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Directed differentiation of cholangiocytes from human pluripotent stem cells

Abstract

Although bile duct disorders are well-recognized causes of liver disease, the molecular and cellular events leading to biliary dysfunction are poorly understood. To enable modeling and drug discovery for biliary disease, we describe a protocol that achieves efficient differentiation of biliary epithelial cells (cholangiocytes) from human pluripotent stem cells (hPSCs) through delivery of developmentally relevant cues, including NOTCH signaling. Using three-dimensional culture, the protocol yields cystic and/or ductal structures that express mature biliary markers, including apical sodium-dependent bile acid transporter, secretin receptor, cilia and cystic fibrosis transmembrane conductance regulator (CFTR). We demonstrate that hPSC-derived cholangiocytes possess epithelial functions, including rhodamine efflux and CFTR-mediated fluid secretion. Furthermore, we show that functionally impaired hPSC-derived cholangiocytes from cystic fibrosis patients are rescued by CFTR correctors. These findings demonstrate that mature cholangiocytes can be differentiated from hPSCs and used for studies of biliary development and disease.

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Figure 1: Characterization of the hepatoblast stage of development in hPSC differentiation cultures.
Figure 2: NOTCH signaling promotes cholangiocyte differentiation from hPSC-derived hepatoblasts.
Figure 3: 3D culture promotes cholangiocyte maturation.
Figure 4: Gene expression profile of hPSCs-derived cholangiocytes compared to sorted cholangiocytes.
Figure 5: Function of hPSC-derived cholangiocytes in vitro.
Figure 6: Generation of cholangiocytes from CF patient iPSCs.
Figure 7: Functional analysis of cholangiocytes from CF patient iPSCs.

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Acknowledgements

We would like to thank J. Rossant, J. Ellis and A.P. Wong (Hospital for Sick Children, Toronto, ON, Canada) for providing CF patient iPSCs (C1 and GM00997). We thank members of the G.K. and A.G. laboratories for discussion and feedback on the manuscript. We would like to thank F. Xu (Advanced Optical Microscopy Facility, University Health Network) for technical assistance with the time-lapse video. In addition, we would also like to thank O. Adeyi and the members of Department Pathology, University Health Network for technical assistance with immunohistochemistry. H9 hESC was obtained from The Wicell Research Institute (Madison, WI, USA), and MSC-iPSC1 cells were obtained from G.Q. Daley (Harvard Stem Cell Institute). This work was supported by funding from the McEwen Centre for Regenerative Medicine and the Canadian Institutes of Health Research MOP133620, (G.K.), the University Health Network Multi-Organ Transplant Program Academic Enrichment Fund (sponsored by Astellas Pharma Canada), Alagille Syndrome Alliance, SickKids Research Institute, Rare Disease Foundation and the Childhood Liver Disease Research and Education Network, U01 DK062453 (Sokol) from the National Institute of Diabetes, Digestive and Kidney Diseases (B.M.K. and A.G.) and the Canadian Institutes of Health Research (MOP:97954 and GPG:102171 to C.E.B.).

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S.O., M.O., G.K., B.M.K. and A.G. conceptualized the study. S.O. and M.O. led the experimental design and development of the differentiation protocol with input from all authors. S.O. and M.O. performed the hPSC differentiation and characterization of hPSC-derived cholangiocytes. M.O., C.E.B., S.A. and S.C. developed the cyst swelling assay for hPSC-derived cholangiocytes. B.L. and M.G. developed and used the ductal antibody for isolation of primary cholangiocytes from human liver. All authors analyzed and interpreted the data. M.O., S.O., G.K., B.M.K. and A.G. prepared the manuscript with contributions and critical revision from all authors.

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Correspondence to Shinichiro Ogawa or Anand Ghanekar.

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The authors declare no competing financial interests.

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Ogawa, M., Ogawa, S., Bear, C. et al. Directed differentiation of cholangiocytes from human pluripotent stem cells. Nat Biotechnol 33, 853–861 (2015). https://doi.org/10.1038/nbt.3294

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