Extracellular Matrix-Dependent Differentiation of Rabbit Tracheal Epithelial Cells in Primary Culture

The differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis. The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.