title: Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.
abstract: Primary sclerosing cholangitis (PSC) is a chronic, idiopathic cholangiopathy. The role of cholangiocytes (biliary epithelial cells) in PSC pathogenesis is unknown and remains an active area of research. Here, through cellular, molecular and next-generation sequencing (NGS) methods, we characterize and identify phenotypic and signaling features of isolated PSC patient-derived cholangiocytes. We isolated cholangiocytes from stage 4 PSC patient liver explants by dissection, differential filtration and immune-magnetic bead separation. We maintained cholangiocytes in culture and assessed for: (i) cholangiocyte, cell adhesion and inflammatory markers; (ii) proliferation rate; (iii) transepithelial electrical resistance (TEER); (iv) cellular senescence; and (v) transcriptomic profiles by NGS. We used two well-established normal human cholangiocyte cell lines (H69 and NHC) as controls. Isolated PSC cells expressed cholangiocyte (eg, cytokeratin 7 and 19) and epithelial cell adhesion markers (EPCAM, ICAM) and were negative for hepatocyte and myofibroblast markers (albumin, α-actin). Proliferation rate was lower for PSC compared with normal cholangiocytes (4 vs 2 days, respectively, P<0.01). Maximum TEER was also lower in PSC compared with normal cholangiocytes (100 vs 145 Ωcm(2), P<0.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all P<0.01). The proportion of cholangiocytes staining positive for senescence-associated β-galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, P<0.01). Finally, NGS confirmed cholangiocyte marker expression in isolated PSC cholangiocytes and extended our findings regarding pro-inflammatory and senescence-associated signaling. In conclusion, we have demonstrated that high-purity cholangiocytes can be isolated from human PSC liver and grown in primary culture. Isolated PSC cholangiocytes exhibit a phenotype that may reflect their in vivo contribution to disease and serve as a vital tool for in vitro investigation of biliary pathobiology and identification of new therapeutic targets in PSC.
authors.affiliation: 1] Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN, USA [2] Center for Cell Signaling in Gastroenterology, Mayo Clinic College of Medicine, Rochester, MN, USA.
authors.forename: James H
authors.identifier: 0000000191041702
authors.initials: JH
authors.lastname: Tabibian
authors.affiliation: 1] Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN, USA [2] Center for Cell Signaling in Gastroenterology, Mayo Clinic College of Medicine, Rochester, MN, USA.
authors.forename: Christy E
authors.initials: CE
authors.lastname: Trussoni
authors.affiliation: 1] Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN, USA [2] Center for Cell Signaling in Gastroenterology, Mayo Clinic College of Medicine, Rochester, MN, USA.
authors.forename: Steven P
authors.initials: SP
authors.lastname: O'Hara
authors.affiliation: 1] Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN, USA [2] Center for Cell Signaling in Gastroenterology, Mayo Clinic College of Medicine, Rochester, MN, USA.
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Splinter
authors.affiliation: Division of Transplantation Surgery, Mayo Clinic, Rochester, MN, USA.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Heimbach
authors.affiliation: 1] Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN, USA [2] Center for Cell Signaling in Gastroenterology, Mayo Clinic College of Medicine, Rochester, MN, USA.
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: LaRusso
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D016922:Cellular Senescence; D015209:Cholangitis, Sclerosing / Q000473:pathology*; D005260:Female; D006801:Humans; D007365:Intercellular Junctions / Q000473:pathology; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged
chemical_list: D015415:Biomarkers