Transcriptomic insights and feeder-free culturing of porcine expanded potential stem cells from cloned embryos

• Successfully derived porcine expanded potential stem cells from cloned embryos (pEPSCsNT) with ~14% efficiency using an optimized culture system. • Transcriptomic analysis revealed that pEPSCsNT closely resemble 8-cell to morula-stage embryos and exhibit a formative pluripotent state with enrichment of EPSC signature genes. • pEPSCsNT demonstrate broad differentiation capacity, including formation of blastocyst-like structures and potential to differentiate into trophoblast lineages, indicating Hippo signaling pathway involvement. • Established a feeder-free culture system using modified medium and 2× Matrigel coating, preserving pluripotency and differentiation potential, which facilitates scalable applications.