FIG. 4.

Prospective isolation of multi-potential airway epithelium-derived progenitors based on expression of α6 integrin. To test the hypothesis that expression of integrin α6 marks progenitor cells in the adult mouse lung, EpCAM^pos integrin α6^high and EpCAM^pos integrin α6^low populations were sorted and plated in 3D Matrigel. (A) FACS strategy: (Ai) total cell population, (Aii) single cell selection, (Aiii) live cell selection. Single live EpCAM^pos integrin α6^high (red) and single live EpCAM^pos integrin α6^low (blue) cells were sorted from trachea (Aiv), airways (Av), and the lung parenchyme (Avi); (Avii) percentage of single live integrin α6^high (red) and α6^low (blue) cells in the EpCAM^pos population sorted from distinct lung regions; mean±SD, n=3 experiments; *P<0.05 versus trachea. (B) Total epithelial colony-forming efficiency (CFE) of EpCAM^pos integrin α6^high (red) and EpCAM^pos integrin α6^low (blue) cells isolated from distinct regions and plated on plastic in low Ca²⁺ medium; mean±SD, n=3 experiments. (C) Representative images of large (left) and small (right) colonies formed by airway EpCAM^pos integrin α6^high cells at day 10 in culture. Cells were fixed and stained with anti-p63 (green) and anti-pan-cytokeratin (white) antibodies following incubation with EdU (red) for 12 h. (D) Cyst-forming efficiency (CFE) in 3D Matrigel was evaluated for GFP^pos EpCAM^pos integrin α6^high (red) and GFP^pos EpCAM^pos integrin α6^low (blue) cells isolated from distinct regions; mean±SD, n=3 experiments; *P<0.05 versus trachea. (D) Representative images of a cyst generated by EpCAM^pos integrin α6^high cells isolated from the airways. Cultures were stained with antibodies against p63 (white), pro-SPC (green), and CCSP (red) at day 18 after plating in 3D Matrigel. (E) Quantification of types of cysts generated by the airway EpCAM^pos integrin α6^high cells based on constituent cell type; bars represent mean±SD, n=3 experiments, 9–12 cysts per experiment. (F) To study the self-renewal potential of airway EpCAM^pos integrin α6^high cells, single cells were recovered from cysts after 18–21 days in culture, mixed with lung fibroblasts, and embedded in Matrigel. (G) The graph shows efficiency of cyst formation (CFE) in primary (P0) and secondary (P1) cultures; results represent mean±SD, n=3 cultures from one of two independent experiments. Scale bars: C, 200 μm; E, 100 μm. n.d., not detectable; *, #=P<0.05 by ANOVA for α6(high) and α6(low), respectively. Color images available online at www.liebertpub.com/scd