Induction And Maintenance Of Human Spinal Cord-Like Neural Stem Cells Mediated By CHIR-99021 And Leukemia Inhibitory Factor

Neural stem cells(NSCs)have valuable therapeutic applications of neurological diseases due to their ability to repair tissues and cross the blood-brain barrier.Therefore,NSCs have become a hot research topic in the field of regenerative medicine and stem cell research.NSCs in different stem cell niche have their own region-specific phenotypes.Preclinical animal studies and clinical trials have proposed that region-specific NSCs have stronger ability to repair and regenerate damaged tissues in the same region,such as the spinal cord region,these specific NSCs are more effective in treating spinal cord injury than brain region-specific NSCs.At present,studies have reported the strategy of obtaining spinal regionspecific NSCs,but the methods are more complicated and costly.This study developed a simple and efficient method to induce spinal-specific NSCs,which is different to the previous reports,that is,the stable NSCs derived from human pluripotent stem cells in chemical defined conditions which contained small molecule CHIR-99021 combined with leukemia inhibitory factor(LIF).The phenotypic characteristics,gene expression patterns and important signaling pathways of CLNSCs have been studied.1.Establishment and identification of CLNSCsHuman pluripotent stem cells were cultured in N2B27 supplement with CHIR-99021 and LIF(CL)to induce CLNSCs.One human embryonic stem cell line and two human induced pluripotent stem cell lines were used in this research.The neural rosette structures which expressed NESTIN and Ncadherin were discovered in 2-3 days of induction,and the neurospheres were formed in 7-10 days.CLNSCs can be cultured for more than 50 generations.Pluripotent markers OCT4 and NANOG were down regulated while early neural markers NESTIN,SOX2 and PAX6 were increased in CLNSCs.Furthermore,CLNSCs be able to differentiate into neurons,oligodendrocytes and astrocytes.The interspecies chimera studies have shown that CLNSCs still express NESTIN and TUBB3 when being injected into E6.5 mouse embryo and cultured for 2 days in vitro,and integrate into the neuroecderm in mouse embryos.2.Spinal cord-specific expression patterns of CLNSCsRNA-seq was performed on the gene expression patterns of CLNSCs,and the results showed that the enrichment of gene expression of CLNSCs is highly correlated with neural fate determination.The results of gene set enrichment analysis(GSEA)showed that CLNSCs were enriched in the GO(gene ontology)term related to the spinal cord,including spinal cord pattern,spinal dorsal/ventral pattern,and spinal cord development.CLNSCs have a region-specific neural stem cell expression pattern,failed express brain tissue-specific NSCs marker genes FOXG1 and OTX2,and increased expression of spinal cord region-specific markers HOXA1-4,HOXA7,HOXB1-4 and HOXC4.3.The role of Wnt signaling pathway on CLNSCsThe KEGG(kyoto encyclopedia of genes and genomes)signaling pathway of CLNSCs is enriched in the significantly change of Wnt signaling pathway.Among them,the ?-catenin degradation complex members AXIN2 and APC2 are highly expressed in the canonical Wnt/?-catenin signaling pathway,and ?-catenin(CTNNB1)expression was weaker than hPSCs.The c-MYC that Wnt/?-catenin target gene transcription level was significantly down-regulated and its immunofluorescence staining was negative.The Wnt/?-catenin inhibitors IWR-1 and XAV939 were added to culture CLNSCs,and stable passaged stem cell line CLINSCs(IWR-1)and CLXNSCs(XAV939)were obtained.CLINSCs and CLXNSCs have similar phenotypes to CLNSCs.OCT4 and NANOG were lower expressed,while neural markers SOX2,NESTIN,PAX6 were highly expressed in CLINSCs and CLXNSCs.They also have the same ability to differentiate into trilineage nerve cells like CLNSCs,indicated that CLNSCs self-renewal does not depend on the activation of canonical Wnt/?-catenin signaling pathway.However,WNT7 A,WNT7B,WNT5 A,and WNT5 B of the non-canonical Wnt signaling pathway are highly expressed in CLNSCs,suggesting that the selfrenewal of CLNSCs may be related to the non-canonical Wnt signaling pathway.All together,this study developed the simple methods to induced human pluripotent stem cells into region-specific NSCs in the serum-free and transgene-free CL culture system.The obtained CLNSCs exhibit spinal cord region specificity,and its not only be used as a research tool for neurodevelopment,but also be used as a cell source for stem cell transplantation to treat neurological diseases,and may has broad prospects in clinical applications in the futher.