TET1 attenuates human pluripotency-related heterogeneities and enhances differentiation capabilities

Human induced pluripotent stem cells (hiPSCs) offer transformative potential for developmental research and therapeutic discovery; however, their variable characteristics are a significant barrier to their broader application. We reprogrammed hiPSCs by adding TET1, a DNA-demethylating dioxygenase, to produce TET1-iPSCs (T-iPSCs) with enhanced epithelialization and differentiation capabilities. A default differentiation survey—designed and implemented in our hiPSC-production pipeline—revealed considerable variation in differentiation capacity across qualitatively pluripotent iPSC clones. By comparing high- differentiation T-iPSCs and conventional iPSCs (C-iPSCs) at the single-cell level, we identified a subpopulation of C-iPSCs in the G0/G1 phase ( CDKN2A -positive) exhibiting low TET1 expression and an elevated extraembryonic mesenchymal gene signature. The extraembryonic mesenchymal gene signature is common to iPSCs and quantitatively correlates to default differentiation results. Deviation of C-iPSCs from the cell-cycling embryonic epithelial gene profile was accompanied by DNA hypermethylation concomitant with TET1 insufficiency and the accompanying derepression of poised and Polycomb-repressed enhancers, leading to the derepression of senescence-associated and extraembryonic genes. TET1-facilitated reprogramming can ameliorate these deviations. This study provides a standardized hiPSC- production pipeline and discovers an aspect of TET1 for establishing human pluripotency by remedying the inherent heterogeneities resulting from C-iPSC reprogramming.