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qBio Seminar Series: Brian Johnson
September 20, 2017 @ 2:00 pm
Pathway driven design, validation and teratogenicity of a 3D microphysiological model of Sonic Hedgehog signaling
Human development is orchestrated by intercellular communication via secreted small molecules. However, in vivo models lack the tractability to interrogate underlying mechanisms and existing platforms for studying these cell-cell interactions in vitro are limited by the constraints of 2 dimensionality. We developed a microplate-based 3D culture device which supports an epithelial monolayer directly overlaid onto a 3D extracellular matrix-embedded mesenchyme, enabling direct 3D epithelial-mesenchymal interaction. Microfluidic devices are cut into commercial microtiter plates, enabled by open microfluidic concepts, support near-linear gravity driven flow, and are well suited for imaging on standard inverted microscopes. To test the practical utility and biological fidelity of this approach, we examined paracrine Sonic Hedgehog (SHH) signaling, which plays critical roles in development of numerous organs. Recapitulating a hallmark pathway response, a live cell dual luciferase reporter assay showed SHH ligand produced from the epithelium generated a reproducible gradient of pathway activity in the adjacent mesenchyme, with high proximal activity decreasing to no activity 500 microns into the gel. SHH pathway activation was efficiently antagonized by small molecule inhibitors of targets in the epithelial secretory (cholesterol/palmitoylation), ECM transport (Hh ligand) and mesenchymal sensing targets (Smoothened, Gli1), supporting the utility of this approach for high-content chemical screening of the complete SHH pathway. Finally, we show that this approach can be tailored to mimic specific biological contexts. To model paracrine signaling in orofacial development, 3D embedded cranial neural crest mesenchymal cells were overlaid with an oral ectodermal monolayer. Observed mesenchymal cell proliferation and active tissue outgrowth recapitulate the cellular and tissue level sequence of normal lip and palate development. Together, these findings demonstrate a novel and practical microphysiological model with broad utility for investigating epithelial mesenchymal interactions and disruptions in development.
This work was supported by funding from NIH/NIDCR (R00DE022010), EPA -Science to Acheive Results (STAR) Center 835737, a T32 CA157322 to BPJ and the University of Wisconsin-Madison.
All qBio sponsored talks take place on Wednesdays at 2:00 p.m. in the 3rd floor Orchard View room of the Discovery Building unless otherwise noted. Talks are open to the public. Access to the room is via the elevator behind Aldo’s Cafe in the Northeast corner of the building.