Polarized expression of cystic fibrosis transmembrane conductance regulator and associated epithelial proteins during the regeneration of human airway surface epithelium in three-dimensional culture

N Castillon, J Hinnrasky, J M Zahm, H Kaplan, N Bonnet, P Corlieu, J M Klossek, K Taouil, A Avril-Delplanque, B Peault, E Puchelle

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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 GFTR 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 epithelia[ 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- secretion after amiloride and forskolin perfusion. After 35 days of culture of spheroids in suspension, a significant increase in Cl- efflux was observed in well-differentiated ciliated cells.

Original languageEnglish
Pages (from-to)989-998
Number of pages10
JournalLaboratory investigation
Volume82
Issue number8
DOIs
Publication statusPublished - Aug 2002

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