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Characterization of Cultured Cholangiocytes Isolated from Livers Laboratory Investigation (2014) 94, 1126–1133 & 2014 USCAP, Inc All rights reserved 0023-6837/14 Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis James H Tabibian1,2,4, Christy E Trussoni1,2,4, Steven P O’Hara1,2, Patrick L Splinter1,2, Julie K Heimbach3 and Nicholas F LaRusso1,2 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, a-actin). Proliferation rate was lower for PSC compared with normal cholangiocytes (4 vs 2 days, respectively, Po0.01). Maximum TEER was also lower in PSC compared with normal cholangiocytes (100 vs 145 Ocm2, Po0.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all Po0.01). The proportion of cholangiocytes staining positive for senescence-associated b-galactosidase was higher in PSC cholangio- cytes compared with NHCs (48% vs 5%, Po0.01). Finally, NGS confirmed cholangiocyte marker expression in isolated PSC cholangiocytes and extended our findings regarding pro-inflammatory and senescence-associated signaling. In conclu- sion, 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. Laboratory Investigation (2014) 94, 1126–1133; doi:10.1038/labinvest.2014.94; published online 21 July 2014 Primary sclerosing cholangitis (PSC) is a chronic, complex, multifocal strictures) and are useful in studying disease pro- idiopathic disease of the bile ducts (ie, cholangiopathy) gression;9–11 however, no single model reflects the multiple characterized by biliary inflammation, periductal fibrosis and and complex features of PSC or the putative mechanisms cholestasis.1–3 Patients with PSC are at increased risk of involved in the etiopathogenesis of human PSC.12–14 end-stage cirrhosis as well as hepatobiliary and colonic Therefore, establishment of primary human cholangiocyte neoplasia.4,5 To date, no effective therapeutic strategies exist cell cultures from PSC livers would represent an essential tool for PSC aside from liver transplantation, which itself is asso- to facilitate mechanistic, in vitro investigations of the PSC ciated with numerous challenges, including recurrent PSC cholangiocyte phenotype and advance current understanding and/or cholangiocarcinoma post-transplantation.2,6–8 Current of PSC.15 animal models of PSC exhibit some of the key features of Although representing only 3% of the total liver cell the human disease (eg, ‘onionskin’ periductal fibrosis and population, previous reports have described isolation and 1Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN, USA; 2Center for Cell Signaling in Gastroenterology, Mayo Clinic College of Medicine, Rochester, MN, USA and 3Division of Transplantation Surgery, Mayo Clinic, Rochester, MN, USA Correspondence: Dr NF LaRusso, MD, Division of Gastroenterology and Hepatology; Center for Cell Signaling in Gastroenterology, Mayo Clinic College of Medicine, 200 First Street SW, 1701 Guggneheim Building, Rochester, MN 55905, USA. E-mail: [email protected] 4Co-first authors. Received 12 March 2014; revised 3 June 2014; accepted 10 June 2014 1126 Laboratory Investigation | Volume 94 October 2014 | www.laboratoryinvestigation.org Isolated, high-purity PSC cholangiocytes JH Tabibian et al culture of (normal) biliary epithelial cells (ie, cholangiocytes). described. PSC cells were grown in media containing DMEM/ Isolating and culturing cholangiocytes from PSC liver, F12 (Sigma-Aldrich, St Louis, MO, USA) supplemented with however, poses considerable challenges given the cholangio- fetal bovine serum (CellGro, Manassas, VA, USA), penicillin/ cyte injury, periductal fibrosis and ductopenia inherent to the streptomycin, vitamin solution, MEM solution, CD lipid disease, and to date there are no validated methods to do concentrate, L-glutamine, soybean trypsin inhibitor, insulin/ so.1,2,16–18 Our objectives in this study were to: (i) establish transferrin/selenium-A, bovine pituitary extract, epidermal 0 methods for high-yield isolation of cholangiocytes from growth factor, 3,3 5-triiodo-L-thyronine, dexamethasone and explanted liver from patients with PSC using serial proteinase forskolin. and hyaluronidase digestion, filtration and immunomagnetic bead purification; (ii) culture and extensively characterize the Polymerase Chain Reaction (PCR) isolated cells to confirm high expression of cholangiocyte- RNA was isolated from primary PSC cells using TRIzol specific markers; and (iii) assess features of PSC and cholan- reagent (Life Technologies), and cDNA was synthesized giocyte injury as previously defined by our laboratory and from the RNA using First Strand cDNA Synthesis kit (Life others, including cellular senescence and the senescence- Technologies). PCR was performed on PSC cDNA for cho- 16,19–21 associated secretory phenotype (SASP). Our methodology langiocyte markers (CK7, CK19, GGT, AQP1 and CFTR), cell allows high-purity (99%) isolation of PSC cholangiocytes, adhesion molecules (EPCAM, ICAM and NCAM, inflam- which appear to exhibit characteristics reflective of PSC matory markers (interleukin (IL)-6 and 8), and non- pathobiology, including the recently appreciated pheno- cholangiocyte markers (albumin, a-smooth muscle actin). menon of cholangiocyte senescent in ex vivo PSC liver Primer sequences used are provided in Supplementary tissue, and that the establishment of these PSC primary Table 1. cholangiocyte isolates will be a valuable tool for studying the pathogenesis of PSC. Immunofluorescence MATERIALS AND METHODS H69 and NHC cells were plated on plastic chamber slides and Cell Isolation PSC cells were plated on collagen-coated chamber slides (BD Cells were isolated from liver explant tissue from a 46-year- Biosciences) and allowed to grow to confluence. Cells were old male patient with stage 4 PSC without cholangiocarci- fixed in a 2% paraformaldehyde solution containing 0.1 M noma through a series of digestion, filtration and bead PIPES, 1 mM EGTA and 3 mM MgSO4 for 10 min at room isolations steps. First, the explant tissue was cut into small, temperature. Cells were washed twice in 1X phosphate-buf- easily digestible pieces using sterile razor blades and then fered saline (PBS) and permeabilized with 0.1% Triton-x 100 incubated in Dulbecco’s modified Eagle’s medium (DMEM) in 1X PBS for 10 min at room temperature. Cells were then solution containing fetal bovine serum, penicillin/strepto- blocked in a 5% goat serum and 1% BSA solution for 1 h at 1 mycin, bovine serum albumin, collagenase and DNase for room temperature, washed and incubated overnight at 4 C 45 min in a shaking water bath at 37 1C. The digested tissue with primary antibodies against CK19 (Sigma) and zonula was filtered through a 100 mM cell strainer with a subsequent occludens 1 (ZO1) (Invitrogen, Grand Island, NY, USA). filtration through a 40 mM cell strainer. Cells contained in the Cells were then washed in PBS plus 0.1% Tween-20 and 40 mM cell strainer were washed with DMEM, and subjected incubated with corresponding fluorophore-conjugated goat to further digestion with a DMEM solution containing secondary antibodies (Invitrogen) for 1 h at room tempera- hyaluronidase for 30 min at 37 1C. The resulting hepatic ture. Cells were washed as before with a final wash in 1X PBS digestant was filtered as described above, and the isolated alone. Coverslips were mounted on the cells using Prolong cells were plated on collagen-coated flasks (BD Biosciences, Gold Antifade Reagent with DAPI (Life Technologies). San Jose, CA, USA) and allowed to grow to confluence. After Immunofluorescence was visualized using laser-scanning reaching confluence, cholangiocyte cells were bead isolated confocal microscopy (Zeiss, Oberkochen, DE, USA). using the Epithelial Enrich magnetic bead isolation kit following the manufacturer’s instructions (Life Technologies, Enzyme-Linked Immunosorbant Assay (ELISA) Grand Island, NY, USA). Of note, cells were also isolated and Basal IL-6 and IL-8 protein levels were measured from PSC, purified using the same techniques from liver explant tissue H69 and NHC cell media supernatants using IL-6 and IL-8 from a 58-year-old female and a 57-year-old male patient, Direct ELISA kits (Invitrogen) following the
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