人脐带间充质干细胞经添加B27的无血清培养基诱导后分化成神经元样细胞

目的 评价人脐带间充质干细胞（HUCMSCs）经添加B27的无血清培养基诱导后向神经元样细胞分化的能力和效率。方法 将第4代HUCMSCs分为4组：A组：血清培养基（SCM）处理14 d；B组：给予SCM+20 ng/mL神经生长因子（NGF）+10 ng/L碱性成纤维细胞生长因子（bFGF）治疗14 d；C组：无血清培养基（SFM）处理14 d；D组：SFM+20 ng/mL NGF+10 ng/mL bFGF处理。每3 d更换各组细胞培养液。倒置显微镜观察神经球生长情况，流式细胞术分析细胞标记物。巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、人重肽神经丝蛋白（NEFH）、神经胶质纤维酸性蛋白（GFAP）通过qRT-PCR和Western blotting进行定量。结果 在诱导前，HUCMSCs表现出丰富的间充质干细胞表面标志物（CD29、CD44和CD105分别为99.5%、49.6%和77.7%）的表达。在第7、10、14天观察神经元样细胞的形成，D组最优，其次是C、B、A组。qRT-PCR和Western blotting结果显示，A、B、C、D组Nestin和GFAP表达逐渐减少，NEFH和NSE表达逐渐增加，A组与其他3组的GFAP、NEFH、Nestin和NSE表达差异有统计学意义（P<0.05）。结论 添加B27的SFM可以有效地将HUMSCs分化为神经元样细胞，添加细胞因子（NGF和bFGF）使分化效果更显著。

Human umbilical cord mesenchymal stem cells differentiate into neuron-like cells afterinduction with B27-supplemented serum-free medium

Objective To evaluate the capacity and efficiency of human umbilical cord mesenchymal stem cells (HUCMSCs) todifferentiate into neuron- like cells after induction with B27- supplemented serum- free medium. Methods HUCMSCs atpassage 4 were cultured for 14 days with serum-containing medium (SCM) (group A), SCM supplemented with 20 ng/mLnerve growth factor (NGF) and 10 ng/mL basic fibroblast growth factor (bFGF) (group B), serum-free medium (SFM) (groupC), or SFM supplemented with 20 ng/mL NGF and 10 ng/mL bFGF. The culture medium were changed every 3 days and thegrowth of the neurospheres was observed using an inverted microscope. The cell markers were analyzed with flow cytometryand the expressions of nestin, neuron- specific enolase (NSE), neurofilament heavy polypeptide (NEFH), and glial fibrillaryacidic protein (GFAP) were quantified by quantitative real-time PCR (qRT-PCR) and Western blotting. Results Beforeinduction, HUCMSCs expressed abundant mesenchymal stem cell surface markers including CD29 (99.5%), CD44 (49.6%) andCD105 (77.7%). Neuron-like cells were observed in the cultures on days 7, 10, and 14, and the cell differentiation was the bestin group D, followed by groups C, B and A. In all the 4 groups, the cellular expressions of nestin and GFAP gradually loweredwhile those of NEFH and NSE increased progressively. The expressions of GFAP, NEFH, nestin and NSE were significantlydifferent between group A and the other 3 groups (P<0.001 or 0.05). Conclusion B27-supplemented SFM effectively induces thedifferentiation of HUCMSCs into neuron- like cells, and the supplementation with cytokines (NGF and bFGF) stronglypromotes the cell differentiation.