Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis

James H. Tabibian, Christy E. Trussoni, Steven P. O'Hara, Patrick L. Splinter, Julie K. Heimbach, Nicholas F La Russo

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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 nextgeneration 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 145ωcm2, Po<.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all Po<.01). The proportion of cholangiocytes staining positive for senescence-associated b-galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, Po<.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.

Original languageEnglish (US)
Pages (from-to)1126-1133
Number of pages8
JournalLaboratory Investigation
Volume94
Issue number10
DOIs
StatePublished - Jan 1 2014

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Sclerosing Cholangitis
Liver
Electric Impedance
Cell Adhesion
Epithelial Cells
Galactosidases
Keratin-7
Keratin-19
Myofibroblasts
Cell Aging
Interleukin-8
Dissection
Actins
Hepatocytes
Albumins
Interleukin-6

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis. / Tabibian, James H.; Trussoni, Christy E.; O'Hara, Steven P.; Splinter, Patrick L.; Heimbach, Julie K.; La Russo, Nicholas F.

In: Laboratory Investigation, Vol. 94, No. 10, 01.01.2014, p. 1126-1133.

Research output: Contribution to journalArticle

Tabibian, James H. ; Trussoni, Christy E. ; O'Hara, Steven P. ; Splinter, Patrick L. ; Heimbach, Julie K. ; La Russo, Nicholas F. / Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis. In: Laboratory Investigation. 2014 ; Vol. 94, No. 10. pp. 1126-1133.
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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 nextgeneration 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 145ωcm2, Po<.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all Po<.01). The proportion of cholangiocytes staining positive for senescence-associated b-galactosidase was higher in PSC cholangiocytes compared with NHCs (48{\%} vs 5{\%}, Po<.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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AU - Splinter, Patrick L.

AU - Heimbach, Julie K.

AU - La Russo, Nicholas F

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N2 - 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 nextgeneration 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 145ωcm2, Po<.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all Po<.01). The proportion of cholangiocytes staining positive for senescence-associated b-galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, Po<.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.

AB - 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 nextgeneration 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 145ωcm2, Po<.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all Po<.01). The proportion of cholangiocytes staining positive for senescence-associated b-galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, Po<.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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