Derivation of primed sheep embryonic stem cells and conversion to an intermediate naïve-like state

Embryonic stem cells (ESCs) derived from the inner cell mass of embryos possess unlimited self-renewal and pluripotency, offering a powerful system to study early development and enable genetic and biotechnological innovation. Although several livestock ESC lines have been reported in recent years, defining culture conditions that support stable, long-term self-renewal and controlled transitions across pluripotent states remains challenging. Here, we report the de novo derivation of sheep (ovine) ESCs (oESCs) from in vivo blastocysts using a chemically defined culture system. The derived cells exhibit morphological and molecular features of primed pluripotency and can be propagated under both feeder-dependent and feeder-free conditions without loss of identity or karyotypic stability. Building on this foundation, we developed enhancer-driven reporter lines that faithfully reflect octamer-binding transcription factor 4 (OCT4) and SRY-box transcription factor 2 (SOX2) transcriptional activity, enabling dynamic visualization of pluripotency and differentiation in live cultures. These reporter systems revealed the responsiveness of oESCs to signaling modulation and provided a functional readout of pluripotency state transitions. When cultured in defined media previously shown to stabilize naïve pluripotency in human ESCs, oESCs adopted a dome-shaped colony morphology, maintained OCT4, SOX2, and Nanog homeobox (NANOG) expression, retained differentiation potential, and exhibited a transcriptomic profile consistent with resetting to an intermediate pluripotent state with naïve-like morphological features. In summary, these findings establish stable ovine ESC lines and demonstrate their plasticity across the pluripotency spectrum, providing a valuable platform for investigating ruminant stem cell biology and advancing livestock biotechnology.