Transcriptional heterogeneity and molecular regulation of totipotency-to-pluripotency transitions in Porcine EPSCs

The molecular characteristics of EPSCs have long been debated. While human EPSCs (hEPSCs) resemble eight-cell and morula-stage embryos, mouse EPSCs (mEPSCs) more closely resemble post-implantation epiblasts. This study explores the developmental potential and molecular characteristics of porcine expanded pluripotent stem cells (pEPSCs), uncovering their unique transcriptional heterogeneity and similarities to both morula and post-implantation epiblast stages. By performing single-cell transcriptomic analysis, three distinct subpopulations (C1, C2, and C3) were identified, corresponding to totipotency, naïve pluripotency, and primed pluripotency. The use of dual fluorescence reporter systems highlighted transitions between these states, resembling embryonic development. Key findings reveal a dynamic balance model of LEUTX and OTX2 in regulating pluripotency transition that OTX2 promotes the shift from totipotency to primed pluripotency by promoting pluripotency and lineage-priming genes, while LEUTX enhances totipotency by activating totipotency genes. LEUTX-overexpressing cells demonstrated enhanced developmental potential and stability in vitro. The study established a conserved framework for understanding state transitions mediated by OTX2 and LEUTX, with implications for regenerative medicine, developmental biology, and highlight the potential of pEPSCs in agricultural and biomedical applications. This study aims to comprehensively profile the chromatin accessibility, transcription factor binding, and gene expression in pig pEPSCs using multiple high-throughput sequencing technologies, including ATAC-seq, ChIP-seq and RNA-seq. The dataset includes pEPSCs samples under various conditions, including wild-type, gene overexpression (e.g., OTX2, LEUTX), and knockout (e.g., OTX2), as well as wild-type and OE_OTX2 4-cell embryo stages.