Figure 4.

Effect of BA-GCs on pluripotency exit. (A) Schematic representation of the experimental procedure (left). ESCs were plated (500 cells/cm²) in DMEM/FBS/2i/LIF medium for 3 days. The resulting domed-shaped colonies were shifted to DMEM/FBS medium supplemented with DMSO, 2i/LIF, fluticasone or budesonide. After 4 days of incubation the cell colonies were imaged or fixed and stained with crystal-violet (right). Scale bar, 150 μm. In the absence of undifferentiating factors, such as the 2i/LIF mix, ESCs proliferate generating irregular flat- instead of round domed-shaped cell colonies. (B) Dose–response activity of CHD-030498 on the morphological transition associated with pluripotency exit. Histogram showing the fraction (%) of flat-shaped cell colonies (left) generated in the presence of budesonide (10 μM, used as a positive control), or CHD-030498 (from 1 to 10 μM). Data are mean ± SD (n = 3; 10 fields/condition). Representative images of crystal violet-stained cell colonies (right). Scale bar, 150 μm. (C) Schematic representation of the experimental procedure (top). Pluripotent ESCs (2i/LIF) were FACS sorted (300 cells/well) on 96-well ultra-low conical plates and incubated in N2B27 ± CHD-030498 (from 1 to 10 μM). After 7 days of incubation the resulting spheroids were imaged and measured. Boxplot diagrams of aggregate diameter at day 7 (left; 10 spheroids/condition), and representative bright-field images (right) of spheroids treated with DMSO (control), or CHD-030498 used at the indicated concentration. Scale bar, 150 μm. (D) Immunofluorescence analysis of E-cadherin (E-cad) expression. Representative confocal images of 8-day-old spheroids, generated as described in (C). Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm.