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Development of a 3D co-culture model using human stem cells for studying embryonic palatal fusion.
Citation:
Wolf, Cynthiaj, C. Becker, K. Das, A. Watkins, D. Belair, AND B. Abbott. Development of a 3D co-culture model using human stem cells for studying embryonic palatal fusion. Teratology Society Annual Meeting, San Antonio, TX, June 25 - 29, 2016.
Impact/Purpose:
This work reflects development of an in vitro three dimensional organotypic model of embryonic tissue fusion, Task 1 of 9.1 Morphogenesis, CSS 12.02 Virtual Tissue Models, to complement and support in silico models and AOPs.
Description:
Morphogenetic tissue fusion is a critical and complex event in embryonic development and failure of this event leads to birth defects, such as cleft palate. Palatal fusion requires adhesion and subsequent dissolution of the medial epithelial layer of the mesenchymal palatal shelves, and is regulated by the growth factors EGF and TGFβ, and others, although the complete regulatory mechanism is not understood. Three dimensional (3D) organotypic models allow us to mimic the native architecture of human tissue to facilitate the study of tissue dynamics and their responses to developmental toxicants. Our goal was to develop and characterize a spheroidal model of palatal fusion to investigate the mechanisms regulating fusion with exposure to growth factors and chemicals in the ToxCast program known to disrupt this event. We present a spheroidal model using human umbilical-derived mesenchymal stem cells (hMSC) spheroid cores cultured for 13 days and then coated with MaxGel™ basement membrane and a layer of human progenitor epithelial keratinocytes (hPEK) (hMSC+hPEK spheroids). We characterized the growth, differentiation, proliferation and fusion activity of the model. Spheroid diameter was dependent on hMSC seeding density, size of the seeding wells, time in culture, and type of medium. hMSC spheroid growth was enhanced with osteogenic differentiation medium. Alkaline phosphatase activity in the hMSC spheroid, indicating osteogenic differentiation, increased in intensity throughout culture to day 14. Preliminary results showed EGF exposure at 2 or 4 ng/ml in hMSC+hPEK spheroid cultures increased cell proliferation by almost 2-fold. In a pilot fusion study, hMSC spheroids when placed in contact began to merge within 8 hrs, while hMSC+hPEK spheroids began to fuse at a later time point, 40-48 hrs, and were completely merged at ~ 4 days. This model will enable us to study the regulation of fusion by manipulation of spheroid activity with growth factors and to evaluate the effects of exposure to ToxCast chemicals associated with cleft palate. Additionally, this model can be implemented in the study of other embryonic fusion events that involve mesenchymal and epithelial tissues. This abstract does not necessarily reflect USEPA policy.