Mechanism Study Of The Function Of Id2 In Promoting Mouse Embryonic Stem Cell Self-renewal

Mouse embryonic stem cells(mESCs)are cells isolated from inner cell mass(ICM)before blastocyst implantation.Under appropriate in vitro c?lture conditions,they not only can proliferate infinitely(self-renewal),but also have the ability to differentiate into various types of cells derived from the three germ layers(pluripotency).Due to these characteristics,they have become an important material for the development of developmental biology and disease model building.Therefore,they have significant economic and social value.Early isolated mouse embryonic stem cells need to be c?ltured in medium containing inactivated feeder cells and fetal bovine serum.An important aspect of embryonic stem cell research is how to maintain its pluripotency under in vitro c?lture conditions.Therefore,after continuous exploration by a large number of researchers,three in vitro c?lture systems suitable for different embryonic stem cell maintenance,such as LIF/serum,N2B27/BMP4 and N2B27/2i,have been established.To study how embryonic stem cells maintain their self-renewal status without differentiation under the c?lture conditions,we should find out the molec?lar mechanism of their self-renewal.After the unremitting efforts of the researchers,some key reg?latory genes and signaling pathways have been identified,such as Tfcp2ll,Nanog,Esrrb and Sox2,LIF/STAT3 and WNT/?-catenin pathways.However,the reg?lation of embryonic stem cells is a complex process by which various genes and signaling pathways interact.It remains unclear how embryonic stem cells maintain an undifferentiated state without differentiation.Therefore,we need to combine cytology and biochemistry with molec?lar biology to further study embryonic stem cells and explore their reg?latory mechanisms of self-renewal and differentiation.It has been previously reported that members of the Id gene family,including Id1,Id2 and Id3,are downstream genes of the BMP4/SMAD signaling pathway in serum-free N2B27 culture and over-expression can partially replace the function of BMP4 in promoting the self-renewal of mouse embryonic stem cells.However,the mechanism by which the Id gene family promotes the self-renewal of mESCs remains unclear.It has also been reported that Id1 gene in serum-containing medium can partially replace the role of LIF by up-reg?lating the expression of Nanog gene while inhibit the expression of mesoderm landmark gene T to maintain mESC self-renewal.However,the role of Id2 and Id3 in mESCs has not been reported.Therefore,our study will focus on the function of Id2 and Id3 in mESCs.Family members of the Id gene have a helix-loop-helix(HLH)domain,which promotes the migration and the cycle of cells.Furthermore,they also can inhibit the differentiation of m?ltiple precursors and delay the cell senescence.Therefore,they may have positive effect on the maintenance of embryonic stem cells.To investigate the effect of Id2 and Id3 in mESCs,we first overexpressed these two genes in 46C mESCs.After validation,the plasmids inserted with the gene of interest were transfected into mESCs by liposome respectively.After drug selection,the established correct cell lines were c?ltured in serum medium in the absence of LIF.After a period of time,mESCs overexpressed Id2 or Id3 still have the shape of embryonic stem cells,while the control cells died or differentiated.Next,we performed immunofluorescent staining to examine the expression of the markers of embryonic stem cells(eg,Nanog,Oct4,Esrrb,Tfcp2l1,etc.).The res?lts showed both Id2 and id3 transfected cells were positive for Nanog and Oct4,and alkaline phosphatase(AP),another pluripotency marker,was also positive,while the control cells were negative.Notably,the self-renewal-promoting effect of Id2 sho?ld be better than the effect of Id3.Therefore,we mainly focus on the gene Id1.We down-reg?late the expression of Id2 gene by shRNA method.The res?lts showed that knockdown of Id2 induced embryonic stem cell differentiation,indicating Id2 is essential for the maintenance of mESCs.Subsequently,we used transcriptome sequencing to screen the downstream target genes of Id2.Sequencing res?lts showed that there were a large number of differentially expressed genes in Id2 expressing cells when compared,with the control cells.After GO analysis,we found that the transcription of c-Myc and n-Myc,two pluripotentcy genes,is up-reg?lated.Their expression levels were further confirmed by qRT,PCR and western blot.Finally,we performed a functional experiment to investigate whether Myc can mediate the function of Id2 in mESCs.The expression of c-Myc and n-Myc was down-reg?lated in Ids-overexpressing mESCs,we found that Id2 mESCs differentiated.Together,these res?lts showed that Id2 depends on c-Myc and n-Myc to maintain the undifferentiated state.Taken together,our res?lts indicate that mESCs overexpressing Id2 and Id3 are able to replace LIF to maintan the self-renewal of embryonic stem cells.Further studies showed that c-Myc and n-Myc,two targets of Id2,can mediate the self-reneneal-promoting effect of Id2 in mESCs.Our res?lts expand the knowledge of the reg?latory network of embryonic stem cell maintenance and are conducive to the basic research and application of stem cells in the future.