Capturing the elusive state of human naïve pluripotency

Resumen

Pluripotent stem cells (PSCs) can transition between cell states in vitro, closely reflecting developmental changes in the early embryo. PSCs can be stabilized in their naïve state by blocking extra-cellular differentiation stimuli involving chemical inhibition of MEK and GSK3 kinases with a two inhibitor cocktail known as “2i”. Mouse 2i-PSCs phenocopy the stable and homogeneous state of undifferentiated pluripotency that exists in vivo at the pre-implantation embryo epiblast. In contrast, PSCs in the absence of 2i adopt a cell identity that mimics the post-implantation epiblast, also known as the primed state. Naïve PSCs are considered the optimal starting state for subsequent applications in cellular therapy, providing high quality cells with full differentiation capacity. However, in the case of human PSCs, the naïve state attained using 2i-based cocktails has turned out to be unstable, rapidly resulting in genetic abnormalities and loss of pluripotency, problems that have been attributed to uncontrolled DNA demethylation caused by MEK inhibition. Towards a solution, we have discovered a novel and simple chemical method to induce stable naïve pluripotency in human PSCs. This method is based on reinforcing the pluripotency transcriptional programs. For this, we have inhibited the CDK8 and CDK19 kinases, which are negative regulators of MEDIATOR, a critical complex for enhancer-driven transcription. We show that chemical inhibition of these kinases (CDK8/19i) efficiently stabilizes PSCs in the naïve state. Importantly, CDK8/19i-naïve human PSCs do not show widespread DNA demethylation, display a normal karyotype and a normal DNA methylation pattern at imprinted loci. They also have the capacity to form teratomas showing differentiation into all three germ layers, a property that is severely compromised in 2i-naïve human PSCs. Moreover, CDK8/19i-naïve human PSCs demonstrate high efficiency in contributing to rabbit blastocysts, in contrast with the low or absent contribution to interspecies chimerism of 2i-based naïve human PSCs. In summary, we have identified CDK8/19 as an actionable regulator of transcription able to stabilize naïve pluripotency without incurring in loss of DNA methylation and genomic instability. The inhibition of CDK8/19 may offer a stable and more homogeneous alternative to primed and 2i-naïve human PSCs. Finally, we have discovered one of the transcriptional mechanisms responsible of cellular identity and plasticity