Study Of Gastrulation In Early Cynomolgus Embryo And Human-monkey Interspecies Chimera

From peri-implantation to gastrulation,mammalian embryos undergo multiple levels cell fate determination,which is one of the key events in the entire embryonic development process.The transition from peri-implantation to gastrulation in mammals requires the specification and organization of the lineage progenitors into a body plan,and any developmental confusion can affect ontology and have a profound impact.Understanding the mechanisms of early embryonic development after implantation in humans is of great importance for both basic developmental biology and regenerative medicine.Studies based mouse models have revealed the molecular mechanisms that control gastrulation.However,there are considerable anatomical,physiological,and developmental differences between mice and humans,limiting the insights derived from mice that are applicable to human embryogenesis,and it is urgent to find animals modle closer to humans as research objects.Human embryonic body researches have improved our understanding of early embryonic related developmental events,such as the specialization of amniotic cells,the formation of the amniotic cavity,and the movement of the gastrulation.But our understanding of molecular mechanisms and cellular events at the embryonic level is still limited.Besides,critical questions remain,including whether primate embryos can be cultured beyond the gastrulation stage in vitro and what the molecular determinants are of embryonic and extraembryonic lineage specification.To address these questions,we established the in vitro culture systerm to support cynomolgus embryos beyond gastrulation and up to 20 days in vitro.The cultured cynomolgus embryos were not only morphologically similar to in vivo embryos,but also underwent periand postimplantation events typical of higher primate species,including embryonic and extraembryonic lineage segregation,bilaminar disc generation,amniotic cavity polarization and yolk sac cavitation,and primordial germ cell-like cell(PGCLC)specification,and got some innovative conclusions.(1)There was a unique expression pattern of the trophoblast marker CDX2 in cynomolgus embryos.On days 8-9 of cynomolgus embryos,CDX2 was co-expressed with OCT4 and GATA6,but this phenomenon was not found in mouse and human embryos.CDX2 is expressed in mice but not in human trophoblastic stem cells,and after E12,CDX2 is expressed only in amniotic epithelial-like cells of cynomolgus embryos.(2)sc RNA-seq and single-cell assay for transposase-accessible chromatin–using sequencing(sc ATAC-seq)were used to analyze transcriptional and accessible chromatin profiles of cynomolgus post-implantation embryos at single-cell resolution,delineating the developmental trajectories of EPI,primitive endoderm(PE),trophectoderm(TE),and PGCs and characterizing transcription factor(TF)regulatory networks and signaling pathway interactions.(3)We found a dynamic process of EPI pluripotency from na(?)ve to primed in nonhuman primate embryos,revealing the role of oxidative phosphorylation pathways in the maintenance of na(?)ve pluripotency.(4)It was revealed that in vitro embryonic PGCLCs coincided with the results of PGC cell specialization in in vivo embryo studies,and amniotic membranes are the source of PGCs in vivo,but the epiblast source is not excluded.In summary,for the first time,primate embryos were cultivated in vitro to the stage of protozoal embryos,and the morphology and gene expression characteristics of the cultured embryos were highly consistent with those of embryos in vivo.Our results shed light on key developmental events and complex molecular mechanisms of early non-human primate embryogenesis after implantation,contributing to the acquisition of mechanistic insights into primate embryogenesis,which may be relevant to human embryonic development.The study of human-animal interspeciecs chimerism has great scientific significance and biomedical potential.Interspecies chimera formation with human pluripotent stem cells(h PSCs)represents a necessary alternative to evaluate h PSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications,including the generation of organs and tissues for transplantation.Genetic and evolutionary distance has an important impact in the production of interspeciecs chimeras,and in-depth study of human and primate embryonic chimeras is expected to decipher the human-animal embryonic chimera growth impediments,looking for ways to break through the barriers.Considering the ethical issues that may be involved in study of chimeric embryos in human-nonhuman primates in vivo,after passing a series of ethical reviews,we conducted a study on the chimeric ability of human extended pluripotent stem cells(hEPSCs)in monkey embryo using in vitro culture system.We found that hEPSCs live,proliferate and contributed to several main lineages(EPI,HYP,and EXMC)in peri-and early postimplantation monkey embryos.The study revealed that hEPS can chimerize into cynomolgus embryos,and can accompany embryonic development into gastrulation,expressing markers of mesoderm and endoderm.Human EPS cells are difficult to differentiate into trophoblast cells in cynomol embryos,but are easier to differentiate into epiblast(EPI)cells,which is different from the results of hEPS cell human-mouse chimera experiments.Through further analysis of cell interaction,the specific interactions between human and monkeys such as EPI cells were revealed.This study answers basic scientific questions such as how heterogeneous chimeric cells interact with each other and how to regulate differences in developmental procedures,and provides new ideas for solving problems such as low heteromeric chimerism efficiency,which has guiding significance for organ regeneration research.