Chemical-induced Differentiation Of Bone Mesenchymal Stem Cells Into Neurons In Vitro

Bone marrow mesenchymal stem cells(BMSCs),as a kind of bone marrow-derived pluripotent stem cells,can differentiate into myocardium,adipocytes,osteoblasts or neuron-like cells in certain conditions.BMSCs are ideal seed cells with wide origin,strong proliferative ability and no ethical controversy.One of their important application prospects is the treatment of neurological disease in replacement of neural stem cells which was limited in source.In this study,we cultured and identificated rat bone marrow-derived mesenchymal stem cells,differentiated BMSCs into neuron-like cells by chemical-induced metheads and researched their sustained differentiation.We also obtained highly purified BMSCs by means of density gradient centrifugation and flow cytometry.These work laid a foundation for the clinical application of bone marrow mesenchymal stem cells in the future.We first compared the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells by two chemical-induced methods: ?-mercaptoethanol(BME)and butylhydroxyanisole(BHA).After cultured into the 4th generation,BMSCs expressed CD44 and CD90 in mass,while CD45 and CD11 b in small number which mainly expressed on hematopoietic cell lines.Being chemical-induced by two methods,the BMSCs became smaller and rounded gradually,formed neuron-like cells with bipolar or multipolar morphology,and expressed nerve cell-related markers of nestin(Nestin)?Glial fibrillary acidic protein(GFAP)and neuron-specific enolase(NSE),but expression level of which in two methods was not consistent.The expression level of GFAP and NSE protein in BHA-induced group was higher than that in BME-induced group,which indicated that BME might be more likely to induce BMSCs differentiating into neuronal precursor stem cells.In the continue culturing process after induced differentiation completely,the expression level of GFAP gene in the BHA-induced group and the Nestin gene in the BME-inducedgroup both decreased gradually,and the morphology of the neuron-like cells was lost.This suggests that the effect of chemical differentiation may be reversible and unsustainable.In addition,we rapidly separated and obtained highly purified rat bone marrow mesenchymal stem cells by density gradient centrifugation and flow cytometry.Firstly bone marrow mesenchymal stem cells were isolated from the rat bone marrow by Ficoll separation;the BMSCs,with many miscellaneous cells,were adhered and growed in community after 5 days of culturing.On the 7th day,the first generation of BMSCs was passeged.Then the P2 BMSCs were labeled with CD271,and the CD271 positive cells were sorted by MoFlo XDP ultrahigh velocity flow cytometry.In this study,we compared the expression level of marker genes in rat bone marrow mesenchymal stem cells after differentiation by two chemical methods,and explored the rapid isolation and culturing methods of highly purified bone marrow mesenchymal stem cells.