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A functional CFTR assay using primary cystic fibrosis intestinal organoids

Abstract

We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro–expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

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Figure 1: Quantification of forskolin-induced mouse organoid swelling.
Figure 2: Forskolin-induced swelling of mouse organoids is Cftr dependent.
Figure 3: Forskolin-induced swelling in human organoids is CFTR dependent.
Figure 4: Chemical CFTR correction in human rectal organoids from subjects with cystic fibrosis.
Figure 5: Differential FIS between organoids from subjects with cystic fibrosis after chemical CFTR restoration.

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Acknowledgements

We thank K. Schneeberger for technical assistance, the Department of Pediatric Gastroenterology of the Wilhelmina Children's Hospital for performing gastroduodenoscopy to obtain intestinal biopsies, K. Tenbrock (Department of Pediatrics, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University) and J.C. Escher (Department of Pediatric Gastroenterology, Erasmus MC–Sophia Children's Hospital) for providing intestinal rest-material, C.J. Kuo (Department of Medicine, Stanford) for providing the R-spondin 1-producing cell line, J. Riordan (Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill) for CFTR-specific monoclonal antibodies, R. Bridges (Department of Rosalind Franklin University of Medicine and Science) and Cystic Fibrosis Foundation Therapeutics for providing CFTR-restoring compounds, P.W. van Leeuwen for assistance with statistical analyses and C.B.M. ten Brink for assistance with Volocity software. This research was partly funded by a grant from the WKZ research fund (OZF-2010) and the Dutch Cystic Fibrosis society (NCFS).

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Authors and Affiliations

Authors

Contributions

J.F.D. performed and designed experiments, interpreted results and wrote the manuscript. C.L.W. performed experiments. H.R.d.J. performed ICMs and interpreted data and reviewers' comments. I.B. isolated rectal biopsies and performed ICMs. H.M.J. included subjects with cystic fibrosis. K.M.d.W.-d.G. included subjects with cystic fibrosis. A.M.B. performed western blot analyses. N.W.M.d.J. performed CFTR mRNA analyses. M.J.C.B. provided mouse Cftr knockout materials. B.J.S. provided mouse Cftr F508del materials. E.E.S.N. interpreted data and reviewers' comments. S.v.d.B. generated reagents for human and mouse organoid cultures. H.C. funded organoid media and interpreted data and reviewers' comments. C.K.v.d.E. obtained funding, interpreted data and reviewers' comments and included subjects with cystic fibrosis. S.M. included healthy control subjects and interpreted data and reviewers' comments. J.M.B. obtained funding, designed experiments, interpreted experiments and wrote the manuscript.

Corresponding authors

Correspondence to Hans Clevers or Jeffrey M Beekman.

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Competing interests

J.M.B., C.K.v.d.E. and J.F.D. are inventors on a patent application related to these findings (PCT/IB2012/057497). H.C. is an inventor on several patents related to these findings (WO2010/090513, WO2012/014076, PCT/IB2012/057497 and PCT/IB2012/052950).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 12607 kb)

Supplementary Video 1

Forskolin-induced swelling of mouse wild-type, Cftr–/–, and F508del-Cftr organoids. (MOV 7590 kb)

Supplementary Video 2

Forskolin-induced swelling of human organoids. (MOV 8939 kb)

Supplementary Video 3

Restoration of FIS in rectal F508del homozygous organoids. (MOV 22654 kb)

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Dekkers, J., Wiegerinck, C., de Jonge, H. et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med 19, 939–945 (2013). https://doi.org/10.1038/nm.3201

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