Isolation of rat and human hepatic cholangiocytes using a peptide derived from a conserved domain of enterobacteria BamA/TamA-like proteins

Abstract

Using the phage display technology, we identified a novel peptide, P11Chol, which preferentially binds to both human and rat cholangiocytes. Peptide P11Chol alignment with protein databases evidenced strong similarities with a highly conserved peptide motif from BamA/TamA-like outer membrane proteins expressed in enterobacteriaceae belonging to Pseudomonadota phylum including Photorhabdus, Providencia, Acinetobacter, Salmonella enterica and Helicobacter pylori species. In addition, we showed that Providencia stuartii bacteria were able to bind to cholangiocytes-like HepaRG cells in vitro and that P11Chol modulated this interaction suggesting the possible involvement of BamA/TamA-like outer membrane proteins in cell adhesion and/or internalization of Providencia stuartii bacteria. Using fluorescent P11Chol peptide, we next developed a flow cytometry procedure to detect and isolate rat and human liver epithelial cells from hepatic cell suspension obtained after collagenase dissociation of liver parenchyma. Three distinct P11Chol-positive rat liver epithelial cell lines (RLEC) were established, which produced functional cholangiocytes capable to form cyst-like structures in vitro and to maintain expression of specific functions in hepatocytes in coculture. The characterization of these three RLEC lines evidenced functional differences that support the concept of small and large cholangiocytes exhibiting different functional phenotypes within the intrahepatic bile tree.