OTX2 inhibits human pluripotent stem cell reprogramming toward 8-cell-like and morula-like states.

In human embryos, major zygotic genome activation (ZGA) initiates at the 8-cell (8C) blastomere stage, marking the start of the ontogenesis program. Recent advancements have shown that primed human pluripotent stem cells (hPSCs) can be reprogrammed to 8C-like cells (8CLCs) with totipotent characteristics in vitro. However, the key regulators driving this transition remain largely unexplored. In this study, we identify OTX2 as a key factor that establishes a repressive barrier to the induction of 8CLCs from primed hPSCs. Our findings reveal that OTX2 deletion greatly enhances the generation of TPRX1-EGFP(+) 8CLCs, which closely resemble the transcriptomic profiles and epigenetic landscape of 8C/morula embryos. Notably, these OTX2-deleted 8CLCs exhibit improved bidirectional differentiation potential and contribute to both embryonic and extraembryonic tissues in chimeric embryos. Mechanistically, OTX2 regulates both naive and totipotent state transition, but exerts its predominant effect on the latter by binding to loci of key 8C-specific regulators. Collectively, our findings define a critical role for OTX2 in regulating totipotency and establish a foundational framework for generating 8CLCs from primed hPSCs in vitro, offering significant insights for stem cell biology and regenerative medicine.