Fig. 4.

FGF2 promotes elaboration of complex 3D salivary organoids in basement membrane. (A) Schematic of initial condition for epithelial clusters cultured with growth factors. (B) E16 epithelial clusters were seeded on porous polycarbonate filters floating on simple medium containing FGF2 or EGF. (C) Schematic of epithelial clusters cultured in basement membrane extract with growth factors. (D) Epithelial clusters were cultured in Matrigel with FGF2 or EGF. ICC and confocal imaging revealed that after 7 days, the EpCAM⁺ epithelium is partially maintained with an increase in vimentin⁺ mesenchyme cells with growth factors, while epithelial clusters grown in Matrigel with FGF2 form complex EpCAM⁺ epithelial organoids with budded structures expressing AQP5. Staining was for EpCAM (epithelium, green), AQP5 (proacinar/acinar cells, red) and vimentin (mesenchyme, cyan) with DAPI (nuclei, blue). (E) Quantitative analysis shows the mean diameter for the epithelial clusters with or without growth factors in Matrigel. (F) Organoids were analyzed for the basal cell marker K14, the apical ductal cell marker K7 and the progenitor cell marker Kit in epithelial clusters after 7 days embedded in Matrigel with FGF2. Staining was for AQP5 (proacinar/acinar cells, red), K14 (basal, cyan), K7 (ductal, cyan) and Kit (progenitor, cyan) with DAPI (nuclei, blue). **P<0.01, ***P<0.001 (one-way ANOVA with Tukey post-hoc test between each condition). n=3 experiments. Scale bars: 50 μm.