Establishment Of Artificial Embryonic Model By Novel Embryonic Stem Cells In Mice

Embryonic stem cells(ESCs)are characterized by their self-renewal ability and pluripotent developmental potential,which endow them with great value in basic research and future clinical application.At present,a variety of mouse,rat and human embryonic stem cells have been successful established.Mouse embryonic stem cells(ESCs)are developmentally closest to the preimplantation inner cell mass(ICM)of blastocysts,with the potential to contribute to all embryonic tissues and the germline,excluding the extra-embryonic tissues in chimeric embryos.There is much research on artificial gametes of mouse,however,there is little evidence regarding artificial embryos in mice.In our study,we attempt to establish mouse artificial embryonic model by octaploid(8N)embryo,and generate blastocyst-derived embryonic stem cells with high potency(Advanced pluripotent stem cells,ASCs).In addition,we reconstruct the artificial embryo by injecting ASCs to 8N embryos.This study is divided into three parts.The main results are as follows:Projectl.Generation of developmental model of mouse octaploid embryoThere is little information regarding mouse octaploid embryonic development and preeise mechanisms contributing to the developmental limitations of octaploid embryos are unknown.To investigate the genetic and epigenetic mechanisms underlying octaploid embryonic development,we generated mouse octaploid embryos and evaluated in vitro/in vivo developmental potential.Here we show that octaploid embryos can develop to the blastocyst stage in vitro,but all fetus are impaired immediately after implantation.Our results indicate that cell lineage specification of octaploid embryo was disorganized.Furthermore,these octaploid embryos showed increased apoptosis/autophagy as well as alterations in epigenetic modifications when compared with diploid embryos.Thus,our cumulative data provide cues for why mouse octaploid embryonic development is limited and why postimplantation development fails.Because ESCs eannot eontribute to placenta,therefore,we try to establish blastocyst derived ASCs with more high potency.Project 2.Induction and identification of novel mouse embryonic stem cellsASCs were derived from blastocysts in two steps using a chemically defined medium supplemented with Activin A(ActA)and basic fibroblast growth factor,followed by culturing in ABCL medium containing ActA,BMP4,CHIR99021 and leukemia inhibitory factor.Notably,ASCs exhibit a distinct transcriptome with the expression of both naive pluripotency genes,as well as mesodermal somatic genes;Eomes,Eras,Tdgf1,Evx1,hand1,Wnt5a and distinct repetitive elements.Importantly,ASCs exhibit a stable hypermethylated epigenome and mostly intact imprints,compared to the hypomethylated epiblast of blastocysts and naive ESCs.Properties of ASCs suggest that they represent cells at an intermediate cellular state between the naive and primed states of pluripotency.ASCs cells provide stem cell sources for further study on artificial embryonic model in mice.Project 3.Assesing pluripotency of ASCs and assembly of artificial embryonic model by ASCsTo compare the key features of pluripotency of ASCs relative to ESCs,we examined expression by immunofluorescence for cMYC and other core pluripotent protein.Excluding the higher expression of cMYC in ASCs versus ESCs,localization of other pluripotent protein was equivalent to that in ESCs.ASCs are developmentally beyond pluripotent cells in the inner cell mass but possess higher potency than EpiSCs.Accordingly,ASCs contributes very efficiently to the fetus,gernline,yolk sac and the placental labyrinth in chimeras.Since they are developmentally more advanced,ASCs do not contribute to the trophoblast.For further stringent tests of potency,ASCs were introduced into tetraploid host blastocysts,where the donor eells eontribute predominantly if not exclusively to the embryo.Then,we injected ASCs cells into 8N embryos and reconstructed artificial embryos.Our reconstructed artificial embryonic models can support the development of preimplantation embryos in vitro,but cannot develop into fetal tissues after implantation.In the future,we will further improve the study of ASCs cells and 8N artificial embryonic model.