SUMMARY:We have previously shown that, in normal human airway tissue, localization of the cystic fibrosis transmembrane conductance regulator (CFTR) can be affected by epithelial maturation, polarity, and differentiation and that CFTR trafficking and apical localization depend on the integrity of the airway epithelium. In this study, we addressed the question of whether the three-dimensional (3-D) organization of adult human airway epithelial cells in suspension culture under rotation, leading to spheroid-like structures, could mimic the in vivo phenomenon of differentiation and polarization. The kinetics of the differentiation, polarity, and formation of the CFTR-ZO-1-ezrin complex was analyzed by transmission, scanning, and immunofluorescence microscopy. Functional activity of the airway surface epithelium was assessed by monitoring the degree of cAMP-stimulated chloride efflux from cultured cells. Our results show that after the initial step of dedifferentiation, characterized by a loss of ciliated cells and disappearance of epithelial subapical CFTR-ezrin-ZO-1 complex, the isolated cells formed 3-D spheroid structures within 24 hours. After 15 days, progressive ciliogenesis was observed and secretory cells could be identified. After 35 days of 3-D culture, ZO-1, CFTR, ezrin, and CD59 were apically or subapically located, and well-differentiated secretory and ciliated cells were identified. CFTR functionality was assessed by analyzing the Cl Ϫ secretion after amiloride and forskolin perfusion. After 35 days of culture of spheroids in suspension, a significant increase in Cl Ϫ efflux was observed in well-differentiated ciliated cells. (Lab Invest 2002, 82:989 -998).

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Airway epithelium stem cells have not yet been prospectively identified, but it is generally assumed that both secretory and basal cells have the capacity to divide and differentiate. Previously, we developed a test for progenitor cells of the human airway epithelium, relying on the transplantation of fetal respiratory tissues into immunodeficient mice. In this study, we hypothesized that airway-repopulating epithelial progenitors can be marked with surface antigens, and we screened an array of such candidate markers, including lectin ligands, the CD44 and CD166 adhesion molecules, and the aquaporin-3 (AQP3) water channel. We observed that AQP3 is selectively expressed on the surface of basal cells, allowing the separation by flow cytometry of AQP3 + basal cells and AQP3 -ciliated and secretory cells. Functional evaluation of sorted cells in vivo showed that AQP3 + cells can restore a normal pseudostratified, mucociliary epithelium as well as submucosal glands. AQP3 -cells are also endowed with a similar potential, although faster engraftment suggests their inclusion of more committed progenitors. These results show that stem cell candidates in the human tracheo-bronchial mucosa can be positively selected with a novel marker but also, for the first time, that epithelial progenitors exist among both basal and suprabasal cell subsets within the human airway. Stem Cells 2005;23:992-1001

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