| Trophectoderm(TE)develops from the outer cells of morula.It is crucial for blastocyst implantation and embryo growth.However,at present,due to the scarcity of embryonic materials,technical difficulties and ethical limitations,it is difficult to directly analyze the human in vivo placenta tissues and derivatives of TE such as the cytotrophoblast,syncytial trophoblast(ST),and extravillous trophoblast(EVT),especially in the first few weeks after blastocyst implantation.Comparative analysis combined with single-cell RNA sequencing(sc RNA seq)has been reported to reveal the evolutionally-conserved characteristics of human and non-human primate(NHP)macaque placenta.Therefore,exploring the important signaling pathways and key regulatory factors in TE development in NHP can provide models and ideas for study the mechanism of human placenta-related diseases.On the other hand,the generation of chimeric animals by complementing early embryos with homologous cells can be used to test the potency of mammalian pluripotent stem cells(PSCs).In NHP,the contribution of donor cells in chimeras is still low due to the induction of naivety in those pluripotent cells are suboptimal and thus the donor cell does not match with the development stage of the host embryo properly,cell heterogeneity.And the donor cells can not match the developmental state of the host embryo.Based on the above problems and feasibilities,this study is mainly divided into the following three parts:First,in order to explore the role of different signaling pathways in human TE development.In this study,we first established a TE enriched gene GATA3 reporter(GFP)H9 cell line(H9-GATA3-GFP)using CRISPR/Cas9 gene editing technology.Using this reporter cell line,we performed a small molecules screening targeting different signaling pathways during na(?)ve hPSCs to hTELCs induction process.Notably,we observed that addition 3μM of WNT signaling activator CHIR 99021(CHIR)significantly inhibited the induction ratio of GATA3~+cells validated by flow cytometry(FACS)and also the expression of TE-related genes downregulated compared to the control tested by RT-q PCR.In addition,activation of WNT signaling pathway in UH10-4CL na(?)ve i PSCs cell line also inhibited the expression of TE related genes.To test whether WNT signaling pathway is necessary for TE induction,we performed CRISPR/Cas9 gene editing technology to generate the mediator of WNT signaling pathwayβ-catenin knock out cell lines using primed hPSC and successfully converted them into na(?)ve state and subsequently,β-catenin knock out na(?)ve PSCs were subjected to TELCs induction.Notably,we observed that hTELCs induction does not requireβ-catenin.We also found that adding CHIR during TELC induction usingβ-catenin knockout cells does not have any adverse effect.Hence,WNT signaling pathway is dispensable in human TELC induction from na(?)ve PSCs.Global gene expression analysis using bulk-RNA seq showed that the amnion-related genes such as ISL1,GABRP,IGFBP3,VTCN1,TFAP2A etc was significantly upregulated in CHIR(WNT signaling activated)group compare to the control in TELC induction.To validate the identity of the amnion-like cells,we performed droplet-based sc RNA seq for 4CL na(?)ve hPSCs,CHIR treated and control cells in TELC induction at day 5.Importantly,the UMAP analysis showed that the amnion-like cells generated by CHIR treated condition clustered tightly with the amnion(non-neuro ectoderm)of human Carnegie 7(CS7).We also tested the chromatin landscape of CHIR treated and control cells in TELC induction at day 5by bulk-ATAC seq.In line with the gene expression observations,we found that the TE-related genes chromatin were less accessible in CHIR treated cells.Altogether,we conclude that the activation of WNT signaling pathway during na(?)ve PSCs to TELCs generates human amnion-like cells(h AMLCs)instead of TELCs.Next,to verify whether this phenomenon is shared between human and NHP such as monkey,we first aim to established optimal na(?)ve monkey embryonic stem cells(na(?)ve cy ESCs)system.We have systematically tested five different na(?)ve including extended pluripotent stem cells culture conditions(4CL,5i LAF,PXGL,LCDM,and RSe T)to generate na(?)ve cy ESCs by examining cell morphology,proliferation,gene expression and chromosome stability,we found that 4CL na(?)ve medium preformed better than other conditions in na(?)ve cy ESCs generation.We next conducted the chimera assay as a gold standard method to verify the pluripotency status and development capacity of 4CL na(?)ve cy ESCs.A live-born chimera monkey and an aborted chimeric fetus with a very high contribution of donor cells produced by 4CL naive medium were generated.Single-nucleus sequencing(sn RNA seq)data identified that 4CL na(?)ve cy ESCs contributed into embryonic lineage such as immune(PBMC and BMC),nerve,heart cells,and also into extraembryonic placental tissue in the chimeric monkey.This study laid a foundation for solving the mechanism of low chimerism rate and non-functional chimerism,and also provided raw materials for the study of naive cy ESCs in vitro to multi-lineage differentiation.Finally,in order to explore the role of WNT signaling pathway on monkey TELCs(cy TELCs),we have successfully induced cy TELCs from 4CL naive cy ESCs.These cy TELCs lost the pluripotent genes NANOG and KLF17 expression and with high level expression of TE-related marker genes such as GATA3 and VGLL1.Importantly,by using this in vitro cy TELCs model,we found that the activation of WNT signaling pathway during cy TELCs induction produces monkey amnion-like cells(cy AMLCs)instead of cy TELCs.This observation was valiated by bulk RNA seq data analysis.In summary,this study identifies the role of WNT signaling pathway in the extraembryonic lineage specification in human and NHP.This study holds immense importance for understanding human and NHP early embryonic development and lays a foundation for the mechanistic study and clinical treatment of pregnancy failure and placenta-related diseases. |
