A universal 6iL/E4 culture system for deriving and maintaining embryonic stem cells across mammalian species

Abstract The derivation of authentic embryonic stem cells (ESCs) across mammalian species remains a major challenge. Here, we report the development of a defined, serum-free culture system, termed 6iL/E4, that enables the derivation and long-term self-renewal of ESCs across diverse mammalian species. Through systematic dissection of signaling pathways, we identified conserved regulatory modules involving GSK3α, WNT, STAT3, PDGFR, and MEK/ERK signaling. The optimized 6iL/E4 conditions support stable derivation and expansion of ESCs from mouse, rat, rabbit, and bovine embryos. For rabbit, ESC derivation required supplementation with the LATS inhibitor TDI-011536 (TDI), and 6iL/TDI-cultured rabbit ESCs exhibited chimera-forming capability. In bovine ESCs, inducible expression of Klf2 and Nanog reinforced pluripotency and promoted in vivo chimeric contribution. Importantly, we demonstrated that 6iL robustly establishes and maintains human pluripotent stem cells in a naïve-like state. These findings reveal conserved principles underlying ESC self-renewal across divergent mammalian species and provide a universal platform for cross-species stem cell research, disease modeling, and biotechnological applications.