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.

Objective Alcoholic hepatitis (AH) is a severe clinical condition that needs novel therapies. The identification of targets for therapy is hampered by the lack of animal models of advanced AH. The authors performed a translational study through a transcriptome analysis in patients with AH to identify new molecular targets. Design Hepatic gene expression profiling was assessed by DNA microarray in patients with AH (n=15) and normal livers (n=7). Functional analysis was assessed by gene set enrichment analysis. Quantitative PCR was performed in patients with AH (n=40), hepatitis C (n=18), non-alcoholic steatohepatitis (n=20) and in mouse models of acute and chronic liver injury. Protein expression was assessed by immunohistochemistry and western blotting. Results Gene expression analysis showed 207 genes >5-fold differentially expressed in patients with AH and revealed seven pathways differentially regulated including ‘cytokine—cytokine receptor interaction’. Several tumour necrosis factor (TNF) superfamily receptors, but not ligands, were overexpressed in AH. Importantly, Fn14 was the only TNF superfamily receptor exclusively upregulated in AH compared with other liver diseases and correlated with both 90-day mortality and severity of portal hypertension. Fn14 protein expression was detected in areas of fibrogenesis and in a population of hepatocytes. Fn14 expression was increased in experimental models of liver injury and was detected in progenitor cells. Conclusion Translational research revealed that TNF superfamily receptors are overexpressed in AH. Fn14, the receptor for TNF-like weak inducer of apoptosis, is selectively upregulated in patients with AH. TNF superfamily receptors could represent a potential target for therapy.

Alcoholic hepatitis (AH) is a severe condition developed in patients with underlying alcoholic liver disease. Ductular reaction has been associated with chronic alcohol consumption but there is no information regarding the extent of liver progenitor cell (LPC) proliferation in AH. The aim of this study was to investigate LPC markers in AH and its correlation with disease severity. Fifty-nine patients with clinical and histological diagnosis of AH were included in the study. LPC markers were assessed by real-time polymerase chain reaction (PCR) and immunohistochemistry. Standard logistic regression analysis and classification and regression trees (CART) analysis were used for statistical analysis. A microarray analysis showed an upregulation of LPC markers in patients with AH. Real-time PCR demonstrated that epithelial cell adhesion molecule (EpCAM), Prominin-1, and Keratin7 were significantly increased in patients with AH compared with normal livers (P 0.01), chronic hepatitis C (P 0.01), and HCV-induced cirrhosis (P 0.01). Immunohistochemistry scores generated for Keratin7 and EpCAM demonstrated a good correlation with gene expression. Keratin7 gene expression correlated with liver failure as assessed by model for endstage liver disease score (r 5 0.41, P 5 0.006) and Maddrey's discriminant function (r 5 0.43, P 5 0.004). Moreover, Keratin7 (OR1.14, P 5 0.004) and Prominin-1 (OR1.14, P 5 0.002), but not EpCAM (OR1.16, P 5 0.06), were identified as independent predictors of 90-day mortality. CART analysis generated an algorithm based on the combination of Keratin7 and EpCAM gene expression that stratified three groups of patients with high, intermediate, and low short-term mortality (89%, 33%, and 6%, respectively; area under the receiver operating curve 0.73, 95% confidence interval 0.60-0.87). Keratin7 expression provided additional discrimination potential to the age, bilirubin, international normalization ratio, creatinine (ABIC) score. Conclusion: LPC markers correlate positively with severity of liver disease and short-term mortality in AH patients. This study suggests that LPC proliferation may be an important feature of AH pathophysiology. (HEPATOLOGY 2012;55:1931-1941

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-dihydrocollidin (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.