Generation Of Self-Renewing Neuromesodermal Progenitors From Human Embryonic Stem Cells

Human embryonic stem cells(hESCs)are pluripotent stem cells(PSCs)that can generate all types of adult cells,and maintain stable self-renewal in vitro.They can generate different types of cells through directional differentiation in vitro,providing a potential cell source for cell replacement therapy,and have a promising potential in regenerative medicine applications.As a part of central nervous system,spinal cord has many functions such as up-and-down conduction nerve conduction,neural signal integration,coordinated motion,etc.Degenerative diseases including muscular atrophic lateral sclerosis,neuromuscular atrophy as well as trauma will lead to spinal cord function loss.Current therapy such as surgical and drug treatment are rarely effective to recover the functions of spinal cord.Thus,cell transplantation now is expected to be a potential method to help patients troubled by spinal cord function loss.Neuromesodermal progenitors(NMPs)can not only differentiate into neurons in the spinal cord,but also produce mesoderm tissues around the spinal cord,which have unique application value in the treatment of spinal cord injury and disease.At present,many effective induction methods have been established to differentiate hPSCs into NMPs,which co-express T and SOX2.However,how to achieve self-renewal of NMPs remains a issue to be solved.This paper using a NMP induction strategy follows the developmental clues in vivo.By using dual Smad inhibition and simultaneously activating WNT and FGF signaling pathways,stable self-renewing NMPs could be successfully obtained,hereafter referred to as srNMPs(self-renewing neuromesodermal progenitors).The cells had the characteristics of neural stem cells of spinal cord.They co-expressed neural marker gene SOX2 and mesodermal marker gene T.Besides,they expressed neural stem cell marker genes NESTIN,NCAD,CD133,proliferation marker gene MKI67 and HOX family genes HOXB8,HOXB9.Under the condition of neural differentiation without adding any morphogens,srNMPs could differentiate into V0_G neurons that were positive for vGLUT2,TACR1,SST,PAX2,HOXB8,HOXB9.After ventralization by exposed to SHH and RA for one week,srNMPs were induced into OLIG2-positive motor neuron precursor cells.After three weeks of neural differentiation,motor neurons co-expressing ISL1,ChAT and vAChT were generated.WNT and BMP4 signaling pathway were activated for dorsalization for one week,and after another three weeks of neural differentiation,srNMPs could differentiate into ATOH1,PAX3,and PAX7-positive dl1interneurons.Above results indicate that srNMPs could respond to induction of dorsal-ventral axis signals,thereby differentiating into dorsal and ventral neurons.NMPs are not only precursor cells of the spinal cord,but also precursor cells of the adjacent paraxial mesoderm.The paraxial mesoderm dominates the formation of somites and can be further differentiated into mesodermal tissue such as cartilage,skeletal muscle and bone.At present,the identification of NMPs is mainly based on the co-expression of SOX2 and T and the potential of differentiating into spinal cord neurons.The differentiation potential of NMPs into mesoderm cells in vitro remains to be verified.In order to demonstrate the mesodermal differentiation potential of srNMPs,we tested the potential of srNMPs through single cell differentation,renal capsule transplantation in vivo differentation,and suspension culture.In single cell differentation experiments,clones formed from single srNMPs spontaneously differentiated into?-SMA-positive mesoderm cells and MAP2-positive neurons in N2B27 basal medium.The transplanted srNMPs could differentiate into?-SMA positive mesoderm cells and MAP2 positive nerve tissue under the renal capsule.In suspension culture experiments,srNMPs spontaneously formed spheres,in which complex structures were found including nerve tissue,cartilage tissue and muscle tissue,which are similar to the spine during embryo development.These results further demonstrate that srNMPs cells are NMPs with mesoderm and neural differentiation potential.This paper also discussed the developmental origin and mechanism of srNMPs.By combining with small molecule inhibitors SB431542,DMH1,and LGK974 to inhibit TGF?,BMP,and WNT signaling pathways,hESCs could be induced into FOXG1-positive frontal neuroepithelial cells,but when WNT and FGF signaling pathways were activated,they could not be induced into NMPs,which demonstrates that NMPs and frontal neuroectodermal cells have different developmental origins.FOXG1-overexpressed cells could be induced into NMPs,which demonstrates that FOXG1 is not the key gene to inhibit frontal neuroectodermal cells to be induced into NMPs.It has been reported that SP8 knockout mice have abnormal spinal development.Therefore,in this study,SP8 knockout hESCs were obtained by CRISPR/Cas9technology.However,the cells could still be successfully induced into NMPs,which proves that SP8 is not a key gene in the induction process of NMPs.In addition,it has been reported that the enhancer of SOX2 gene in NMPs is different from ESCs and neuroepithelial cells.The expression of SOX2 in NMPs is regulated by enhancer N1,and the expression of SOX2 in ESCs and neuroepithelial cells is regulated by enhancer N2.To figuring out whether there are differences between the methylation of SOX2enhancers N1 and N2 of human ESCs,NE and NMPs,we detected it by methylation sequencing.The results showed that the methylation sites of the enhancers N1 and N2 of SOX2 were not methylated,so the difference in expression activities of SOX2 enhancers N1 and N2 was not caused by methylation regulation.In summary,this study successfully obtained self-renewable srNMPs.These cells co-expressed genes SOX2 and T,could stably passage for more than 30 generations in vitro and maintain the stable cell phenotype.The cells with spinal cord neural stem cells molecular characteristics could differentiate into dorsal and ventral neurons of the spinal cord in response to dorsal-ventral differentiation signals,and differentiate into neurons and mesodermal cells.Suspension cultured srNMPs could spontaneously form a cell mass containing nerve tissue,muscle tissue and cartilage tissue,which is similar to a developing spine.The srNMPs established in this study are expected to have promising applications in cell therapy for spinal cord function loss and in research about regulation mechanisms of NMPs differentiation fate selection.