Correction of defective CFTR/ENaC function and tightness of cystic fibrosis airway epithelium by amniotic mesenchymal stromal (stem) cells

Abstract

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F-actin content was increased in co-cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co-cultures had a tendency of increased expression of occludin and ZO-1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC-CFBE co-cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC-CFBE co-cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co-culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.

Keywords: CFTR; ENaC; actin; amniotic membrane; cell therapy; mesenchymal stromal cells; tight junctions.

Fig. 1

Co-cultures show a reorganization of actin cytoskeleton. CFBE cells (A), hAMSC-CFBE co-cultures at 1:5 (B), 1:10 (C), 1:20 (D) ratios, hAMSCs (E) and 16HBE cells (F) grown on Transwells for 6 days were stained with TRITC-phalloidin for F-actin and counterstained with DAPI for the nuclei; scale bar = 20 μm. F-actin content was obtained in the same culture conditions, detected by an actin polymerization assay and expressed as fluorescence intensity normalized to protein content of each sample (G). Data are expressed as mean ± SEM of three experiments.

Fig. 2

Co-cultures display a reorganized pattern of ZO-1. 16HBE (A), CFBE (B), hAMSCs (C), hAMSC-CFBE co-cultures at 1:5 (D), 1:10 (E), 1:20 (F) ratios, grown on Transwells for 6 days were stained with FITC-conjugated anti-ZO-1 antibody and counterstained with propidium iodide for nuclei. Intercellular ZO-1 pattern is indicated by white arrows; scale bar = 10 μm.

Fig. 3

Co-cultures determine a decrease in paracellular permeability to dextrans. FITC-dextrans, 10s (A and C) or 2000s (B and D), were added to the apical side of monolayers and after 10, 30 and 50 min., the apparent permeability (pAPP) was calculated by measuring the fluorescence in the basal medium. Results obtained in direct co-cultures are shown in (A) and (B), while those obtained in indirect co-cultures are shown in (C) and (D). Data are expressed as mean ± SEM of three experiments.

Fig. 4

Co-cultures are characterized by a corrected CFTR protein expression. (A) Representative Western blot of a typical experiment of CFTR protein expression in hAMSC-CFBE co-cultures. The lysates of 16HBE and CFBE/wtCFTR are shown in the first and last lane, respectively, as a reference to the location of Band C and Band B of wtCFTR. The same membrane was probed with anti β-tubulin to confirm that protein loading was the same across the gel. (B) The histogram summarizes the relative change in the expression of Band C in co-culture with respect to CFTR protein Band C expression in 16HBE cells normalized as 1. Results represent means ± SEM of three independent experiments.

Fig. 5

Co-cultures show resumption of CFTR-dependent chloride efflux. (A) CFTR-dependent chloride efflux was evaluated in 16HBE, CFBE, hAMSCs and hAMSC-CFBE at different ratios (1:5, 1:10 and 1:20) obtained both in direct and separate co-cultures. Data are expressed as mean ± SEM of 4–9 independent experiments. Statistical comparison was made with respect to the values obtained in CFBE cells. (B) CFTR-dependent chloride efflux was evaluated in CFBE-F508del, hAMSCs and hAMSC-CFBE-F508del at different ratios (1:5, 1:10 and 1:20) in direct co-cultures. Data are expressed as mean ± SEM of five independent experiments. Statistical comparison was made with respect to the values obtained in CFBE-F508del cells.

Fig. 6

Co-cultures show a reduction in fluid hyper-absorption phenotype. Apical fluid re-absorption was studied after incubation for 24 hrs in the presence or absence of camostat (30 μM). (A) 16HBE, CFBE and hAMSCs were grown on Transwells for 6 days and assayed for fluid absorption for further 24 hrs in the presence or absence of camostat (c). (B) Direct co-cultures at the ratios of 1:5, 1:10 and 1:20 were studied as in (A). (C) Indirect co-cultures at the ratios of 1:5, 1:10 and 1:20 were studied as in (A).