Citation:

Wolf, C. AND C. Becker. Multicellular organoid model of human palatal fusion and response to VPA. Teratology Society Annual meeting, San Diego, California, June 22 - 26, 2019.

Impact/Purpose:

Cleft palate and other orofacial clefts are one of the most common birth defects, affecting 1 in 700 births worldwide. 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 pahtways involved in the fusion process and whether exogenous chemical exposure interferes with the fusion process. We developed an enhanced model and test suspect cleft palate teratogens on fusion of these palate organoids.

Description:

Tissue fusion occurs in various parts of the developing embryo to complete proper development. Disruption of the fusion process, whether by genetic control or chemical exposure, can lead to malformations, such as cleft palate. Our laboratory previously developed an organotypic model of the palatal shelves that includes human mesenchymal stem cells (M) and epithelial progenitor cells (hPEKs) and documented effects of chemicals such as Valproic acid (VPA) on fusion. Here, we developed a 3-cell type (3CT) model to include the endothelial component of the embryonic palate and compared the effects of VPA on fusion. Spheroids were generated by seeding a co-suspension of M and HUVECs (human umbilical vein endothelial cells; V) at ratios of 1:2, 1:1 or 2:1 in M:V growth media into agarose dishes. The next day, M medium in the mixture was replaced by osteogenic differentiation medium (O) medium and spheroids were cultured to day 7. M spheroids were generated in M growth medium and cultured in O medium on days 1 - 7. All spheroids were coated with hPEKs on d7 to become organoids. Organoids were placed in contact on d8 and cultured in co-culture media with VPA for 2 days. A cell suspension ratio of 2:1 V:M resulted in V localizing to the perimeter of the spheroid. A ratio of 1:2 V:M resulted in endothelial cells interspersed among M, as in vivo. 3CT organoids expressed genetic markers for each cell type, vimentin, keratin17, VWF; an indicator of osteogenic differentiation, RUNX2; and components of the signaling pathway mediating fusion including BMP2, and TGFβ2 receptor. VPA exposure inhibited fusion in the 2CT model at 10 µM with no cytotoxicity, and preliminary data suggest VPA inhibited fusion in the 3CT model with an altered concentration-response. These results indicate the 3CT palate organoid model is a functional model, representative of the embryonic palate and may be more appropriate for fusion assessment than the previous model. Work is in progress to identify other signaling pathway components that mediate palatal fusion and test other suspect teratogens. This abstract does not necessarily reflect EPA policy.

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