Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets [scRNA-Seq]

Matrigel®/BME®, a solubilized basement membrane-like preparation, is especially rich in the Extra-Cellular Matrix (ECM) protein laminin-111. This hydrogel supports the formation and long-term growth of epithelial 3D organoids from adult stem cells (ASC)1,2. Laminin111 activates integrin β1 complexes, thus preventing anoikis and driving epithelial polarization. Here, we address if a bacterial integrin-activating protein can mimic Matrigel/BME. Enteropathogenic Yersinia bacteria invade human gut epithelial cells using their outer membrane protein Invasin. This protein binds and activates a diversity of integrin β1 complexes, including the laminin111-specific α6β1 receptor. A recombinant 25 kDa C-terminal integrin-binding fragment of Invasin allowed adhesion of gut epithelial cells, when coated on culture plates. Upon addition of organoid growth factor medium, the epithelial cells grew out in 2D and could be expanded and passaged long-term. Polarization, junction formation and generation of various intestinal cell types (i.e. enterocytes, goblet cells, Paneth cells, and enteroendocrine cells) was stable over time. Sustained expansion of multiple other human-, mouse-, and even snake epithelia was accomplished under comparable conditions. The 2D culturing format holds advantages over the 3D ‘in gel’ format in terms of imaging, accessibility of basal and apical domains and automation for high throughput screening approaches. Invasin represents a fully defined, affordable, versatile, and animal-free complement to Matrigel/BME. Ileum N39 triple reporter line (Goblet cells; MUC2-GFP, enteroendocrine (EEC) cells; CHGA-iRFP and Paneth cells: DEFA5-dsRED) obtained from44 were passaged on the integrin-binding domain of Invasin (5 µg/ml) in transwell system with 3 µm pore size (Greiner Bio-one, 662630) using expansion medium. Differentiation was induced using a 2-step differentiation protocol. The first step is to add 500 µl of patterning medium to the lower compartment of the transwell system for 7 days (refresh medium every 2 days with patterning medium), the upper compartment is just air. The second step is to induce Paneth cells, 500 µl maturation medium (as described in44) was added to the lower compartment of the transwell system, with just air in the upper compartment. This culture condition was maintained for 7 days with refreshing maturation medium in the lower compartment every day.