Organotypic Cultures Of Rat Pulmonary Epithelial-Cells - Morphologic Differentiation

Surfactant is synthesized by Type II pneunocytes which arise by differentiation of pre-Type II cells. In the present study, differentiation of pre-Type II cells was characterized in organotypic cultures of lung epithelial cells isolated on day 18 and 19 of gestation. Cells were incubated in 10% carbon-stripped fetal calf serum on collagen pads for 3–4 days in presence and absence of (1μM) T3 and/or hydrocortisone (HC); epithelial cells were then plated in rnonolayers. Morphologic differentiation was assessed after isolation from the collagen matrix. Synthesis and secretion of surfactant apolipoproteins A (apo A) was used as a biochemical marker of differentiation. Epithelial cells from cultures in carbon stripped media were poorly differentiated consisting of uniform populations of cells in clumps with abundant cytoplasmic polyribosomes and glycogen. Cells (co-cultured with fibroblasts) in the presence of HC with and without T3, demonstrated marked differentation of purified epithelial cells. Morphologic markers of differentiation included: 1) acinar structures, microvillous lined lumens, defined junctional complexes surrounded by spindle cells, 2) loss of cytoplasmic glycogen, 3) appearance of lamellar bodies and tubular myelin. Synthesis and secretion of apo A was demonstrated by 2D-IEF-SDS-PAGE. Differentiation increased with time of culture, was enhanced by T3 and hydrocortisone, maximal effects on differentiation observed with both hormones. We demonstrate in vivo differentiation of Type II cells in organotypic culture and stimulatory effects of HC and T3 on Type II cell differentiation.