FIG. 4.
The clonogenic activity of cells isolated from the homeostatic lung. (A) Total lung and sorted CD49f/EpCAMhi cells were cultivated on mitotic-inactivated mouse embryonic fibroblasts (MEFs) and colony-forming units (CFUs) were assessed at day 10 (n=4 for all experiments). The lung cell CFU activity was statistically higher in fractions (FRs) 2 and 3 (P<0.001; normal font). CD49f/EpCAMhi cell clonogenicity was significantly higher than total lung cells in all fractions (P<0.01; not shown), with FR3 displaying the greatest CFU capacity (P<0.05; italics). (B–E) Control mice were injected with BrdU, and after 1 h euthanized. Lungs were harvested and cells dissociated, separated by density, and processed for immunofluorescence. Micrographs of FR4 cells labeled for (B) DAPI nuclear stain (blue); (C) Ki-67 (green); (D) BrdU (red); and (E) overlay. Scale bar=50 micron. (F) Transparent image of a cluster of putative epithelial (progenitor and more differentiated) cells isolated from FR3 and labeled for (G) DAPI (blue); (H) SCGB1A1 (green); (I) SFTPC (red); and (J) overlay. Scale bar=20 micron. (K–O) Clonogenicity of SFTPC-positive cells. Micrographs of fraction 4 cells incubated for an additional 3 days, treated with BrdU for 1 h, and prepared for immunolabeling. (K) Transparent light; (L) DAPI nuclear stain (blue); (M) BrdU (green); (N) SFTPC (red); and (O) overlay. Scale bar=50 micron. Color images available online at www.liebertpub.com/scd