Induced Pluripotent Stem Cells From Subjects With Primary Sclerosing Cholangitis Develop a Senescence Phenotype Following Biliary Differentiation.

Primary sclerosing cholangitis (PSC) is a chronic fibroinflammatory disease of the biliary tract characterized by cellular senescence and periportal fibrogenesis. Specific disease features that are cell intrinsic and either genetically or epigenetically mediated remain unclear due in part to a lack of appropriate, patient-specific, in vitro models. Recently, our group developed systems to create induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs) and biliary epithelial organoids (cholangioids). We use these models to investigate whether PSC cholangiocytes are intrinsically predisposed to cellular senescence. Skin fibroblasts from healthy controls and subjects with PSC were reprogrammed to pluripotency, differentiated to cholangiocytes, and subsequently grown in three-dimensional matrigel-based culture to induce formation of cholangioids. RNA sequencing (RNA-seq) on iDCs showed significant differences in gene expression patterns, including enrichment of pathways associated with cell cycle, senescence, and hepatic fibrosis, that correlate with PSC. These pathways also overlapped with RNA-seq analysis on isolated cholangiocytes from subjects with PSC. Exome sequencing on the subjects with PSC revealed genetic variants of unknown significance in the genes identified in these pathways. Three-dimensional culture revealed smaller size, lack of a central lumen, and increased cellular senescence in PSC-derived cholangioids. Congruent with this, PSC-derived iDCs showed increased secretion of the extracellular matrix molecule fibronectin as well as the inflammatory cytokines interleukin-6, and chemokine (C-C motif) ligand 2. Conditioned media (CM) from PSC-derived iDCs more potently activated hepatic stellate cells compared to control CM. Conclusion: We demonstrated efficient generation of iDCs and cholangioids from patients with PSC that show disease-specific features. PSC cholangiocytes are intrinsically predisposed to cellular senescence. These features are unmasked following biliary differentiation of pluripotent stem cells and have functional consequences in epithelial organoids.

Self-Organogenesis from 2D Micropatterns to 3D Biomimetic Biliary Trees.

BACKGROUND AND AIMS: Globally, liver diseases account for 2 million deaths per year. For those with advanced liver disease the only curative approach is liver transplantation. However, less than 10% of those in need get a liver transplant due to limited organ availability. To circumvent this challenge, there has been a great focus in generating a bioengineered liver. Despite its essential role in liver functions, a functional biliary system has not yet been developed. In this framework, exploration of epithelial cell self-organogenesis and microengineering-driven geometrical cell confinement allow to envision the bioengineering of a functional biomimetic intrahepatic biliary tract. APPROACH: three-dimensional (3D) bile ducts were built in vitro by restricting cell adhesion to two-dimensional (2D) patterns to guide cell self-organization. Tree shapes mimicking the configuration of the human biliary system were micropatterned on glass slides, restricting cell attachment to these areas. Different tree geometries and culture conditions were explored to stimulate self-organogenesis of normal rat cholangiocytes (NRCs) used as a biliary cell model, either alone or in co-culture with human umbilical endothelial cells (HUVECs). RESULTS: Pre-seeding the micropatterns with HUVECs promoted luminogenesis with higher efficiency to yield functional branched biliary tubes. Lumen formation, apico-basal polarity, and preservation of the cholangiocyte phenotype were confirmed. Moreover, intact and functional biliary structures were detached from the micropatterns for further manipulation. CONCLUSION: This study presents physiologically relevant 3D biliary duct networks built in vitro from 2D micropatterns. This opens opportunities for investigating bile duct organogenesis, physiopathology, and drug testing.

Microfluidic confinement enhances phenotype and function of hepatocyte spheroids.

A number of cell culture approaches have been described for maintenance of primary hepatocytes. Forming hepatocytes into three-dimensional (3-D) spheroids is one well-accepted method for extending epithelial phenotype of these cells. Our laboratory has previously observed enhanced function of two-dimensional (2-D, monolayer) hepatocyte cultures in microfluidic devices due to increased production of several hepato-inductive growth factors, including hepatocyte growth factor (HGF). In the present study, we wanted to test a hypothesis that culturing hepatocyte spheroids (3-D) in microfluidic devices will also result in enhanced phenotype and function. To test this hypothesis, we fabricated devices with small and large volumes. Both types of devices included a microstructured floor containing arrays of pyramidal wells to promote assembly of hepatocytes into spheroids with individual diameters of ~100 µm. The hepatocyte spheroids were found to be more functional, as evidenced by higher level of albumin synthesis, bile acid production, and hepatic enzyme expression, in low-volume compared with large-volume devices. Importantly, high functionality of spheroid cultures correlated with elevated levels of HGF secretion. Although decay of hepatic function (albumin secretion) was observed over the course 3 wk, this behavior could be abrogated by inhibiting TGF-ß1 signaling. With TGF-ß1 inhibitor, microfluidic hepatocyte spheroid cultures maintained high and stable levels of albumin synthesis over the course of 4 wk. To further highlight utility of this culture platform for liver disease modeling, we carried out alcohol injury experiments in microfluidic devices and tested protective effects of interleukin-22: a potential therapy for alcoholic hepatitis.

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Cholangiocyte autophagy contributes to hepatic cystogenesis in polycystic liver disease and represents a potential therapeutic target.

Polycystic liver disease (PLD) is a group of genetic disorders with limited treatment options and significant morbidity. Hepatic cysts arise from cholangiocytes exhibiting a hyperproliferative phenotype. Considering that hyperproliferation of many cell types is associated with alterations in autophagy, we hypothesized that autophagy is altered in PLD cholangiocytes, contributes to hepatic cystogenesis, and might represent a potential therapeutic target. We employed functional pathway cluster analysis and next-generation sequencing, transmission electron microscopy, immunofluorescence confocal microscopy, and western blotting to assess autophagy in human and rodent PLD cholangiocytes. A three-dimensional culture model was used to study the effects of molecular and pharmacologic inhibition of autophagy on hepatic cystogenesis in vitro, and the polycystic kidney disease-specific rat, an animal model of PLD, to study the effects of hydroxychloroquine, a drug that interferes with the autophagy pathway, on disease progression in vivo. Assessment of the transcriptome of PLD cholangiocytes followed by functional pathway cluster analysis revealed that the autophagy-lysosomal pathway is one of the most altered pathways in PLD. Direct evaluation of autophagy in PLD cholangiocytes both in vitro and in vivo showed increased number and size of autophagosomes, lysosomes, and autolysosomes; overexpression of autophagy-related proteins (Atg5, Beclin1, Atg7, and LC3); and enhanced autophagic flux associated with activation of the cAMP-protein kinase A-cAMP response element-binding protein signaling pathway. Molecular and pharmacologic intervention in autophagy with ATG7 small interfering RNA, bafilomycin A1 , and hydroxychloroquine reduced proliferation of PLD cholangiocytes in vitro and growth of hepatic cysts in three-dimensional cultures. Hydroxychloroquine also efficiently inhibited hepatic cystogenesis in the polycystic kidney disease-specific rat. CONCLUSION: Autophagy is increased in PLD cholangiocytes, contributes to hepatic cystogenesis, and represents a potential therapeutic target for disease treatment. (Hepatology 2018;67:1088-1108).

Development and characterization of cholangioids from normal and diseased human cholangiocytes as an in vitro model to study primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ~5000 cells with a central lumen of ~150 µm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ~80%. Cholangioids exposed to hydrogen peroxide exhibited cellular senescence and the senescence-associated secretory phenotype (SASP; increased IL-6, p21, SA-ß-Gal, yH2A.x and p16 expression). Furthermore, cholangioids derived from NHC-sen or PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-senescent NHC. We observed that NHC-sen and PSC cholangioids release greater number of extracellular vesicles (EVs) compared to controls. Moreover, conditioned media from NHC-sen cholangioids resulted in an ~2-fold increase in macrophage migration. In summary, we developed a method to generate normal and diseased cholangioids, characterized them morphologically and functionally, showed that they can be induced to senescence and SASP, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.

ETS Proto-oncogene 1 Transcriptionally Up-regulates the Cholangiocyte Senescence-associated Protein Cyclin-dependent Kinase Inhibitor 2A.

Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory cholangiopathy (disease of the bile ducts) of unknown pathogenesis. We reported that cholangiocyte senescence features prominently in PSC and that neuroblastoma RAS viral oncogene homolog (NRAS) is activated in PSC cholangiocytes. Additionally, persistent microbial insult (e.g. LPSs) induces cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a) expression and senescence in cultured cholangiocytes in an NRAS-dependent manner. However, the molecular mechanisms involved in LPS-induced cholangiocyte senescence and NRAS-dependent regulation of CDKN2A remain unclear. Using our in vitro senescence model, we found that LPS-induced CDKN2A expression coincided with a 4.5-fold increase in ETS1 (ETS proto-oncogene 1) mRNA, suggesting that ETS1 is involved in regulating CDKN2A This idea was confirmed by RNAi-mediated suppression or genetic deletion of ETS1, which blocked CDKN2A expression and reduced cholangiocyte senescence. Furthermore, site-directed mutagenesis of a predicted ETS-binding site within the CDKN2A promoter abolished luciferase reporter activity. Pharmacological inhibition of RAS/MAPK reduced ETS1 and CDKN2A protein expression and CDKN2A promoter-driven luciferase activity by ∼50%. In contrast, constitutively active NRAS expression induced ETS1 and CDKN2A protein expression, whereas ETS1 RNAi blocked this increase. Chromatin immunoprecipitation-PCR detected increased ETS1 and histone 3 lysine 4 trimethylation (H3K4Me3) at the CDKN2A promoter following LPS-induced senescence. Additionally, phospho-ETS1 expression was increased in cholangiocytes of human PSC livers and in the Abcb4 (Mdr2)-/- mouse model of PSC. These data pinpoint ETS1 and H3K4Me3 as key transcriptional regulators in NRAS-induced expression of CDKN2A, and this regulatory axis may therefore represent a potential therapeutic target for PSC treatment.

MicroRNAs in the Cholangiopathies: Pathogenesis, Diagnosis, and Treatment.

The cholangiopathies are a group of liver diseases resulting from different etiologies but with the cholangiocyte as the primary target. As a group, the cholangiopathies result in significant morbidity and mortality and represent one of the main indications for liver transplant in both children and adults. Contributing to this situation is the absence of a thorough understanding of their pathogenesis and a lack of adequate diagnostic and prognostic biomarkers. MicroRNAs are small non-coding RNAs that modify gene expression post-transcriptionally. They have been implicated in the pathogenesis of many diseases, including the cholangiopathies. Thus, in this review we provide an overview of the literature on miRNAs in the cholangiopathies and discuss future research directions.

Two α-dicarbonyls downregulate migration, invasion, and adhesion of liver cancer cells in a p53-dependent manner.

BACKGROUND: Hepatocellular carcinoma accounts for more than 600,000 deaths per year due to it being a highly invasive tumor. The α-dicarbonyl, methylglyoxal demonstrates efficacy at reducing tumor burden, however the anti-cancerous activities of 3-deoxyglucosone, have never been studied. AIMS: To determine the anti-cancerous potential of methylglyoxal and 3-deoxyglucosone on liver tumor cells. METHODS: The in vitro effects of methylglyoxal and 3-deoxyglucosone were studied by investigating migration, invasion, and adhesion of Huh-7, HepG2, and Hep3B cells. RESULTS: 3-Deoxyglucosone inhibited migration of Huh-7 and HepG2 cells. Methylglyoxal decreased migration of HepG2 cells. Additionally, 3-deoxyglucosone and methylglyoxal impaired invasion, and adhesion of Huh-7 and HepG2 cells. In Hep3B cells, a p53 null cell line, 3-deoxyglucosone and methylglyoxal had no effect on migration, invasion, or adhesion. However, both compounds inhibited invasion of wild-type p53 transfected Hep3B cells. Silencing of p53 in Huh-7 and HepG2 cells abrogated the effects of the α-dicarbonyls on cell invasion. 3DG and MG did not alter p53 total protein but promoted nuclear translocation of p53. CONCLUSIONS: These studies suggest that 3-deoxyglucosone and methylglyoxal impair invasion, migration, and adhesion of hepatocellular carcinoma. The effects of both compounds on cell invasion are dependent on p53 and imply that α-dicarbonyls could be efficacious in the treatment of p53-expressing invasive liver tumors.

Extrahepatic cholangiocyte cilia are abnormal in biliary atresia.

OBJECTIVES: Biliary atresia (BA) is a rapidly progressive form of biliary fibrosis affecting neonates. We previously reported that primary cilia on the intrahepatic cholangiocytes of patients with both syndromic and nonsyndromic BA were structurally abnormal. Our objective was to determine whether extrahepatic cholangiocytes in human biliary atresia, intrahepatic and extrahepatic cholangiocytes of rhesus rotavirus (RRV)-infected neonatal mice, and RRV-infected primary neonatal extrahepatic cholangiocytes also demonstrate ciliary abnormalities. METHODS: The livers of neonatal BALB/c mice injected with RRV that developed jaundice, human extrahepatic bile duct samples obtained at time of hepatoportoenterostomy, and RRV-infected primary neonatal cholangiocytes were stained with antibodies against acetylated α tubulin to identify primary cilia. RESULTS: Extrahepatic cholangiocytes from RRV-treated mice demonstrated minimal loss of primary cilia at day 3 but almost complete loss at day 8 and partial loss at day 12. No changes were seen in mouse intrahepatic bile ducts at any of the time points. In the human BA samples, primary cilia were almost completely absent from extrahepatic duct cholangiocytes. There were, however, abundant cilia in the peribiliary glands adjacent to extrahepatic ducts in the BA sample. Cilia in RRV-infected primary neonatal cholangiocytes were significantly decreased compared with controls. CONCLUSIONS: Primary cilia are selectively lost from neonatal extrahepatic but not intrahepatic cholangiocytes after RRV infection in BALB/c mice. The cilia are also decreased in RRV-infected primary cholangiocytes and the extrahepatic ducts from human patients with BA. This suggests that ciliary abnormalities are part of the pathophysiology of BA.