Study On Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Neuronlike Cells By GM1 And Growth Factors

Objectives Bone marrow mesenchymal stem cells(BMSCs) are a group of pluripotent stem cells which can continue to self-renew, proliferation and have multi-lineage differentiation potential. BMSCs effectively differentiate into neuron-like cells by basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF), and growth factors have little toxic side effects on cells. Monosialotetrahexosyl ganglioside(GM1) is important component of neurons membrane, and it is essential for the differentiation and maturation of neurons. Meanwhile, damaged neurons can be protected and repaired by GM1. This study is designed to approach a way to more efficiently induce BMSCs into neuron-like cells by GM1 and growth factors differentiatly and supply experimental evidence for cell replacement therapy to neurodegenerative disease.Methods Bone marrow mononuclear cells were obtained by density gradient centrifugation, then primary culture and subculture was proceeded, and the morphological changes of cells were observed. Whereafter, surface markers(CD29,CD105,CD34,CD45) of the 3rd passage BMSCs were identified by flow cytometry. Meanwhile, pluripotency of BMSCs were identified. Subsequently, BMSCs were divided into four group in order to induce into neuron-like cells with different inducers. Group A used growth factors(bFGF+EGF) alone, group B used GM1 alone, group C used GM1 and growth factors(bFGF+EGF) and group D acted as a control group. Respectively, the positive rate of Nestin, β-Tubulin Ⅲ and GFAP were detected by immunocytochemistry before and after induction and the data is statistically analyzed by SPSS18.0.Results After bone marrow mononuclear cells inoculated for 24 h, most cells had been adherent. Then the first full dose exchanged for 48 h, and we saw that several bone marrow mononuclear cells were scattered. On the 5th to 6th day for primary culture, short spindle cells, polygonal cells and squamous cells of colonies were formed. On the 8th to 11 th day, the cells growed faster and became fusiform. On the 14 th to 16 th day, the cells spreaded to 80%- 90% of the bottom of the culture bottle with swirling growth. The morphology of the passage cells were similar to primary cells, but the latent period of growth was much shorter. The results of the 3rd passage BMSCs of surface markers were CD29(+), CD105(+), CD34(-), CD45(-) by flow cytometry. Meanwhile, the BMSCs could be induced into osteoblasts and adipocytes. Before BMSCs were induced into neuron-like cells, the rate of Nestin, β-TubulinⅢ and GFAP were negative in four group. On the 2nd to 4th day for induction, the Nestin positive rate of group A, B and C showed an upward trend, and that group C was much higher than group A, B and D. On the 6th to 8th day for induction, the Nestin positive rate of group A, B and C decreased, however, the β-TubulinⅢand GFAP positive rate showed a significantly upward trend. and that group C was higher than group A and B. All in all, the Nestin, β-TubulinⅢand GFAP positive rate of group C were higher than group A, B and D.Conclusion 1. By using combination of density gradient centrifugation and adherent cultivation approach, high purity BMSCs can be obtained, and the viability of cells is high, so the cells are suitable for further experimental research. 2. GM1 and growth factors(bFGF+EGF) can more effectively induce BMSCs into neuron-like cells in comparison with growth factors(bFGF+EGF) or GM1 alone.