We previously demonstrated that coexpressing retinoic acid (RA) receptor gamma and liver receptor homolog-1 (LRH1 or NR5A2) with OCT4, MYC, KLF4, and SOX2 (4F) rapidly reprograms mouse embryonic fibroblast cells (MEFs) into induced pluripotent stem cells (iPSCs). Here, we further explore the role of RA in reprogramming and report that the six factors (6F) efficiently and directly reprogram MEFs into integration-free iPSCs in defined medium (N2B27) in the absence of feeder cells. Through genetic and chemical approaches, we find that RA signalling is essential, in a highly dose-sensitive manner, for MEF reprogramming. The removal of exogenous RA from N2B27, the inhibition of endogenous RA synthesis or the expression of a dominant-negative form of RARA severely impedes reprogramming. By contrast, supplementing N2B27 with various retinoids substantially boosts reprogramming. In addition, when coexpressed with LRH1, RA receptors (RARs) can promote reprogramming in the absence of both exogenous and endogenously synthesized RA. 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

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