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.

Figure 1. Isolated PSC cholangiocytes express biliary epithelial markers at comparable levels to normal cholangiocytes

A) Isolated PSC cholangiocytes express typical biliary epithelial cell markers CK7, CK19, and GGT in addition to aquaporin and cell adhesion markers that are also expressed in normal cholangiocyte cell lines (semi-quantitative PCR). The exception to the similar expression profile between PSC and normal cholangiocytes is CFTR (known to be upregulated in response to liver injury), which is present in PSC cholangiocytes but absent in H69 or NHC cell lines. B) Isolated PSC cholangiocytes demonstrate CK19 (cholangiocyte marker) positivity as indicated by green immunofluorescence staining. C) Over 99% of cholangiocytes isolated from PSC liver explants (as well as established normal cholangiocyte cell lines) express the cholangiocyte marker CK19, indicating a highly purified population of PSC cholangiocytes.

Figure 2. Isolated PSC cholangiocytes exhibit abnormal morphological features, diminished tight junction protein expression, and impaired barrier integrity

A) Confluent normal human cholangiocyte (NHC) cell line form a tight monolayer after 7 days in culture, as compared to isolated PSC cholangiocytes (B), which are slow to become confluent after 7 days in culture and appear irregular and larger in size as compared to NHCs (both 20x magnification). C) Cultured NHCs form tight junctions with neighboring cells, as indicated by expression of tight junction marker, Z01 (green), whereas cultured PSC cholangiocytes display fewer than normal tight junctions between cells as indicated by decreased ZO1 expression. D) Transepithelial electrical resistance (TEER) was measured in normal cholangiocytes, H69 and NHC, and isolated PSC cholangiocytes to determine formation and integrity of tight junctions between cells. Isolated PSC cholangiocytes exhibit significantly lower TEER readings compared to both H69 and NHC cell lines from days 2 to 5 (p<0.01), indicating impaired tight junctions in the former.

Figure 3. Isolated PSC cholangiocytes demonstrate features of cellular senescence

NHCs (A) and PSC cholangiocytes (B) were stained (blue) for SA-β-gal expression, a marker of cellular senescence. C) Quantitation of blue-stained cells reveals a significantly higher proportion of SA-β-gal positive cholangiocytes in PSC compared to normal (p<0.001). D) Cell counting demonstrates that isolated PSC cholangiocytes have a slower growth rate as compared to NHC and H69 cells. E) Doubling times of isolated PSC cholangiocytes compared to NHC and H69 cells.

Figure 4. Isolated PSC cholangiocytes express increased levels of inflammatory markers

A) Unstimulated PSC cells secrete significantly higher amounts of the cytokine, IL-6, (up to a 9-fold difference) compared to unstimulated H69 and NHC cells as determined by ELISA analysis on cell culture supernate. B) PSC cells also have a significantly higher basal secretion of the chemokine, IL-8, as compared to H69 (a 10-fold difference) and NHC cells (a 5-fold difference). C) Semi-quantitative PCR demonstrates an increase in IL-6 and IL-8 mRNA expression in the PSC cells over H69 and NHC cells. D) Quantitative PCR confirms message increase by showing a 20-fold increase in IL-6 mRNA expression and a 45-fold increase in IL-8 mRNA expression in PSC versus normal human cholangiocytes in a basal state.

Figure 5. Next-generation sequencing confirms cholangiocyte marker expression and extends findings regarding the pro-fibroinflammatory and senescence-associated features of isolated PSC cholangiocytes

We examined transcriptomic profiles in PSC cholangiocytes derived from three separate livers as well as H69 and NHC controls by high-throughput NGS. A) Cholangiocyte cytoskeletal markers (CK7, β-actin) are abundantly expressed in both PSC cholangiocytes and normal cholangiocyte cell lines, while non-cholangiocyte markers (α-actin, albumin) are minimally expressed, consistent with an enriched cholangiocyte population. B) Corroborating the immunofluorescence, PCR, and ELISA findings, NGS-based mRNA expression of multiple inflammatory cytokines and chemokines is markedly increased in isolated PSC cholangiocytes compared to controls. C) Markers of senescence (p21) and senescence-associated secretory phenotype (PAI-1, IGFBP5, and MMP3) are consistently increased in isoated PSC cholangiocytes. Key: PSC1, PSC2, and PSC3 correspond to a 46 year-old male, 58 year-old female, and 57 year-old male patient with PSC, respectively, as described in the methods section.

Figure 5. Next-generation sequencing confirms cholangiocyte marker expression and extends findings regarding the pro-fibroinflammatory and senescence-associated features of isolated PSC cholangiocytes

We examined transcriptomic profiles in PSC cholangiocytes derived from three separate livers as well as H69 and NHC controls by high-throughput NGS. A) Cholangiocyte cytoskeletal markers (CK7, β-actin) are abundantly expressed in both PSC cholangiocytes and normal cholangiocyte cell lines, while non-cholangiocyte markers (α-actin, albumin) are minimally expressed, consistent with an enriched cholangiocyte population. B) Corroborating the immunofluorescence, PCR, and ELISA findings, NGS-based mRNA expression of multiple inflammatory cytokines and chemokines is markedly increased in isolated PSC cholangiocytes compared to controls. C) Markers of senescence (p21) and senescence-associated secretory phenotype (PAI-1, IGFBP5, and MMP3) are consistently increased in isoated PSC cholangiocytes. Key: PSC1, PSC2, and PSC3 correspond to a 46 year-old male, 58 year-old female, and 57 year-old male patient with PSC, respectively, as described in the methods section.

Figure 5. Next-generation sequencing confirms cholangiocyte marker expression and extends findings regarding the pro-fibroinflammatory and senescence-associated features of isolated PSC cholangiocytes

We examined transcriptomic profiles in PSC cholangiocytes derived from three separate livers as well as H69 and NHC controls by high-throughput NGS. A) Cholangiocyte cytoskeletal markers (CK7, β-actin) are abundantly expressed in both PSC cholangiocytes and normal cholangiocyte cell lines, while non-cholangiocyte markers (α-actin, albumin) are minimally expressed, consistent with an enriched cholangiocyte population. B) Corroborating the immunofluorescence, PCR, and ELISA findings, NGS-based mRNA expression of multiple inflammatory cytokines and chemokines is markedly increased in isolated PSC cholangiocytes compared to controls. C) Markers of senescence (p21) and senescence-associated secretory phenotype (PAI-1, IGFBP5, and MMP3) are consistently increased in isoated PSC cholangiocytes. Key: PSC1, PSC2, and PSC3 correspond to a 46 year-old male, 58 year-old female, and 57 year-old male patient with PSC, respectively, as described in the methods section.