title: Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells.
abstract: The morphogenesis and movement of bile canaliculi (BC) are not well understood. This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful. We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC-like structures developed between rising/piled-up cells. We investigated how BC-like structures developed with maturation of SHs and whether the structures were functionally active as BC. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Immunocytochemistry and immunoblotting for BC proteins, such as ectoATPase, 5'-nucleotidase, dipeptidylpeptidase IV, and multidrug-resistance associated protein 2, were examined and time-lapse microscopy was used for the observation of BC contractions. Secretion of bilirubin into the reconstructed BC was also observed. The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins were localized in the intercellular space that coincided with BC-like structures reconstructed between rising/piled-up cells. Tight junction-associated protein ZO-1 was also expressed along the BC-like structures. Bilirubin added to the medium were secreted into BC-like structure and accumulated without leakage. Time-lapse microscopy showed continuous contractions of reconstructed BC. In conclusion, BC-like structures reconstructed by SHs may be functional with membrane polarity, secretory ability, and motility. These results show that this culture system may suitable for investigating the mechanism of the formation of BC and their functions.
authors.affiliation: Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Sudo
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Ikeda
authors.forename: Shinichi
authors.initials: S
authors.lastname: Sugimoto
authors.forename: Keisuke
authors.initials: K
authors.lastname: Harada
authors.forename: Koichi
authors.initials: K
authors.lastname: Hirata
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tanishita
authors.forename: Yohichi
authors.initials: Y
authors.lastname: Mochizuki
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Mitaka
mesh_terms: D000818:Animals; D001648:Bile Canaliculi / Q000502:physiology* / Q000648:ultrastructure*; D001663:Bilirubin / Q000378:metabolism; D015153:Blotting, Western; D002450:Cell Communication / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology*; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000096:biosynthesis; D016253:Microscopy, Immunoelectron; D009940:Organoids / Q000502:physiology* / Q000648:ultrastructure*; D010750:Phosphoproteins / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology; D019108:Tight Junctions / Q000648:ultrastructure; D062826:Zonula Occludens-1 Protein
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D001663:Bilirubin