Study Of The Establishment Of Feeder-free Human Extended Pluripotent Stem Cells And Their Metabolic And Epigenetic Characteristics

In mammals,there are two different cell types in early embryo development:pluripotent cells and totipotent cells.Totipotent cells have the developmental potential to develop into the whole embryo,including embryos and extraembryonic tissues;On the other hand,pluripotent cells can only form the embryonic lineage that can develop into most organs.The two types of cells correspond to different stages of development which is caused by fate decisions at the early stages of embryonic development,during which totipotent cells gradually lose totipotency and transform into pluripotency.The established embryonic stem cell(ESC)lines represent the pluripotent state,but this earlier state of pluripotency has long been a subject that scientists are trying to explore.In 2017,Professor Hongkui Deng and his colleagues established a cocktail medium called "LCDM" through the screening of small molecule compounds,which can transform conventional mouse and human pluripotent embryonic stem cells into "extended pluripotency" stem cells(EPSCs),and the EPSC has the bidirectional chimeric ability,which can generate embryonic tissue and extra-embryonic tissues,including yolk sac and placenta.However,the system is based on feeder cells,which introduces lots of uncertain components,which may cause great trouble to the potential clinical application,Moreover,it also limits the study of the specific molecular mechanism and molecular characteristics in the process of EPSC transformation.Therefore,this dissertation attempts to optimize and establish a feeder-free "extended pluripotency" stem cell culture system,and further explore the epigenetic and metabolic mechanism regulating the EPSC transformation and maintenance.In this dissertation,a feeder-free EPSC(i.e.,ffEPSC)transformation and culture system was established,and the extended pluripotency of the ffEPSCs was comprehensively evaluated and determined at the molecular characteristics,chimeric ability,and transcriptome levels.The results showed that ffEPSC has higher level of preimplantation gene expression and lower level of post-implantation gene expression than conventional ESC.At the same time,human zygotic genome activation genes are also significantly upregulated in ffEPSC.Human endogenous retroviral HERVK is significantly activated in ffEPSC.Moreover,the bidirectional contribution ability of ffEPSCs to the embryonic and extraembryonic lineage is verified by the chimerism experiment of single cell injection into mouse 8-cell embryos and teratoma formation experiment.The results showed that ffEPSCs could develop into embryonic and extraembryonic lineage cells in vivo and express the corresponding lineage marker,both in the blastocyst stage and E13.5 stage of chimeric embryos.The spontaneous differentiation also proved that ffEPSCs have the ability to contribute bidirectionally to embryonic and extraembryonic lineages,and demonstrated their multilineage differentiation potential in vitro by inducing the differentiation of ffEPSCs to neural precursor cells and pancreatic β cells.This dissertation further explores the epigenetic and metabolic characteristics of ffEPSCs.Quantitative analysis of multiple histone modifications showed that ffEPSCs exhibit a higher global H3K27me3 modification level than ESC.However,the addition of the H3K27me3 methyltransferase inhibitor GSK126 can speed up the conversion process of ffEPSC transition.Further Ch IP-seq analysis showed that although the global H3K27me3 level is higher in ffEPSCs,the level of H3K27me3 in the coding gene region in ffEPSC is significantly lower than that of ESC.In particular,H3K27me3 at the BMP4 locus is significantly reduced in ffEPSC,accompanied by active transcription of BMP4.This is consistent with BMP4’s ability to facilitate the transition of PSCs from primed to na(?)ve pluripotency,and our results also show that the addition of BMP4 can accelerate the transition from ESCs to ffEPSCs.In addition,through transcriptome data analysis and Seahorse experiment,this dissertation proves that there is a metabolic remodeling that ffEPSC has the lower glycolysis level than ESC.Importantly,inhibition of the glycolysis process has a favorable effect on the state of ffEPSC,that is,the addition of glycolysis inhibitors during the culture process of ffEPSCs can promote the maintenance of ffEPSCs.In summary,this dissertation first establishes a feeder-free culture condition to obtain human ffEPSCs,which have the extended pluripotency of embryonic and extraembryonic chimerism.At the same time,we further explore the dynamics of epigenetic and metabolism characteristics in ffEPSC conversion and maintenance,demonstrating the importance of H3K27me3 and glycolysis in extended pluripotency conversion and maintenance.With relevant small molecule inhibitors,we have further optimized the ffEPSC conversion and culture system.Our research provides possibilities for mechanistic studies and further applications of extended pluripotent cells,as well as new insights into the transition between different pluripotent states.