Journal
LABORATORY INVESTIGATION
Volume 94, Issue 10, Pages 1126-1133Publisher
ELSEVIER SCIENCE INC
DOI: 10.1038/labinvest.2014.94
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Funding
- National Institutes of Health [AI089713, DK57993, DK007198]
- PSC Partners Seeking a Cure
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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, alpha-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 Omega cm(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 beta-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.
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