Abstract
BACKGROUND: Generating expanded potential stem cells from cloned porcine embryos (pEPSCs(NT)) represents a notable advancement in regenerative medicine and agricultural biotechnology. However, challenges, including low derivation efficiency, limited understanding of transcriptomic features, and unknown feasibility of culturing under feeder-free conditions, remain. This study aimed to generate pEPSCs using blastocysts derived from parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer (SCNT) using a modified culture system. METHODS: We derived pEPSC(NT) lines using an optimized culture system. We characterized the pEPSC lines from all three origins by analyzing pluripotent marker expression, performing karyotyping, and assessing their differentiation potential into the three germ layers. Furthermore, we performed a comparative transcriptomic analysis using in vivo and cloned embryo data, with a major focus on cell lines derived from SCNT (pEPSCs(NT)). We optimized feeder-free culture conditions for the pEPSC(NT) line and derived the pEPSC(NT) lines using an optimized culture system with an efficiency of ~ 14%. RESULTS: The cells were closely correlated with 8-cell to morula-stage embryos and exhibited significant enrichment of EPSC signature genes, suggesting a unique pluripotent state relatively close to the naïve state, specifically within a formative state. The pEPSCs(NT) possessed broad differentiation capacity, indicative of Hippo signaling pathway enrichment, blastocyst-like structure formation ability, and potential differentiation into trophoblast lineage cells. CONCLUSIONS: Our modified culture medium combined with the 2× Matrigel coating system facilitated the transition to feeder-independent culture conditions. These findings facilitate the establishment of a feeder-free culture system while preserving pluripotency and differentiation potential.