Isolation of cells that retain differentiated functions in vitro: properties of clonally isolated type II alveolar pneumonocytes

Abstract

The terminal respiratory passages of mammalian lung consist of numerous cell types including: ciliated epithelial, goblet, Clara, argentaffin, mucous, endothelial, fibroblast, smooth muscle, macrophage, mast, type I and type II alveolar cells. This cellular heterogeneity persists when pulmonary tissue is enzymatically dissociated into single cells, but in suspensions of single, viable cells, when cloned, each cell has the potential of generating a clonal cell population, thus assuring purity of cell type. This was the reason for making an attempt to isolate a population of type II alveolar epithelial cells from the terminal alveoli of rats. Type II cells synthesize, store, and secrete a surface-active phospholipid (the lung surfactant) which coats the terminal respiratory passage ways and lowers surface tension at the air alveolar interface and thus aids in stabilizing the air sacs. Application of the clonal culture techniques to primary cell suspensions of normal adult rat lung resulted in the isolation of 4 cell strains (L-1, L-2, L-3, L-4), which appear to have originated from type II alveolar pneumocytes. These 4 strains possess 2 ultrastructural markers which are present in type II cells of the whole lung: osmiophilic lamellar bodies and peroxisomes. Osmiophilic lamellar bodies are the cellular organelles in which the pulmonary surfactant is stored prior to secretion onto the alveolar surface. Peroxisomes are organelles possessing catalase activity and are also present in all type II cells. One clone, L-2, synthesizes a highly saturated lecithin which is the major component of the pulmonary surfactant. The pathway of lecithin synthesis utilized by L-2 cells in vitro is the same as that used by type II cells in the whole lung, and this function (monitored by conversion of labeled precursors into lecithin) is easily quantified in cultured cells. L-2 cells grown in monolayer culture retain the normal (female rat) diploid karyotype after 50 population doublings, and they continue to have an epithelial aspect. Osmiophilic lamellar bodies are present in the cytoplasm of the L-2 cells throughout 50 population doublings in vitro. Ultrastructurally these organelles resemble similar structures present in the type II cells of the intact rat lung. Occasionally lamellar bodies are observed being secreted from the L-2 cells into the culture medium. This process resembles the secretion of lamellar bodies into the alveolar space by type II cells in the lung parenchyma. Monolayer cultures of L-2 cells produce lecithin via the same biosynthetic pathways as used by the type II cells in the intact lung. There are two de novo pathways for lecithin synthesis: pathway I, the choline incorporation route, and pathway II, the phosphatidylethanolamine methylation route. Isotopic measurement of these two pathways in L-2 cells reveals predominance of pathway I, the choline route. Conversion of 14C choline and 14C methionine to lecithin is a function of incubation time. The linear generation of radioactive lecithin product can be maintained for at least 5 hr and the choline incorporation route was 200-400 times more active than the methionine incorporation pathway.