Study On The Mechanism Of Action Of ID1/3 In The Maintenance And Exit Of Pluripotency Of Human Embryonic Stem Cell

Background:Human embryonic stem cells(hESCs)are a kind of pluripotent stem cells that can be cultured in vitro continuously and stably and have the differentiation potential of three germ layer tissue cells.After the stimulation of appropriate conditions,a variety of cells with clinical application prospects can be differentiated,which lays a solid foundation for the development of regenerative medicine and provides good materials for the research of developmental biology.According to the early research on mouse embryonic stem cells and human embryonic stem cells,embryonic stem cells are generally divided into naive and primed states.The embryonic stem cells in naive state are similar to the inner cell mass of preimplantation blastocyst,while the embryonic stem cells in primed state are more similar to the epiblast cells of post implantation embryo.The inherent differences between naive and primed embryonic stem cells lead to their different characteristics and application prospects.Therefore,at present,many researchers are committed to inducing the traditional human embryonic stem cells from primed state to naive state in order to maximize the application value of human embryonic stem cells.To give full play to the application potential of embryonic stem cells,we must first understand how their fate is determined,that is,how the self-renewal of cells is maintained,and how cells exit pluripotency and differentiate into specific cell lineages.Naive and primed stem cells have different regulatory mechanisms of pluripotency maintenance and dissolution.Naive hESCs maintains their undifferentiated state mainly through LIF/STAT3 and Wnt/β-catenin signaling pathways,while primed maintains their pluripotency mainly through FGF/ERK and TGFβ/Smad2/3.In addition,many studies have shown that BMP4/Smad1/5 and Insulin/JNK also play an important regulatory role in the process of pluripotency maintenance or dissolution of embryonic stem cells.ID1 and ID3 belong to the HLH protein family.They cooperate with each other and play an important regulatory role in embryo development.ID1 and ID3 can be activated by signaling pathways including BMP4/Smad1/5 in embryonic stem cells,but the role of ID1 and ID3 in maintaining self-renewal of human primed and naive embryonic stem cells is still unclear.Objective:Objective to explore the regulatory role of ID1 and ID3 in regulating the maintenance of self-renewal and pluripotency dissolution of human embryonic stem cells(hESCs),so as to provide new ideas for a comprehensive understanding of the maintenance mechanism of self-renewal and cell fate selection of hESCs,and lay a foundation for the maximum application value of hESCs.Methods:(1)The effects of single and double knocks of ID1 and ID3 on the survival and proliferation of primed embryonic stem cells were observed;(2)The effects of single and double knocks of ID1 and ID3 on pluripotency maintenance of primed stem cells,pluripotency dissolution and three germ layer differentiation during EB differentiation were observed;(3)The role of ID1 over-expression in pluripotency maintenance and differentiation of primed embryonic stem cells;(4)Single cell sequencing was used to reveal the mechanism of ID1/3 regulating self-renewal of primed embryonic stem cells;(5)To establish the culture system of human naive embryonic stem cells,and to observe the effect of single and double knockout of ID1/3 on self-renewal and maintenance of human naive embryonic stem cells;(6)Observe the difference of differentiation of trophoblast stem cells between embryonic stem cell control line and ID1/3 knockout line in naive state;(7)Combined with single-cell sequencing data and related reports,the regulatory mechanism of ID1/3 on self-renewal of human embryonic stem cells in Naive state was analyzed.Results:In the primed and naive embryonic stem cells,the double knockout of ID1 and ID3 reduced the viability of single cells.After Y27632 was added,the viability of cells recovered.In addition,double knockout of ID1/3 can prolong G1 phase and shorten S phase of ES cell cycle.The knockout of ID1/3 decreased the expression of pluripotency marker genes in primed and naive embryonic stem cells,especially in naive embryonic stem cells.In the early stage of EB differentiation,ID1/3 knockout will make the pluripotent gene expression of embryonic stem cells in primed state down regulated rapidly,that is,promote the pluripotent dissolution of embryonic stem cells,at the same time,the expression of ectoderm related genes increased.At the late stage of EB differentiation,ID1/3 knockout restricted the mesoendoderm differentiation of EB cells.In the process of differentiation from embryonic stem cells to trophoblast embryonic stem cells in naive state,double knockout of ID1/3 limits the differentiation of trophoblast embryonic stem cells.Single cell sequencing and subsequent Western blot analysis showed that ID1/3 knockout decreased AKT phosphorylation in ES cells,while ID1 overexpression promoted AKT phosphorylation.Combined with the existing reports,ID1/3 can regulate the maintenance of self-renewal of primed and naive embryonic stem cells through AKT signaling pathway.In addition,ID1/3 can directly interact with TCF3 to promote the expression of pluripotent marker genes of embryonic stem cells in naive state.Conclusion:It has been found that ID1/3 plays an important role in regulating pluripotency maintenance and dissolution of human embryonic stem cells in primed and naive state through AKT signaling pathway.