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Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation

Nature Biotechnology volume 33, pages 845–852 (2015)Cite this article

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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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Figure 1: Generation of CPs from hiPSCs.
Figure 2: Generation of CLCs from CPs.
Figure 3: Functional characterization of CLC organoids.
Figure 4: Activin and Notch signaling are essential for CLC organoid formation.
Figure 5: CLC organoids respond to secretin and somatostatin stimuli and reproduce the effects of somatostatin analog (octreotide) in polycystic liver disease in vitro.
Figure 6: Modeling CF liver disease in vitro using hiPSCs derived from patients with CF.

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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

  1. Miguel Cardoso de Brito and Pedro Madrigal: These authors contributed equally to this work.

  2. Nicholas R F Hannan and Ludovic Vallier: These authors share senior authorship for this work.

Authors and Affiliations

  1. Department of Surgery, Wellcome Trust-Medical Research Council Stem Cell Institute, Anne McLaren Laboratory, 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. Division of Cancer, Norwegian PSC Research Center, 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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  1. Fotios Sampaziotis
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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.

Corresponding author

Correspondence to Ludovic Vallier.

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Competing interests

L.V is a founder and shareholder of DefiniGEN.

Integrated supplementary information

Supplementary Figure 1 Comparison between CLCs and hESC-derived cholangiocytes (hESC-Chol) generated by Dianat et al.

(a-d) Comparison of the transcriptional profiles of CLCs, hESC-Chol. and primary Common Bile Duct (CBD) cholangiocytes. (a, b) Hierarchical clustering (Euclidean distance, complete linkage method) of the transcriptional signatures of hESC-Chol (Dianat et al.), CBD and CLCs for (a) n= 14939 probes (b) n= 15954 genes. For each probe (a) and gene (b), standard scores (z-scores) indicating the probe (a) or gene (b) level expression relative to standard deviations above the mean for each sample were used. Color code break points were spaced equally between zmin=-0.5 and zmax=1. (c, d) Heatmap showing the hierarchically clustered Spearman correlation matrix resulting from the pair-wise comparisons of probe (c) or gene (d) expression values (z-scores). CLCs show higher correlation to CBD cholangiocytes than hESC-derived cholangiocytes. (e) Percentage of overlapping functions (POF) between primary CBD cholangiocytes and the two differentiation protocols, hepatoblast or hIPSCs for top 1000 expression-based ranked genes. Number of significant GO terms (Bonferroni adjusted p < 0.05): CLCs: 545; Primary: 449; hESC-Chol: 527; HBs: 521; hPSC: 488. (f) Percentage of CLCs responding to calcium stimulation through ATP or acetylcholine, n=4. Error bars represent SD. (g) Percentage of CLC organoids responding to secretin or somatostatin stimulation, n=4. Error bars represent SD.

Supplementary Figure 2 Generation of hepatocyte-like cells from iPSC-derived bipotent hepatoblasts.

(a) IF analyses demonstrating the expression of key hepatocyte markers as indicated. Scale bars correspond to 100μm. (b) QPCR analyses demonstrating the expression of genes characteristic for hepatocytes. Center line, median; box, interquartile range; whiskers, range. Asterisks represent statistically significant differences (2-tailed t-test, ***P<0.001, ****P<0.0001). (c) Hepatocytes differentiated from iPS derived bipotent hepatoblasts exhibit cytochrome P450 activity, characteristic of primary human hepatocytes, n=9, P<0.0001 (2-tailed t-test).

Supplementary Figure 3 Activin and retinoic acid in the absence of BMP4 signaling suppress hepatic lineage commitment of bipotent hepatoblasts

(a) IF analyses demonstrating suppression of the hepatic/hepatoblast marker AFP in response to activin stimulation and the opposite effect of the activin signaling pathway inhibitor SB-431542. Scale bars correspond to 100μm. (b) IF analyses demonstrating expression of the hepatoblast (HB) marker TBX3 in response to BMP4 stimulation of bipotent hepatoblasts and the opposite effect of the BMP4 signaling pathway inhibitor noggin. Scale bars correspond to 100μm. (c) QPCR analyses demonstrating sustained expression of genes encoding hepatic and hepatoblast markers in response to BMP4 stimulation and inhibition of activin signaling through SB-431542 (SB) and suppression of the same markers in the presence of activin and retinoic acid (RA). Center line, median; box, interquartile range; whiskers, range. Asterisks represent statistically significant differences between HBs and other conditions (one-way ANOVA with Dunnett correction for multiple comparisons, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Supplementary Figure 4 Cell number expansion and differentiation efficiency for the generation of CLC organoids from hIPSCs

(a) Flow cytometry analyses demonstrating the percentage of cells expressing characteristic markers for key stages of CLC differentiation. The final population of CLCs is characterized in 3 lines (bottom row): A1ATD-1, FSPS11B and CF2. (b) Number of cells expressing appropriate markers for each key stage of CLC differentiation, demonstrating the generation of 57.28x106 mature (CK7+Sox9+) CLCs from a starting population of 1x106 hIPSCs. Error bars represent SD, n=3.

Supplementary Figure 5 The method for differentiation of iPSCs to CLCs is efficient across different lines.

(a) IF analyses demonstrating the expression of key biliary markers in the hIPSC line FSPS11B. Scale bar correspond to 100μm (b) QPCR analyses demonstrating the expression genes encoding early and mature biliary markers in CLCs derived from FSPS11B hIPSC-cells. Asterisks represent statistical significance (2-tailed t-test, ***P<0.001, ****P<0.0001), n=e, error bars represent SD. (c) GGT activity of CLCs derived from FSPS11B-hIPSCs cells. Center line, median; box, interquartile range; whiskers, range. Asterisks represent statistical significance (one-way ANOVA with Dunnett correction for multiple comparisons, ****P<0.0001), n=3. The data shown are complementary to figures 1,2,5 and 6 demonstrating the characterization of CLC organoids.

Supplementary Figure 6 Expression of key markers during CLC differentiation.

Heatmap demonstrating the expression of selected genes in hIPSCs, hepatoblasts (HBs), CLCs and Common Bile Duct cholangiocytes (CBD). For each probe, standard scores (z-scores) indicate the differential expression measured in number of standard deviations from the average level across all the samples.

Supplementary Figure 7 CLCs express the ileal bile acid transporter (iSBT/ASBT) but not the bile salt export pump (BSEP) physiologically expressed in hepatocytes.

(a) QPCR analyses for the expression of ASBT and BSEP in CLCs and hIPSC-derived hepatocyte-like cells (HLCs). Center line, median; box, interquartile range; whiskers, range. Asterisks represent statistical significance (one-way ANOVA with Tuckey correction for multiple comparisons, ***P<0.001, ****P<0.0001), n=3. (b) IF analyses demonstrating the expression of ASBT on the luminal surface of a cystic organoid. Scale bars: 100μM, L: Lumen

Supplementary Figure 8 The Notch signaling pathway is activated during the hepatoblast (HB) to cholangiocyte progenitor (CP) transition.

IF analyses demonstrating nuclear localization of the cleaved Notch Intracellular Domain (NICD) in CPs.

Supplementary Figure 9 Validation of PLD-hIPSCs.

(a) Sanger sequencing of PLD-hIPSCs demonstrating the presence of 2 heterozygous mutations in the PKHD1 gene.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Tables 3–6, Supplementary Note and Supplementary Methods (PDF 2880 kb)

Supplementary Table 1

Raw gene expression data (CT values) corresponding to the QPCR analyses in Figures 1d, 5f and 6a (XLSX 27 kb)

Supplementary Table 2

Microarray gene expression data corresponding to the heat map in Figure 2e (XLSX 965 kb)

Supplementary Table 7

Statistical analysis information (test statistic, degrees of freedom, statistical significance for each test) (XLSX 20 kb)

Bile acid export assay: CLF export (MOV 1273 kb)

Bile acid export assay: FITC control (MOV 368 kb)

Increase in intracellular calcium in response to acetylcholine stimulation (MOV 1196 kb)

Increase in intracellular calcium in response to ATP (MOV 967 kb)

Increasing organoid size in response to secretin (MOV 2466 kb)

Decreasing organoid size in response to somatostatin (MOV 902 kb)

Decreasing organoid size in response to octreotide (MOV 2466 kb)

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Sampaziotis, F., Cardoso de Brito, M., Madrigal, P. et al. Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation. Nat Biotechnol 33, 845–852 (2015). https://doi.org/10.1038/nbt.3275

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