Human Alveolar Type II Cells: Stimulation of DNA Synthesis by Insulin and Endothelial Cell Growth Supplement

Abstract

Proliferation of type II cells is important for the recovery of the alveolar epithelium after acute lung injury. However, the factors that regulate the proliferation of human type II cells are unknown. Human alveolar type II cells were isolated from resected lung by dissociation with porcine pancreatic elastase and crystalline trypsin and purified by density-gradient centrifugation and serial differential adherence. The purity of the type II cells in the final adherent preparation was 84.4 +/- 1.1% type II cells by alkaline phosphatase and 87.7 +/- 2.8% by cytokeratin (n = 7). The medium MCDB-151 with 0.4% fetal bovine serum (FBS) was used to demonstrate the stimulatory effect of individual growth factors. Under these conditions, thymidine incorporation was stimulated by insulin, epidermal growth factor, endothelial cell growth supplement (ECGS), and acidic and basic fibroblast growth factors. Cholera toxin did not stimulate thymidine incorporation. The most effective stimulation was by the combination of insulin and ECGS. The incorporation of bromodeoxyuridine was used to identify the proportion of cells that were active in DNA synthesis. Insulin and ECGS increased the percentage of cells that incorporated bromodeoxyuridine from 8.5 +/- 1.3% to 21.3 +/- 2.4% (n = 6). Mitotic figures were seen in smears prepared from cultures incubated with insulin and ECGS. This observation was confirmed by electron microscopy, which demonstrated type II cells in metaphase. Increasing the concentration of FBS or human serum in the culture medium to 10% decreased the stimulatory effect of insulin and ECGS.(ABSTRACT TRUNCATED AT 250 WORDS)