Fetal type 2 pneumocytes form alveolar‐like structures and maintain long‐term differentiation on extracellular matrix

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Abstract
We investigated the effects of reconstituted basement membrane (a crude extract of the Engelbreth‐Holm‐Swarm tumor) on type 2 pneumocyte differentiation during long‐term culture. Cells were derived from mature 29 d fetal rabbits. Morphology was studied by light and electron microscopy. On thin gel, the cells initially segregated into clumps; they were cuboidal with apical microvilli and contained lamellar bodies, but dedifferentiated by 8 d. On thick gel, epithelial cells associated into spherical clusters surrounding a central lumen. These alveolarlike structures persisted at least 22 d. The cells were cuboidal and had lamellar bodies and intercellular tight junctions; they exhibited polarity, with apical microvilli facing the lumen, basally located nuclei, and gel matrix abutting the basal surface. In contrast, cells cultured on plastic formed colonies, then a monolayer, but dedifferentiated 5–7 d after plating. [14C]Acetate was used to label newly synthesized phospholipids. The amount of disaturated phosphatidylcholine (DSPC), expressed as a percentage of total phosphatidylcholine (PC), was used as an indicator of surfactant lipid production; percentage DSPC synthesized by cells cultured on thick gel did not change significantly, from 55 ± 3 at 3 d, to 63 ± 2 at 22 d in culture. DSPC synthesized by cells cultured on plastic decreased from 57 ± 1% at 3 d to 45 ± 2% at 22 d (p < 0.001), which is consistent with the morphologic evidence of dedifferentiation. Synthesis of total PC compared with total phospholipid did not vary with either time in culture or substrate. This study emphasizes the importance of a complex extracellular matrix for maintenance of type 2 pneumocyte differentiation. The system should prove useful for studying the interaction of these cells with basement membrane, including the role of events occurring at the cell surface in modulating expression of a differentiated phenotype.