Study On Dynamic Development Of Pre-gastrulation Human Embryos

With the development of industrial level and the improvement of living standard,poor living environments,unhealthy eating habits and increasing survival pressures have led to a rapid decline in the quality of sperm and oocyte of childbearing age couples,the infertility rate has become an important issue of reproductive health.On the seventh day of embryonic development,a human embryo needs to be implanted into the uterus to survive and develop.Due to the lack of materials and technologies,how the embryo develops in the uteru and the key cellular and molecular events that at this stage remains a black box.The failure of embryo implantation and abnormal development of embryo at this stage are the main causes of early miscarriage.Therefore,the study of human pre-gastrulation embryonic development will provide important theoretical basis to understand the origin of life and explore the dynamic change process and molecular regulation mechanism of early embryonic development.Two studies in 2016 established an in vitro culture system for human embryos after implantation based on the in vitro culture of mouse embryos,which was able to culture the embryos up to 13 days on a 2D basis and observed some lineage separations.However,there are some important defects in this 2D system.For example,the 2D embryos are flat and lack the 3D topological structure in vivo.Although 2D cultivated trophoblast after 12 days is still alive,the whole embryo structure collapse and appeared developmental disorder.Many cell types(amniotic epithelial),cavity(amniotic cavity,yolk sac)and structure(basement membrane,anterior-posterior axis,primitive streak)cannot be clearly observed in 2D conditions.Therefore,these defects limit the ability of the 2D system to truly simulate the development of embryo in vivo and to reveal the developmental events and mechanisms of the embryo at this stage.Based on these defects,we carry out the study of this paper.With strict ethical permission and the patient’s informed consent,by improving the medium and culture methods,we developed a three-dimensional(3D)human blastocyst culture system using clinically donated embryos.Using this system,the human blastocyst was first cultured to the primitive streak stage under 3D conditions.These 3D embryos can highly simulate the development of embryos in vivo,including the embryonic disc,amnion,basal membrane,and the unique secondary yolk sac,anterior and posterior axes,and primitive streak which cannot be produced by 2D conditions.Using this system,we investigated and revealed the key developmental events of the human pre-gastrulation embryo:(1)Revealed the dynamic and molecular regulatory networks of the lineage differentiation and development of the epiblast,hypoblast,and trophoblas through the analysis of the transcriptional expression profiles of a large number of single cells.(2)Amniotic epithelial(AME)is the first type of cell line isolated from mesodermal cells.Unlike rodents,human AME occurs before the formation of the primitive streak,but its characteristics and molecular mechanism are unclear.We found that AME significantly down-regulated the pluripotent gene,and its formation was significantly correlated with the loss of the basement membrane.Comparing to the epiblast,it had a unique molecular expression profile.(3)We illuminated the differentiation of cytotrophoblast,extravillous cytotrophoblast and syncytiotrophoblast after embryo implantation for the first time,as well as the signaling pathways and transcription factors that cause differentiation,revealing that the function of extravillous cytotrophoblast in early embryo is different from that in the middle and late placenta.(4)We revealed that the transition from naive to primed state will be rapid after implantation of the epibalst cells(PSCs,pluripotent stem cells).PSCs remained relatively stable from implantation to day 14,its development and transformation are determined by the coordination of different pluripotent factors.(5)Through transcriptome analysis of human and non-human primate embryos,we found that there were significant differences in metabolism between human and non-human primate epiblast cells,and they were conservative in maintaining pluripotency of stem cells and in key molecules and signaling pathways of development.The original in vitro 3D culture system of human embryo elaborated the developmental events such as the separation of lineage,the formation of amniotic cavity,the separation of amniotic epithelium,the formation of yolk sac,the generation of embryo polarity and the formation of primitive streak in the process of embryo development.In this paper,we answered the scientific questions about the maintance and transiton of EPI pluripotency state,the formation of AME,the differentiation of Tr B.This work provides a new research model for the early embryo development and stem cell research,provides theoretical basis for the tissue and organ regeneration research,provides theoretical supports to understand implantation failure and early abortion,provides research strategy and research direction in the treatment of infertility and the solution of the problems existing in assisted reproductive technology.