The development of complex in vitro hepatic systems and artificial liver devices has been hampered by the lack of reliable sources for relevant cell types, such as hepatic stellate cells (HSCs). Here ...we report efficient differentiation of human pluripotent stem cells into HSC-like cells (iPSC-HSCs). iPSC-HSCs closely resemble primary human HSCs at the transcriptional, cellular, and functional levels and possess a gene expression profile intermediate between that of quiescent and activated HSCs. Functional analyses revealed that iPSC-HSCs accumulate retinyl esters in lipid droplets and are activated in response to mediators of wound healing, similar to their in vivo counterparts. When maintained as 3D spheroids with HepaRG hepatocytes, iPSC-HSCs exhibit a quiescent phenotype but mount a fibrogenic response and secrete pro-collagen in response to known stimuli and hepatocyte toxicity. Thus, this protocol provides a robust in vitro system for studying HSC development, modeling liver fibrosis, and drug toxicity screening.
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•A protocol mimicking liver development enables generation of HSCs from hPSCs•iPSC-HSCs possess properties similar to those of their in vitro counterparts•iPSC-HSCs can form liver spheroids when aggregated with hepatocytes•Liver spheroids can be used to model fibrosis and liver toxicity
Coll et al. describe efficient differentiation of human pluripotent stem cells into hepatic stellate cell-like cells (iPSC-HSCs). iPSC-HSCs share phenotypic and functional features with primary HSCs and are useful for studying HSC development and for the generation of 3D in vitro liver systems for toxicity assessment and fibrosis modeling.
MicroRNAs (miRNAs) are well-known regulators of disease pathogenesis and have great potential as biomarkers and therapeutic targets. We aimed at profiling miRNAs in alcoholic hepatitis (AH) and ...identifying miRNAs potentially involved in liver injury.
MiRNA profiling was performed in liver samples from patients with AH, alcohol liver disease, non-alcoholic steatohepatitis, HCV disease and normal liver tissue. Expression of miRNAs was assessed in liver and serum from patients with AH and animal models. Mimic and decoy miR-182 were used in vitro and in vivo to evaluate miR-182's biological functions.
MiRNA expression profile in liver was highly altered in AH and distinctive from alcohol-induced cirrhotic livers. Moreover, we identified a set of 18 miRNAs predominantly expressed in AH as compared with other chronic liver conditions. Integrative miRNA-mRNA functional analysis revealed the association of AH-altered miRNAs with nuclear receptors, IGF-1 signalling and cholestasis. Interestingly, miR-182 was the most highly expressed miRNA in AH, which correlated with degree of ductular reaction, disease severity and short-term mortality. MiR-182 mimic induced an upregulation of inflammatory mediators in biliary cells. At experimental level, miR-182 was increased in biliary cells in mice fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet but not upregulated by alcohol intake or fibrosis. Inhibition of miR-182 in DDC-fed mice reduced liver damage, bile acid accumulation and inflammatory response.
AH is characterised by a deregulated miRNA profile, including miR-182, which is associated with disease severity and liver injury. These results highlight the potential of miRNAs as therapeutic targets and biomarkers in AH.
Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic ...injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1β). However, HNF1β expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)‐positive hepatocytes. In order to investigate the role of HNF1β‐expressing cells we used a tamoxifen‐inducible Hnf1βCreER/R26RYfp/LacZ mouse to lineage‐trace Hnf1β+ biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1β+ cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1β+ cells to hepatocyte was detected. We next assessed the contribution of Hnf1β+‐derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl‐1,4‐dihydro‐collidin (DDC)‐diet and a choline‐deficient ethionine‐supplemented (CDE)‐diet. The contribution of Hnf1β+ cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC‐diet treatment, mice fed with a CDE‐diet showed a small population of hepatocytes derived from Hnf1β+ cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome‐wide expression profile of Hnf1β+‐derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways. Conclusion: HNF1β+ biliary duct cells are the origin of LPC. HNF1β+ cells do not contribute to hepatocyte turnover in the healthy liver, but after certain liver injury, they can differentiate to hepatocytes contributing to liver regeneration. (Hepatology 2014;60:1367–1377)
Background and Aims
Ductular reaction (DR) expands in chronic liver diseases and correlates with disease severity. Besides its potential role in liver regeneration, DR plays a role in the ...wound‐healing response of the liver, promoting periductular fibrosis and inflammatory cell recruitment. However, there is no information regarding its role in intrahepatic angiogenesis. In the current study we investigated the potential contribution of DR cells to hepatic vascular remodeling during chronic liver disease.
Approach and Results
In mouse models of liver injury, DR cells express genes involved in angiogenesis. Among angiogenesis‐related genes, the expression of Slit2 and its receptor Roundabout 1 (Robo1) was localized in DR cells and neoangiogenic vessels, respectively. The angiogenic role of the Slit2–Robo1 pathway in chronic liver disease was confirmed in ROBO1/2−/+ mice treated with 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine, which displayed reduced intrahepatic neovascular density compared to wild‐type mice. However, ROBO1/2 deficiency did not affect angiogenesis in partial hepatectomy. In patients with advanced alcohol‐associated disease, angiogenesis was associated with DR, and up‐regulation of SLIT2–ROBO1 correlated with DR and disease severity. In vitro, human liver‐derived organoids produced SLIT2 and induced tube formation of endothelial cells.
Conclusions
Overall, our data indicate that DR expansion promotes angiogenesis through the Slit2–Robo1 pathway and recognize DR cells as key players in the liver wound‐healing response.
Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the ...miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.
Fatty-acid-binding proteins (FABPs) are small intracellular proteins that coordinate lipid-mediated processes by targeting metabolic and immune response pathways. The aim of the study was to ...investigate plasma FABPs levels and their relationship with clinical outcomes in cirrhosis. Plasma levels of L-FABP1(liver and kidney), I-FABP2(intestine), and A-FABP4(adipocyte and macrophages) were measured in 274 patients with decompensated cirrhosis. Hepatic gene expression of FABPs was assessed in liver biopsies from patients with decompensated cirrhosis and in liver cell types from mice with cirrhosis. Immunohistochemistry of A-FABP4 in human liver biopsy was also performed. Plasma levels of FABPs were increased in patients with decompensated cirrhosis compared to those of healthy subjects (L-FABP1: 25 (17-39) vs 10 (9-17) ng/mL p = 0.001, I-FABP2: 1.1 (0.5-2.1) vs 0.6 (0.4-1) ng/mL p = 0.04 and A-FABP4: 37 (20-68) vs 16 (11-33) ng/mL p = 0.002), respectively. Increased A-FABP4 levels were associated with complications of cirrhosis, acute-on-chronic liver failure and poor survival. Hepatic A-FABP4 gene expression was upregulated in decompensated cirrhosis. Macrophages were the main liver cell that over-expressed A-FABP4 in experimental cirrhosis and increased A-FABP4 was found in macrophages of human biopsies by immunohistochemistry. A-FABP4 levels are increased in decompensated cirrhosis and correlate with poor outcomes. Liver macrophages appear to be the main source of A-FABP4 in decompensated cirrhosis.
Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the ...role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs (miRNAs) circulate in several body fluids and can be useful biomarkers. The aim of this study was to identify blood-circulating miRNAs associated with cirrhosis progression and ...acute-on-chronic liver failure (ACLF).
Using high-throughput screening of 754 miRNAs, serum samples from 45 patients with compensated cirrhosis, decompensated cirrhosis, or ACLF were compared with those from healthy individuals (n = 15). miRNA levels were correlated with clinical parameters, organ failure, and disease progression and outcome. Dysregulated miRNAs were evaluated in portal and hepatic vein samples (n = 33), liver tissues (n = 17), and peripheral blood mononuclear cells (PBMCs) (n = 16).
miRNA screening analysis revealed that circulating miRNAs are dysregulated in cirrhosis progression, with 51 miRNAs being differentially expressed among all groups of patients. Unsupervised clustering and principal component analysis indicated that the main differences in miRNA expression occurred at decompensation, showing similar levels in patients with decompensated cirrhosis and those with ACLF. Of 43 selected miRNAs examined for differences among groups, 10 were differentially expressed according to disease progression. Moreover, 20 circulating miRNAs were correlated with model for end-stage liver disease and Child-Pugh scores. Notably, 11 dysregulated miRNAs were associated with kidney or liver failure, encephalopathy, bacterial infection, and poor outcomes. The most severely dysregulated miRNAs (i.e. miR-146a-5p, miR-26a-5p, and miR-191-5p) were further evaluated in portal and hepatic vein blood and liver tissue, but showed no differences. However, PBMCs from patients with cirrhosis showed significant downregulation of miR-26 and miR-146a, suggesting a extrahepatic origin of some circulating miRNAs.
This study is a repository of circulating miRNA data following cirrhosis progression and ACLF. Circulating miRNAs were profoundly dysregulated during the progression of chronic liver disease, were associated with failure of several organs and could have prognostic utility.
Circulating miRNAs are small molecules in the blood that can be used to identify or predict a clinical condition. Our study aimed to identify miRNAs for use as biomarkers in patients with cirrhosis or acute-on-chronic liver failure. Several miRNAs were found to be dysregulated during the progression of disease, and some were also related to organ failure and disease-related outcomes.
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•Circulating miRNAs are dysregulated with cirrhosis progression and in patients with ACLF.•Patient decompensation is associated with important changes in the levels of circulating miRNAs.•A total of 11 circulating miRNAs were identified as associated with organ failure and 7 with poor outcome.•The miRNAs most dysregulated during cirrhosis progression were miR-146a, miR-26a, and miR-191.•miR-146a was dysregulated in PBMCs of patients with decompensated cirrhosis vs. compensated cirrhosis.
Objectives
Alcoholic hepatitis (AH) is a severe condition characterized by a marked inflammatory response and high short-term mortality. Endothelial dysfunction (ED) is an early event in vascular and ...inflammatory disorders. The aim of this study is to evaluate ED in AH patients.
Methods
Prognostic value of ED biomarkers was evaluated in patients with severe AH (
n
= 67), compensated alcoholic cirrhosis (
n
= 15), heavy drinkers without liver disease (
n
= 15) and controls (
n
= 9), and in a validation cohort of 50 patients with AH. Gene expression of ED markers was analyzed in liver tissue.
Results
Plasma levels of ED markers such as vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin and von Willebrand factor (vWF) increased along alcohol-related liver disease (ALD) progression. Intergroup analysis showed a significant increase of these markers in AH patients. In addition, VCAM-1 showed a positive correlation with Maddrey, MELD and ABIC scores and inflammation parameters (i.e. C-reactive protein and LPS levels). Importantly, levels of VCAM-1 were higher in patients with increased mortality and were independently associated with short-term survival (90-day) when adjusted by ABIC score. These results were confirmed in an independent cohort of AH patients. In addition, severe AH patients showed altered hepatic expression of ED markers.
Conclusions
In this study we show that advanced ALD and particularly severe AH is associated with an increase of ED biomarkers, which correlate with patient outcomes. These results suggest that ED may be a pathogenic event in AH and highlight endothelial factors as potential biomarkers in AH.
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