Figure 2: Undifferentiated ESC produce low levels of RA, and increase this capacity during spontaneous differentiation as EBs. (A) A stable clone of ESC (ESC-RARE-Luc) carrying the Luciferase reporter gene under the control of retinoic acid response elements (RARE) was treated for 8 hours with conditioned medium from undifferentiated ESC (ESC+LIF) or EBs at different stages of differentiation. Where indicated, retinol was added to medium during conditioning. UC: unconditioned medium. Relative Light Unit (RLU) values are represented after background subtraction. Background was measured using wild type E14 ESC treated with UC-medium. Data represent average ±SD of three independent experiments. (B) Retinol supplementation increases spontaneous EBs differentiation in medium supplemented with Charcoal/ Dextran treated FBS. Differentiation was evaluated by RT-qPCR analysis of Brachyury. Values are normalized to β-Actin expression and represented as fold change compared with untreated EBs at day 3 after formation. Vehicle alone (Ethanol) was used as a control. Data represent average ±SD of three independent experiments. *, P<0.05 (n = 3).