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Anti-neoplastic and Anti-epileptic Agents Inhibit Fusion in 3D Organotypic Human Palate Fusion Model
Citation:
Fitzpatrick, H. AND C. Wolf. Anti-neoplastic and Anti-epileptic Agents Inhibit Fusion in 3D Organotypic Human Palate Fusion Model. Birth Defects Research and Prevention 63rd Annual Meeting, Charleston, SC, June 24 - 28, 2023.
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 the improved organotypic model for human palate fusion and testing of the model with signaling pathway inhibitors.
Description:
Palatogenesis is the multistage process by which the oral palate is formed. The final event, fusion of the palatal shelves and disintegration of the epithelial seam, is of particular interest as a point of failure which leads to cleft palate. Key limitations of animal models for investigation of human processes led our lab to produce an in vitro human stem cell organoid model utilizing three different cell types that successfully mimic human palate fusion. Here, we designed a 3-cell-type organotypic palate model and investigated whether ToxCast chemicals Topiramate (TPM) and Nocodazole (NDZ), suspected of being cleft palate teratogens, could impede fusion in our model. TPM is an anti-epileptic and one of the most prescribed drugs in the US. NDZ is an anti-neoplastic agent which binds actin, preventing microtubule polymerization which often has the effect of arresting cells in G2 or M. Umbilical-derived human mesenchymal stem cells (hMSCs) and human umbilical vascular endothelial cells (HUVECs) are passaged and combined at a ratio of 2:1 in agarose microwells to form spheroids. Spheroids are cultured in osteogenic differentiation medium for seven days, after which, they are collected, combined with human primary epithelial keratinocytes (hPEKs), and placed on an orbital shaker for 6hrs to facilitate coating. The spheroids, now ‘organoids’, are used the next day for experimentation. We exposed groups of organoids placed in contact to varying concentrations of TPM and NDZ in culture medium and measured their fusion over three days. TPM showed inhibition of fusion at 100µM (p<0.05) and NDZ showed inhibition of fusion at 0.5µM, 1µM, and 5µM (p<0.05, p<0.05, and p<0.001, respectively). Neither chemical produced changes in membrane integrity or esterase activity, however, 1µM and 5µM NDZ (p<0.005 and p<0.0001) reduced ATP production, which may indicate cytotoxicity, or a metabolic effect induced by cell cycle arrest. The sensitivity of our model to known and suspected cleft palate teratogens shows that it is a useful tool for evaluating chemical effects and can be adapted to higher throughput screening. This abstract does not necessarily reflect EPA policy.