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 initiation of the ontogenesis program. Recent advancements have demonstrated that human pluripotent stem cells (hPSCs) can revert to 8C-like cells (8CLCs) with totipotency in vitro. However, the key regulators involved this transition remain largely unexplored. In this study, we identified OTX2 as a novel regulator repressing the induction of 8C-like cells (8CLCs) from hPSCs. Our findings reveal that OTX2 depletion facilitates the generation TPRX1-EGFP+ totipotent-like stem cells (TLSCs), which closely resemble the transcriptomic profile and chromatin accessibility of 8C blastomeres. Notably, TLSCs with OTX2 depletion exhibit bidirectional embryonic and extraembryonic differentiation potential in forming chimeric embryos. OTX2 influences totipotent state transition by binding at loci of key 8CLC regulator, which function in parallel with DUX4. Collectively, our findings reveal a previously unrecognized function of OTX2 in regulating totipotency and establish a new framework for generating TLSCs from hPSCs in vitro, offering significant insights for stem cell biology and regenerative medicine.