Highly cooperative chimeric super-SOX induces naïve pluripotency across species [RNA-Seq]
Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission demonstrated only for mice and rats.  By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a remarkable gain-of-function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-grade OSKM iPSCs capable of supporting the development of healthy all-iPSC mice.  Sox2/Oct4 dimerization emerged as the core driver of naïve pluripotency with its levels diminished upon priming. Transient overexpression of Sox2-17 and Klf4 (S*K cocktail) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naïve reset in mammals. Overall design: To investigate the effects of SOX2AV and SOX2-17 on the fidelity of reprogramming, we generated and characterized 30 hiPSC lines using episomal reprogramming of new-born foreskin (young, Y) and 56-year-old male dermal (old, O, AG04148) fibroblasts. For episomal reprogramming 5x10^5 fibroblasts were nucleofected with 3 µg of plasmid DNA mix: pCXLE-SOX2-P2A-KLF4 (Addgene ID 193292), pCXLE-SOX2AV-p2a-KLF4 (Addgene ID193291) pCXLE-SOX2-17-P2A-KLF4 (Addgene ID 193290), pCXLE-L-MYC-F2A-LIN28 (ML, Addgene ID 27080), pCXLE-hOCT4-shTP53 (Addgene ID 27077), pCXWB-EBNA1 (Addgene ID 37624) using Lonza NHDF Nucleofector kit (U-23 program), and plated in ROCKi-containing fibroblast media on a CF1 feeder layer. All the selected clonal iPSC lines were integration-free with normal karyotypes. The hiPSC samples were collected at passage 10-12, at day 3 after plating on Matrigel (Cornig) in StemFlex media (Gibco).