| Somatic cell nuclear transfer(SCNT),cell fusion,reprogramming techniques or change the culture conditions can change the fate of cell,and even to reshape its functions,such as somatic cells can be reprogrammed to the status of pluripotent stem cells.Mammalian pluripotent stem cells(PSCs)are defined as naive-and primed-state according to their varieties in cellular,molecular,epigenetic and functional states,respectively.nPSCs had attracted much attention in clinical application because of its easy for genetic engineering and regeneration of functional tissues and organs in vitro or in vivo.Thus maintaining their naive state effectively is of great significance to basic and clinical research of stem cells.Numerous studies shows that naive PSCs had more high mitochondrial activity and self-renewal ability than Primed ESCs,and these two kinds of pluripotent stem cells had significant differences in DNA methylation.In previous studies,our laboratory group obtain Naive stem cells from the monkey Primed ESCs by changing the culture system as the first in the world.There had not significantly genetic differences through the spectrum analysis of gene expression between the two cell lines.But naive pluripotent stem cell methyltransferase DNMT3A(DNA methyltransferase 3a)and DNMT3B(DNA methyltransferase 3b)gene expressed at low levels,it reveals that the two kinds of stem cells had different levels of DNA methylation.But we still not clear whether DNA methylation plays an important role in reprogramming andhigh level metabolism of Naive-like.In this paper,we use of CRSPR/Cas9 technology to knocked out DNMT3A and DNMT3B gene which are two significant different methyltransferase gene between the Primed Cells and Pluripotent Stem Naive Pluripotent Stem Cells,We obtained the DNMT3A and DNMT3B gene knock stable cell lines.then study the efficiency of reprogramming of primed state differentiate into naive state after DNMT3A and DNMT3B knockout,we found that DNMT3A and DNMT3B knockdown increased(P?0.05)the conversion of Naive-like nPSCs significantly,these illustrate thatthe low level of DNA methylation were in favor of naive-like reprogramming and promote Naive-like nPSCs metabolism ability.The brain is one of the most complex organs of the human body,its not only contains a variety of types of cells but also the development mechanism is so complex that we had not clear yet.Recently,studies show that many neurodegenerative diseases,such as the neural tube defects,microcephaly,epilepsy,autism,Rett syndrome,mental retardation and other diseases,that all due to fetal brain cells development or function abnormalities.Although people had a certain understanding of these diseases,westill lack of the research on brain development,which led to we still not clear the pathogenesis of these diseases.Neural stem cells(NSCs)can self-renewal and differentiate into various types of brain cells inthe central nervous system,such as neurons,astrocytes and oligodendrocytes pluripotent cells,and has strong migration.Neural stem cells are not only the basis of brain development,neural regeneration and brain evolution but also the research methods of drug screening and neurodegenerative diseases.Therefore,the research on the development and differentiation of neural stem cells has become the basis for drug therapy of neurological diseases and the study of brain development.In recent years,numerous studies had shown that use of specific growth factors and small chemical molecules can induce neural stem cells differentiate into specific cells,which can effectively repair damaged nerve tissue after transplantation with the development of stem cells technology and regenerative medicine research.Neural stem cells provide a new way of therapy central nervous system degenerative diseases,including Parkinson's disease(PD),spinal cord injury(SCI),multiple sclerosis(MS)and other diseases,which brought the hope for patients with nervous system diseases.Embryonic stem cells(ESCs)as a unique resource that can differentiate into varieties of different cell types in vitro theoretically.Embryonic stem cells provides a basic model for research cell fate selection,differentiation and basic development process,and the pluripotent of embryonic stem cells provides a new method in drug screening and cell transplantation.Bcecause of embryonic stem cells technology is widely used in the field of biomedical,so is very necessary for our to set up a reliable experimental method for control of embryonic stem cell self-renewal and differentiation in laboratory.In this experiment,we inducing embryonic stem cells differentiate into neuroepithelial cells(NESCs)by a simple adherent culture method,the neuroepithelial cells had the ability to self-renewal and differentiation into functional offspring cells and had strong migration ability.Neural stem cells in brain development process mainly through two different developmental stages,namely neuroepithelial stem cells(NESCs)and radial glial precursor cells(RGPCs).NESCs refers to the neural stem cells from the neural plate to the neural tube stage,its the earliest neural stem cells which have strong proliferation and differentiation ability and can produce the whole brain nerve cells and high purity(>80%)neurons.NESCs is transient in vivo,and its abnormal proliferation and differentiation will lead to neural tube and brain development defects.When the neural tube is closed,NESCs can differentiate into RGPCs,then the differential RGPCs will further proliferate and differentiate to produce neurons in different brain regions to complete the normal development of the brain.Therefore,the conversion of NESCs to RGPCs(NESCs-RGPCs)is an important event in the development of the brain.It is of great scientific value to study the developmental mechanism of the brain and the defect of brain development.However,this transformation is a rapid process,and the previously established neural stem cell system is difficult to stable long-term culture of NESCs and achieve accurate "NESCs-RGPCs"transformation,so it is difficult to accurately study on this process in vivo.Recently,our laboratory had developed a new,simple,finite elements,robust culture system which include:use monkey or human embryonic stem cells and induced pluripotent stem cells(iPS)can directional differentiate to stable culture system for high purity neuroepithelial stem cells.In this system,we found that NESCs can achieve "NESCs-RGPCs" transformation.The transformation of these preliminary studies on "NESCs-RGPCs" provides us a unique cell model.In this study,NESC culture media removing CHIR99021(Wnt signal activator)CHIR99021,NESCs can be quickly converted to RGPCs,shown that the Wnt signal pathway plays a key role to maintain the self-renewal of NESCs and "NESCs-RGPCs" transformation.We have built up a robust and stable system:to achieve a single neuroepithelial stem cell clonal expansion to development miniature neural tube structure,and in vitro,the cells can be differentiated into functional neurons to transplanted into the monkey brain and regenerate axons,through RNA sequencing analysisthe relationship between the differentiation of neurons in early stage later,RGPCs,and NESCs-ND,we demonstrate that neural tube formation and self-renewal of neuroepithelial stem cell relies on mitochondrial high energy metabolism and Wnt signaling pathways.Neuroepithelial play a decisive role in the development of telencephalon,and neuroepithelial differentiated into radial glial progenitor cells(RGPCs)through the Notch signaling pathway and Wnt signaling pathway regulation of this change preciselyIn this experiment,we use chemical drugs AZD3965 to inhibition of glycolysis.AZD3965 is an inhibitor of monocarboxylate transporter 1(MCT1)to prevent the transport of lactate into and out of cells.The inhibition of MCT1 by AZD3965 to inhibit the energy metabolism of neural stem cell to analysis of difference in the metabolites and signal pathways and other aspects after energy metabolism was suppressed. |
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