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Development of an organotypic stem cell model for the study of human embryonic palatal fusion
Wolf, Cynthiaj, D. Belair, C. Becker, K. Das, Judy Schmid, AND B. Abbott. Development of an organotypic stem cell model for the study of human embryonic palatal fusion. BIRTH DEFECTS RESEARCH PART B: DEVELOPMENTAL AND REPRODUCTIVE TOXICOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, , 1322-1334, (2018). https://doi.org/10.1002/bdr2.1394
Virtual Tissue Models are uniquely positioned to capture the connectivity between different scales of biological organization. The focus of the Morphogenetic Fusion Task is to develop a complex 3-D culture model to mimic embryonic fusion events required during development. Morphogenetic fusion is a complex process having contributions from multiple cell types and cell behaviors and is sensitive to chemical disruption. The spheroid model is being developed to evaluate responses to chemicals and perturbation in molecular initiating events or key events involved in fusion-related phenotypes (birth defects). This foundational work describes the development of the model, investigation into fusion and the response to the growth factor EGF as would occur in vivo.
Cleft palate (CP) is a common birth defect, occurring in an estimated 1 in 1000 births worldwide. The secondary palate is formed by paired palatal shelves that grow toward each other, appose, attach and fuse. CP can result from disruption of any of these processes. The palatal shelves basically consist of a mesenchymal tissue core covered with a layer of epithelial cells. One of the mechanisms that can cause CP is failure of fusion, i.e., failure to remove the epithelial seam between the palatal shelves to allow the mesenchyme to merge and form a continuous palate. This process requires complex interactions between mesenchymal and epithelial cells, and signaling components such as growth factors. Epidermal growth factor (EGF) plays an important role in palate growth and differentiation, while it may impede fusion. We developed a 3D organotypic model using human mesenchymal and epithelial stem cells to mimic human embryonic palatal shelves, and tested its functional relevance by monitoring the effects of human EGF (hEGF) on proliferation and fusion. Spheroids were generated from human umbilical-derived mesenchymal stem cells (hMSCs) directed down an osteogenic lineage by culture medium and evaluated for osteogenic differentiation. Heterotypic spheroids, or organoids, were constructed by coating hMSC spheroids with MaxGel™ extracellular matrix solution followed by a layer of human progenitor epithelial keratinocytes (hPEK). Organoids were incubated in co-culture medium with or without hEGF and assessed for cell proliferation and spheroid pairs were assessed for time to fusion. Osteogenic differentiation in hMSC spheroids was highest by day 13. hEGF delayed fusion of heterotypic organoids after 12 and 18 hours of contact. hEGF increased proliferation in organoids at 4 ng/ml, and proliferation was detected in hPEKs alone on microcarrier beads, suggesting a potential mechanism for delayed fusion by hEGF. Our results show that this model of human palatal fusion consisting of a core of differentiated hMSCs with a hPEK outer layer appropriately mimics the morphology of the developing human palate and responds to hEGF as expected. Future studies will focus on using the organoid model to evaluate the effects of teratogenic chemicals on palatal fusion, and validating the results.