The Role And Mechanism Of Wnt/β-Catenin Signaling Pathway In Regulating Differentiation Into Neural Stem Cells From Induced Pluripotent Stem Cells In Vitro

Objective Mouse induced pluripotent stem cells(iPS cells)were induced to differentiate into neural stem cells in vitro.Meantime,the expression changes of Wnt/β-catenin signaling pathway’s related factors were researched in the process of neural differentiation to explore its role and the mechanism.Methods Firstly,the cultivation and identification of mouse iPS cells:the cells were identified by alkaline phosphatase staining,qRT-PCR,immunofluorescence and embryoid bodies forming experiment method after cultivation in vitro.Secondly,the induced neural differentiation system was set up,and the expression levels of Wnt/β-catenin signaling pathway’s related factors were researched:Mouse iPS cells were induced to differentiate into neural stem cells in vitro by N2B27 medium and RA,and the expression levels of Nestin,Sox2 and βⅢ Tubulin in neural stem cells derived from iPS cells(iPS-NSCs)were detected,the ability of proliferation and differentiation in vitro of iPS-NSCs were evaluated as well;the expression changes of the specific markers of neural stem cells Nestin and Sox2,the specific marker of early neuron βⅢ Tubulin and pluripotency factor Oct3/4 were researched,as well as the Wnt/β-catenin signaling pathway’s related factors β-catenin,Axin2 and Wnt3a in the process of neural differentiation.Thirdly,the role and the mechanism of Wnt/β-catenin signaling pathway in the process of neural differentiation of mouse iPS cells:the cells were divided into normal control group,Wnt3a group,DKK-1 group,CHIR99021 group and IWR-1 group to regulate the Wnt/β-catenin signaling pathway in the process of neural differentiation;meantime,the expression level changes ofβ-catenin and Nestin were investigated in each group;the proliferation of iPS-NSCs in each group were assessd using suspension culture method after regulating Wnt/β-catenin signaling pathway in the process of neural differentiation to study its role in iPS-NSCs’proliferation.Results Firstly,the cultivation and identification of mouse iPS cells:the cells exhibited clonal-generating capability,alkaline phosphatase positive expression,high expression five pluripotency related factor Oct3/4,Sox2,c-Myc,Klf4 and Nanog(P<0.05),could form embryoid bodies which could express three layers’ proteins at the same time,which meaned the cells had the multipotent differentiation potential.Secondly,the induced neural differentiation system was set up,and the expression levels of Wnt/β-catenin signaling pathway’s related factors were researched:the iPS-NSCs attained by N2B27 medium and RA induction expressed Nestin,Sox2 and βⅢ Tubulin,and had the ability of proliferation and differentiation in vitro;the expression of neural stem cells marker Nestin and early neuron specific marker βⅢ Tubulin both increased,and the pluripotency factor Oct3/4 decreased in the process of neural differentiation(P<0.05);the expression of Wnt/β-catenin signaling pathway’s related factors β-catenin and Axin2 both decreased(P<0.05).Thirdly,the role and the mechanism of Wnt/β-catenin signaling pathway in the process of neural differentiation of mouse iPS cells:Wnt3a treatment could up-regulate the expression of β-catenin,and DKK-1 and IWR-1 treatment could down-regulate it(P<0.05);the expression of Nestin decreased after Wnt3a treatment,and the expression of Nestin increased after DKK-1 and IWR-1 treatment(P<0.05);the proliferation ability of iPS-NSC were weaker after Wnt3a treatment,the cells occurred serious apoptosis after CHIR99021 treatment,and iPS-NSCs had stronger proliferation ability after DKK-1 and IWR-1 treatment in the process of neural differentiation.Conclusion Firstly,mouse iPS cells exhibited clonal-generating capability,alkaline phosphatase positive expression,high expression five pluripotency related factor Oct3/4,Sox2,c-Myc,Klf4 and Nanog,had the multipotent differentiation potential,and cultured cells could stably grew and passage using the cultivation system in this study.Secondly,mouse iPS cells could be successfully induced to differentiate into neural stem cells by N2B27 medium and RA,the iPS-NSCs had the ability of proliferation and tend to differentiate into neurons.Thirdly,the Wnt/β-catenin signaling pathway’s related factorsβ-catenin and Axin2 were high expression in mouse iPS cells,and decreased in the process of neural differentiation,suggesting that Wnt/β-catenin signaling pathway was inhibited.Fourthly,the differentiation efficiency of neural differentiation reduced after Wnt3a treatment to up-regulate Wnt/β-catenin signaling pathway in the process of neural differentiation,and increased after DKK-1 and IWR-1 treatment to down-regulate Wnt/β-catenin signaling pathway in the process of neural differentiation,which showed Wnt/β-catenin signaling pathway play a negative regulation role in neural differentiation of mouse iPS cells.Fifthly,the proliferation ability of iPS-NSCs were weaker after Wnt3a treatment to up-regulate Wnt/β-catenin signaling pathway in the process of neural differentiation,and iPS-NSCs had stronger proliferation ability after DKK-1 and IWR-1 treatment in the process of neural differentiation,inducating Wnt/β-catenin signaling pathway also could down-regulate the proliferation ability of iPS-NSCs.