in volume, and myotube nucleuses were also increased. The newly formed myotubes and spindle-shaped fibroblasts were distributed in parallel interval. The immunohistochemistry of BMSCs revealed that cells were positive for CD44, with dark brown granules in the cytoplasm, especially around the nucleus, but they were negative for CD34. The immunohistochemistry of induced BMSCs demonstrated that cells were positive for desmin and skeletal muscle myosins. These findings indicate that myogenic differentiation factors and 5-azacytidine could induce the oriented differentiation of BMSCs into skeletal cells, with the presence of positive expression of desmin and skeletal muscle myosins.

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