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: To examine whether stable reprogramming of the NSC genome could be achieved without continued transgene expression, fusion experiments were performed using ESΔN-iNanog and ESΔN-iEsrrb cells. Nanog−/− RCNβH(t) NSCs were transfected with a CAG-driven TdTomato-IRES-hygromycinR construct. RCNβH(t) Red NSCs were fused with ESΔN-iNanog or ESΔN-iEsrrb cells (Figure 5A) and primary hybrids were replated in blasticidin and hygromycin. In the absence of doxycycline, only a small number of hybrid colonies were obtained (Table S5); these were predominantly differentiated (Figure 5B) and could not be expanded. In contrast, Nanog and Esrrb induction resulted in the formation of self-renewing AP-positive colonies (Figures 5B and S6G). Nanog induction promoted reprogramming at high frequency (∼300 colonies/million NSCs fused) as previously reported (Silva et al., 2006, 2009). In contrast, Esrrb overexpression resulted in a 10-fold lower reprogramming efficiency (Table S5). These differences were not due to altered fusion efficiencies, since similar results were obtained after replating sorted primary hybrids (Figure S6F; Table S6). Nonetheless, all reprogrammed hybrid lines could be expanded and cultured over multiple passages. Cells were then maintained or released from doxycycline and passaged in the presence or absence of G418 (to select for transcription from Nanog; Figure 5A). Hybrid lines could be serially passaged without continued Esrrb or Nanog in G418 (Figure 5C). In the absence of G418 selection, hybrid cells could be propagated without continued Esrrb or Nanog induction but had an increased tendency to differentiate, similar to Nanog−/− ESCs (Chambers et al., 2007). This propensity was eliminated upon induction of Esrrb, identifying a further common feature between Esrrb and 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.