Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening

Naive human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional primed'' hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Here, we describe the results from a high-throughput screen using similar to 3,000 well-annotated compounds to identify essential signaling requirements for naive human pluripotency. We report that MEK1/2 inhibitors can be replaced during maintenance of naive human pluripotency by inhibitors targeting either upstream (FGFR, RAF) or downstream (ERK1/2) kinases. Naive hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naive resetting in combination with PKC, ROCK, and TNKS inhibitors and activin A. This work demonstrates that induction and maintenance of naive human pluripotency are governed by distinct signaling requirements.

Automated summary

Through a high-throughput screen, essential signaling requirements for maintaining naive human pluripotency were identified, showing that MEK1/2 inhibitors can be replaced by inhibitors targeting upstream or downstream kinases during maintenance. Dual inhibition of MEK and ERK, along with other inhibitors and activin A, promotes primed-to-naive resetting efficiently.