Capturing totipotency in human cells through spliceosomal repression

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency. [Display omitted] •ZLCs are transiently reprogrammed from human ESCs/iPSCs through splicing inhibition•Differing from 8CLCs, ZLCs silence pluripotent genes and activate distinct ZGA genes•ZLCs convert into hTBLCs with pre-ZGA characters after long-term passaging•hTBLCs pass through ZLC stage to parallelly generate EPI-, PrE-, and TE-like lineages Two states of totipotency could be captured using splicing inhibition as a handle to reprogram human pluripotent stem cells. The first transient represents a distinct ZGA-like state that could subsequently transition into stable, totipotent blastomere-like cells that express pre-ZGA-specific genes.