Figure 3.
Retinoic acid (RA) signalling promotes mouse embryonic fibroblast cell (MEF) reprogramming in the defined condition. (A): The effect of withdrawing VA (retinol) from N2B27/leukemia inhibitory factor (LIF) on 4F and 6F reprogramming. *, p < .05; **, p < .01. (B): Reprogramming MEFs in medium containing retinoids. *, p < .05; **, p < .01. (C): The retinoic acid receptor gamma agonist CD437 (25.0 nM) increases AP+ colony numbers by 4F plus LRH1 in −VA medium. (D): RA signalling activated by various retinoids in a luciferase assay. (E): Quantitative real‐time PCR analysis of gene expression in MEFs cultured in N2B27/LIF in the presence of retinoids for 2 days. These genes include pluripotency‐related genes (c‐Myc, Klf4), Rars (α, β, γ) and genes involved in retinol metabolism (Adh1, Raldh2, Cyp26a1, and Stra6). The expression levels are shown relative to Gapdh and normalized to embryonic stem cells. (F): Proliferation of MEFs in retinoids. MEFs (1 × 105) were plated in N2B27/LIF with or without vitamin A or ATRA. Cells were counted daily for 6 days. Δ, p < .05; ΔΔ, p < .01: ATRA 100.0 nM compared with −VA; *, p < .05; **, p < .01: ATRA 1.0 µM compared with −VA. In the MEF reprogramming experiments, AP+ colonies were scored on day 18 after transfection. The experiments were repeated at least three times, and the error bars shown standard deviations from the mean of triplicate determinations in one representative experiment. Abbreviations: AP, alkaline phosphatase; ATRA, all‐trans retinoic acid; 9cRA, 9‐cis‐retinoic acid; LRH1, liver receptor homolog‐1; VA, vitamin A.