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Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells.

Festuccia N, Osorno R, Halbritter F, Karwacki-Neisius V, Navarro P, Colby D, Wong F, Yates A, Tomlinson SR, Chambers I - Cell Stem Cell (2012)

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.

View Article: PubMed Central - PubMed

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.

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Esrrb Can Reprogram Nanog−/− Somatic Cells to Naive Pluripotency(A) Experimental scheme used to derive pre-iPSCs and to induce completion of reprogramming.(B) Morphology and Nanog:GFP expression in pre-iPSΔN-iEsrrb cells cultured in the absence of doxycycline (top) or in doxycycline/5-azacytidine for 3 days (bottom).(C) FACS plots of viral transgene expression (dsRed) and Nanog:GFP in pre-iPSΔN-iNanog (iN) or pre-iPSΔN-iEsrrb (iE) cells treated with doxycycline/5-azacytidine as indicated. Percentages of cells positive for Nanog:GFP are shown.(D) Q-PCR of endogenous genes in ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells and derivative NSCs, pre-iPSCs, and iPSCs. Primers do not detect transgenes. Nanog primers bind to intron I, which remains in all targeted cells. All cell lines were maintained without doxycycline for at least three passages. mRNA levels (normalized to TBP) are relative to expression in NSΔN-iEsrrb cells (Olig2) or ESΔN-iEsrrb cells (all other genes). Error bars: iPSCs: standard deviation of gene expression in three independent clones. ESC, pre-iPSC, and NSC lines: standard deviation of gene expression in three independent experiments.(E) Q-PCR of retroviral transgenes in ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells and derivative NSCs, pre-iPSCs, and iPSCs. Primers do not detect endogenous transcripts. mRNA levels (normalized to TBP) are relative to expression in pre-iPSΔN-iEsrrb cells. Error bars: standard deviation of expression values in three independent experiments.(F) Morphology, dsRed, and Nanog:GFP expression in iPSΔN-iEsrrb cells cultured on gelatin without doxycycline for three passages.(G) Midgestation embryo obtained from blastocyst injection of iPSΔN-iEsrrb cells transfected with a ubiquitously expressed TdTomato transgene (right); control embryo (left).See also Table S7.
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fig6: Esrrb Can Reprogram Nanog−/− Somatic Cells to Naive Pluripotency(A) Experimental scheme used to derive pre-iPSCs and to induce completion of reprogramming.(B) Morphology and Nanog:GFP expression in pre-iPSΔN-iEsrrb cells cultured in the absence of doxycycline (top) or in doxycycline/5-azacytidine for 3 days (bottom).(C) FACS plots of viral transgene expression (dsRed) and Nanog:GFP in pre-iPSΔN-iNanog (iN) or pre-iPSΔN-iEsrrb (iE) cells treated with doxycycline/5-azacytidine as indicated. Percentages of cells positive for Nanog:GFP are shown.(D) Q-PCR of endogenous genes in ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells and derivative NSCs, pre-iPSCs, and iPSCs. Primers do not detect transgenes. Nanog primers bind to intron I, which remains in all targeted cells. All cell lines were maintained without doxycycline for at least three passages. mRNA levels (normalized to TBP) are relative to expression in NSΔN-iEsrrb cells (Olig2) or ESΔN-iEsrrb cells (all other genes). Error bars: iPSCs: standard deviation of gene expression in three independent clones. ESC, pre-iPSC, and NSC lines: standard deviation of gene expression in three independent experiments.(E) Q-PCR of retroviral transgenes in ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells and derivative NSCs, pre-iPSCs, and iPSCs. Primers do not detect endogenous transcripts. mRNA levels (normalized to TBP) are relative to expression in pre-iPSΔN-iEsrrb cells. Error bars: standard deviation of expression values in three independent experiments.(F) Morphology, dsRed, and Nanog:GFP expression in iPSΔN-iEsrrb cells cultured on gelatin without doxycycline for three passages.(G) Midgestation embryo obtained from blastocyst injection of iPSΔN-iEsrrb cells transfected with a ubiquitously expressed TdTomato transgene (right); control embryo (left).See also Table S7.

Mentions: The ability of Esrrb to substitute for Nanog during transcription-factor-based induced pluripotency (Takahashi and Yamanaka, 2006) was next tested. Nanog is strictly required for completion of this process with Nanog−/− cells stalling in an intermediate, pre-iPSC state in which they acquire the morphology and growth factor dependence of ESCs but do not express endogenous pluripotency genes or silence retroviral transgene expression (Silva et al., 2009). NSCs were generated from ESΔN-iNanog and ESΔN-iEsrrb ESCs and passaged ten times in NSC medium. These lines express the NSC marker Olig2 and Sox2 but not other pluripotency factors (Figure 6D). NSΔN-iNanog and NSΔN-iEsrrb cells were infected with retroviral vectors encoding Oct4, Klf4, c-Myc, and dsRed (to monitor LTR silencing upon completion of reprogramming; Figure 6A). Colonies resembling pre-iPSCs emerged at day 5 postinfection and could be maintained on feeders without reactivating Nanog:GFP (Figure 6B). Other pluripotency genes remained silenced and viral transgenes were expressed (Figures 6D and 6E). pre-iPSΔN-iNanog and pre-iPSΔN-iEsrrb cells were then treated with doxycycline to activate the Nanog or Esrrb transgenes. This was performed with or without 5-azacytidine, which has been shown to promote reprogramming (Huangfu et al., 2008) and facilitate the pre-iPSC to iPSC transition (Theunissen et al., 2011). Nanog induction in pre-iPSΔN-iNanog cells led to the emergence of Nanog:GFP+ cells by day 6 (Figure 6C). Strikingly, Esrrb induction resulted in faster, more pronounced reactivation of Nanog:GFP. For both pre-iPSΔN-iNanog and pre-iPSΔN-iEsrrb, G418-resistant, Nanog:GFP+ colonies could be picked and expanded without feeders or doxycycline. The resulting iPSΔN-iNanog and iPSΔN-iEsrrb lines resembled the parental ESC lines morphologically, were Nanog:GFP+/dsRed– (Figure 6F), expressed endogenous pluripotency genes, and had silenced the viral transgenes (Figures 6D and 6E). Blastocyst injection of iPSΔN-iEsrrb cells resulted in contribution to midgestation embryos (Figure 6G; Table S7). These results demonstrate that Esrrb can drive completion of reprogramming in the absence of Nanog, indicating that Esrrb can substitute for Nanog in the acquisition of pluripotency.


Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells.

Festuccia N, Osorno R, Halbritter F, Karwacki-Neisius V, Navarro P, Colby D, Wong F, Yates A, Tomlinson SR, Chambers I - Cell Stem Cell (2012)

Esrrb Can Reprogram Nanog−/− Somatic Cells to Naive Pluripotency(A) Experimental scheme used to derive pre-iPSCs and to induce completion of reprogramming.(B) Morphology and Nanog:GFP expression in pre-iPSΔN-iEsrrb cells cultured in the absence of doxycycline (top) or in doxycycline/5-azacytidine for 3 days (bottom).(C) FACS plots of viral transgene expression (dsRed) and Nanog:GFP in pre-iPSΔN-iNanog (iN) or pre-iPSΔN-iEsrrb (iE) cells treated with doxycycline/5-azacytidine as indicated. Percentages of cells positive for Nanog:GFP are shown.(D) Q-PCR of endogenous genes in ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells and derivative NSCs, pre-iPSCs, and iPSCs. Primers do not detect transgenes. Nanog primers bind to intron I, which remains in all targeted cells. All cell lines were maintained without doxycycline for at least three passages. mRNA levels (normalized to TBP) are relative to expression in NSΔN-iEsrrb cells (Olig2) or ESΔN-iEsrrb cells (all other genes). Error bars: iPSCs: standard deviation of gene expression in three independent clones. ESC, pre-iPSC, and NSC lines: standard deviation of gene expression in three independent experiments.(E) Q-PCR of retroviral transgenes in ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells and derivative NSCs, pre-iPSCs, and iPSCs. Primers do not detect endogenous transcripts. mRNA levels (normalized to TBP) are relative to expression in pre-iPSΔN-iEsrrb cells. Error bars: standard deviation of expression values in three independent experiments.(F) Morphology, dsRed, and Nanog:GFP expression in iPSΔN-iEsrrb cells cultured on gelatin without doxycycline for three passages.(G) Midgestation embryo obtained from blastocyst injection of iPSΔN-iEsrrb cells transfected with a ubiquitously expressed TdTomato transgene (right); control embryo (left).See also Table S7.
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fig6: Esrrb Can Reprogram Nanog−/− Somatic Cells to Naive Pluripotency(A) Experimental scheme used to derive pre-iPSCs and to induce completion of reprogramming.(B) Morphology and Nanog:GFP expression in pre-iPSΔN-iEsrrb cells cultured in the absence of doxycycline (top) or in doxycycline/5-azacytidine for 3 days (bottom).(C) FACS plots of viral transgene expression (dsRed) and Nanog:GFP in pre-iPSΔN-iNanog (iN) or pre-iPSΔN-iEsrrb (iE) cells treated with doxycycline/5-azacytidine as indicated. Percentages of cells positive for Nanog:GFP are shown.(D) Q-PCR of endogenous genes in ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells and derivative NSCs, pre-iPSCs, and iPSCs. Primers do not detect transgenes. Nanog primers bind to intron I, which remains in all targeted cells. All cell lines were maintained without doxycycline for at least three passages. mRNA levels (normalized to TBP) are relative to expression in NSΔN-iEsrrb cells (Olig2) or ESΔN-iEsrrb cells (all other genes). Error bars: iPSCs: standard deviation of gene expression in three independent clones. ESC, pre-iPSC, and NSC lines: standard deviation of gene expression in three independent experiments.(E) Q-PCR of retroviral transgenes in ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells and derivative NSCs, pre-iPSCs, and iPSCs. Primers do not detect endogenous transcripts. mRNA levels (normalized to TBP) are relative to expression in pre-iPSΔN-iEsrrb cells. Error bars: standard deviation of expression values in three independent experiments.(F) Morphology, dsRed, and Nanog:GFP expression in iPSΔN-iEsrrb cells cultured on gelatin without doxycycline for three passages.(G) Midgestation embryo obtained from blastocyst injection of iPSΔN-iEsrrb cells transfected with a ubiquitously expressed TdTomato transgene (right); control embryo (left).See also Table S7.
Mentions: The ability of Esrrb to substitute for Nanog during transcription-factor-based induced pluripotency (Takahashi and Yamanaka, 2006) was next tested. Nanog is strictly required for completion of this process with Nanog−/− cells stalling in an intermediate, pre-iPSC state in which they acquire the morphology and growth factor dependence of ESCs but do not express endogenous pluripotency genes or silence retroviral transgene expression (Silva et al., 2009). NSCs were generated from ESΔN-iNanog and ESΔN-iEsrrb ESCs and passaged ten times in NSC medium. These lines express the NSC marker Olig2 and Sox2 but not other pluripotency factors (Figure 6D). NSΔN-iNanog and NSΔN-iEsrrb cells were infected with retroviral vectors encoding Oct4, Klf4, c-Myc, and dsRed (to monitor LTR silencing upon completion of reprogramming; Figure 6A). Colonies resembling pre-iPSCs emerged at day 5 postinfection and could be maintained on feeders without reactivating Nanog:GFP (Figure 6B). Other pluripotency genes remained silenced and viral transgenes were expressed (Figures 6D and 6E). pre-iPSΔN-iNanog and pre-iPSΔN-iEsrrb cells were then treated with doxycycline to activate the Nanog or Esrrb transgenes. This was performed with or without 5-azacytidine, which has been shown to promote reprogramming (Huangfu et al., 2008) and facilitate the pre-iPSC to iPSC transition (Theunissen et al., 2011). Nanog induction in pre-iPSΔN-iNanog cells led to the emergence of Nanog:GFP+ cells by day 6 (Figure 6C). Strikingly, Esrrb induction resulted in faster, more pronounced reactivation of Nanog:GFP. For both pre-iPSΔN-iNanog and pre-iPSΔN-iEsrrb, G418-resistant, Nanog:GFP+ colonies could be picked and expanded without feeders or doxycycline. The resulting iPSΔN-iNanog and iPSΔN-iEsrrb lines resembled the parental ESC lines morphologically, were Nanog:GFP+/dsRed– (Figure 6F), expressed endogenous pluripotency genes, and had silenced the viral transgenes (Figures 6D and 6E). Blastocyst injection of iPSΔN-iEsrrb cells resulted in contribution to midgestation embryos (Figure 6G; Table S7). These results demonstrate that Esrrb can drive completion of reprogramming in the absence of Nanog, indicating that Esrrb can substitute for Nanog in the acquisition of pluripotency.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus