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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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).

Author information

Affiliations

  1. Department of Pediatric Pulmonology, Wilhelmina Children's Hospital, University Medical Center, Utrecht, The Netherlands.

    • Johanna F Dekkers
    • , Karin M de Winter-de Groot
    • , Arianne M Brandsma
    • , Nienke W M de Jong
    • , Cornelis K van der Ent
    •  & Jeffrey M Beekman
  2. Center for Molecular and Cellular Intervention, Wilhelmina Children's Hospital, University Medical Center, Utrecht, The Netherlands.

    • Johanna F Dekkers
    • , Caroline L Wiegerinck
    • , Sabine Middendorp
    •  & Jeffrey M Beekman
  3. Department of Immunology, Wilhelmina Children's Hospital, University Medical Center, Utrecht, The Netherlands.

    • Johanna F Dekkers
    • , Arianne M Brandsma
    • , Nienke W M de Jong
    •  & Jeffrey M Beekman
  4. Department of Pediatric Gastroenterology, Wilhelmina Children's Hospital, University Medical Center, Utrecht, The Netherlands.

    • Caroline L Wiegerinck
    • , Edward E S Nieuwenhuis
    •  & Sabine Middendorp
  5. Department of Gastroenterology and Hepatology, Erasmus MC, Rotterdam, The Netherlands.

    • Hugo R de Jonge
    •  & Marcel J C Bijvelds
  6. Department of Pulmonology, University Medical Center, Utrecht, The Netherlands.

    • Inez Bronsveld
  7. Department of Pediatric Pulmonology, Erasmus MC, Rotterdam, The Netherlands.

    • Hettie M Janssens
  8. Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands.

    • Bob J Scholte
  9. Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands.

    • Stieneke van den Brink
    •  & Hans Clevers
  10. University Medical Center, Utrecht, The Netherlands.

    • Stieneke van den Brink
    •  & Hans Clevers

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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.

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).

Corresponding authors

Correspondence to Hans Clevers or Jeffrey M Beekman.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7

Videos

  1. 1.

    Supplementary Video 1

    Forskolin-induced swelling of mouse wild-type, Cftr–/–, and F508del-Cftr organoids.

  2. 2.

    Supplementary Video 2

    Forskolin-induced swelling of human organoids.

  3. 3.

    Supplementary Video 3

    Restoration of FIS in rectal F508del homozygous organoids.

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DOI

https://doi.org/10.1038/nm.3201

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