Pulmonary Epithelial Cell Proliferation in Primary Culture of Alveolar Type II Cells

Abstract

A small subpopulation of pulmonary epithelial cells (PE) proliferates in low-density primary culture of alveolar type II cells and forms colonies of cells that could be passaged for several generations and that in some respects maintain a differentiated phenotype of the alveolar type II cells. At this time it is not known if these cells are some form of progenitor epithelial cells or type II cells that are not fully differentiated in vitro. The proliferation of the PE cells was dependent on serum, alveolar macrophage-conditioned medium, and insulin being included in the culture medium. Under these conditions, approximately 0.5-1.0% of the seeded cells that adhered to the culture dishes were capable of forming colonies. Efficiency of colony formation increased to 5-10% in subsequent passages. PE cells maintained a high level (> 40%) of saturated phosphatidylcholine (PC) as a percentage of total PC throughout the culture period (> 28 days). However, the saturated PC content was not constant throughout the long-term culture period and the subsequent passages (41.3% at 29 days and 37.3% in the 3rd passage). These cells also contained numerous lamellar bodies and were able to bind the Maclura pomifera lectin. PE cells also expressed cytokeratin No. 19, as well as alkaline phosphatase activity, both possible markers for differentiated type II cells. However, PE cell synthesized low levels of Pg (approximately 2%), were squamous, and tended to form multiple strata, unlike the cuboidal type II cells in vivo. The cells did not exhibit immunocytochemically demonstrable surfactant-associated protein A (SP-A). Additional factors and culture requirements may be necessary for complete maturation of cultured PE cells. This was demonstrated by culturing PE cells on EHS matrix. Aggregates of cells surrounding a central lumen were formed after a few hours in culture and were maintained for 20 days. The cells contained lamellar bodies and some intercellular junctions. PE cells can be regarded as a highly selected subpopulation of pulmonary epithelial cells that concomitantly maintain proliferation and aspects of differentiated alveolar type II cells in long-term culture.

Growth of putative progenitors of type II pneumocytes in culture of human cystic fibrosis alveoli

“…Furthermore, under some conditions of culture, type II pneumocytes will proliferate (Tanswell et al, 1991). It has been suggested that this proliferation is due to a sub-population of type II pneumocytes and that stem cells are present in heterogeneous populations of type II pneumocytes (Kalina et al, 1993). In the case of the cystic fibrosis pulmonary tissue studied here, progenitor cells of type II pneumocytes may well have proliferated under our culture conditions.…”

Influence of Air Exposure Treatment on Alveolar Type II Epithelial Cells Cultured on Extracellular Matrix.

“…They do not proliferate in the culture system. On the other hand, type II cells are cuboidal and serve as the stem cell for the alveolar epithelium, with potential to proliferate and to assume type I phenotype (3, 16, 18,25).For this reason, investigators have used isolated type II cells to study alveolar cell biology, biochemistry, and molecular biology (2,4,12,13,21,24,25 (2,12,13). Briefly, 21-to 28-day-old male Wistar rats were anesthetized with diethyl ether, and the pulmonary circulation was per fused with a balanced salt solution, and the trachea was cannulated.…”

“…Type II cells produce and secrete surfactant protein, a unique phospholipid and protein mixture which serves to lower the surface tension in alveoli. In previous studies, type II cells have been immersed in culture mediumand not exposed to air (4,12,21). Wedemonstrated that type II cells exhibit improved maintenance of structural and functional differentiation when cultured in a collagen gel matrix (21).…”

“…For this reason, investigators have used isolated type II cells to study alveolar cell biology, biochemistry, and molecular biology (2,4,12,13,21,24,25 (2,12,13). Briefly, 21-to 28-day-old male Wistar rats were anesthetized with diethyl ether, and the pulmonary circulation was per fused with a balanced salt solution, and the trachea was cannulated.…”

“…For this reason, investigators have used isolated type II cells to study alveolar cell biology, biochemistry, and molecular biology (2,4,12,13,21,24,25). Type II cells produce and secrete surfactant protein, a unique phospholipid and protein mixture which serves to lower the surface tension in alveoli.…”