Abstract

The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.

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Acknowledgements

The authors would like to thank the Cambridge BRC hiPSCs core facility for the derivation of the cystic fibrosis hiPSC line, P.-A. Tsagkaraki for her help with the generation of the manuscript figures and statistical analyses, J. Skepper, L. Carter and the University of Cambridge Advanced Imaging Centre for their help with electron microscopy, C. McGee and the Wellcome Trust Sanger Institute for their help with microarray data processing and analysis, B. McLeod for IT support and S. Brown for technical support and advice. This work was funded by European Research Council starting grant Relieve IMDs (L.V., N.H.), the Cambridge Hospitals National Institute for Health Research Biomedical Research Center (L.V., N.H., F.S.), the Evelyn Trust (N.H.), the European Union FP7 grant TissuGEN (M.C.d.B.) and core support grant from the Wellcome Trust and UK Medical Research Council to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. F.S. has been supported by an Addenbrooke's Charitable Trust Clinical Research Training Fellowship and a joint UK Medical Research Council–Sparks Clinical Research Training Fellowship.

Author information

Author notes

    • Miguel Cardoso de Brito
    •  & Pedro Madrigal

    These authors contributed equally to this work.

    • Nicholas R F Hannan
    •  & Ludovic Vallier

    These authors share senior authorship for this work.

Affiliations

  1. Wellcome Trust-Medical Research Council Stem Cell Institute, Anne McLaren Laboratory, Department of Surgery, University of Cambridge, Cambridge, UK.

    • Fotios Sampaziotis
    • , Miguel Cardoso de Brito
    • , Pedro Madrigal
    • , Alessandro Bertero
    • , Filipa A C Soares
    • , Nicholas R F Hannan
    •  & Ludovic Vallier
  2. Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, UK.

    • Fotios Sampaziotis
    • , Kourosh Saeb-Parsy
    •  & J Andrew Bradley
  3. Wellcome Trust Sanger Institute, Hinxton, UK.

    • Pedro Madrigal
    •  & Ludovic Vallier
  4. Norwegian PSC Research Center, Division of Cancer, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

    • Elisabeth Schrumpf
    • , Espen Melum
    •  & Tom H Karlsen
  5. K.G. Jebsen Inflammation Research Centre, Research Institute of Internal Medicine, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

    • Elisabeth Schrumpf
    • , Espen Melum
    •  & Tom H Karlsen
  6. Institute of Clinical Medicine, University of Oslo, Oslo, Norway.

    • Elisabeth Schrumpf
    •  & Tom H Karlsen
  7. Department of Hepatology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

    • Fotios Sampaziotis
    •  & William T H Gelson
  8. Department of Histopathology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

    • Susan Davies
  9. Child Health Clinical Academic Grouping, King's Health Partners, Denmark Hill Campus, London, UK.

    • Alastair Baker
  10. Department of Medicine, Division of Gastroenterology and Hepatology, University of Cambridge, Cambridge, UK.

    • Arthur Kaser
  11. Department of Medicine, School of Clinical Medicine, University of Cambridge, Cambridge, UK.

    • Graeme J Alexander

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Contributions

F.S.: design and concept of study, execution of experiments and data acquisition, development of protocols and validation, generation, collection and interpretation of data, production of figures, manuscript writing, editing and final approval of manuscript. M.C.d.B., F.A.C.S.: technical support, execution of experiments. P.M.: bioinformatics and statistical analyses. A. Bertero: bioinformatics analyses. K.S.-P., E.S., E.M.: primary tissue provision. T.H.K., J.A.B., W.T.H.G., S.D., A. Baker, A.K., G.J.A.: critical revision of the manuscript for important intellectual content. N.R.F.H.: design and concept of study, study supervision, generation and interpretation of data, editing and final approval of manuscript. L.V.: design and concept of study, study supervision, interpretation of data, editing and final approval of manuscript.

Competing interests

L.V is a founder and shareholder of DefiniGEN.

Corresponding author

Correspondence to Ludovic Vallier.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9, Supplementary Tables 3–6, Supplementary Note and Supplementary Methods

Excel files

  1. 1.

    Supplementary Table 1

    Raw gene expression data (CT values) corresponding to the QPCR analyses in Figures 1d, 5f and 6a

  2. 2.

    Supplementary Table 2

    Microarray gene expression data corresponding to the heat map in Figure 2e

  3. 3.

    Supplementary Table 7

    Statistical analysis information (test statistic, degrees of freedom, statistical significance for each test)

Videos

  1. 1.

    Bile acid export assay: CLF export

  2. 2.

    Bile acid export assay: FITC control

  3. 3.

    Increase in intracellular calcium in response to acetylcholine stimulation

  4. 4.

    Increase in intracellular calcium in response to ATP

  5. 5.

    Increasing organoid size in response to secretin

  6. 6.

    Decreasing organoid size in response to somatostatin

  7. 7.

    Decreasing organoid size in response to octreotide

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DOI

https://doi.org/10.1038/nbt.3275

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