Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells.
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.
Affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
- Cellular Reprogramming*
- Embryo, Mammalian/cytology*
- Germ Layers/cytology*
- Induced Pluripotent Stem Cells/cytology*/metabolism
- Receptors, Retinoic Acid/metabolism*
- Signal Transduction*
- Transcription Factors
- Wnt Signaling Pathway
- beta Catenin/metabolism
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stem1926-fig-0005: Retinoic acid (RA) signalling in EpiSC reprogramming. (A): RA signalling as measured by a luciferase assay in EpiSCs cultured in N2B27/activin/FGF2 with VA and other retinoids. The activities are shown relative to that of Renilla luciferase and normalized to that of EpiSCs in −VA. (B): A diagram of EpiSC reprogramming in 2i/LIF. (C): The effects of retinoids on reprogramming Oct4‐GFP reporter EpiSCs to induced pluripotent stem cells by LRH1. *, p < .05; **, p < .01. Scale bar = 200 µm. (D): RA signalling at various stages of EpiSC reprogramming. **, p < .01. (E): The effects of expressing RARG (under the CMV early enhancer/chicken β actin promoter) on reprogramming EpiSCs by LRH1. Note that even small amounts of Rarg PB transposon have deleterious effects. *, p < .05; **, p < .01. (F): Blocking LRH1‐mediated EpiSC reprogramming by ligand depletion (citral), RA receptor inhibition (CD2665) or expression of RARA‐DN. Reprogramming was partially restored by 0.1 nM ATRA. Oct4‐GFP+ colonies were scored on day 9 after transfection. *, p < .05; **, p < .01. In all the experiments, Oct4‐GFP+ colonies were scored on day 9 after transfection. 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: ATRA, all‐trans retinoic acid; 9cRA, 9‐cis‐retinoic acid; EpiSCs, epiblast stem cells; FGF2, fibroblast growth factor 2; LIF, leukemia inhibitory factor; LRH1, liver receptor homolog‐1; RARA‐DN, dominant‐negative form of RARA; RARG, retinoic acid receptor gamma; VA, vitamin A.
EpiSCs expressed much lower levels of RARs than MEFs (Supporting Information Fig. S4). Nevertheless, retinoids, such as retinol, ATRA, 9cRA, and CD437, substantially increased RARE luciferase reporter activities in EpiSC, and removing VA from N2B27 considerably decreased the activities (Fig. 5A). Remarkably, a much lower concentration of retinoids was needed to induce robust luciferase activities in EpiSCs. For example, 10.0 nM ATRA in MEFs efficiently activated the reporter, whereas 0.1 nM was able to robustly activate the reporter in EpiSCs. Importantly, retinoids at these low concentrations in EpiSCs did not substantially change the expression of most key genes involved in pluripotency or differentiation (Supporting Information Fig. S6A).