Engineering Epithelial–Mesenchymal Microtissues to Study Cell–Cell Interactions in Development

Abstract

Intercellular signaling drives human development, but there is a paucity of in vitro models that recapitulate important tissue architecture while remaining operationally simple and scalable. As an example, formation of the upper lip and palate requires the orchestrated proliferation and fusion of embryonic facial growth centers and is dependent on paracrine epithelial-mesenchymal signaling through multiple pathways including the Sonic Hedgehog (SHH), transforming growth factor-beta (Tgf-β), bone morphogenic protein (BMP), and epidermal growth factor (EGF) pathways. We have developed a robust, throughputcompatible microphysiological system to model intercellular signaling including epithelial-mesenchymal interactions that is useful for studying both normal and abnormal orofacial development. We describe the construction and operation of an engineered microplate created using CNC micromilling of 96-well microtiter plates capable of containing up to 20 epithelial-mesenchymal microtissues. A dense threedimensional mesenchyme is created by embedding cells (O9-1, 3T3) in a biomimetic hydrogel. An epithelial layer is then overlayed on the microtissue by loading cells in engineered microchannels that flank the microtissue. The result is an engineering epithelial-mesenchymal interface that is both on and perpendicular to the imaging plane making it suitable for high-content imaging and analysis. The resulting microtissues and device are compatible with diverse analytical techniques including fluorescent and luminescent cell health and enzymatic reporter assays, gene expression analyses, and protein staining. This tractable model and approach promise to shed light on critical processes in intercellular signaling events in orofacial development and beyond.

The role of Hedgehog and Notch signaling pathway in cancer

“…Notch and Hedgehog (Hh) were first described in Drosophila by Thomas Hunt Morgan in 1917 and by Nusslein-Volhard and Wieschaus in 1980 [1,2]. Both signaling pathways exist widely in vertebrates and invertebrates, are highly primordial and conserved in evolution, and are intimately involved in the fate determination of cells [3,4], differentiation of tissues [5], development of organs [2,[6][7][8][9], formation of embryos [10,11] and homeostasis in organisms [12]. Abnormalities in the modulation of both pathways during embryonic development may contribute to malformations [13][14][15] and disorders [16][17][18][19].…”