Highly efficient generation of blastocyst-like structures from mouse totipotent blastomere-like cells

Mammalian embryogenesis begins with a totipotent zygote. Early embryogenesis can be recapitulated by aggregated extended pluripotent stem cells (EPSCs) in a 3D culture system. However, the efficiency of generating blastoids is low and whether other reported totipotent stem cells retained a similar capacity remains unknown. Here we show that spliceosomal repressed totipotent blastomere-like cells (TBLCs) form blastocyst-like structures when aggregated in 3D microwells with around 80% efficiency. TBLC-blastoids resemble blastocysts in morphology and cell-lineage allocation, show similar transcriptional profile with natural blastocysts and contain more TE cells and fewer undefined intermediate cells compared to blastoids from EPSCs. Moreover, TBLC-blastoids can develop beyond the implantation stage in vitro and induce decidualization after transferred into uterus. In summary, we supply an alternative cell type to generate ameliorated blastoids highly efficiently for studying early mouse embryogenesis. RNA-seq were performed on mESCS and mCMG-TBLCs with mCMG ESCs in ES medium plus 2.5 nM PlaB on feeders for over 6 passages; scRNA-seq were performed on TBLC-blastoids.