Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells.
Bottom Line: Moreover, Esrrb can reprogram Nanog(-/-) EpiSCs and can rescue stalled reprogramming in Nanog(-/-) pre-iPSCs.Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent ESC self-renewal.These findings are consistent with the functional placement of Esrrb downstream of Nanog.
Affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, Scotland.Show MeSH
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Mentions: Nanog is dispensable for the establishment and maintenance of primed pluripotency (Osorno et al., 2012) but is required for the acquisition of naive pluripotency, since somatic Nanog−/− cells cannot be converted into fully reprogrammed iPSCs (Silva et al., 2009). To determine whether Esrrb could revert EpiSCs to an ESC state in the absence of Nanog, ESΔN-iNanog and ESΔN-iEsrrb ESCs were converted into EpiSC lines (EpiΔN-iNanog and EpiΔN-iEsrrb) by passaging in Activin/FGF (Guo et al., 2009). This allowed comparative investigation of the abilities of Nanog and Esrrb to impose an ESC identity by simply applying doxycycline and removing Activin/Fgf. AP-positive Epi-iPSC colonies were obtained following induction of Nanog and, to our surprise, also following Esrrb induction (Figure 4A). However, whereas Esrrb induced EpiSC reprogramming with greater efficiency than Nanog in wild-type cells, the opposite was observed in Nanog−/− cells (Figures 4A and 4B), suggesting that Nanog is required for maximal Esrrb efficacy. AP-positive colonies were obtained after as little as 24 hr exposure to doxycycline of both EpiΔN-iNanog and ESΔN-iEsrrb cells with a clear correlation between the doxycycline treatment period and the number of Epi-iPSC colonies obtained (Figures 4A and 4B). Esrrb-induced Epi-iPSΔN-iEsrrb clones were picked and expanded in the absence of further transgene induction and had reacquired expression of Sox2, Klf2, and Tbx3 and downregulated Fgf5 (Figure 4C). Importantly, Epi-iPSΔN-iEsrrb cells reacquired both ESC morphology and levels of Nanog:GFP similar to those in ESCs (Figure 4D). Epi-iPSΔN-iEsrrb cells could also form self-renewing AP-positive colonies in BMP/LIF and 2i/LIF (Figure 4E). Consistent with these findings, Epi-iPSΔN-iEsrrb cells injected into blastocysts produced adult chimeras (Figure 4F; Table S3). These results demonstrate that Esrrb can functionally substitute for the hitherto unique capacity of Nanog to reprogram Nanog−/− cells to naive pluripotency.
Affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, Scotland.