Tabibian James H, Trussoni Christy E, O'Hara Steven P, Splinter Patrick L, Heimbach Julie K, LaRusso Nicholas F
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.
Division of Transplantation Surgery, Mayo Clinic, Rochester, MN, USA.
Lab Invest. 2014 Oct;94(10):1126-33. doi: 10.1038/labinvest.2014.94. Epub 2014 Jul 21.
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.
原发性硬化性胆管炎（PSC）是一种慢性特发性胆管病。胆管细胞（胆管上皮细胞）在PSC发病机制中的作用尚不清楚，仍是一个活跃的研究领域。在此，我们通过细胞、分子和下一代测序（NGS）方法，对分离出的PSC患者来源的胆管细胞的表型和信号特征进行了表征和鉴定。我们通过解剖、差异过滤和免疫磁珠分离从4期PSC患者肝脏外植体中分离出胆管细胞。我们将胆管细胞培养并评估：（i）胆管细胞、细胞黏附及炎症标志物；（ii）增殖率；（iii）跨上皮电阻（TEER）；（iv）细胞衰老；以及（v）通过NGS检测转录组图谱。我们使用两种成熟的正常人胆管细胞系（H69和NHC）作为对照。分离出的PSC细胞表达胆管细胞标志物（如细胞角蛋白7和19）和上皮细胞黏附标志物（EPCAM、ICAM），而肝细胞和成肌纤维细胞标志物（白蛋白、α-肌动蛋白）呈阴性。与正常胆管细胞相比，PSC细胞的增殖率较低（分别为4天和2天，P<0.01）。与正常胆管细胞相比，PSC细胞的最大TEER也较低（100 vs 145 Ωcm²，P<0.05）。与NHC和H69相比，白细胞介素-6（IL-6）和IL-8（蛋白质和mRNA）均升高（所有P<0.01）。与NHC相比，PSC胆管细胞中衰老相关β-半乳糖苷酶染色阳性的胆管细胞比例更高（48% vs 5%，P<0.01）。最后，NGS证实了分离出的PSC胆管细胞中胆管细胞标志物的表达，并扩展了我们关于促炎和衰老相关信号传导的研究结果。总之，我们已经证明可以从人PSC肝脏中分离出高纯度胆管细胞并进行原代培养。分离出的PSC胆管细胞表现出一种表型，这可能反映了它们在体内对疾病的影响，并可作为体外研究胆管病理生物学和鉴定PSC新治疗靶点的重要工具。
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