Phenotypic Analysis of BrdU Label-Retaining Cells during the Maturation of Conducting Airway Epithelium in a Porcine Lung

Figure 9

The expression of putative epithelial stem/progenitor cell markers and sphere formation assay of primary porcine bronchial epithelial cells. Freshly isolated primary porcine bronchial epithelial cells were cultured on cover slides in BEGM medium for 24 h prior to be employed for IF assay using indicated primary antibodies. (a) Immunofluorescence staining for keratin 5 (Krt5) (green) revealed that the majority of primary porcine bronchial epithelial cells expressed Krt5. (b) Immunofluorescence staining for c-Kit (CD117) (green) suggested that a subset of primary porcine bronchial epithelial cells expressed c-Kit. (c) Immunofluorescence staining for CD49f (ITGA) (green) showed that a subpopulation of primary porcine bronchial epithelial cells expressed CD49f. Nuclear stained with DAPI (blue). (d, e) Colocalization of Krt5 and c-Kit or CD49f in primary porcine bronchial epithelial cells. The colocation of putative lung epithelial stem/progenitor cell markers in primary porcine bronchial epithelial cells was ascertained by IF assay using indicated antibodies. Results showed that the coexpression of keratin 5 (green) and CD49f (red) (d) or c-Kit (red) (e) in a subset of keratin 5-positive cells. Nuclear stained with DAPI (blue). (f–h) Flow cytometry analysis of primary porcine bronchial epithelial cells cultured on a collagen precoated 100 mm dish in BEGM medium for 48 h and stained with fluorescence dye-labeled respective antibodies to Krt5 and CD49f or cultured in Matrigel for a 3D sphere formation assay. (f) A representative flow cytometry quadrant plot of primary porcine bronchial epithelial cells stained with FITC anti-Krt5 and APC anti-CD49f antibodies showed the portions of CD49f-positive (Q1, 0.3%), Krt5/CD49f double positive (Q2, 0.2%), Krt5-positive (Q3, 38.3%), and Krt5-negative (Q4, 61.2%) cells. (g, h) Representative images of the 3D culture of sphere formation assay showed spheres of primary porcine bronchial epithelial cells cultured for 7 days in Matrigel. G and H were enlarged insets in the corresponding selected areas of (b) and (c). Bars in (a–e) = 50 μm in all images; bars in (g–h) = 100 μm.

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