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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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Expression of Esrrb Reverts EpiSCs to Chimera Competency(A) AP-positive colony formation by Epi-iPSCs. EpiSCs expressing polyoma large T-antigen were transfected with episomal vectors encoding empty vector (EV), Nanog, Klf4, or Esrrb, plated in the indicated medium containing puromycin, and stained for AP after 7 days. Error bars: standard deviation (n = 3).(B) Morphology of primary Epi-iPSC colonies formed after transfection of the respective episomal vector and culture in the indicated medium for 7 days.(C) Morphology and Nanog:GFP expression of primary Epi-iPSC colonies formed after transfection of the respective episomal vector and culture in FCS/LIF/GMEMβ for 7 days.(D) FACS analysis of Pecam1 expression 7 days after transfection of the indicated DNAs. TNG/T ESCs (blue) and EpiSCs (gray) were used as controls for Pecam1 expression.(E) mRNA expression in E14/T EpiSC and Epi-iPSC colonies expanded in the absence of selection after episomal expression of Esrrb and medium switch into FCS/LIF/GMEMβ. Error bars: standard deviation of gene expression in three independent experiments.(F) Chimeric mouse obtained from blastocyst injection of Esrrb-induced Epi-iPSCs.See also Figure S5 and Table S3.
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fig3: Expression of Esrrb Reverts EpiSCs to Chimera Competency(A) AP-positive colony formation by Epi-iPSCs. EpiSCs expressing polyoma large T-antigen were transfected with episomal vectors encoding empty vector (EV), Nanog, Klf4, or Esrrb, plated in the indicated medium containing puromycin, and stained for AP after 7 days. Error bars: standard deviation (n = 3).(B) Morphology of primary Epi-iPSC colonies formed after transfection of the respective episomal vector and culture in the indicated medium for 7 days.(C) Morphology and Nanog:GFP expression of primary Epi-iPSC colonies formed after transfection of the respective episomal vector and culture in FCS/LIF/GMEMβ for 7 days.(D) FACS analysis of Pecam1 expression 7 days after transfection of the indicated DNAs. TNG/T ESCs (blue) and EpiSCs (gray) were used as controls for Pecam1 expression.(E) mRNA expression in E14/T EpiSC and Epi-iPSC colonies expanded in the absence of selection after episomal expression of Esrrb and medium switch into FCS/LIF/GMEMβ. Error bars: standard deviation of gene expression in three independent experiments.(F) Chimeric mouse obtained from blastocyst injection of Esrrb-induced Epi-iPSCs.See also Figure S5 and Table S3.

Mentions: It has been shown that Nanog or Klf4 overexpression can reprogram EpiSCs to ESC pluripotency (Guo et al., 2009; Silva et al., 2009). Therefore, the abilities of Nanog, Esrrb, and Klf4 to mediate the reversion of EpiSCs to an ESC state were compared. Episomal expression of Nanog, Esrrb, or Klf4, coupled with removal of Activin/Fgf, could induce reversion of EpiSCs to an ESC-like state (Figure 3A). Esrrb displayed a higher reprogramming efficiency than Nanog or Klf4 (Figure 3A). Furthermore, Nanog and Esrrb allowed AP-positive colony formation in all conditions (Figure 3A), whereas Klf4 could only revert EpiSCs to ESC pluripotency when combined with LIF/2i (Figure 3A). Primary Epi-iPSC colonies displayed an undifferentiated morphology (Figure 3B) and in FCS/LIF/GMEMβ, Nanog and Esrrb, but not Klf4, induced the re-expression of Nanog:GFP (Figure 3C) and Pecam1 (Figure 3D), a cell surface marker expressed in the inner cell mass (ICM)/ESCs and downregulated in the epiblast/EpiSCs (Hayashi et al., 2008; Robson et al., 2001). To further characterize the Esrrb-induced Epi-iPSCs, clones were picked and expanded in FCS/LIF/GMEMβ. Expression of Nanog, Sox2, Klf4, Klf2, and Tbx3 were restored to ESC levels, while expression of the early marker of differention Fgf5 was reduced (Figure 3E). Injections of the Esrrb-reverted Epi-iPSCs into blastocysts produced adult chimeras, indicating that enforced Esrrb expression can restore chimera-forming potential to EpiSCs (Figure 3F; Table S3).


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)

Expression of Esrrb Reverts EpiSCs to Chimera Competency(A) AP-positive colony formation by Epi-iPSCs. EpiSCs expressing polyoma large T-antigen were transfected with episomal vectors encoding empty vector (EV), Nanog, Klf4, or Esrrb, plated in the indicated medium containing puromycin, and stained for AP after 7 days. Error bars: standard deviation (n = 3).(B) Morphology of primary Epi-iPSC colonies formed after transfection of the respective episomal vector and culture in the indicated medium for 7 days.(C) Morphology and Nanog:GFP expression of primary Epi-iPSC colonies formed after transfection of the respective episomal vector and culture in FCS/LIF/GMEMβ for 7 days.(D) FACS analysis of Pecam1 expression 7 days after transfection of the indicated DNAs. TNG/T ESCs (blue) and EpiSCs (gray) were used as controls for Pecam1 expression.(E) mRNA expression in E14/T EpiSC and Epi-iPSC colonies expanded in the absence of selection after episomal expression of Esrrb and medium switch into FCS/LIF/GMEMβ. Error bars: standard deviation of gene expression in three independent experiments.(F) Chimeric mouse obtained from blastocyst injection of Esrrb-induced Epi-iPSCs.See also Figure S5 and Table S3.
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fig3: Expression of Esrrb Reverts EpiSCs to Chimera Competency(A) AP-positive colony formation by Epi-iPSCs. EpiSCs expressing polyoma large T-antigen were transfected with episomal vectors encoding empty vector (EV), Nanog, Klf4, or Esrrb, plated in the indicated medium containing puromycin, and stained for AP after 7 days. Error bars: standard deviation (n = 3).(B) Morphology of primary Epi-iPSC colonies formed after transfection of the respective episomal vector and culture in the indicated medium for 7 days.(C) Morphology and Nanog:GFP expression of primary Epi-iPSC colonies formed after transfection of the respective episomal vector and culture in FCS/LIF/GMEMβ for 7 days.(D) FACS analysis of Pecam1 expression 7 days after transfection of the indicated DNAs. TNG/T ESCs (blue) and EpiSCs (gray) were used as controls for Pecam1 expression.(E) mRNA expression in E14/T EpiSC and Epi-iPSC colonies expanded in the absence of selection after episomal expression of Esrrb and medium switch into FCS/LIF/GMEMβ. Error bars: standard deviation of gene expression in three independent experiments.(F) Chimeric mouse obtained from blastocyst injection of Esrrb-induced Epi-iPSCs.See also Figure S5 and Table S3.
Mentions: It has been shown that Nanog or Klf4 overexpression can reprogram EpiSCs to ESC pluripotency (Guo et al., 2009; Silva et al., 2009). Therefore, the abilities of Nanog, Esrrb, and Klf4 to mediate the reversion of EpiSCs to an ESC state were compared. Episomal expression of Nanog, Esrrb, or Klf4, coupled with removal of Activin/Fgf, could induce reversion of EpiSCs to an ESC-like state (Figure 3A). Esrrb displayed a higher reprogramming efficiency than Nanog or Klf4 (Figure 3A). Furthermore, Nanog and Esrrb allowed AP-positive colony formation in all conditions (Figure 3A), whereas Klf4 could only revert EpiSCs to ESC pluripotency when combined with LIF/2i (Figure 3A). Primary Epi-iPSC colonies displayed an undifferentiated morphology (Figure 3B) and in FCS/LIF/GMEMβ, Nanog and Esrrb, but not Klf4, induced the re-expression of Nanog:GFP (Figure 3C) and Pecam1 (Figure 3D), a cell surface marker expressed in the inner cell mass (ICM)/ESCs and downregulated in the epiblast/EpiSCs (Hayashi et al., 2008; Robson et al., 2001). To further characterize the Esrrb-induced Epi-iPSCs, clones were picked and expanded in FCS/LIF/GMEMβ. Expression of Nanog, Sox2, Klf4, Klf2, and Tbx3 were restored to ESC levels, while expression of the early marker of differention Fgf5 was reduced (Figure 3E). Injections of the Esrrb-reverted Epi-iPSCs into blastocysts produced adult chimeras, indicating that enforced Esrrb expression can restore chimera-forming potential to EpiSCs (Figure 3F; Table S3).

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