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Effects of BMPr and ROCK inhibition in a 3D organotypic human palate fusion model
Citation:
Fitzpatrick, H. AND C. Wolf. Effects of BMPr and ROCK inhibition in a 3D organotypic human palate fusion model. Birth Defects Research and Prevention Annual meeting, Vancouver, British Columbia, CANADA, June 25 - 29, 2022.
Impact/Purpose:
The contribution of teratogenic chemicals to cleft palate in humans is difficult to determine and has been limited to epidemiological studies, for which data is hard to obtain. Developing in vitro models using human stem cells allows us to study the contribution of chemicals on cleft palate in the human and allows more efficient testing of chemicals in medium throughput assays. The current study describes an improved organotypic model for human palate fusion and testing of the model with signaling pathway inhibitors.
Description:
Cleft palate is one of the most common birth defects in the United States, effecting an estimated 1 in 1700 births. Palatogenesis is a multistage process that includes fusion, the dissolution of the palatal shelves’ medial edge epithelium (MEE) allowing the mesenchymal cores to merge. Fusion involves numerous biochemical and genetic regulatory pathways including Bone Morphogenetic Proteins (BMPs) and Rho-activated kinase (ROCK). ROCK mediates cellular processes including contraction, adhesion, migration, and apoptosis, on which dissolution of the MEE is partly dependent. We used a 3D heterotypic human palate organoid model to evaluate the effects of Y-27632, a ROCK inhibitor, and K02288, a BMP-receptor inhibitor, on organoid fusion and cytotoxicity. We hypothesized that these antagonists would inhibit organoid fusion when compared to controls. Spheroids were generated from umbilical-derived human mesenchymal stem cells (hMSCs) and human umbilical vascular endothelial cells (HUVECs) by seeding onto agarose microwells at a ratio of 2:1 respectively and culturing in osteogenic differentiation medium. After seven days of culture, spheroids were differentiated as confirmed by alkaline phosphatase and coated with human primary epithelial keratinocytes (hPEKs), completing the organoid model. Organoids were cultured for 3 days with 1µM, 5µM, and 10µM concentrations of Y-27632 or K02288. At 5µM and 10 µM, K02288 inhibited fusion (p< 0.0013 and p< 0.0005, respectively) and increased ATP content (p<0.05). Y-27632 did not inhibit fusion at any concentration, but increased ATP content with increasing concentration (trend, p=0.0508) and at higher concentrations, often resulted in apparent proliferation and migration of hPEKs. Neither compound exhibited cytotoxic effects. These results demonstrate the importance of BMP signaling in our model. Inhibition of the ROCK pathway appears to have effects other than disruption of fusion in this model. Further research is needed to determine the action of ROCK signaling in our model. This abstract does not necessarily reflect EPA policy.