Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells

Authors

  • Noushin Dianat,

    1. INSERM, U972, Paul Brousse Hospital, Villejuif, France
    2. Université Paris Sud, UMR-S 972, Villejuif, France
    3. IFR 93, Bicêtre Hospital, Kremlin-Bicêtre, France
    4. DHU Hepatinov, Paul Brousse Hospital, Villejuif, France
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  • Hélène Dubois-Pot-Schneider,

    1. INSERM, UMR-S 991, Pontchaillou Hospital, Rennes, France
    2. University of Rennes 1, Rennes, France
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  • Clara Steichen,

    1. INSERM, U972, Paul Brousse Hospital, Villejuif, France
    2. Université Paris Sud, UMR-S 972, Villejuif, France
    3. IFR 93, Bicêtre Hospital, Kremlin-Bicêtre, France
    4. DHU Hepatinov, Paul Brousse Hospital, Villejuif, France
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  • Christophe Desterke,

    1. INSERM, U972, Paul Brousse Hospital, Villejuif, France
    2. Université Paris Sud, UMR-S 972, Villejuif, France
    3. DHU Hepatinov, Paul Brousse Hospital, Villejuif, France
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  • Philippe Leclerc,

    1. IFR 93, Bicêtre Hospital, Kremlin-Bicêtre, France
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  • Aurélien Raveux,

    1. INSERM, U972, Paul Brousse Hospital, Villejuif, France
    2. Université Paris Sud, UMR-S 972, Villejuif, France
    3. IFR 93, Bicêtre Hospital, Kremlin-Bicêtre, France
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  • Laurent Combettes,

    1. DHU Hepatinov, Paul Brousse Hospital, Villejuif, France
    2. INSERM UMR-S 757, UPS-Orsay, Orsay, France
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  • Anne Weber,

    1. INSERM, U972, Paul Brousse Hospital, Villejuif, France
    2. Université Paris Sud, UMR-S 972, Villejuif, France
    3. IFR 93, Bicêtre Hospital, Kremlin-Bicêtre, France
    4. DHU Hepatinov, Paul Brousse Hospital, Villejuif, France
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  • Anne Corlu,

    Corresponding author
    1. INSERM, UMR-S 991, Pontchaillou Hospital, Rennes, France
    2. University of Rennes 1, Rennes, France
    • Address reprint requests to: Anne Corlu, Ph.D., INSERM, UMR-S 991, Pontchaillou Hospital, Rennes F-35033, France. E-mail: anne.corlu@inserm.fr; or Anne Dubart-Kupperschmitt, M.D., INSERM, U972, Paul Brousse Hospital, Villejuif, F-94807, France. anne.dubart@inserm.fr; fax: +33 (0)1 47 26 03 19, +33 (0)2 99 54 01 37.

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    • Senior co-authors.

  • Anne Dubart-Kupperschmitt

    Corresponding author
    1. INSERM, U972, Paul Brousse Hospital, Villejuif, France
    2. Université Paris Sud, UMR-S 972, Villejuif, France
    3. IFR 93, Bicêtre Hospital, Kremlin-Bicêtre, France
    4. DHU Hepatinov, Paul Brousse Hospital, Villejuif, France
    • Address reprint requests to: Anne Corlu, Ph.D., INSERM, UMR-S 991, Pontchaillou Hospital, Rennes F-35033, France. E-mail: anne.corlu@inserm.fr; or Anne Dubart-Kupperschmitt, M.D., INSERM, U972, Paul Brousse Hospital, Villejuif, F-94807, France. anne.dubart@inserm.fr; fax: +33 (0)1 47 26 03 19, +33 (0)2 99 54 01 37.

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    • Senior co-authors.


  • Potential conflict of interest: Nothing to report.

  • N.D., H.D.P.S., and C.S. were supported by fellowships from Région Ile de France (DIM Stem Pôle), Contrat plan état région Bretagne (axe biothérapie) and AFM respectively. The work was supported by FP7-HEALTH.2011.1.4-2-278152 "InnovaLiv," ANR-2010-RFCS-004 "Liv-iPS," the FEDER (Fonds Européen de Développement Régional), the Contrat projets état région Bretagne and the Ligue Contre le Cancer-Comité d'Ile-et-Vilaine.

Abstract

Cholangiocytes are biliary epithelial cells, which, like hepatocytes, originate from hepatoblasts during embryonic development. In this study we investigated the potential of human embryonic stem cells (hESCs) to differentiate into cholangiocytes and we report a new approach, which drives differentiation of hESCs toward the cholangiocytic lineage using feeder-free and defined culture conditions. After differentiation into hepatic progenitors, hESCs were differentiated further into cholangiocytes using growth hormone, epidermal growth factor, interleukin-6, and then sodium taurocholate. These conditions also allowed us to generate cholangiocytes from HepaRG-derived hepatoblasts. hESC- and HepaRG-derived cholangiocyte-like cells expressed markers of cholangiocytes including cytokeratin 7 and osteopontin, and the transcription factors SOX9 and hepatocyte nuclear factor 6. The cells also displayed specific proteins important for cholangiocyte functions including cystic fibrosis transmembrane conductance regulator, secretin receptor, and nuclear receptors. They formed primary cilia and also responded to hormonal stimulation by increase of intracellular Ca2+. We demonstrated by integrative genomics that the expression of genes, which signed hESC- or HepaRG-cholangiocytes, separates hepatocytic lineage from cholangiocyte lineage. When grown in a 3D matrix, cholangiocytes developed epithelial/apicobasal polarity and formed functional cysts and biliary ducts. In addition, we showed that cholangiocyte-like cells could also be generated from human induced pluripotent stem cells, demonstrating the efficacy of our approach with stem/progenitor cells of diverse origins. Conclusion: We have developed a robust and efficient method for differentiating pluripotent stem cells into cholangiocyte-like cells, which display structural and functional similarities to bile duct cells in normal liver. These cells will be useful for the in vitro study of the molecular mechanisms of bile duct development and have important potential for therapeutic strategies, including bioengineered liver approaches. (Hepatology 2014;60:700–714)

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