Abstract: Objective To evaluate the capacity and efficiency of human umbilical cord mesenchymal stem cells (HUCMSCs) to differentiate into neuron- like cells after induction with B27- supplemented serum- free medium. Methods HUCMSCs at passage 4 were cultured for 14 days with serum-containing medium (SCM) (group A), SCM supplemented with 20 ng/mL nerve growth factor (NGF) and 10 ng/mL basic fibroblast growth factor (bFGF) (group B), serum-free medium (SFM) (group C), or SFM supplemented with 20 ng/mL NGF and 10 ng/mL bFGF. The culture medium were changed every 3 days and the growth of the neurospheres was observed using an inverted microscope. The cell markers were analyzed with flow cytometry and the expressions of nestin, neuron- specific enolase (NSE), neurofilament heavy polypeptide (NEFH), and glial fibrillary acidic protein (GFAP) were quantified by quantitative real-time PCR (qRT-PCR) and Western blotting. Results Before induction, HUCMSCs expressed abundant mesenchymal stem cell surface markers including CD29 (99.5%), CD44 (49.6%) and CD105 (77.7%). Neuron-like cells were observed in the cultures on days 7, 10, and 14, and the cell differentiation was the best in group D, followed by groups C, B and A. In all the 4 groups, the cellular expressions of nestin and GFAP gradually lowered while those of NEFH and NSE increased progressively. The expressions of GFAP, NEFH, nestin and NSE were significantly different between group A and the other 3 groups (P<0.001 or 0.05). Conclusion B27-supplemented SFM effectively induces the differentiation of HUCMSCs into neuron- like cells, and the supplementation with cytokines (NGF and bFGF) strongly promotes the cell differentiation.

Key words: human umbilical cord mesenchymal stem cells, neuron-like cells, differentiation, serum-free medium