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Epithelial progenitor cells are dependent upon a complex 3D niche to promote their proliferation and differentiation during development, which can be recapitulated in organoids. The specific requirements of the niche remain unclear for many cell types, including the proacinar cells that give rise to secretory acinar epithelial cells that produce saliva. Here, using ex vivo cultures of E16 primary mouse submandibular salivary gland epithelial cell clusters, we investigated the requirement for mesenchymal cells and other factors in producing salivary organoids in culture. Native E16 salivary mesenchyme, but not NIH3T3 cells or mesenchymal cell conditioned medium, supported robust protein expression of the progenitor marker Kit and the acinar/proacinar marker AQP5, with a requirement for FGF2 expression by the mesenchyme. Enriched salivary epithelial clusters that were grown in laminin-enriched basement membrane extract or laminin-111 together with exogenous FGF2, but not with EGF, underwent morphogenesis to form organoids that displayed robust expression of AQP5 in terminal buds. Knockdown of FGF2 in the mesenchyme or depletion of mesenchyme cells from the organoids significantly reduced AQP5 levels even in the presence of FGF2, suggesting a requirement for autocrine FGF2 signaling in the mesenchyme cells for AQP5 expression. We conclude that basement membrane proteins and mesenchyme cells function as niche factors in salivary organoids.

FGF2-dependent mesenchyme and laminin-111 are niche factors in salivary gland organoids