A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species

The derivation of authentic embryonic stem cells (ESCs) from diverse mammalian species offers valuable opportunities for advancing regenerative medicine, studying developmental biology, and enabling species conservation. Here, we report the development of a robust, serum-free culture system, termed 6iL/E4 that enables the derivation and long-term self-renewal of ESCs from multiple mammalian species, including mouse, rat, bovine, rabbit, and human. Using systematic signaling pathway analysis, we identified key regulators--including GSK3, STAT3, PDGFR, BRAF, and LATS--critical for ESC maintenance across species. Additionally, inducible expression of KLF2 and NANOG enhances the naive pluripotency and chimeric potential of bovine ESCs. The E4 medium also supports stable ESC growth while minimizing lineage bias. 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 biotechnology applications. In BriefWang et al. developed 6iL/E4, a serum-free system sustaining ESCs from mouse, rat, bovine, rabbit, and human. These findings reveal conserved fundamental mechanisms governing ESC self-renewal across diverse mammalian species. HighlightsO_LIDeveloped 6iL/E4 system for ESC derivation across five mammalian species. C_LIO_LIPDGFR signaling inhibition as critical for ESC derivation across species. C_LIO_LIE4 medium improves ESC maintenance and avoids neural bias of traditional N2B27. C_LIO_LIInducible KLF2/NANOG enhances naive pluripotency and chimera formation in bovine. C_LI