Effect of BA-GCs on gastruloid development. (A) Schematic representation of the experimental design. Pluripotent ESCs (2i/LIF) were FACS sorted (300 cells/well) on 96-well ultra-low conical plates and incubated in N2B27 ± CHD-032201 or CHD-030498 (10 and 1 μM). (B,C) Boxplot diagrams of aggregate diameter at day 2 (left; 10 aggregates/condition), and of gastruloid elongation index (middle) and representative bright-field images (right) of gastruloids treated with DMSO (control), CHD-032201 or CHD-030498 at 10 (B) and 1 μM (C) (10 gastruloids/condition). Scale bar, 150 μm. (D) Immunofluorescence analysis of pluripotency (Oct4, Nanog) and differentiation (Cdx2, brachyury, Sox2) marker expression. Representative confocal images of 5-day-old aggregates/gastruloids, untreated or treated with budesonide (10 μM, positive control) or with CHD-030498 (1 μM). Nuclei were counterstained with DAPI (blue). Scale bar, 150 μm. (E) Dose-dependent effect of CHD-030498, added at 1, 0.5, 0.25 and 0.125 μM, from T0 to 120 h. The elongation index is shown (10 gastruloids/condition). Scale bar, 150 μm.