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The lack of adequate human in vitro models that recapitulate the cellular composition and response of the human liver to injury hampers the development of anti-fibrotic drugs. The goal of this study was to develop a human spheroid culture model to study liver fibrosis by using induced pluripotent stem cell (iPSC)-derived liver cells. iPSCs were independently differentiated towards hepatoblasts (iHepatoblasts), hepatic stellate cells (iHSCs), endothelial cells (iECs) and macrophages (iMΦ), before assembly into free floating spheroids by culturing cells in 96-well U-bottom plates and orbital shaking for up to 21 days to allow further maturation. Through transcriptome analysis, we show further maturation of iECs and iMΦ, the differentiation of the iHepatoblasts towards hepatocyte-like cells (iHeps) and the inactivation of the iHSCs by the end of the 3D culture. Moreover, these cultures display a similar expression of cell-specific marker genes ( CYP3A4, PDGFRβ, CD31 and CD68 ) and sensitivity to hepatotoxicity as spheroids made using freshly isolated primary human liver cells. Furthermore, we show the functionality of the iHeps and the iHSCs by mimicking liver fibrosis through iHep-induced iHSC activation, using acetaminophen. In conclusion, we have established a reproducible human iPSC-derived liver culture model that can be used to mimic fibrosis in vitro as a replacement of primary human liver derived 3D models. The model can be used to investigate pathways involved in fibrosis development and to identify new targets for chronic liver disease therapy., (Creative Commons Attribution license.)

Thirty-five years ago, precision-cut liver slices (PCLS) were described as a promising tool and were expected to become the standard in vitro model to study liver disease as they tick off all characteristics of a good in vitro model. In contrast to most in vitro models, PCLS retain the complex 3D liver structures found in vivo, including cell-cell and cell-matrix interactions, and therefore should constitute the most reliable tool to model and to investigate pathways underlying chronic liver disease in vitro. Nevertheless, the biggest disadvantage of the model is the initiation of a procedure-induced fibrotic response. In this review, we describe the parameters and potential of PCLS cultures and discuss whether the initially described limitations and pitfalls have been overcome. We summarize the latest advances in PCLS research and critically evaluate PCLS use and progress since its invention in 1985.

Background: The current diagnosis of early-stage liver fibrosis often relies on a serological or imaging-based evaluation of the stage of fibrosis, sometimes followed by an invasive liver biopsy procedure. Novel non-invasive experimental diagnostic tools are often based on markers of hepatocyte damage, or changes in liver stiffness and architecture, which are late-stage characteristics of fibrosis progression, making them unsuitable for the diagnosis of early-stage liver fibrosis. miRNAs control hepatic stellate cell (HSC) activation and are proposed as relevant diagnostic markers. Methods: We investigated the possibility of circulating miRNAs, which we found to be dysregulated upon HSC activation, to mark the presence of significant liver fibrosis (F ≥ 2) in patients with chronic alcohol abuse, chronic viral infection (HBV/HCV), and non-alcoholic fatty liver disease (NAFLD). Results: miRNA-profiling identified miRNA-451a, miRNA-142-5p, Let-7f-5p, and miRNA-378a-3p to be significantly dysregulated upon in vitro HSC activation, and to be highly enriched in their extracellular vesicles, suggesting their potential use as biomarkers. Analysis of the plasma of patients with significant liver fibrosis (F ≥ 2) and no or mild fibrosis (F = 0-1), using miRNA-122-5p and miRNA-29a-3p as positive control, found miRNA-451a, miRNA-142-5p, and Let-7f-5p, but not miRNA-378a-3p, able to distinguish between the two patient populations. Using logistic regression analysis, combining all five dysregulated circulating miRNAs, we created the miRFIB-score with a predictive value superior to the clinical scores Fibrosis-4 (Fib-4), aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio, and AST to platelet ratio index (APRI). The combination of the miRFIB-score with circulating PDGFRβ-levels further increased the predictive capacity for the diagnosis of significant liver fibrosis. Conclusion s -scores are accurate tools for the diagnosis of significant liver fibrosis in a heterogeneous patient population.: The miRFIB- and miRFIB p -scores are accurate tools for the diagnosis of significant liver fibrosis in a heterogeneous patient population., Competing Interests: The authors declare no conflict of interest.

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl4- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.

Background and Aims: Chronic liver damage causes hepatic stellate cell (HSC) activation and contraction, leading to intrahepatic microvascular and structural changes. In vitro endothelin-1 (ET-1)-induced contraction of HSCs can be reduced by somatostatin (SST); however, intrahepatic in vivo effects have never been studied., Methods: Sinusoidal diameter was measured by intravital fluorescence microscopy in carbon tetrachloride (CCl(4)) and control mice before and after an intravenous (IV) bolus and after 0, 5, 10 and 15 min of an IV infusion of saline, 8 microg/kg/h SST or 8 microg/kg/h octreotide., Results: The baseline sinusoidal diameter in CCl(4) mice (3.01+/-0.05 microm) was significantly smaller than that in controls (4.37+/-0.06 microm). The sinusoidal diameter increased significantly in both groups after a bolus (27, 16% respectively) and following 5 min of SST IV infusion (28, 14% respectively). The percentage increase was significantly higher in CCl(4) mice as compared with controls. This dilatory effect continued for at least 15 min. SST did not influence the mean arterial blood pressure (MAP) and portal venous inflow. In none of the groups did octreotide or saline have any influence on sinusoidal diameters, MAP and portal venous inflow., Conclusions: Sinusoidal diameter in cirrhotic mice is significantly smaller than that in controls. SST causes significant sinusoidal dilation following a bolus and for at least 15 min of IV infusion. Octreotide does not have any influence on liver sinusoids. These results demonstrate for the first time the in vivo dilatory effect of SST on liver sinusoids.

Previous studies have shown antifibrotic effects of somatostatin. Since hepatic stellate cells (HSC) express somatostatin receptors and play a key role in hepatic fibrogenesis, we investigated the in vitro antifibrotic effect of somatostatin on rat HSC. At day 12 after isolation, cells were exposed to different concentrations of somatostatin (10(-6)-10(-9) mol l(-1)). mRNA expression of collagen types I and III, and of smooth muscle alpha-actin (alpha-SMA) was analysed by Northern blotting. At 10(-9) mol l(-1), somatostatin significantly reduced mRNA expression of collagen I (72.3 +/- 10.7%; 95% confidence interval (95% CI): 45.5-99.0), collagen III (79.0 +/- 4.5%; 95% CI: 67.6-90.4) and alpha-SMA (65.7 +/- 5.9%; 95% CI: 51.1-80.2), as compared to control normalized at 100%. These results were confirmed by quantitative RT-PCR. Cycloheximide experiments indicated that somatostatin has no direct transcriptional effect.Using immunoprecipitation, we demonstrated that somatostatin also decreased de novo synthesis of collagen I (73 +/-10%; 95% CI: 48-98%), collagen III (65 +/- 13%; 95% CI: 33-97%) and alpha-SMA (47 +/- 9%; 95% CI: 25-69%). Remarkably, at higher concentrations, somatostatin did not suppress collagen mRNA expression nor de novo protein synthesis. We ascribe this observation to desensitization of the cells for somatostatin. Cell proliferation, as measured by 5-bromo-2'-deoxyuridine labelling, was not altered by somatostatin. No significant effect on the intermediate and actin cytoskeleton were detected by immunohistochemistry and Western blotting. Our findings imply that in vivo antifibrotic effects of somatostatin could result partially from a direct action of somatostatin on HSC, but other, in vivo effects are probably also involved.

After receiving information from afferent nerves, the hypothalamus sends signals to peripheral organs, including the liver, to keep homeostasis. There are two ways for the hypothalamus to signal to the peripheral organs: by stimulating the autonomic nerves and by releasing hormones from the pituitary gland. In order to reveal the involvement of the autonomic nervous system in liver function, we focus in this study on autonomic nerves and neuroendocrine connections between the hypothalamus and the liver. The hypothalamus consists of three major areas: lateral, medial, and periventricular. Each area has some nuclei. There are two important nuclei and one area in the hypothalamus that send out the neural autonomic information to the peripheral organs: the ventromedial hypothalamic nucleus (VMH) in the medial area, the lateral hypothalamic area (LHA), and the periventricular hypothalamic nucleus (PVN) in the periventricular area. VMH sends sympathetic signals to the liver via the celiac ganglia, the LHA sends parasympathetic signals to the liver via the vagal nerve, and the PVN integrates information from other areas of the hypothalamus and sends both autonomic signals to the liver. As for the afferent nerves, there are two pathways: a vagal afferent and a dorsal afferent nerve pathway. Vagal afferent nerves are thought to play a role as sensors in the peripheral organs and to send signals to the brain, including the hypothalamus, via nodosa ganglia of the vagal nerve. On the other hand, dorsal afferent nerves are primary sensory nerves that send signals to the brain via lower thoracic dorsal root ganglia. In the liver, many nerves contain classical neurotransmitters (noradrenaline and acetylcholine) and neuropeptides (substance P, calcitonin gene-related peptide, neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, glucagon, glucagon-like peptide, neurotensin, serotonin, and galanin). Their distribution in the liver is species-dependent. Some of these nerves are thought to be involved in the regulation of hepatic function as well as of hemodynamics. In addition to direct neural connections, the hypothalamus can affect metabolic functions by neuroendocrine connections: the hypothalamus-pancreas axis, the hypothalamus-adrenal axis, and the hypothalamus-pituitary axis. In the hypothalamus-pancreas axis, autonomic nerves release glucagon and insulin, which directly enter the liver and affect liver metabolism. In the hypothalamus-adrenal axis, autonomic nerves release catecholamines such as adrenaline and noradrenaline from the adrenal medulla, which also affects liver metabolism. In the hypothalamus-pituitary axis, release of glucocorticoids and thyroid hormones is stimulated by pituitary hormones. Both groups of hormones modulate hepatic metabolism. Taken together, the hypothalamus controls liver functions by neural and neuroendocrine connections., (Copyright 2004 Wiley-Liss, Inc.)

Background & Aims: Hepatic stellate cells (HSCs) are considered therapeutic targets to decrease portal hypertension. To elucidate some of the hemodynamic effects of somatostatin (SST) on portal pressure, the presence and function of SST receptors (SSTRs) on HSCs were investigated., Methods: SSTR messenger RNA expression, and SSTR presence was investigated using reverse-transcription polymerase chain reaction, real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The function of SSTRs was studied by examining the effects of SST and specific SSTR agonists on endothelin-1(ET-1)-induced HSC contraction., Results: Specific amplicons for SSTR subtypes 1, 2, and 3 were demonstrated in rat liver and in activated HSCs. The presence of SSTR subtypes 1, 2, and 3 was confirmed by Western blotting. With immunohistochemistry, a strong staining of HSCs was obtained for SSTR subtypes 1, 2, and 3 in CCl4-treated rats, but not in normal rat liver. Incubation of HSCs on collagen gels with buffer, 10(-8) mol/L SST, and 2 x 10(-8) mol/L ET-1 resulted in collagen surface area decreases of 5.5% +/- 3.3%, 6.8% +/- 4.4%, and 49.8% +/- 8.3%, respectively. Relative contraction of gels preincubated with 10(-8) mol/L SST followed by 2 x 10(-8) mol/L ET-1 or vice versa as compared with maximal contraction (100%) with 2 x 10(-8) mol/L ET-1 were 72.6% +/- 17.9% and 76.2% +/- 12.6%, respectively (P < 0.05). SSTR agonist 1, but not SSTR agonist 2 or 3, was able to counteract the contractile effect of ET-1. CONCLUSIONA: Activated rat HSCs bear SSTR subtypes 1, 2, and 3. SST causes significant partial inhibition of ET-1-induced contraction of activated HSCs, mainly by stimulation of SSTR subtype 1.

A 21-year-old woman with Crohn's disease of the colon developed a skin rash after 3 weeks of treatment with sulfasalazine. Administration of sulfasalazine was discontinued. When mesalazine was instituted 1 week later, she developed a severe hypersensitivity reaction characterized by fever, diarrhea, skin rash with subsequent desquamation, marked atypical lymphocytosis, and severe hepatotoxicity. Recovery was complete. The clinical and biological features as well as liver pathology of this case bear a striking resemblance to earlier reports of hypersensitivity reaction with severe hepatotoxicity to sulfasalazine. The authors urge caution when mesalazine is given to a patient with known hypersensitivity to sulfasalazine.