Generation Of Self-renewing Neural Stem Cells From Human Embryonic Stem Cells

Human embryonic stem cells(hESCs)can self-renew in vitro,and have the potential to give rise to any cell types of the human body.hESCs could be induced to differentiate into specific cells,which could be valuable cell source to treatment disease.Therefore,hESCs have important application in regenerative medicine.Many neurodegenerative diseases usually involved neuronal loss,such as dopaminergic neuron loss involved in Parkinson's disease(PD),and the damage of basal cholinergic neurons in the Alzheimer's disease(AD).Cell transplantation is expected to improve the pathological state of neurodegenerative diseases.The generation of specific neurons by directed differentiation from human pluripotent stem cells(hPSCs)will provide important support for cell therapy of neurodegenerative diseases.Small molecules that modulate stem cell fate and function offer significant opportunities to realize the therapeutic potential of stem cells.They are more convenient to use,and their effects can be fine-tuned by varying their concentrations and combinations.In the previously study,we found hESCs could be induced to primitive neural stem cells by simultaneously inhibiting glycogen synthase kinase 3(GSK3),transforming growth factor ?(TGF?)and Notch signaling.However,the neuroepithelia obtained by this method were posteriorized probably due to the activation of Wnt signaling by GSK3 inhibition.In this study,hESC lines H1 and HUES9 were cultured in serum-free and feeder free condition.The hESC colonies had clear border and compact.The individual cell had large nucleus and few cytoplasm by light-microscope.The cells expressed octamer-binding transcription factor 4(OCT4)by immunofluorescence assay,indicating that hESCs were maintained in an undifferentiated state.Combination of small molecular TGF? inhibitor(SB431542)and Notch inhibitor(RO4929097)induces neural conversion of hESCs to generate paired box protein 6(PAX6)+,Nestin+ cells.We found that GSK3 inhibitor(CHIR99021)at low concentration and TGF? inhibitor(SB431542)can maintain the long-term self-renewal of above-mentioned neural precursors.In both of early and late passage,the cells were stably positive for forebrain genes forkhead box protein G1(FOXG1)and orthodenticle homeobox 2(OTX2),neural stem cell marker PAX6 and Nestin,and cell proliferative marker Ki67,suggesting the cell population were forebrain neural stem cells(fNSCs).Importantly,fNSCs were able to respose to the venral patterninginduced by sonic hedgehog(SHH),and generated ventral forebrain gamma-aminobutyric acid(GABA)neurons which expressing microtubule-associated protein 2(MAP2)and NK2 homeobox 1(NKX2.1).However,PAX6 positivity may indicate dorsal identity of fNSCs.We next sought to generate ventral forbrain neural stem cells(vfNSCs)from hESC under condition including SHH,its activator SAG,and TGF? inhibitor(SB431542).After neural induction under above method,we found a condition including GSK3 inhibitor(CHIR99021),SHH,and TGF? inhibitor(SB431542)was sufficient to maintain the cell population demonstrating vfNSC phenotypes.The cells were positive for forebrain genes FOXG1 and OTX2,neural stem cell marker nestin,and ventral forebrain gene NKX2.1.In addition,the combination of small molecular Wnt inhibitor(XAV-939),Notch inhibitor(RO4929097),and SHH agonist,could efficiently induce these vfNSCs to generate NKX2.1+ and insulin gene enhancer protein 1(ISL1)+ ventral forebrain neurons which were also positive for MAP2,GABA and glutamic acid decarboxylase 65(GAD65),suggesting the production of inhibitory forebrain interneurons.This study provides a hESC differentiation strategy,which could be used as a platform for studying forebrain development in vitro.In addition,the generation of self-renewing cell source of forebrain inhibitory interneurons also could be useful for cell transplantation therapy of neurodegenerative diseases such as Alzheimer's disease and Huntington's diseases.