Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (7): 1019-1025.doi: 10.3969/j.issn.2095-4344.2166

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Mechanism of bone marrow mesenchymal stem cells differentiation into functional neurons induced by glial cell line derived neurotrophic factor

Zhu Xuefen1, Huang Cheng1, Ding Jian1, Dai Yongping1, Liu Yuanbing1, Le Lixiang1, Wang Liangliang1, Yang Jiandong2   

  1. 1Rugao People’s Hospital, Rugao 226500, Jiangsu Province, China; 2Department of Spine Surgery, Northern Jiangsu People’s Hospital, Yangzhou 225000, Jiangsu Province, China
  • Received:2020-02-12 Revised:2020-02-22 Accepted:2020-04-03 Online:2021-03-08 Published:2020-12-08
  • Contact: Yang Jiandong, Chief physician, Associate professor, Master’s supervisor, Department of Spine Surgery, Northern Jiangsu People’s Hospital, Yangzhou 225000, Jiangsu Province, China
  • About author:Zhu Xuefen, Rugao People’s Hospital, Rugao 226500, Jiangsu Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81071466; the Science and Education Leading Talents Program for Invigorating Health of Yangzhou during 13th Five-Year Plan Period, No. [2018]6; the Science and Technology Program of Nantong City, No. GJZ16044; the Science and Technology Program of Rugao City, No. SRG(15)3015

Abstract: BACKGROUND: Glial cell line derived neurotrophic factor (GDNF) plays an important role in inducing the differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro and promoting the regeneration of neuron axons.
OBJECTIVE: To observe BMSCs differentiation induced by over-expression of GDNF gene, detect synaptic function of cells and expression of Wnt signaling pathway components after differentiation, and preliminarily explore the mechanism of BMSCs differentiation into mature neurons.
METHODS:  Rat BMSCs were isolated and cultured, and further divided into recombinant adenovirus-containing GDNF gene transfection group (Ad-GDNF-BMSCs), adenovirus transfection control group (Ad-BMSCs), and untransfected control group. The relative expression of GDNF gene in BMSCs of each group was detected by Q-PCR, and the expression of β-catenin, cyclin D1, NeuN and MAP-2 was detected by immunofluorescence technology in each group. High K+ stimulation induced cell depolarization response after differentiation, and FM4-64 marks synaptic vesicle activity of differentiated cells.
RESULTS AND CONCLUSION: (1) The adenovirus-transfected gene had no significant negative effect on the proliferation of BMSCs. BMSCs could express endogenous GDNF gene continuously and at high levels. (2) Under the induction of GDNF gene, BMSCs could express neuron-specific protein NeuN after 3 days cultivation in vitro. The expression of β-catenin protein also could be detected in the cytoplasm of cells. After 7 days cultivation in vitro culture, BMSCs expressed the mature neuronal marker protein MAP-2, and the cell body contracted significantly. Neuron axon-like structures appeared around the cell body. Moreover, β-catenin and cyclin D1 were respectively detected in the cell cytoplasm and the nucleus, while the expression levels of NeuN, MAP-2, β-catenin, and cyclin D1 were not observed in Ad-BMSCs and untransfected control groups, and the cells remained spindle-shaped. (3) After 11 days of in vitro culture, the cells in the Ad-GDNF-BMSCs group showed typical neuronal processes or axons and connected to each other into a network structure, which could be labeled with FM4-64 to show red fluorescence, and induced by high K+ stimulation to induce action potentials in the cells. Synaptic vesicle activity in posterior axons showed FM4-64 red fluorescence gradually decaying. Under the same conditions, cells in the Ad-GDNF-BMSCs group and untransfected control group did not present FM4-64 fluorescently labeled synaptic vesicle activity. (4) Continuous GDNF induction can promote BMSCs differentiated into mature neurons with synaptic cycle function, and may be carried out through the classic Wnt/β-catenin signaling pathway.

Key words: stem cells, bone marrow mesenchymal stem cells, neurons, factors, synapses, axons, pathways

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