You are here:
Engineered three-dimensional multicellular culture model to recapitulate morphogenetic fusion using human cells
Citation:
Belair, D. Engineered three-dimensional multicellular culture model to recapitulate morphogenetic fusion using human cells. To be Presented at Gordon Research Conference, Biddeford, ME, June 25 - July 01, 2016.
Impact/Purpose:
Gordon Research Conference- Signal Transduction by Engineered Extracellular Matrices, Biddeford, Maine June 25, 2016- July 1, 2016
Description:
Tissue fusion during early mammalian development requires crosstalk between multiple cell types. For example, paracrine signaling between palatal epithelial cells and palatal mesenchyme mediates the fusion of opposing palatal shelves during embryonic development. Fusion events in developmental processes including heart development, neural tube closure, and palatal fusion are dependent on epithelial-mesenchymal interactions (EMIs) and specific signaling pathways that have been elucidated largely using gene knockout mouse models. A broad analysis of literature using ToxRefDB identified 63 ToxCast chemicals associated with cleft palate in animal models. However, the influence of these and other putative teratogens on human palatal fusion has not been examined in depth due to the lack of in vitro models incorporating EMIs between human cell types. We sought to engineer the stratified mesenchymal and epithelial structure of the developing palate in vitro using spheroid culture of human Wharton’s Jelly mesenchymal stem cells (hMSC). hMSC spheroids exhibited uniform size over time (175 ± 21 µm mean diameter) that was proportional to starting cell density. Further, hMSCs in spheroid culture exhibited increased alkaline phosphatase activity and increased expression of bglap and runx2 after 7 days of culture in osteo-induction medium, which suggests that spheroid culture together with osteo-induction medium supports osteogenic differentiation. We developed a novel procedure to coat osteogenic hMSC spheroids with human primary epidermal keratinocyte progenitor cells (hPEKp) and demonstrated that heterotypic cell spheroids exhibited increased tgfb3 expression relative to hPEKp or hMSC alone. This suggests that co-culture promotes the functional specification of cultured epithelial cells. Finally, we examined the fusion of heterotypic cell spheroids using cell tracking dyes together with confocal microscopy and confirmed the ability of hMSC/hPEKp spheroids to fuse by examining the disappearance of hPEKp from the interface of opposing spheroids. Engineered hMSC spheroids with coatings of prototypical extracellular matrix (ECM) proteins further demonstrated that ECM identity may influence the fusion of hMSC/hPEKp spheroids as measured by differential expression of developmental genes. Analysis of adverse outcome pathways related to palate fusion points to an EGF/TGFβ3 switch that could be a target for cleft palate teratogens, and this prediction agrees with our results wherein both egf and egfr expression were upregulated during spheroid fusion. We hypothesize that engineered hMSC/hPEKp spheroids will enable interrogation of signaling pathways crucial to morphogenetic fusion for predictive toxicology. This organotypic model can be broadly applied to model morphogenetic fusion of other tissues exhibiting EMIs. This abstract does not necessarily reflect EPA policy.