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

Ge Guo 0 Austin Smith ) 0 0 Wellcome Trust Centre for Stem Cell Research and Department of Biochemistry, University of Cambridge , Tennis Court Road, Cambridge CB2 1QR , UK SUMMARY In rodents, the nave 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 nave 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. INTRODUCTION Mouse embryonic stem (ES) cells and post-implantation epiblast stem cells (EpiSCs) are considered to represent two phases in the ontogeny of the pluripotent epiblast (Rossant, 2008). ES cells are obtained from the initial nave population that emerges in the mature blastocyst (Brook and Gardner, 1997; Evans and Kaufman, 1981; Nichols et al., 2009). They contribute extensively to chimaeras, demonstrating retention of early epiblast identity and associated unbiased developmental potential. EpiSCs are derived by culturing post-implantation egg cylinder epiblast (Brons et al., 2007; Tesar et al., 2007). They can differentiate into some cell types in culture, including germ cells (Hayashi and Surani, 2009), and can produce teratomas. However, they colonise blastocyst chimaeras poorly, if at all (Guo et al., 2009; Tesar et al., 2007). Intriguingly, stem cells derived from human embryos show features more in common with rodent EpiSCs than with ES cells (Nichols and Smith, 2009; Rossant, 2008). ES cells and EpiSCs express the central pluripotency factors Oct4 (Pou5f1 Mouse Genome Informatics) and Sox2 at similar levels, but diverge in other important respects. EpiSCs do not express, or have lower levels than ES cells, of many pluripotencyassociated transcription factors, and they show upregulation of early lineage specification markers (Guo et al., 2009; Hanna et al., 2009; Tesar et al., 2007). They also exhibit a differentiated epigenetic feature in female cells: the decoration of one X chromosome with the silencing mark H3K27me3 (Guo et al., 2009; Silva et al., 2003). EpiSCs do not respond productively to the ES cell self-renewal cytokine leukaemia inhibitory factor (LIF) (Brons et al., 2007) and they cannot be propagated in fully defined ES cell culture conditions, known as 2i, in which the mitogen-activated protein kinase (Erk; Mapk1) cascade and glycogen synthase kinase 3 (Gsk3) are selectively inhibited (Guo et al., 2009; Ying et al., 2008). ES cells are maintained in a relatively homogenous ground state by application of these two inhibitors (Silva and Smith, 2008; Wray et al., 2010; Ying et al., 2008). EpiSCs, by contrast, appear to be variable both between and within cell lines. This might reflect both their origin from heterogeneous egg cylinder populations and the use of undefined culture environments, which may either select for, or induce, particular phenotypes. EpiSC-like cells derived on feeders in serum or serum replacement have been reported to acquire ES cell-like features without genetic manipulation (Bao et al., 2009; Greber et al., 2010). This is proposed to represent epigenetic reversion or dedifferentiation. However, the potential heterogeneity and developmental status of these EpiSC cultures are uncertain. Furthermore, EpiSCs on feeders can differentiate into primordial germ cells that may then undergo conversion into embryonic germ (EG) cells (Hayashi and Surani, 2009). Consequently, it is unclear what process underlies such spontaneous conversion. Importantly, for the present study, when EpiSCs are derived and maintained in defined conditions using fibroblast growth factor (FGF) and activin without feeders, they do not spontaneously acquire ES cell properties (Brons et al., 2007) and on transfer to 2i they very rarely form undifferentiated colonies (Guo et al., 2009). Robust conversion can be triggered, however, by transfection with single genes: either Klf4, Klf2 or Nanog (Guo et al., 2009; Hall et al., 2009; Hanna et al., 2009; Silva et al., 2009). Reprogramming is dependent upon withdrawal of the EpiSC selfrenewal factors FGF and activin, and is promoted by 2i in combination with LIF (Yang et al., 2010). Interestingly, despite their closer developmental proximity to nave pluripotency and their endogenous expression of Oct4 and Sox2, the efficiency of generating induced pluripotent stem (iPS) cells from transfected EpiSCs in defined culture is not demonstrably higher than from fibroblasts. Therefore, in addition to illuminating features of pluripotency and developmental restriction, characterising the transition from EpiSC to iPS cell might also contribute to an understanding of somatic cell reprogramming. To identify factors that can surmount the molecular roadblock between EpiSCs and ground state pluripotency we undertook a genome-wide screen. MATERIALS AND METHODS Cell culture EpiSCs derived from E5.5 mouse embryos (Guo et al., 2009) were cultured on fibronectin in N2B27 medium (Ying and Smith, 2003) with activin A (20 ng/ml) and FGF2 (12.5 ng/ml) prepared in-house. ES cells and iPS cells were cultured in 2i/LIF medium (Ying et al., 2008) comprising N2B27 with MEK inhibitor (1 M PD0325901), Gsk3 inhibitor (3 M Chir99021) and 200 units/ml LIF (Smith, 1991). LIF/BMP4 medium is N2B27 with LIF (100 units/ml) and 5 ng/ml BMP4 (R&D Systems). Immunostaining and blastocyst injection (...truncated)