Differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells in vitro:Induction of Wnt3a signaling molecules / 中国组织工程研究

ABSTRACT

BACKGROUND:Wnt signaling pathway is the key to regulation of cell proliferation and differentiation.The evidence suggests that this pathway participates in the neural precursor cell proliferation,differentiation and determination of the regulation of cell fate.At present,the Wnt signaling pathway effects on the mesenchymal stem cells(MSCs)differentiated into neuron-like cells have not been reported.OBJECTIVE:To seek Wnt signal molecule that promotes the differentiation of MSCs into neuron-like cells.METHODS:MSCs were isolated from the femur of Sprague Dawley rats in vitro using the density gradient centrifugation,and then cultured.Following passage,cell surface markers CD29,CD44,CD34 and CD45 were measured using morphology and flow cytometry.These cells were selected and evaluated.Using neurotrophic factor and basic fibroblast growth factor combined with Wnt3a and Wnt5a induction scheme,effects of Wnt3a and Wnt5a during the differentiation of MSCs into neuron-like cells were compared utilizing immunohistochemistry and reverse transcription-polymerase chain reaction(RT-PCR).Simple culture of basic fibroblast growth factor served as controls.RESULTS AND CONCLUSION:Following culture and passage of MSCs,cells adhered to the wall,showing even spindle shape.Flow cytometry showed great expression of CD29 and CD44 and low expression of CD34 and CD45.Following Wnt3a induction,cells were positive for nestin,neuron specific enolase,but no significantly expressed glial fibrillary acidic protein.Following induction,cell viability was good.In the Wnt5a induction and control groups,weakly positive expression of nestin was found,but neuron specific enolase and glial fibrillary acidic protein were negative.RT-PCR results demonstrated that nestin expression was detected in the Wnt3a induction group before and after induction.Significant amplified bands for neuron specific enolase were detected at day 5 following induction,and became more significant at day 10.Weak bands for glial fibrillary acidic protein were visible at day 10 following induction.These indicated that Wnt3a can promote the differentiation of MSCs into neuron-like cells in vitro.