Summary As the quintessential reprogramming model, OCT3/4, SOX2, KLF4, and c-MYC re-wire somatic cells to achieve induced pluripotency. Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms. Employing transposons, we systematically assessed cellular and molecular hallmarks of mouse somatic cell reprogramming by various polycistronic cassettes. Reprogramming responses varied in the extent of initiation and stabilization of transgene-independent pluripotency. Notably, the cassettes employed one of two KLF4 variants, differing only by nine N-terminal amino acids, which generated dissimilar protein stoichiometry. Extending the shorter variant by nine N-terminal amino acids or augmenting stoichiometry by KLF4 supplementation rescued both protein levels and phenotypic disparities, implicating a threshold in determining reprogramming outcomes. Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant. Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets.
Graphical Abstract
Highlights • Reprogramming vectors inconsistently employ one of two unappreciated Klf4 variants • Polycistronic cassettes encoding Klf4 N-terminal variants drive distinct stoichiometry • Reprogramming initiation and stabilization are sensitive to Klf4 protein levels • Accordingly, gene expression elicited by public vectors forms two distinct clusters
In this article, Woltjen and colleagues reveal the unwitting employment of two different Klf4 cDNAs to construct somatic cell reprogramming vectors. Cloned in a polycistronic cassette, each variant produces different KLF4 protein levels, impacting reprogramming phenotypes. Moreover, comparative gene expression during iPSC derivation bifurcates based on the Klf4 cDNA used, underpinning the importance of factor stoichiometry in defining reprogramming outcomes.