Multicellular organoid model of human palatal fusion and investigation of signaling pathways
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The human palate forms by fusion of two separate palatal shelves during embryonic development. Disruption of this process can lead to cleft palate. Our laboratory previously developed a 2-cell type (2CT) organotypic model of the palatal shelves that includes human mesenchymal stem cells (M) and human epithelial progenitor cells (hPEKs) to study palatal fusion. Here, we developed a 3-cell type (3CT) model to include the endothelial component of the embryonic palate, investigated the signaling pathways regulating palatal fusion in both models, and compared their fusion responses to Valproic Acid (VPA). Spheroids were generated by seeding M or a co-suspension of M and human umbilical vein endothelial cells (V) at 2:1 in M:V growth media into agarose dishes. The next day (d1), M medium alone or in the mixture was replaced by osteogenic differentiation (O) medium. Spheroids were cultured to day 7 (d7) and coated with hPEKs on overnight to become organoids. Organoids were placed in contact with each other on d8 (d0 of fusion), cultured in co-culture medium with inhibitors or VPA for 2 days, and imaged on days 0, 1, and 2 for fusion analysis. Untreated organoids were collected on d8 for PCR analysis and immunohistochemistry. 2CT and 3CT organoids expressed genes for each model’s cell type, vimentin, cytokeratin17, PECAM; extracellular matrix protein collagen IV; osteogenic marker RUNX2; and components of signaling pathways mediating fusion including BMP2, E-Cadherin, Irf6, ROCK1, TGFβ3 and TGFβ2r. Collagen IV, vimentin, cytokeratin 17, and PECAM-1 were also identified by immunocytochemistry. Fusion was inhibited by an FGF inhibitor (CH5183284) in 3CT organoids at lower concentrations (10 µM) compared to 2CT organoids (20 µM), while fusion was not significantly inhibited by a BMP inhibitor (K02288) in either model. Fusion was inhibited by VPA in 3CT organoids at lower concentrations (1 µM) compared to 2CT organoids (10 µM). These results suggest the 3CT model may be a more sensitive model for the study of human palatal fusion. This work does not necessarily reflect USEPA policy.