FIG. 3.

A fraction of airway-derived epithelial cells gives rise to p63^pos CCSP^pos pro-SPC^pos cysts in 3D culture and subcutaneous Matrigel implants. (A) Scheme: EpCAM^pos cells were isolated from distinct anatomical regions of EGFP mouse lungs, mixed with wild-type fibroblasts, and embedded in Matrigel. Cells were either cultured in insert plates (top) or injected under the skin of NSG mice (bottom). (B) Representative images of GFP^pos cysts at day 18 in culture. (C) Cyst-forming efficiency (CFE) by EpCAM^pos cells from trachea, airways, and lung parenchyme of EGFP mice; mean±SD; n=3 experiments. (D) Representative immunohistochemistry images of cysts in culture (left panels) or in subcutaneous implants (right panels) stained with antibodies against p63 (white), pro-SPC (green), and CCSP (red). The labels on the right side of the images indicate the source of epithelial cells. (E) Percentage of cyst types based on expression of differentiation markers; bars represent mean+SD, n=3 experiments, 4–25 cysts per culture. (F) At day 5 postplating, airway epithelial cells were labeled with BrdU for 5 h, fixed, and stained with antibodies against p63, CCSP, or pro-SPC (red) and BrdU (blue). (G) Quantification of p63^pos, CCSP^pos, and pro-SPC^pos cells (red bars) in the pool of BrdU-incorporating cells at day 5 in culture. Scale bars: B, 100 μm; D, 20 μm; § indicates image acquired from the same region at a different optical plane, F, 20 μm. *, P<0.05 by ANOVA; n.d., not detectable. Color images available online at www.liebertpub.com/scd