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Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.
Citation:
Belair, D., Cynthiaj Wolf, K. Das, A. Watkins, AND B. Abbott. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events. Presented at Society of Toxicology Meeting, New Orleans, LA, March 13 - 17, 2016.
Impact/Purpose:
This abstract will be presented at the Society of Toxicology Meeting March 13-17,2016, New Orleans, LA
Description:
Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling pathways elucidated using gene knockout mouse models. A broad analysis of literature, ToxRefDB, and ToxCast identified 63 chemicals that are related to cleft palate. However,the influence of these putative teratogens on human palatal fusion has not been studied due to the lack of in vitro models. We sought to engineer the stratified mesenchymal and epithelial structure of the developing palate in vitro via organotypic culture of human mesenchymal stem cell (hMSC) spheroids coated with a single layer of human primary epidermalkeratinocytes (hPEKp). hMSC spheroids exhibited uniform size over time (175 ± 21 µm mean diameter) proportional to starting cell density. Further, we developed a novel procedure to coat hMSC spheroids homogeneously with a single layer of hPEKp cells using a seeding ratio of 0.1-0.2 hPEKp per hMSC, and hMSC/hPEKp spheroids expressed mesenchymal markers (vim+, C044+, CD105+, CD34-) and epithelial markers (krt17+, itga6+) via qRT-PCR. Analysis of adverse outcome pathways related to palate fusion points to an EGF/TGFj33 switch that could be a target for cleft palate teratogens, and both egf and egfr were expressed by hMSC/hPEKp spheres. Finally, hMSCs and hPEKp cells expressed a model protein,GFP, using non-viral transfection. We hypothesize that hMSC/hPEKp spheroids together with genetic engineering approaches will enable interrogation of signaling pathways crucial to morphogenetic fusion for predictive toxicology. The organotypic culture model described here can be broadly applied to model morphogenetic fusion of other stratified mesenchymal and epithelial tissues. This abstract does not necessarily reflect EPA policy.