Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

ABSTRACT The discovery of induced pluripotent stem cell (iPSC) technology by Shinya Yamanaka has truly enabled the stem cell field. After 16 years of intense research, the delivery methods and culture media have improved but the original factors—Oct4, Sox2, Klf4, and Myc (OSKM)—remain central for driving reprogramming. Here we define structural elements in chimeric Sox2/Sox17 transcription factors that rescued the ability of nonfunctional Oct factors to induce pluripotency. Most importantly, we discovered a single amino acid swap in the DNA-binding domain of Sox2, A61V, that stabilizes the Sox/Oct heterodimer on DNA through hydrophobic interaction with Oct. The highly cooperative Sox2 AV mutant enables iPSC generation with Oct4 orthologs, such as Oct2 and Oct6, as well as rescues otherwise detrimental Oct4 mutants and domain deletions. Sox2 AV has a dramatic effect on the cell fate reset, significantly improving the developmental potential of OSKM iPSCs. Moreover, by swapping multiple beneficial elements of Sox17 into Sox2 we have built a chimeric super-SOX factor—Sox2-17—that delivers unprecedented reprogramming efficiency and kinetics in five tested species. Sox2-17 enhances five-, four-, and three-factor reprogramming up to hundreds of times, enables two-factor generation of human iPSCs, and allows integration-free reprogramming of otherwise non-permissive aged human, non-human primate, and cattle fibroblasts. Our study demonstrates that a complete developmental reset requires both robust activation of regulatory elements controlled by the canonical SoxOct motif and limiting cellular proliferation driven by Oct4 and Myc. A high level of Sox2 expression and Sox2/Oct4 heterodimerization emerge as the key determinants of high-grade pluripotency that fades along the naïve-to-primed continuum. Transient expression of SK cocktail can restore the naivety, providing a powerful technology to induce more complete developmental reset in pluripotent cells across species.