The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct–like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.
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The authors would like to thank J. Skepper, L. Carter and the University of Cambridge Advanced Imaging Centre for their help with electron microscopy; E. Farnell and the University of Cambridge, Cambridge Genomic Services for their help with microarray data processing and analysis; A. Petrunkina and the NIHR Cambridge BRC Cell Phenotyping Hub for their help with cell sorting; K. Burling and the MRC MDU Mouse Biochemistry Laboratory (MRC_MC_UU_12012/5) for processing mouse serum samples; and R. El-Khairi for her help with IF images, R. Grandy for his help with providing relevant references, the Cambridge Biorepository for Translational Medicine for the provision of human tissue used in the study; D. Trono (Ecole Polytechnique Federale de Lausanne) for the gift of the plasmids used for the generation of GFP-expressing ECOs and B. McLeod for IT support. The monoclonal antibody TROMA-III, developed by R. Kemler, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa.
This work was funded by ERC starting grant Relieve IMDs (281335; L.V., N.R.F.H.), the Cambridge Hospitals National Institute for Health Research Biomedical Research Centre (L.V., N.R.F.H., S. Sinha., F.S.), the Evelyn Trust (N.H.) and the EU FP7 grant TissuGEN (M.C.D.B.) and was supported in part by the Intramural Research Program of the NIH/NIAID (R.L.G., C.A.R.). F.S. has been supported by an Addenbrooke's Charitable Trust Clinical Research Training Fellowship and a joint MRC–Sparks Clinical Research Training Fellowship. (MR/L016761/1) A.W.J. and A.E.M. acknowledge support from EPSRC (EP/L504920/1) and an Engineering for Clinical Practice Grant from the Department of Engineering, University of Cambridge. J.B. was supported by a BHF Studentship (Grant FS/13/65/30441).
L. Vallier is a founder and shareholder of DefiniGEN. The remaining authors have nothing to disclose.
Supplementary Figures 1–14 and Supplementary Tables 1 and 3–5 (PDF 3856 kb)
Microarray gene expression data corresponding to the heat map in Figure 1d. (XLSX 817 kb)
Magnetic resonance cholangio-pancreatography (MRCP; sagital plane) of an extrahepatic biliary injury mouse 104 d after biliary reconstruction with an ECO-populated scaffold. (MP4 596 kb)
T1-weighed magnetic resonance imaging (coronal plane) of an extrahepatic biliary injury mouse 104 d after biliary reconstruction with an ECO-populated scaffold. (MP4 2803 kb)
T2-weighed magnetic resonance imaging (coronal plane) of an immunocompromised mouse 1 month after biliary reconstruction with an ECO-populated scaffold. (MP4 1128 kb)
Time-lapse images demonstrating the generation of a fully grown organoid from a single cell. (AVI 109 kb)
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Sampaziotis, F., Justin, A., Tysoe, O. et al. Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids. Nat Med 23, 954–963 (2017). https://doi.org/10.1038/nm.4360
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