Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt‐agonistic R‐spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ...ever‐expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1‐based cultures, and develop into budding cyst‐like structures (organoids) that expand five‐fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi‐potentiality.
The establishment of conditions for long‐term culture and expansion of adult, bi‐potent pancreas progenitors may facilitate novel and tailored therapeutic approaches.
Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers ...therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2
, CLEC5A
, MARCO
liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.
Non-alcoholic steatohepatitis (NASH) is an increasing cause of chronic liver disease characterized by steatosis, inflammation, and fibrosis which can lead to cirrhosis, hepatocellular carcinoma, and ...mortality. Quantitative, noninvasive methods for characterizing the pathophysiology of NASH at both the preclinical and clinical level are sorely needed. We report here a multiparametric magnetic resonance imaging (MRI) protocol with the fibrogenesis probe Gd-Hyd to characterize fibrotic disease activity and steatosis in a common mouse model of NASH. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) to induce NASH with advanced fibrosis. Mice fed normal chow and CDAHFD underwent MRI after 2, 6, 10 and 14 weeks to measure liver T1, T2*, fat fraction, and dynamic T1-weighted Gd-Hyd enhanced imaging of the liver. Steatosis, inflammation, and fibrosis were then quantified by histology. NASH and fibrosis developed quickly in CDAHFD fed mice with strong correlation between morphometric steatosis quantification and liver fat estimated by MRI (r = 0.90). Sirius red histology and collagen quantification confirmed increasing fibrosis over time (r = 0.82). Though baseline T1 and T2* measurements did not correlate with fibrosis, Gd-Hyd signal enhancement provided a measure of the extent of active fibrotic disease progression and correlated strongly with lysyl oxidase expression. Gd-Hyd MRI accurately detects fibrogenesis in a mouse model of NASH with advanced fibrosis and can be combined with other MR measures, like fat imaging, to more accurately assess disease burden.
Syngeneic, immunocompetent allograft tumor models recapitulate important aspects of the tumor microenvironment and have short tumor latency with predictable growth kinetics, making them useful for ...trialing novel therapeutics. Here, we describe surgical techniques for orthotopic and heterotopic pancreatic ductal adenocarcinoma (PDAC) tumor implantation and characterize phenotypes based on implantation site.Mice (
=8 per group) were implanted with 10
cells in the pancreas or flank. Hy15549 and Han4.13 cell lines were derived from primary murine PDAC (Ptf1-Cre; LSL-KRAS-G12D; Trp53 Lox/+) on C57BL/6 and FVB strains, respectively. Single-cell suspension and solid tumor implants were compared. Tumors were treated with two intravenous doses of FOLFIRINOX and responses evaluated.All mice developed pancreatic tumors within 7 days. Orthotopic tumors grew faster and larger than heterotopic tumors. By 3 weeks, orthotopic mice began losing weight, and showed declines in body condition requiring euthanasia starting at 4 weeks. Single-cell injection into the pancreas had near 100% engraftment, but solid tumor implant engraftment was ∼50% and was associated with growth restriction. Orthotopic tumors were significantly more responsive to intravenous FOLFIRINOX compared with heterotopic tumors, with greater reductions in size and increased apoptosis. Heterotopic tumors were more desmoplastic and hypovascular. However, drug uptake into tumor tissue was equivalent regardless of tumor location or degree of fibrosis, indicating that microenvironment differences between heterotopic and orthotopic tumors influenced response to therapy.Our results show that orthotopic and heterotopic allograft locations confer unique microenvironments that influence growth kinetics, desmoplastic response and angiogenesis. Tumor location influences chemosensitivity to FOLFIRINOX and should inform future preclinical trials.This article has an associated First Person interview with the first author of the paper.
Acute kidney injury in decompensated cirrhosis has limited therapeutic options, and novel mechanistic targets are urgently needed. Angiopoietin‐2 is a context‐specific antagonist of Tie2, a receptor ...that signals vascular quiescence. Considering the prominence of vascular destabilization in decompensated cirrhosis, we evaluated Angiopoietin‐2 to predict clinical outcomes. Serum Angiopoietin‐2 was measured serially in a prospective cohort of hospitalized patients with decompensated cirrhosis and acute kidney injury. Clinical characteristics and outcomes were examined over a 90‐day period and analyzed according to Angiopoietin‐2 levels. Primary outcome was 90‐day mortality. Our study included 191 inpatients (median Angiopoietin‐2 level 18.2 interquartile range 11.8, 26.5 ng/mL). Median Model for End‐Stage Liver Disease (MELD) score was 23 17, 30 and 90‐day mortality was 41%. Increased Angiopoietin‐2 levels were associated with increased mortality (died 21.9 13.9, 30.3 ng/mL vs. alive 15.2 9.8, 23.0 ng/mL; P < 0.001), higher Acute Kidney Injury Network stage (stage I 13.4 9.8, 20.1 ng/mL vs. stage II 20.0 14.1, 26.2 ng/mL vs. stage III 21.9 13.0, 29.5 ng/mL; P = 0.002), and need for renal replacement therapy (16.5 11.3, 23.6 ng/mL vs. 25.1 13.3, 30.3 ng/mL; P = 0.005). The association between Angiopoietin‐2 and mortality was significant in unadjusted and adjusted Cox regression models (P ≤ 0.001 for all models), and improved discrimination for mortality when added to MELD score (integrated discrimination increment 0.067; P = 0.001). Conclusion: Angiopoietin‐2 was associated with mortality and other clinically relevant outcomes in a cohort of patients with decompensated cirrhosis with acute kidney injury. Further experimental study of Angiopoietin/Tie2 signaling is warranted to explore its potential mechanistic and therapeutic role in this population.
Hepatocellular carcinoma (HCC) is a highly morbid condition with lack of effective treatment options. HCC arises from chronically inflamed and damaged liver tissue; therefore, chemoprevention may be ...a useful strategy to reduce HCC incidence. Several reports suggest that epigallocatechin gallate (EGCG), extracted from green tea, can suppress liver inflammation and fibrosis in animal models, but its role in HCC chemoprevention is not well established. In this study, male Wistar rats were injected with diethylnitrosamine at 50 mg/kg for 18 weeks to induce cirrhosis and HCC, and EGCG was given in drinking water at a concentration of 0.02%. Clinically achievable dosing of EGCG was well-tolerated in diethylnitrosamine-injured rats and was associated with improved serum liver markers including alanine transaminase, aspartate transaminase, and total bilirubin, and reduced HCC tumor formation. Transcriptomic analysis of diethylnitrosamine-injured hepatic tissue was notable for increased expression of genes associated with the Hoshida high risk HCC gene signature, which was prevented with EGCG treatment. EGCG treatment also inhibited fibrosis progression, which was associated with inactivation of hepatic stellate cells and induction of the senescence-associated secretory phenotype. In conclusion, EGCG administered at clinically safe doses exhibited both chemopreventive and antifibrotic effects in a rat diethylnitrosamine liver injury model.
A comparative analysis of mouse and human pancreatic development may reveal common mechanisms that control key steps as organ morphogenesis and cell proliferation and differentiation. More ...specifically, understanding beta cell development remains an issue, despite recent progress related to their generation from human embryonic and induced pluripotent stem cells. In this study, we use an integrated approach, including prospective isolation, organ culture, and characterization of intermediate stages, and report that cells from human and mouse fetal pancreas can be expanded in the long term and give rise to hollow duct-like structures in 3D cultures. The expanded cells express a combination of markers (E-cadherin, PDX1, NKX6-1, SOX9, and HNF1β) that reveals pancreatic progenitor identity. Proliferation of embryonic progenitors was stimulated by the Wnt agonist R-spondin1 (RSPO1), FGF10, and EGF. This combination of growth factors allowed maintaining human fetal pancreatic progenitors in culture for many passages, a finding not reported previously. Importantly, in the absence of EGF, proliferation was reduced, while endocrine differentiation was significantly enhanced. We conclude that modulation of EGF signaling affects in vitro expansion and differentiation of progenitors from embryonic pancreas of both mice and man.
Patients with simple steatosis (SS) and nonalcoholic steatohepatitis can develop progressive liver fibrosis, which is associated with liver-related mortality. The mechanisms contributing to liver ...fibrosis development in SS, however, are poorly understood. SS is characterized by hepatocellular free fatty acid (FFA) accumulation without lobular inflammation seen in nonalcoholic steatohepatitis. Because the Hippo signaling transcriptional coactivator YAP1 (YAP) has previously been linked with nonalcoholic fatty liver disease (NAFLD)–related fibrosis, we sought to explore how hepatocyte FFAs activate a YAP-mediated profibrogenic program.
We analyzed RNA sequencing data from a GEO DataSet (accession: GSE162694) consisting of 143 patients with NAFLD. We also performed immunohistochemical, immunofluorescence, immunoblot, and quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) in liver specimens from NAFLD subjects, from a murine dietary NAFLD model, and in FFA-treated hepatic spheroids and hepatocytes.
YAP-target gene expression correlated with increasing fibrosis stage in NAFLD patients and was associated with fibrosis in mice fed a NAFLD-inducing diet. Hepatocyte-specific YAP deletion in the murine NAFLD model attenuated diet-induced fibrosis, suggesting a causative role of YAP in NAFLD-related fibrosis. Likewise, in hepatic spheroids composed of Huh7 hepatoma cells and primary human hepatic stellate cells, Huh7 YAP silencing reduced FFA-induced fibrogenic gene expression. Notably, inhibition of p38 mitogen-activated protein kinase could block YAP activation in FFA-treated Huh7 cells.
These studies provide further evidence for the pathological role of YAP in NAFLD-associated fibrosis and that YAP activation in NAFLD may be driven by FFA-induced p38 MAPK activation.
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