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Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells

Abstract

The difficulty in isolating and propagating functional primary cholangiocytes is a major limitation in the study of biliary disorders and the testing of novel therapeutic agents. To overcome this problem, we have developed a platform for the differentiation of human pluripotent stem cells (hPSCs) into functional cholangiocyte-like cells (CLCs). We have previously reported that our 26-d protocol closely recapitulates key stages of biliary development, starting with the differentiation of hPSCs into endoderm and subsequently into foregut progenitor (FP) cells, followed by the generation of hepatoblasts (HBs), cholangiocyte progenitors (CPs) expressing early biliary markers and mature CLCs displaying cholangiocyte functionality. Compared with alternative protocols for biliary differentiation of hPSCs, our system does not require coculture with other cell types and relies on chemically defined conditions up to and including the generation of CPs. A complex extracellular matrix is used for the maturation of CLCs; therefore, experience in hPSC culture and 3D organoid systems may be necessary for optimal results. Finally, the capacity of our platform for generating large amounts of disease-specific functional cholangiocytes will have broad applications for cholangiopathies, in disease modeling and for screening of therapeutic compounds.

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Figure 1: Generation of cholangiocyte-like cells (CLCs) from human pluripotent stem cells (hPSCs).
Figure 2: Derivation of CLCs from embryonic stem (ES) cells.
Figure 3: Immunofluorescence analyses demonstrating the expression of characteristic markers at key stages of CLC differentiation.
Figure 4: Flow cytometry analyses demonstrating the expression of characteristic markers at key stages of CLC differentiation.
Figure 5: Functional properties of CLC organoids.

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Acknowledgements

This work was funded by ERC starting grant Relieve IMDs (L.V., N.R.F.H.), the Cambridge Hospitals National Institute for Health Research Biomedical Research Center (L.V., N.R.F.H., F.S.), the Evelyn trust (N.R.F.H.) and the EU Fp7 grant TissuGEN (M.C.d.B.). F.S. was supported by an Addenbrooke's Charitable Trust Clinical Research Training Fellowship and a joint MRC-Sparks Clinical Research Training Fellowship.

The authors thank the Cambridge BRC hIPSCs core facility for the derivation of the Cystic Fibrosis hIPSC line, P. Materek (Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge Stem Cell Institute, Anne McLaren Laboratory, Department of Surgery, University of Cambridge) for the provision of cells used as negative controls, D. Ortmann for his input into the design of the figures and P.-A. Tsagkaraki for her help with the generation of the manuscript figures and statistical analyses.

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Contributions

F.S.: design and concept of study, execution of experiments and data acquisition, development of protocols and validation, collection of data, production of figures, manuscript writing and editing, and final approval of the manuscript. M.C.d.B., I.G. and A.B.: execution of experiments, collection and provision of data. N.R.F.H.: design and concept of study, editing and final approval of the manuscript. L.V.: design and concept of the study, editing and final approval of the manuscript. M.C.d.B., I.G. and A.B. contributed equally to this work. L.V. and N.R.F.H. jointly directed this work, contributing equally.

Corresponding author

Correspondence to Ludovic Vallier.

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Competing interests

L.V. is a founder and shareholder of DefiniGEN. The remaining authors have nothing to disclose.

Integrated supplementary information

Supplementary Figure 1 Morphology of CLC organoids

CLC organoids exhibit a typical cystic or branching tubular morphology. The black arrow indicates a tubular organoid, while the white arrow indicates a branching point. Scale bars: 100μm

Supplementary Figure 2 Gating strategy used for the flow cytometry analyses demonstrated in Figure 4.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 412 kb)

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Sampaziotis, F., de Brito, M., Geti, I. et al. Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells. Nat Protoc 12, 814–827 (2017). https://doi.org/10.1038/nprot.2017.011

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