Cholangiocyte organoids can repair bile ducts after transplantation in the human liver

Abstract

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.

Fig. 1. Transcriptional profiling of primary cholangiocytes.

(A) Schematic representation of the methodology used for single cell RNA sequencing (scRNAseq). (B) UMAP plot (7295 primary cells, n=10 individuals) illustrating distinct primary cholangiocyte populations in different regions of the biliary tree. (C-D) Immunofluorescence images (C) and UMAP representation of normalized gene expression (D) of primary cholangiocytes illustrating differential expression of representative region markers. Scale bars: 50μm. (E) Heatmap of top 100 differentially expressed genes (DEGs) in pseudotime (Data S2) demonstrating a gradual transition in the transcriptional profile of cholangiocytes between different regions of the biliary tree. PRI, Primary; IHD, IntraHepatic Ducts; CBD, Common Bile Duct; GB, Gallbladder; P, Pangreas; D, Duodenum.

Fig. 2. Cholangiocyte Organoid (CO) identity is controlled by niche stimuli.

(A) UMAP (35,603 cells) of primary cholangiocytes and their corresponding organoids before and after gallbladder bile treatment, illustrating similarities between different region organoids and changes in their signature in response to bile. PRI, Primary; IHD, IntraHepatic Ducts; CBD, Common Bile Duct; GB, Gallbladder; ORG, Organoids; BTO, Bile-Treated Organoids. (B) Heatmap of top 100 Differentially Expressed Genes (DEGs) between primary regions, organoids and BTOs (Data S1–S2), illustrating that organoids lose regional differences and upregulate culture-related genes, but re-acquire gallbladder markers following bile treatment. (C-D) QPCR (C) (n=4 samples per group; center line, median; box, interquartile range (IQR); whiskers, range; housekeeping gene, HMBS; #P>0.05, **P<0.01, ***P<0.001, ****P<0.0001); and immunofluorescence (D) demonstrating upregulation of gallbladder markers and bile acid target genes following treatment with chenodeoxycholic acid (CDA), in the absence of the FXR inhibitor Z-GS. Z-GS, Z-guggulsterone. Scale bars, 50μm.

Fig. 3. Cholangiocyte organoids (COs) rescue cholangiopathy following transplantation and assume an identity corresponding to the site of engraftment.

(A) Experimental outline schematic. (B) Kaplan-Meier curve (Table S1: number of animals at risk) demonstrating animal rescue following gallbladder organoids injection; P=0.0018(**), log-rank test. (C) Magnetic Resonance Cholangiopancreatography (MRCP) demonstrating rescue of cholangiopathy following organoid injection. (D) Immunofluorescence demonstrating engraftment of Red Fluorescent Protein (RFP)-expressing gallbladder organoids in portal triads, with upregulation of intrahepatic (SOX4) markers. Scale bars; yellow, 50μm; white, 100μm. PV, portal vein.

Fig. 4. Cholangiocyte organoids (COs) engraft in a human liver receiving Normothermic Perfusion (NMP) and improve bile properties.

(A) Schematic representation of the technique for organoid injection and (B) photograph of the NMP circuit used. BD, Bile Duct; GB, Gallbladder; HA, Hepatic Artery; PV, Portal Vein; IVC, Inferior Vena Cava; L, Liver RFP, Red Fluorescent Protein; P, pump; O, oxygenator; PRC, Packed Red Cells. (C) Flow cytometry revealing absence of RFP cells in the perfusate. (D) Immunofluorescence revealing engraftment of RFP gallbladder organoids with upregulation of intrahepatic (SOX4) and loss of gallbladder (SOX17) markers. Scale bars, 50μm. (E) Organoid injection improves bile pH and choleresis. ***P<0.001. N=3 NMP livers. Each measurement is represented by a different data point, each organ is represented by a different symbol.