A genome-wide screen in EpiSCs identifies Nr5a nuclear receptors as potent inducers of ground state pluripotency

In rodents, the naive early epiblast undergoes profound morphogenetic, transcriptional and epigenetic changes after implantation. These differences are maintained between blastocyst-derived embryonic stem (ES) cells and egg cylinder-derived epiblast stem cells (EpiSCs). Notably, ES cells robustly colonise chimaeras, whereas EpiSCs show little or no contribution. ES cells self-renew independently of mitogenic growth factors, whereas EpiSCs require fibroblast growth factor. However, EpiSCs retain the core pluripotency factors Oct4 and Sox2 and the developmental barrier dividing them from unrestricted pluripotency can be surmounted by a single reprogramming factor. This provides an opportunity to identify molecules that can reset the naive state. We undertook a forward genetic screen for effectors of EpiSC reprogramming, employing piggyBac transposition to activate endogenous gene expression at random and selecting for undifferentiated colonies in the absence of growth factor signalling. Three recovered clones harboured integrations that activate the closely related orphan nuclear receptor genes Nr5a1 and Nr5a2. Activity of Nr5a1 and Nr5a2 was confirmed by direct transfection. Reprogrammed colonies were obtained without transgene integration and at 10-fold higher frequency than with other single factors. Converted cells exhibited the diagnostic self-renewal characteristics, gene expression profile and X chromosome activation signature of ground state pluripotency. They efficiently produced adult chimaeras and gave germline transmission. Nr5a receptors regulate Oct4 transcription but this is insufficient for reprogramming. Intriguingly, unlike previously identified reprogramming molecules, Nr5a receptors play no evident role in ES cell self-renewal. This implies a different foundation for their capacity to reset pluripotency and suggests that further factors remain to be identified.