Figure 4.

In vitro culture model for propagation of epithelial progenitor cells. Culture conditions were optimized using a Lin^neg/EpCAM^pos cell population of β-actin/GFP transgenic mice. Representative images of Day 6 cultures of cells seeded in growth factor–reduced Matrigel and cultured under (A) basic medium (BM), (B) BM and in the presence of mouse lung fibroblast cells (MLg), or (C) BM supplemented with SB431542 in the presence of MLg cells (1 × 10⁴ cells/condition). (D) Analysis of cytokeratin (CK)-18 expression within GFP^pos colonies by dual immunofluorescence analysis. Photomicrographs demonstrate CK-18–immunoreactive cells (D), GFP-immunoreactive cells (E), and a merged image showing coexpression of CK-18 and GFP (F). DAPI is included as a nuclear counterstain (blue). (G) Analysis of clonality of epithelial colonies. Lin^neg EpCAM^pos cells were isolated from mice expressing ubiquitous GFP (green signal) or TdTomato (red signal) and cocultured in the presence of MLg cells and SB431542. No mixing of red and green fluorescent cells is observed within individual colonies, indicating their clonal origin. (H) Quantitative analysis of the colony-forming ability of Lin^neg/EpCAM^pos cells in BM, BM+MLg, and BM+MLg+SB431542. Cultures of GFP⁺ cells (input cell number: 5,000) from lineage tracing mice, (I) CCSPcreER, (J) SPC-GFP, and (K) FoxJ1-GFP were photographed at Day 6. Scale bars: 200 μm for A, B, C, G, I, J, and K; 100 μm for F.