Valproic Acid Enhances Reprogramming Efficiency And Neuronal Differentiation On Small Molecules Staged-Induction Neural Stem Cells:Suggested Role Of MTOR Signaling

BackgroundThe discovery of neural stem cells(NSCs)provides a new approach of cell therapy for many neurological diseases with loss of neuron.However,current research is concerned with the problem of the source of NSCs.The research on the source of exogenous neural stem cells is mainly focused on somatic-induced NSCs,which are safe and efficient sources of NSCs because of their short induction period and low risk of tumor formations without pluripotent stem cells.The inhibition or activation of specific cellular pathways by small molecules(SM)induces somatic cells to be NSCs,which is currently the most promising source of NSCs.Our research screened seven small molecules through literature reading and previous experiments,and found that adding seven small molecules together would cause cell death.Therefore,we further consulted the literature,combining the rules of somatic cell reprogramming to NSCs,the characteristics of SM related pathways and the results of previous experiments.We used different SMs at different stages.to reduce the number of SMs at each stage and improve the induction efficiency to obtain small molecules staged-induction neural stem cells(SMSINS).In addition,removing one of SMs in the induction program,we found that valproic acid(VPA)showed the most important role in enhancing the induction efficiency of NSCs and neuronal differentiation,possibly with VPA activated mTOR signaling.Therefore,we hypothesized that VPA can significantly increase the induction efficiency of induced neural stem cell(iNSCs)and neuronal differentiation by the activation of mTOR signaling.Objectives1.Explore the induction program of small molecules and obtain a safe and efficient source of NSCs.2.Explore the role and mechanism of VPA regulation of mTOR signaling on induced neural stem cells.MethodsPrimary mouse embryonic fibroblasts(MEFs)were extracted and induced with the induction protocol(MEFs were treated with LDN193189,SB431542 on dayl and day2.CHIR99021,DAPT,and VPA were used on day3 and day4.CHIR99021,DAPT were used on day5 and day6.Shh and Purmorphamine were used to improved neural differentiation on day7 and day8.Then,all small molecules were withdrawn on the last 2 days.).Thereafter,immunostaining and flow cytometry were used to detect expression of NSCs marker protein,and the ability of SMSINS to differentiate into neurons,astrocytes,and oligodendrocytes was identified.Cell was divided into the following groups:SMSINS,-VPA,SMSINS+rapamycin and-VPA+MHY1485.Proliferative capacity,expression level of mTOR signaling,NSCs marker protein and neuron marker protein were detected by CCK8,Western blot and immunofluorescence,respectively.Results1.SMSINS could express the typical NSC markers Nestin and Sox2,and stably passage.It has the ability to spontaneously differentiate and induce differentiation into neurons,astrocytes and oligodendrocytes in vitro,as well as induce differentiation into dopaminergic,cholinergic and GABAergic neurons.2.For the role of each SM in the induction efficiency,the effect of VPA was most significant,followed by DAPT,LDN193189,Purmorphamine/Shh,CHIR99021 and SB431542.3.VPA could activate mTOR signaling.Inhibition of mTOR signaling reduced induction efficiency and neuronal differentiation,but activated mTOR signaling partly increased them.Conclusions1.MEFs can be induced into NSCs by the method of small molecules stage induction.2.The VPA-mediated mTOR signaling pathway may enhance reprogramming efficiency and neuronal differentiation of SMSINS.