Construction And Identification Of BDNF Overexpressed Human Umbilical Cord Mesenchymal Stem Cell-derived Dopaminergic-like Neurons

BackgroundParkinson's disease(PD)is the second most common neurodegenerative disease of the central nervous system with incidence second only to Alzheimer's disease(AD).It is mainly characterized by motor symptoms such as bradykinesia,resting tremor,rigidity and postural instability.In the late stage of PD,cognitive impairment and even dementia may occur,leading to a decline in living ability or even disability,bringing a heavy burden to families and society.However,current treatments can only improve symptoms but can't delay or block the progressive degeneration of dopaminergic neurons.Human umbilical cord mesenchymal stem cells(h UC-MSCs)have significant potentials of proliferation,self-renewal and high differentiation,which can be induced to differentiate into neurons and glial cells.Thus,h UC-MSCs have been widely used in the stem cell-based therapy of neurodegenerative diseases because of their characteristics such as abundant sources,easy separation,easy culture,immunoregulation,paracrine,low immunogenicity,non-tumorigenicity,and limited ethical issues.Studies have shown that h UC-MSCs can be induced to differentiate into mature dopaminergic-like neurons in vitro,and h UC-MSCs-derived dopaminergic-like neurons transplantation is better than simple h UC-MSCs transplantation.Therefore,h UC-MSCs-derived dopaminergic neuron transplantationmight be one of the ideal treatments for PD cell replacement therapy.Studies have shown that only 5%-10% of transplanted dopaminergic cells can survive during transplantation [1],which is far from sufficient to produce a significant therapeutic effect on PD.Therefore,how to promote the survival of transplanted neurons has become an urgent and difficult problem to be solved in the cell replacement therapy of PD.Studies have shown that transplantation of h UC-MSCs modified by vector-mediated gene transfection with fibroblast growth factor-20 or human vascular endothelial growth factor can significantly improve the behavioral symptoms in PD model rats,which is more effective than h UC-MSCs transplantation alone[2,3],suggesting that certain gene-transduced h UC-MSCs can produce better therapeutic results.Brain-derived neurotrophic factor(BDNF)is one of the most common neurotrophic factors in the human brain.It binds tyrosine kinase B(Trk B)with high affinity,stimulates the mechanism of pro-survival,and plays important roles in the survival,development,proliferation and differentiation of neurons.Meanwhile,BDNF can promote the differentiation of stem cells into neurons and promote their maturation.However,the effect of BDNF overexpressed h UC-MSCs-derived dopaminergic-like neurons on PD is still unclear.ObjectiveTo explore the possible role and mechanism of BDNF overexpressed h UC-MSCs-derived dopaminergic-like neurons in vitro.MethodsThe MSCs in the umbilical cord tissue of healthy full-term cesarean section were isolated and subcultured by tissue block method,and the P3 generation cellswere induced to differentiate into dopaminergic-like neurons.The expression of Neu N and ?-tubulin-III and the co-expression of TH and Nurr1 were detected by immunofluorescence assay.h UC-MSCs-derived dopaminergic-like neurons were transfected with lentivirus-mediated green fluorescent protein(GFP)-empty vector,GFP-BDNF overexpressing vector and BDNF si RNA,respectively.The experiments were divided into five groups: h UC-MSCs group,dopaminergic-like neurons group,dopaminergic-like neurons + empty vector group,dopaminergic-like neurons +overexpressing BDNF vector group,dopaminergic-like neurons + BDNF si RNA group.The expression of GFP and the co-expression of Nurr1 and TH in each group were examined by immunofluorescence assay.Western blot was used to detect the expression of TH,Nurr1,PI3 K,Akt,p-Akt,Tr KB and BDNF in each group.Results1.Immunofluorescence showed that the expression of Neu N and ?-tubulin-III after induction increased significantly than that before induction(P<0.01).The double-labeled co-expression of TH and Nurr1 after induction also increased significantly than that before induction(P < 0.01).2.Results from immunofluorescence suggested that cells in the dopaminergic-like neurons + empty vector group and the dopaminergic-like neurons + overexpressing BDNF vector group showed obvious expression of GFP.No GFP expression was observed in the h UC-MSCs group,the dopaminergic-like neurons group and the dopaminergic-like neurons + BDNF si RNA group.3.Results showed that the co-expression of TH and Nurr1 in the dopaminergic-like neurons group,the dopaminergic-like neurons + empty vector group,the dopaminergic-like neurons + BDNF group,the dopaminergic-like neurons + BDNF si RNA group was significantly higher than that in the h UC-MSCs group(P<0.01).There was no significant difference of the co-expression of TH and Nurr1 between the dopaminergic-like neurons group and the dopaminergic-like neurons + empty vector group.The co-expression of TH and Nurr1 in the dopaminergic-like neurons +BDNF group was significantly higher than that in the dopaminergic-like neurons,the dopaminergic-like neurons + empty vector group and the dopaminergic-like neurons+ BDNF si RNA group(P<0.01).The co-expression of TH with Nurr1 in the dopaminergic-like neurons + BDNF si RNA group was significantly lower than that in the dopaminergic-like neurons and the dopaminergic-like neurons + empty vector group(P<0.01).4.Results from Western blot showed that the expression of TH,Nurr1,PI3 K,Akt,p-Akt and Tr KB in the dopaminergic-like neurons group,the dopaminergic-like neurons + empty vector group,the dopaminergic-like neurons + BDNF group,the dopaminergic-like neurons + BDNF si RNA group was significantly higher than that in the h UC-MSCs group(P<0.01).The expression of BDNF in the dopaminergic-like neurons group,the dopaminergic-like neurons + empty vector group,the dopaminergic-like neurons + BDNF group was significantly increased compared with that in the h UC-MSCs group(P<0.01).However,we observed no difference in the expression of BDNF between the h UC-MSCs group and the dopaminergic-like neurons + BDNF si RNA group.There was no significant difference in the expression of TH,Nurr1,PI3 K,Akt,p-Akt,Tr KB and BDNF between the dopaminergic-like neurons group and the dopaminergic-like neurons + empty vector group.The expression of TH,Nurr1,PI3 K,Akt,p-Akt,Tr KB and BDNF in the dopaminergic-like neurons+BDNF group was significantly higher than that in the dopaminergic-like neurons,the dopaminergic-like neurons + empty vector group and the dopaminergic-like neurons + BDNF si RNA group(P<0.01).The expression of TH,Nurr1,PI3 K,Akt,p-Akt,Tr KB and BDNF in the dopaminergic-like neurons +BDNF si RNA group was significantly lower than that in the dopaminergic-like neurons and the dopaminergic-like neurons +empty vector group(P<0.01).Conclusions1.h UC-MSCs can be successfully induced to differentiate into dopaminergic-like neurons in vitro.2.The overexpression of BDNF can promote the transformation and maturation of h UC-MSCs-derived dopaminergic-like neurons,which may be related to the up-regulatation of the PI3K/ Akt signaling pathway.