Isolation of human umbilical cord mesenchymal stem cells and their differentiation into dopaminergic neurons / 中华神经医学杂志

ABSTRACT

Objective To investigate the methods of isolation of human umbilical cord mesenchymal stem cells (MSCs) in Wharton' s jelly and the differentiation of MSCs into dopaminergic neurons. Methods The umbilical cord mesenchymal tissue was scraped off from the Wharton's jelly,and then, collagenase Ⅳ was employed to isolate the MSCs. The isolated cells were primarily cultured in DMEM/F12 medium containing 10% FBS. Inverted microscopy was used to observe the cytomorphology, and flow cytometry was employed to detect the cell surface antigens and the cell cycle.We evaluated the cell viability using CCK8 kit. Two-step method was employed to induce the MSCs of the P3 generation to differentiate into dopaminergic neurons, and immunocytochemistry and Western blotting were used to detect the expressions of neuron specific enolase (NSE) and tyrosine hydroxylase (TH) 3, 6 and 9 d after the induction. Results The isolated MSCs showed fibroblast-like shape, with parallel arrangement and vortex-like growth. MSCs of the P3 generation expressed CD73, CD29, CD44and CD105, but did not express CD34, CD45, CD106 and HLA-DR. The doubling time in the exponential phase was at the 48th h of culture, and 91.13% cells were under G0-G1. These cells had similar morphology of the neurons. The immunocytochemical assay showed that the NSE and TH positive rates were 19.5% and 8.9% on the 9th d of induction; and Western blotting showed that MSCs obviously expressed NSE and weakly expressed TH.Conclusion MSCs can be isolated from the umbilical cord mesenchymal tissue, and be induced to differentiate into dopaminergic neurons in vitro.