Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells.
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.
Affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.Show MeSH
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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.
Affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.