Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell ...infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Fibroproliferative diseases are driven by dysregulated tissue repair responses and are a major cause of morbidity and mortality because they affect nearly every organ system. Type 2 cytokine ...responses are critically involved in tissue repair; however, the mechanisms that regulate beneficial regeneration versus pathological fibrosis are not well understood. Here, we have shown that the type 2 effector cytokine interleukin-13 simultaneously, yet independently, directed hepatic fibrosis and the compensatory proliferation of hepatocytes and biliary cells in progressive models of liver disease induced by interleukin-13 overexpression or after infection with Schistosoma mansoni. Using transgenic mice with interleukin-13 signaling genetically disrupted in hepatocytes, cholangiocytes, or resident tissue fibroblasts, we have revealed direct and distinct roles for interleukin-13 in fibrosis, steatosis, cholestasis, and ductular reaction. Together, these studies show that these mechanisms are simultaneously controlled but distinctly regulated by interleukin-13 signaling. Thus, it may be possible to promote interleukin-13-dependent hepatobiliary expansion without generating pathological fibrosis.
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•Type 2 cell-mediated fibrosis and regeneration are independently regulated by IL-13•Pathological fibrosis is driven by direct IL-13 signaling in PDGFRB+ fibroblasts•IL-13 stimulates hepatobiliary progenitor cells and cholangiocytes to proliferate•IL-13 regulates lipogenesis, bile acid synthesis, and biliary-dependent steatosis
Fibroproliferative diseases will affect nearly half of the global population and result in significant loss of quality of life due to comorbidities. In this work, Wynn and colleagues demonstrate that the type 2 cytokine interleukin-13 signals through distinct cellular pathways to simultaneously drive hepatic regeneration, fibrosis, ductular reaction, cholestasis, and steatosis.
Background and Aims
Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for ...the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown.
Approach and Results
Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine‐rich repeat–containing G‐protein‐coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional‐specific markers corresponding to their location of origin. Of particular interest, down‐regulation of biliary markers and up‐regulation of cell‐cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions.
Conclusions
Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.
Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms. In a cross-sectional study of asthmatics of varying severity (n = 51), endobronchial ...tissue gene expression analysis revealed three major patient clusters: TH2-high, TH17-high, and TH2/17-low. TH2-high and TH17-high patterns were mutually exclusive in individual patient samples, and their gene signatures were inversely correlated and differentially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) and TH17 signatures, we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in a preclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells and neutrophilic inflammation in the lung. However, neutralization of IL-13 and IL-17 protected mice from eosinophilia, mucus hyperplasia, and airway hyperreactivity and abolished the neutrophilic inflammation, suggesting that combination therapies targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes.
Nonalcoholic fatty liver disease (NAFLD) is now the most common progressive liver disease in developed countries and is the second leading indication for liver transplantation due to the extensive ...fibrosis it causes. NAFLD progression is thought to be tied to chronic low-level type 1 inflammation originating in the adipose tissue during obesity; however, the specific immunological mechanisms regulating the progression of NAFLD-associated fibrosis in the liver are unclear. To investigate the immunopathogenesis of NAFLD more completely, we investigated adipose dysfunction, nonalcoholic steatohepatitis (NASH), and fibrosis in mice that develop polarized type 1 or type 2 immune responses. Unexpectedly, obese interleukin-10 (IL-10)/IL-4-deficient mice (type 1-polarized) were highly resistant to NASH. This protection was associated with an increased hepatic interferon-γ (IFN-γ) signature. Conversely, IFN-γ-deficient mice progressed rapidly to NASH with evidence of fibrosis dependent on transforming growth factor-β (TGF-β) and IL-13 signaling. Unlike increasing type 1 inflammation and the marked loss of eosinophils seen in expanding adipose tissue, progression of NASH was associated with increasing eosinophilic type 2 liver inflammation in mice and human patient biopsies. Finally, simultaneous inhibition of TGF-β and IL-13 signaling attenuated the fibrotic machinery more completely than TGF-β alone in NAFLD-associated fibrosis. Thus, although type 2 immunity maintains healthy metabolic signaling in adipose tissues, it exacerbates the progression of NAFLD collaboratively with TGF-β in the liver.
There continues to be a major need for more effective inflammatory bowel disease (IBD) therapies. IL-13Rα2 is a decoy receptor that binds the cytokine IL-13 with high affinity and diminishes its ...STAT6-mediated effector functions. Previously, we found that IL-13Rα2 was necessary for IBD in mice deficient in the anti-inflammatory cytokine IL-10. Here, we tested for the first time a therapeutic antibody specifically targeting IL-13Rα2. We also used the antibody and Il13ra2
mice to dissect the role of IL-13Rα2 in IBD pathogenesis and recovery. Il13ra2
mice were modestly protected from induction of dextran sodium sulfate (DSS)-induced colitis. Following a 7-day recovery period, Il13ra2
mice or wild-type mice administered the IL-13Rα2-neutralizing antibody had significantly improved colon health compared to control mice. Neutralizing IL-13Rα2 to increase IL-13 bioavailability promoted resolution of IBD even if neutralization occurred only during recovery. To link our observations in mice to a large human cohort, we conducted a phenome-wide association study of a more active variant of IL-13 (R130Q) that has reduced affinity for IL-13Rα2. Human subjects carrying R130Q reported a lower risk for Crohn's disease. Our findings endorse moving anti-IL-13Rα2 into preclinical drug development with the goal of accelerating recovery and maintaining remission in Crohn's disease patients.
Fibrosis is characterized by progressive replacement of normal tissue by extracellular matrix. Diagnosis relies on biopsies as noninvasive methods for detection and quantification of fibrosis are ...still limited. This work aimed to address the ability of 2 molecular magnetic resonance (MR) probes, EP-3533 and Gd-Hyd, to identify fibrosis and fibrogenesis, respectively, independently of the presence of underlying inflammation in a mouse model of chronic liver disease caused by infection with Schistosoma mansoni.
METHODSThree groups of mice that develop either mild type 2 inflammation and fibrosis (wild type), severe fibrosis with exacerbated type 2 inflammation (Il10Il12bIl13ra2), or minimal fibrosis with marked type 1 inflammation (Il4ra) after infection with S. mansoni were imaged using both probes for determination of signal enhancement. Schistosoma mansoni–infected wild-type mice developed chronic liver fibrosis.
RESULTSThe liver MR signal enhancement after either probe administration was significantly higher in S. mansoni–infected wild-type mice compared with naive animals. The S. mansoni–infected Il4ra mice presented with little liver signal enhancement after probe injection despite the presence of substantial inflammation. Schistosoma mansoni–infected Il10Il12bIl13ra2 mice presented with marked fibrosis, which correlated to increased signal enhancement after injection of either probe.
CONCLUSIONSBoth MR probes, EP-3533 and Gd-Hyd, were specific for fibrosis in this model of chronic liver disease regardless of the presence or severity of the underlying inflammation. These results, in addition to previous findings, show the potential application of both molecular MR probes for detection and quantification of fibrosis from various etiologies.
An increased incidence of dilated cardiomyopathy and atrial thrombosis was noted in a breeding colony of BALB/c mice deficient in IL4 receptor α. The condition affected mice of both sexes and of ...various ages, and extensive testing (microbiology, serology, histopathology) failed
to ascertain the cause. Transmission electron microscopy of heart samples showed structural defects in the myocardial intercalated disks, characterized by unorganized and heavily convoluted arrangement with lower density and less prominent desmosomes and adherens junctions, widening of the
intercellular space, myofibrillar lysis adjacent to intercalated disks, occasional sarcomere lysis with marked myofiber degeneration, vacuolation, accumulation of cell debris, and myelin figures. The intercalated disk contains cell adhesion molecules that form cell junctions, allowing contraction
coupling of cardiomyocytes and the electrical and mechanical connection between cardiac fibers. Thus, defects at this level result in poor myocardial contraction, intracardiac blood stagnation, and consequently cardiac dilation with clinical signs of heart failure. The background strain or,
potentially, the Cre-loxP-mediated recombination system used to create these mice may have contributed to the elevated incidence of cardiomyopathy and atrial thrombosis in this colony. Due to the backcrossing breeding scheme used, we cannot discount the emergence and colonywide dissemination
of a spontaneous mutation that affects the intercalated disk. This report underscores the importance of carefully monitoring genetically modified mice colonies for unexpected phenotypes that may result from spontaneous or unintended mutations or enhanced strain background pathology.
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