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Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells.

Yang J, Wang W, Ooi J, Campos LS, Lu L, Liu P - Stem Cells (2015)

Bottom Line: Remarkably, the reprogramming of epiblast stem cells into embryonic stem cell-like cells also requires low levels of RA, which can modulate Wnt signalling through physical interactions of RARs with β-catenin.These results highlight the important functions of RA signalling in reprogramming somatic cells and primed stem cells to naïve pluripotency.Stem Cells 2015;33:1390-1404.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

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Related in: MedlinePlus

Reprogramming mouse embryonic fibroblast cells (MEFs) by 6F is both retinoic acid (RA) receptor ligand‐dependent and ligand‐independent. (A): MEFs were reprogrammed by 4F alone, 4F plus either RARG or LRH1, or 4F plus both RARG and LRH1 (6F) in +VA and −VA medium. Significantly more AP+ colonies are obtained by 6F, even in −VA. (B): Blocking RA signalling by citral (50.0 μM) or CD2665 (1.0 μM) as measured in a luciferase assay. (C): Blocking 4F‐ or 6F‐mediated MEF reprogramming by citral (50.0 μM). Adding ATRA rescues reprogramming. Note that 6F produces substantially more AP+ colonies in the presence of citral compared with the other conditions. (D): The blocking of MEF reprogramming by RARA‐DN. (E): The effects of blocking RA signalling by either citral (50.0 μM) or RARA‐DN on MEF reprogramming. Note that adding citral and expressing RARA‐DN almost completely blocked reprogramming. Adding ATRA at 10.0 nM could partially rescue the block. In the MEF reprogramming experiments, AP+ colonies were scored on day 18 after transfection. *, p < .05; **, p < .01. The experiments were repeated at least three times, and the error bars show standard deviations from the mean of triplicate determinations in one representative experiment. Abbreviations: AP, alkaline phosphatase; ATRA, all‐trans retinoic acid; LRH1, liver receptor homolog‐1; RARA‐DN, dominant‐negative form of RARA; RARG, retinoic acid receptor gamma; VA, vitamin A.
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stem1926-fig-0004: Reprogramming mouse embryonic fibroblast cells (MEFs) by 6F is both retinoic acid (RA) receptor ligand‐dependent and ligand‐independent. (A): MEFs were reprogrammed by 4F alone, 4F plus either RARG or LRH1, or 4F plus both RARG and LRH1 (6F) in +VA and −VA medium. Significantly more AP+ colonies are obtained by 6F, even in −VA. (B): Blocking RA signalling by citral (50.0 μM) or CD2665 (1.0 μM) as measured in a luciferase assay. (C): Blocking 4F‐ or 6F‐mediated MEF reprogramming by citral (50.0 μM). Adding ATRA rescues reprogramming. Note that 6F produces substantially more AP+ colonies in the presence of citral compared with the other conditions. (D): The blocking of MEF reprogramming by RARA‐DN. (E): The effects of blocking RA signalling by either citral (50.0 μM) or RARA‐DN on MEF reprogramming. Note that adding citral and expressing RARA‐DN almost completely blocked reprogramming. Adding ATRA at 10.0 nM could partially rescue the block. In the MEF reprogramming experiments, AP+ colonies were scored on day 18 after transfection. *, p < .05; **, p < .01. The experiments were repeated at least three times, and the error bars show standard deviations from the mean of triplicate determinations in one representative experiment. Abbreviations: AP, alkaline phosphatase; ATRA, all‐trans retinoic acid; LRH1, liver receptor homolog‐1; RARA‐DN, dominant‐negative form of RARA; RARG, retinoic acid receptor gamma; VA, vitamin A.

Mentions: We observed in the previous experiments that the removal of VA from N2B27/LIF drastically reduced the colony number in 4F reprogramming, but only a relatively mild colony number reduction was observed in 6F reprogramming (Fig. 3A). Moreover, supplementation of CD437 did not change the kinetics of Rex1 reactivation in 4F reprogramming, unlike the early Rex1 reactivation observed in 6F reprogramming. These results indicated that some of the positive effects of RARG and LRH1 on reprogramming must be mediated by a RA‐ or RAR ligand‐independent mechanism. To further dissect these possibilities, we expressed 4F using the constitutive CAG promoter but controlled Rarg expression in a Dox‐inducible manner. The expression of exogenous RARG increased AP+ colony numbers two‐ to threefold across all the tested Dox induction concentrations (Fig. 4A and Supporting Information Fig. S3A), indicating that VA in the standard N2B27 likely limits further increases in RA signalling. Moreover, in −VA medium, exogenous RARG was no longer able to boost MEF reprogramming (Fig. 4A and Supporting Information Fig. S3A). Therefore, the effect of expressing RARG alone (no endogenous LRH1 in MEFs) is primarily dependent on VA or its metabolites, that is, ligand‐dependent. Similarly, the promoting effect of RARA alone on reprogramming was also VA‐dependent (Supporting Information Fig. S3B). Interestingly, expressing LRH1 alone also promoted 4F reprogramming in +VA medium (Fig. 4A and Supporting Information Fig. S3B), as shown in previous reports using serum‐containing media 46. However, again the effect was largely absent when −VA medium was used (Fig. 4A and Supporting Information Fig. S3B). Therefore, LRH1 promotes reprogramming likely through interaction with the relatively low levels of endogenous RARs in MEFs. At these levels of RARs, the effects of exogenous LRH1 appear to be primarily dependent on the RAR ligands.


Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells.

Yang J, Wang W, Ooi J, Campos LS, Lu L, Liu P - Stem Cells (2015)

Reprogramming mouse embryonic fibroblast cells (MEFs) by 6F is both retinoic acid (RA) receptor ligand‐dependent and ligand‐independent. (A): MEFs were reprogrammed by 4F alone, 4F plus either RARG or LRH1, or 4F plus both RARG and LRH1 (6F) in +VA and −VA medium. Significantly more AP+ colonies are obtained by 6F, even in −VA. (B): Blocking RA signalling by citral (50.0 μM) or CD2665 (1.0 μM) as measured in a luciferase assay. (C): Blocking 4F‐ or 6F‐mediated MEF reprogramming by citral (50.0 μM). Adding ATRA rescues reprogramming. Note that 6F produces substantially more AP+ colonies in the presence of citral compared with the other conditions. (D): The blocking of MEF reprogramming by RARA‐DN. (E): The effects of blocking RA signalling by either citral (50.0 μM) or RARA‐DN on MEF reprogramming. Note that adding citral and expressing RARA‐DN almost completely blocked reprogramming. Adding ATRA at 10.0 nM could partially rescue the block. In the MEF reprogramming experiments, AP+ colonies were scored on day 18 after transfection. *, p < .05; **, p < .01. The experiments were repeated at least three times, and the error bars show standard deviations from the mean of triplicate determinations in one representative experiment. Abbreviations: AP, alkaline phosphatase; ATRA, all‐trans retinoic acid; LRH1, liver receptor homolog‐1; RARA‐DN, dominant‐negative form of RARA; RARG, retinoic acid receptor gamma; VA, vitamin A.
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stem1926-fig-0004: Reprogramming mouse embryonic fibroblast cells (MEFs) by 6F is both retinoic acid (RA) receptor ligand‐dependent and ligand‐independent. (A): MEFs were reprogrammed by 4F alone, 4F plus either RARG or LRH1, or 4F plus both RARG and LRH1 (6F) in +VA and −VA medium. Significantly more AP+ colonies are obtained by 6F, even in −VA. (B): Blocking RA signalling by citral (50.0 μM) or CD2665 (1.0 μM) as measured in a luciferase assay. (C): Blocking 4F‐ or 6F‐mediated MEF reprogramming by citral (50.0 μM). Adding ATRA rescues reprogramming. Note that 6F produces substantially more AP+ colonies in the presence of citral compared with the other conditions. (D): The blocking of MEF reprogramming by RARA‐DN. (E): The effects of blocking RA signalling by either citral (50.0 μM) or RARA‐DN on MEF reprogramming. Note that adding citral and expressing RARA‐DN almost completely blocked reprogramming. Adding ATRA at 10.0 nM could partially rescue the block. In the MEF reprogramming experiments, AP+ colonies were scored on day 18 after transfection. *, p < .05; **, p < .01. The experiments were repeated at least three times, and the error bars show standard deviations from the mean of triplicate determinations in one representative experiment. Abbreviations: AP, alkaline phosphatase; ATRA, all‐trans retinoic acid; LRH1, liver receptor homolog‐1; RARA‐DN, dominant‐negative form of RARA; RARG, retinoic acid receptor gamma; VA, vitamin A.
Mentions: We observed in the previous experiments that the removal of VA from N2B27/LIF drastically reduced the colony number in 4F reprogramming, but only a relatively mild colony number reduction was observed in 6F reprogramming (Fig. 3A). Moreover, supplementation of CD437 did not change the kinetics of Rex1 reactivation in 4F reprogramming, unlike the early Rex1 reactivation observed in 6F reprogramming. These results indicated that some of the positive effects of RARG and LRH1 on reprogramming must be mediated by a RA‐ or RAR ligand‐independent mechanism. To further dissect these possibilities, we expressed 4F using the constitutive CAG promoter but controlled Rarg expression in a Dox‐inducible manner. The expression of exogenous RARG increased AP+ colony numbers two‐ to threefold across all the tested Dox induction concentrations (Fig. 4A and Supporting Information Fig. S3A), indicating that VA in the standard N2B27 likely limits further increases in RA signalling. Moreover, in −VA medium, exogenous RARG was no longer able to boost MEF reprogramming (Fig. 4A and Supporting Information Fig. S3A). Therefore, the effect of expressing RARG alone (no endogenous LRH1 in MEFs) is primarily dependent on VA or its metabolites, that is, ligand‐dependent. Similarly, the promoting effect of RARA alone on reprogramming was also VA‐dependent (Supporting Information Fig. S3B). Interestingly, expressing LRH1 alone also promoted 4F reprogramming in +VA medium (Fig. 4A and Supporting Information Fig. S3B), as shown in previous reports using serum‐containing media 46. However, again the effect was largely absent when −VA medium was used (Fig. 4A and Supporting Information Fig. S3B). Therefore, LRH1 promotes reprogramming likely through interaction with the relatively low levels of endogenous RARs in MEFs. At these levels of RARs, the effects of exogenous LRH1 appear to be primarily dependent on the RAR ligands.

Bottom Line: Remarkably, the reprogramming of epiblast stem cells into embryonic stem cell-like cells also requires low levels of RA, which can modulate Wnt signalling through physical interactions of RARs with β-catenin.These results highlight the important functions of RA signalling in reprogramming somatic cells and primed stem cells to naïve pluripotency.Stem Cells 2015;33:1390-1404.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

Show MeSH
Related in: MedlinePlus