LEUTX and OTX2 orchestrate totipotency-to-pluripotency transitions and heterogeneity in pEPSCs

The molecular characteristics of expanded pluripotent stem cells (EPSCs) remain debated. Human EPSCs (hEPSCs) resemble embryos at the 8-cell to morula stage, while mouse EPSCs (mEPSCs) resemble post-implantation epiblasts. This study examines the developmental potential and molecular features of porcine EPSCs (pEPSCs), revealing their distinct transcriptional heterogeneity and dual resemblance to both morula and post-implantation epiblast stages. Single-cell transcriptomic analysis identified three subpopulations (C1, C2, and C3) in pEPSCs, corresponding to totipotency-like, naive pluripotency-like, and primed pluripotency-like. Using the NANOG-tdTomato/SOX2-EGFP reporter system, we further characterized this heterogeneity, uncovering a continuum from totipotency to primed pluripotency across subpopulations C1 to C3, which parallels transitions observed in early embryonic development. Additionally, we identified OTX2 and LEUTX as key regulators in this process, with OTX2 promoting pluripotency and LEUTX enhancing totipotency and blastoid formation. This study enhances our understanding of mammalian stem cells and highlights the potential of pEPSCs in agricultural and biomedical applications.