Citation:

Belair, D., Cynthiaj Wolf, C. Wood, S. Moorefield, C. Becker, H. Ren, A. Fisher, AND B. Abbott. Effects of chemicals and pathway inhibitors on a human in vitro model of secondary palatal fusion. Society of Toxicology 57th Annual Meeting, San Antonio, Texas, March 11 - 15, 2018.

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 to study. 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 test suspect cleft palate teratogens on fusion of these palate organoids.

Description:

The mechanisms of tissue and organ formation during embryonic development are unique, but many tissues like the iris, urethra, heart, neural tube, and palate rely upon common cellular and tissue events including tissue fusion. Few human in vitro assays exist to study human embryonic developmental processes, and there is a need for an in vitro tool to study development events like tissue fusion. Cleft palate can result from failure of fusion of the palatal shelves and is the most common craniofacial birth defect in humans. In this study, we engineered a model system that recapitulates the cellular architecture and events of palate fusion and allows us to assess the influence of suspected teratogens, growth factors, and pathway inhibitors. Spheroids mimicking the palatal mesenchyme were formed from human Wharton’s jelly stromal cells (HWJSCs), which were differentiated to an osteogenic lineage over 7 days to mimic the osteogenic phenotype of embryonic palate. Differentiated HWJSC spheroids expressed genes associated with ECM production, angiogenesis, cell adhesion, and skeletal system development. Spheroids were covered with human neonatal epidermal keratinocyte progenitor cells (HPEKp) to mimic the cellular architecture of the palate, and spheroids less than 275 µm in diameter were optimal for promoting at least 50% coverage over the spheroid surface with HPEKp. Pharmacological inhibitors against known morphogenesis pathways identified the BMP pathway as critical for fusion and the EGF, FGF, HGF, and IGF pathways as critical for both fusion and survival of palatal organoids. We compared these findings with an epithelial wound healing assay and observed that EGF, IGF, and BMP inhibition reduced epithelial cell migration by at least 50%, suggesting that these pathways are involved in epithelial cell morphogenesis. We studied 12 cleft palate teratogens (identified from rodent models) in the in vitro fusion model and identified that tretinoin and tributyltin chloride interfered with the fusion and survival of palatal organoids, while valproic acid and triamcinolone interfered with the fusion of palatal organoids in a dose-dependent fashion. Finally, although we have established involvement of the EGF pathway in spheroid fusion, we did not observe disruption of spheroid fusion by exposure to TCDD, a palate teratogen known to disrupt EGF signaling, either dosed by itself or with added EGF, which suggests that spheroid fusion may be differently sensitive to EGF-disrupting chemicals compared to palate fusion. Our studies demonstrate the feasibility of using human stem cells and three-dimensional co-culture to mimic the cellular and molecular events of palate fusion.

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