Cell Therapy For Cerebral Ischemia Mice By Transplantation Of Forebrain Neural Stem Cells Derived From Human Embryonic Stem Cells

Background:Human pluripotent stem cells(h PSCs)are self-renewing pluripotent stem cells with the potential to differentiate into any types of functional cells.Therefore,therapeutically valuable cells can be obtained from h PSCs in vitro.Neural stem cells and neurons with specific function derived from h PSCs have important application value in cell therapy for neurological diseases.Purposes:Cerebral ischemia is one of the major diseases causing disability and death.The irreversible death of neurons will occur at the injury site.At present,there is no effective therapeutics in clinical practice.Human embryonic stem cells(h ESCs)are a kind of pluripotent stem cells that can stably self-renew in vitro and differentiate into various types of cell.We intended to obtain forebrain neural stem cells(FNSCs)from h ESCs by directed differentiation in vitro,and then explore the effects of FNSCs transplantation treating cerebral ischemia mice.Methods:1.In vitro experiments:FNSCs and forebrain neurons derived from h ESCs were obtained by small molecule induced differentiation,and identified by immunofluorescence staining and other techniques.CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)gene editing technology was used to construct FOXG1 gene knockout h ESCs cell line to explore gene's effects on forebrain differentiation.2.In vivo experiments:Severe combined immunodeficiency(SCID)mice were randomly divided into sham operation group,model group,and cell transplantation group.Model of cerebral ischemia was constructed by middle cerebral artery embolization(MCAO).Seven days after cerebral ischemia,EGFP-labeled FNSCs were transplanted into the injured cerebral cortex of the cell transplantation group,and the model group was injected with an equal volume of phosphate buffer saline(PBS).After5 weeks,behavioral tests were performed to evaluate the effects of FNSCstransplantation on motor function.Immunofluorescence staining was used to detect the survival and differentiation of transplanted FNSCs in vivo.Results:1.Immunofluorescence staining showed that FNSCs were derived from h ESCs under a chemically defined condition.The FNSCs expressed forebrain markers FOXG1and OTX2,and neural stem cell markers NESTIN,SOX2 and PAX6.2.The FNSCs could spontaneously differentiate into cortical neurons expressing TBR1and BRN2,markers of cortical neurons,and NEUN,a marker of mature neurons.3.FOXG1 gene knockout h ESCs cell line was successfully constructed by using CRISPR/Cas9 technology.Immunofluorescence staining and quantitative RT-PCR showed that the deletion of FOXG1 gene did not affect the differentiation of forebrain neural stem cells from h ESCs.4.Motor function of cerebral ischemia mice was significantly improved by FNSCs transplantation.The transplanted FNSCs could survive in vivo and differentiate into TUJ1~+neurons evidenced by immunofluorescencent staining.Conclusions:1.Self-renewing FNSCs derived from h ESCs were successfully obtained.And the FNSCs were able to differentiate into forebrain cortical neurons.2.FOXG1 is not a key gene that regulates forebrain neural differentiation in early neural induction process,.3.Motor function was improved after transplantation of FNSCs into cerebral ischemic injury mice.