Myogenic differentiation factors and 5-azacytidine induce the differentiation of rat bone marrow mesenchymal stem cells into skeletal muscle cells in vitro / 中国组织工程研究

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 calls(BMSCs)into skeletal muscle cells.METHODS:Following isolation and culture,passage 3 BMSCs were induced to differentiate in vitro by a combination of5-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 calls,thin and fiat polygonal cells,polygonal cells,and triangle-shaped cells were observed.After 12 days,calls confluenced and covered the whole bottom of culture flask,with slightly altered morphology of BMSCs.After 5-azacytidine induction,some calls 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 calls 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 calls 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.