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Characterization of peribiliary gland-constituting cells based on expression of Trop2 in mouse biliary tract

松井, 理司 東京大学 DOI:10.15083/0002001451

2021.09.08

概要

The bile duct, a tubular epithelial tissue, plays an important role in the drainage of bile from the liver into the small intestine. Based on histology and embryology, the bile duct is classified into the intrahepatic bile duct (IHBD) and the extrahepatic bile duct (EHBD). While IHBD forms an intricate tree-like network in the liver parenchyma, EHBD forms luminal structure that links IHBD to the duodenum. EHBD has many accessory glands, namely “peribiliary glands (PBGs)”. PBG is composed of heterogeneous cell populations such as mucus and pancreatic enzyme-producing epithelial cells, while it is known to constitute niches for multi-potential stem/progenitor cells, called “biliary tree stem/progenitor cells (BTSCs)”, in human EHBD. BTSC shows a similar gene expression profile to liver stem/progenitor cell (LPC) and pancreatic progenitor cell, having a potential for differentiating into hepatocytes, mature biliary epithelial cells (BECs) and pancreatic islets. However, there is no applicable method to isolate PBG-constituting cells from the EHBD.

Therefore, the role and nature of PBGs in the mouse EHBD remains unclear. The objective of this study is to establish the method for isolating and characterizing PBG-constituting cells in the mouse EHBD.

In the present study, I found that trophoblast cell surface protein 2 (Trop2) was expressed in the luminal epithelium of mouse EHBD exclusively, but not in the PBG. Based on the differential expression profile of Trop2, the lumen-forming biliary epithelial cells (LBECs) and PBG-constituting biliary epithelial cells (PBECs) were isolated for further characterization by gene expression analysis, immunostaining, and assays of colony and organoid formation.

Gene expression profiling revealed that the isolated mouse PBECs expressed several genes characteristic of human PBGs, fetal pancreatic progenitor and intestinal tuft cells. In the colony formation assay, PBECs showed significantly higher colony formation capacity than LBECs. The expanded PBECs showed up-regulation of Trop2 expression and down-regulation of human PBG-related genes in the 2D culture condition. In the 3D organoid formation assay, PBECs gave rise to a cysts structure with epithelial polarity, showing the gene expression patterns similar to LBECs.

Finally, I examined the expression pattern of Trop2 during EHBD regeneration after bile duct ligation (BDL), a severe cholestasis model. After BDL, the luminal epithelium was severely injured and damaged LBECs were peeled off from the lumen. On the other hand, PBECs proliferated and re-expressed Trop2 in PBGs upon EHBD injury. Next, I compared the colony formation capacity between Trop2+ and Trop2- BECs after BDL and showed that the colony formation capacity of Trop2+ BECs was dramatically increased after EHBD injury.

Taking these in vitro and in vivo data together, PBGs contain progenitor-like cells with high capacity for proliferation, which supply new LBECs during biliary regeneration. Thus, Trop2 is a useful marker to investigate the pathophysiological roles and characteristics of PBGs in biliary diseases.

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