The Epigenetic Regulation Of Novel Pluripotency State Of Human Stem Cells

Objective: Extended pluripotent stem(EPS)cell is a new type of human pluripotent stem cells established by Hongkui Deng laboratory of Peking University in 2017.EPS cell expresses typical pluripotent transcription factors,such as NANOG,OCT4,SOX2.Meanwhile,it also expressed up-regulated transcription factors related to inner cell mass in early embryo,such as KLF4 and DNMT3 L.EPS cell is distinctive from the primed human ES cell and the na?ve mouse ES cell in the chimeric ablility.EPS cell exhibited unexpectedly broader developmental plasticity,not only contributing to embryonic tissues,but also to extraembryonic tissues such as the placenta.However,other cells can only contribute to embryonic tissues.Therefore,EPS cell is irreplaceable compared with other pluripotent stem cells in terms of both developmental potential and research significance.However,the conversion mechanism or specific molecular markers are not clear at present.The established culture system is based on feeder cells,and the complicated and unclear components also hinder further molecular mechanism dissection.Therefore,this project is aiming to optimize the culture system of EPS cell and investigate the underlying epigenetic regulation mechanism.Epigenetics refers to the information that can be inherited between daughter cells without changing the DNA sequence,and causes heritable changes through epigenetic mechanisms,such as DNA and histone modifications.It not only affects gene expression,regulation and inheritance,but also plays an extremely important role in development,cancer biology and regenerative medicine.H3K27me3 refers to the trimethylation of lysine 27 on histone H3,which can regulate transcriptional repression.They are generally deposited in the region of promoters and are mainly decorates promoters of developmental genes,such as Hox and Sox genes.Here the epigenetic differences of H3K27me3 between human ES celland EPS cell are determined,and the functional role of epigenetic hindrance between ESC and EPSC is dissected through chemical inhibitors and gene editing for key epigenetic enzymes.Methods: The EPS cell cultivation is optimized based on feeder-free system,and determined by immunofluorescence,flow cytometry fluorescence analysis,real-time fluorescent quantitative PCR and RNA sequencing to assess the expression profile of pluripotency genes and the dynamic changes between ES cells and EPS cell.Injected GFP-labeled human EPS cell into an eight-cell(8C)stage mouse embryo is to verify the chimeric ability of EPS cell in feeder-free system.Then,H3K27me3-related chemical compounds GSK126,GSKJ1 and GSKJ4 are added separately to determine whether H3K27me3 is an epigenetic barrier of EPS cell transition from ES cells,and whether the change of H3K27me3 is conducive to the conversion of ES cell to EPS cell through the domed-clonal morphology and corresponding molecular indexes.Results: The EPS cell can be adapted to Matrigel-based feeder-free system.The expression levels of pluripotent genes NANOG and OCT4 in EPS cell were lower than ES cell.Meanwhile,KLF4 and DNMT3 L of pre-implantation genes in early development were upregulated,DUSP6 and ZIC2 of post-implantation genes in late development were downregulated.Human EPS cell showed superior species chimeric efficiency,and the molecular index CDX2 representing trophoectoderm was co-located with GFP in embryo.Along with the conversion from ES cell to EPS cell,H3K27me3 methyltransferase EZH2 expression level is up-regulated,while demethylase is down-regulated.Both FACS and Western blot results showed that H3K27me3 increased gradually during the conversion process.The inhibitor experiment showed that the addition of chemical inhibitor of H3K27me3 methyltransferase EZH2 was conducive to the conversion of ES cell to EPS cell,while the addition of inhibitor of H3K27me3 demethylase hindered the conversion.Meanwhile,HUES8 with KDM6 A deletion could not be converted to EPS cell.Conclusion: First,the conversion and maintenance of human ES cell to EPS cell is feasible in matrigel-based feeder-free conditions.Compared with conventional ES cell,EPS cell corresponds to earlier development in embryo,both in terms of molecular indexes and functional chimerism.Second,the level of H3K27me3 in EPS cell was higher than that in ES cell,but it was an obstructive epigenetic modification for the conversion of ES cell to EPS cell.These data provide an experimental basis for the studies of dynamic changes of H3K27me3 in early human embryos in vitro.