Establishment of Rabbit Formative-like Pluripotent Cells by Bidirectional Modulation of WNT Signaling Pathway

Abstract

The generation of embryonic stem cells (ESCs) with full developmental capacity and robust chimera-forming potential remains a major bottleneck for most non-rodent mammalian species including rabbits, an important animal both for biomedicine and agriculture. Formative pluripotency is a unique intermediate state bridging the naive and primed pluripotent states, which is defined by distinct molecular signatures, intrinsic competence for primordial germ cell (PGC) specification, and unique developmental adaptability to early embryonic microenvironments. Here, we established a novel culture medium termed KFiC, which could bidirectionally and coordinately modulate the WNT/{beta}-catenin signaling pathway via combined treatment of WNT activator CHIR99021 and inhibitor IWR-1. The KFiC culture system enables efficient derivation and stable long-term self-renewal of rbESCs with consistent formative-like pluripotent characteristics. KFiC-cultured rbESCs (KFiC-rbESCs) form compact, undifferentiated colonies, maintain intact genomic stability, and recapitulate the transcriptional landscape of pre-implantation epiblast cells, accompanied by sustained high expression of the core formative pluripotency marker OTX2. Functionally, KFiC-rbESCs possess efficient PGC differentiation competence and extensively contribute to embryonic development after microinjection into rabbit morulae, displaying their superior developmental plasticity. Notably, the KFiC system can stabilize an intermediate formative-like pluripotent state in mouse, human, and porcine pluripotent stem cells, and KFiC-adapted mouse ESCs retain potent chimera-forming ability. Collectively, this study establishes a reliable culture platform for generating and maintaining formative-like rbESCs. This cross-species compatible system provides a promising foundation for further optimizing naive-level pluripotency in rbESCs and facilitates the generation of genetically engineered rabbit disease models.