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

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 STAT3, GSK3α, PDGF, RAF and LATS—critical for ESC maintenance across species.

Additionally, inducible expression of KLF2 and NANOG enhances the naïve pluripotency and chimeric potential of bovine ESCs. The E4 basal medium also supports stable ESC growth while minimizing lineage bias. These findings support the evolutionary conservation of pluripotency signaling and provide a universal platform for cross-species stem cell research, disease modeling, and biotechnology applications.