Stability Of Imprinting And Differentiation Capacity In Naive Human Cells Induced By Chemical Inhibition Of Cdk8 And Cdk19

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naive and primed states are the best characterized pluripotency states. Naive pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naive PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naive human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naive human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naive human PSCs. We report that long-term cultured CDK8/19i-naive human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naive human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naive state. Interestingly, long-term cultured CDK8/19i-naive human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naive human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naive state that preserves imprinting and potency over long-term culture.