Citation:

Wolf, C., C. Becker, J. Smith, AND C. Wood. Comparison of multicellular organoid models of human palatal fusion. Society of Toxicology, San Diego, CA, March 27 - 31, 2022.

Impact/Purpose:

The study of the etiology of cleft palate in the human is difficult. Study has been restricted to in vivo studies in rodents or in vitro organ culture of rodent palates. Culture of spheroids and heterotypic organoids generated from human stem cells allows us to model palatal fusion in humans with a 3D human palate model, and address questions into the signaling pathways involved in the fusion process and whether exogenous chemical exposure interferes with the fusion process. We developed an organoid model and tested suspect cleft palate teratogens on fusion of these palate organoids. Here we develop an enhanced model and test the suitability of this new model for studying human palate fusion. 

Description:

Cleft palate (CP) is a birth defect that occurs in nearly 1 in 1000 human births worldwide. The mammalian palate is formed by fusion of two palatal shelves consisting of mesenchymal tissue surrounded by an epithelial layer. Disruption of this fusion process can lead to CP. This process differs between human and rodents and study in human requires an in vitro model. Our laboratory previously developed a 2-cell type (2CT) organotypic model of human palatal shelves that includes human mesenchymal stem cells (M) and human progenitor epithelial keratinocytes (EK) 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 several chemicals and fusion signaling inhibitors. Spheroids were generated by seeding M in M growth medium or a co-suspension of 2:1 M and human umbilical vein endothelial cells (V) in 2:1 M:V growth media into agarose dishes. The next day (d1), media was replaced by osteogenic differentiation medium (O) or 2:1 O:V medium. Osteogenic differentiation in cultured spheroids was confirmed by alkaline phosphatase activity. Spheroids were cultured for 7 days and coated with EKs overnight to become organoids. Newly formed organoids were placed in contact with each other (d0 of fusion), cultured with chemical for 2 days, and imaged on days 0, 1, and 2 for fusion analysis. Untreated organoids were collected for PCR analysis and immunohistochemistry of model components. Cell types and fusion signaling components in the 2CT and 3CT organoids were confirmed by expression of cell type markers vimentin, cytokeratin17, PECAM-1[HD1] ; osteogenic marker RUNX2; and fusion signaling components 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 FGFr inhibitor (CH5183284) and a TGFβ2r inhibitor (LY21761) in 3CT organoids at lower concentrations (10 µM) compared to 2CT organoids (20 µM), by day 2. Fusion was also inhibited by Valproic Acid 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.

Record Details: