Fig. 4. Inhibition of SMAD2/3 signaling disrupts morphology and differentiation of endometrial epithelial organoids.

a Schematic showing the strategy used to isolate uterine epithelium from control and Smad2/3 cKO mice for the culture of epithelial organoids. Organoids from control mice were grown in the presence or absence of the TGFβ receptor (ALK4/ALK5/ALK7) inhibitor, A83-01 for 5 passages. b, c Phase contrast images of cystic (b, round-shaped) or dense (c, lobular shaped) organoids. d–f Quantification of cystic vs. dense organoids across the three conditions, control + vehicle (d), control + A83-01 (e), and Smad2/3 cKO (f). Values displayed as percentage of total organoids from n = 3 mice per group over 5 passages, mean ± SEM. g–i Phase contrast imaging of the organoids from control mice grown in the absence (g) or presence (h) of A83-01, and from Smad2/3 cKO mice (i). j–l H&E stained cross sections of organoids from control mice (j), control mice treated with the A83-01 inhibitor (k), and from Smad2/3 cKO mice (l). m–r Cross sections of endometrial organoids from control mice cultured in the absence (m, p) or presence of A83-01 (n, q) or from Smad2/3 cKO mice (o, r). The organoids were immunostained with the epithelial cell marker antibody, cytokeratin 8 (CK8, red) and the mucin 1 antibody (m–o, MUC1, green), or with CK8 (red) and the glandular cell marker, FOXA2 (p–r, green). These experiments were performed in organoids derived from at least three mice per group.