The Study On Differentiation Of The Human Umbilical Cord Stem Cells Into Neuron-like Cells

BackgroundAs the intense research of HUCMSCs, more and more results show that HUCMSCs can differentiate into neuron-like cells successfully, and it plays a great role in animal models of nervous system diseases. But there are still a lot of problems:the biological characteristics of HUCMSCs are not very clear; although a variety of induction methods can obtain s neuron-like cells successfully, the induction efficiency is not higher, and it exists a great limit in acquisition of a single species of neurons; induction of HUCMSCs was not clear in the mechanism of neuron-like cells, stem cell therapy of the central nervous system disease is still in the experimental animals.ObjectivesIn order to obtain the neuron-like cells from the HUCMSCs and compare the effects of different induction methods, HUCMSCs are treated by three different induction methods, this may provide evidence for the treating of the neurodegenative diseases using HUCMSCs-derived neuron-like cells.Methods1. Culture and identification of the HUCMSCs:HUCMSCs were cultured in DMEM+10%FBS for proliferation and passage. When conflucency reach to80-90%, the cells were passaged according to1:2or1:3. The phenotype identification of HUCMSCs was detected by Flow cytometry (FCM).2. The induction of HUCMSCs:three induction methods were used to differentiate the HUCMSCs into neuron-like cells:(1) low sugar DMEM+10%FBS+20%Human fetal brain tissue extracts(HFBTE);(2) bovine neuronal stem cell culture medium+20%HFBTE;(3)low sugar DMEM+10%FBS+bFGF+DMSO+insulin+EGF.3. The expression of neuronal markers and neurotrophic factors were detected by immunofluorescence, RT-PCR and Western blotting in HUCMSCs.Results 1. The results of FCM showed that the expression of CD73, CD90and CD105in HUCMSCs are positive (>95%), and the expression of CD14, CD34, CD45, CD79a, HLA-DR are negative (<2%).2. The results of RT-PCR and Western blotting showed that HUCMSCs can express neuronal markers NSE, GFAP, and dopamine neurons transcription factors Nurrl, Wnt-1and En-1.3. The results of RT-PCR demonstrated that HUCMSCs can express neurotrophic factors BDNF, GDNF, VEGF, LIF, NGF and NT-3.4.Western blotting showed that, compared to untreated the HUCMSCs (control group), HUCMSCs were treated by DMEM+10%FBS+20%HFBTE for3days and5days, neuronal markers NSE, GFAP, beta3-tubulin, DAT and TH increased obviously; the results of Immunofluorescence showed that HUCMSCs were treated by DMEM+10%FBS+20%HFBTE for3days, the positive rate of NSE is18.32±4.07%, GFAP is16.94±7.56%, Nestin is14.47±3.20%, LMX1B is7.17±3.28%, DBH is1.81±1.07%, DAT is5.65±0.93%; at day5, the positive rate of NSE is22.08±2.75%, GFAP is17.83±5.55%, Nestin is23.82±6.05%, LMX1B is11.98±2.71%, DBH is11.83±2.75%, DAT is13.86±5.50%respectively.5. RT-PCR and Western blotting detection showed that, HUCMSCs were treated by bovine neuronal stem cell culture medium+20%HFBTE for3days and5days, the expression of neuronal markers (Nestin, NSE, S100B, En-1) and neurotrophic factor (BDNF, GDNF, VEGF, LIF, NGF and NT-3) increases obviously;6. Similarly, low sugar DMEM+10%FBS+insulin+bFGF+DMSO+EGF induced HUCMSCs for7days, the expression of neuronal markers (GBX2, GFAP and DAT) and neurotrophic factors (BDNF, GDNF, VEGF, LIF, NGF and NT-3) increase significantly.ConclusionsNeuron-like cells were obtained from the HUCMSCs treated by low sugar DMEM+10%FBS+20%HFBTE, low sugar DMEM+20%HFBTE and low sugar DMEM+10%FBS+bFGF+DMSO+insulin+EGF, which provides evidences for further clinical application.