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 embryonic stem cells (sESCs) 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 OCT4 and SOX2 transcriptional activity, enabling dynamic visualization of pluripotency and differentiation in live cultures. These reporter systems revealed the responsiveness of sESCs 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, sESCs adopted dome-shaped colony morphology, maintained OCT4, SOX2, and NANOG expression, retained differentiation potential, and exhibited a transcriptomic profile consistent with resetting to an intermediate pluripotent state with naïve-like morphological features. These findings establish stable sheep ESC lines and demonstrate their plasticity across the pluripotency spectrum, providing a valuable platform for investigating ruminant stem cell biology and advancing livestock biotechnology.