stabilizing-hepatocellular-phenotype-using-optimized-synthetic

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

University of Edinburgh

Reply to Elastin-based molecular MRI of liver fibrosis.

Recruitment of trainees into clinical academic medicine remains an area of concern across the globe, with clinical academics making up a dwindling proportion of the medical workforce. To date, few approaches have emphasised early medical student research involvement as a solution to the decline of the clinician-scientist.

Twelve tips for teachers to encourage student engagement in academic medicine.

Tissue progenitor cells are an attractive target for regenerative therapy. In various organs, bone marrow cell (BMC) therapy has shown promising preliminary results, but to date no definite mechanism has been demonstrated to account for the observed benefit in organ regeneration. Tissue injury and regeneration is invariably accompanied by macrophage infiltration, but their influence upon the progenitor cells is incompletely understood, and direct signaling pathways may be obscured by the multiple roles of macrophages during organ injury. We therefore examined a model without injury; a single i.v. injection of unfractionated BMCs in healthy mice. This induced ductular reactions (DRs) in healthy mice. We demonstrate that macrophages within the unfractionated BMCs are responsible for the production of DRs, engrafting in the recipient liver and localizing to the DRs. Engrafted macrophages produce the cytokine TWEAK (TNF-like weak inducer of apoptosis) in situ. We go on to show that recombinant TWEAK activates DRs and that BMC mediated DRs are TWEAK dependent. DRs are accompanied by liver growth, occur in the absence of liver tissue injury and hepatic progenitor cells can be isolated from the livers of mice with DRs. Overall these results reveal a hitherto undescribed mechanism linking macrophage infiltration to DRs in the liver and highlight a rationale for macrophage derived cell therapy in regenerative medicine.

Bone marrow injection stimulates hepatic ductular reactions in the absence of injury via macrophage-mediated TWEAK signaling.

Faithfully recapitulating human physiology "in a dish" from a renewable source remains a holy grail for medicine and pharma. Many procedures have been described that, to a limited extent, exhibit human tissue-specific function in vitro. In particular, incomplete cellular differentiation and/or the loss of cell phenotype postdifferentiation play a major part in this void. We have developed an interdisciplinary approach to address this problem, using skill sets in cell biology, materials chemistry, and pharmacology. Pluripotent stem cells were differentiated to hepatocytes before being replated onto a synthetic surface. Our approach yielded metabolically active hepatocyte populations that displayed stable function for more than 2 weeks in vitro. Although metabolic activity was an important indication of cell utility, the accurate prediction of cellular toxicity in response to specific pharmacological compounds represented our goal. Therefore, detailed analysis of hepatocellular toxicity was performed in response to a custom-built and well-defined compound set and compared with primary human hepatocytes. Importantly, stem cell-derived hepatocytes displayed equivalence to primary human material. Moreover, we demonstrated that our approach was capable of modeling metabolic differences observed in the population. In conclusion, we report that pluripotent stem cell-derived hepatocytes will model toxicity predictably and in a manner comparable to current gold standard assays, representing a major advance in the field.

Developing high-fidelity hepatotoxicity models from pluripotent stem cells.

Active myofibroblast (MF) contraction contributes significantly to the increased intrahepatic vascular resistance that is the primary cause of portal hypertension (PHT) in cirrhosis. We sought proof of concept for direct therapeutic targeting of the dynamic component of PHT and markers of MF activation using short-term administration of the peptide hormone relaxin (RLN). We defined the portal hypotensive effect in rat models of sinusoidal PHT and the expression, activity, and function of the RLN-receptor signaling axis in human liver MFs. The effects of RLN were studied after 8 and 16 weeks carbon tetrachloride intoxication, following bile duct ligation, and in tissue culture models. Hemodynamic changes were analyzed by direct cannulation, perivascular flowprobe, indocyanine green imaging, and functional magnetic resonance imaging. Serum and hepatic nitric oxide (NO) levels were determined by immunoassay. Hepatic inflammation was assessed by histology and serum markers and fibrosis by collagen proportionate area. Gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting and hepatic stellate cell (HSC)-MF contractility by gel contraction assay. Increased expression of RLN receptor (RXFP1) was shown in HSC-MFs and fibrotic liver diseases in both rats and humans. RLN induced a selective and significant reduction in portal pressure in pathologically distinct PHT models, through augmentation of intrahepatic NO signaling and a dramatic reduction in contractile filament expression in HSC-MFs. Critical for translation, RLN did not induce systemic hypotension even in advanced cirrhosis models. Portal blood flow and hepatic oxygenation were increased by RLN in early cirrhosis. Treatment of human HSC-MFs with RLN inhibited contractility and induced an antifibrogenic phenotype in an RXFP1-dependent manner.

Relaxin modulates human and rat hepatic myofibroblast function and ameliorates portal hypertension in vivo.

Pericytes are ubiquitous perivascular cells that have recently attracted interest as potential myofibroblast precursors. In turn, myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis. Given the worldwide burden of fibrotic disease and paucity of therapeutic options available to halt its progression, elucidating the origins of myofibroblasts is of prime importance. The advent of genetic strategies that permit fate-mapping of specific cell populations through permanent and heritable expression of reporter proteins has begun to shed light on the source of the fibrogenic myofibroblast. Here we discuss recent studies in multiple organs that highlight the central role of pericytes in the origins of fibrosis.

Origins of fibrosis: pericytes take centre stage.

ECAT-V: where clinical and research training meet.

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding α(v) integrin subunit because various α(v)-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of β₃, β₅ or β₆ integrins or conditional loss of β₈ integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the α(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of α(v)-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all α(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs.

Despite major progress in the knowledge and management of human liver injury, there are millions of people suffering from chronic liver disease. Currently, the only cure for end-stage liver disease is orthotopic liver transplantation; however, this approach is severely limited by organ donation. Alternative approaches to restoring liver function have therefore been pursued, including the use of somatic and stem cell populations. Although such approaches are essential in developing scalable treatments, there is also an imperative to develop predictive human systems that more effectively study and/or prevent the onset of liver disease and decompensated organ function. We used a renewable human stem cell resource, from defined genetic backgrounds, and drove them through developmental intermediates to yield highly active, drug-inducible, and predictive human hepatocyte populations. Most importantly, stem cell-derived hepatocytes displayed equivalence to primary adult hepatocytes, following incubation with known hepatotoxins. In summary, we have developed a serum-free, scalable, and shippable cell-based model that faithfully predicts the potential for human liver injury. Such a resource has direct application in human modeling and, in the future, could play an important role in developing renewable cell-based therapies.

Accurate prediction of drug-induced liver injury using stem cell-derived populations.

Liver fibrosis results from the co-ordinated actions of myofibroblasts and macrophages, a proportion of which are of bone marrow origin. The functional effect of such bone marrow-derived cells on liver fibrosis is unclear. We examine whether changing bone marrow genotype can down-regulate the liver's fibrotic response to injury and investigate mechanisms involved. Proteinase activated receptor 1 (PAR1) is up-regulated in fibrotic liver disease in humans, and deficiency of PAR1 is associated with reduced liver fibrosis in rodent models. In this study, recipient mice received bone marrow transplantation from PAR1-deficient or wild-type donors prior to carbon tetrachloride-induced liver fibrosis. Bone marrow transplantation alone from PAR1-deficient mice was able to confer significant reductions in hepatic collagen content and activated myofibroblast expansion on wild-type recipients. This effect was associated with a decrease in hepatic scar-associated macrophages and a reduction in macrophage recruitment from the bone marrow. In vitro, PAR1 signalling on bone marrow-derived macrophages directly induced their chemotaxis but did not stimulate proliferation. These data suggest that the bone marrow can modulate the fibrotic response of the liver to recurrent injury. PAR1 signalling can contribute to this response by mechanisms that include the regulation of macrophage recruitment.

Proteinase activated receptor 1 mediated fibrosis in a mouse model of liver injury: a role for bone marrow derived macrophages.

Fibrosis is a highly conserved and co-ordinated protective response to tissue injury. The interaction of multiple pathways, molecules and systems determines whether fibrosis is self-limiting and homeostatic, or whether it is uncontrolled and excessive. Immune cells have been identified as key players in this fibrotic cascade, with the capacity to exert either injury-inducing or repair-promoting effects. A multi-organ approach was recently suggested to identify the core and regulatory pathways in fibrosis, with the aim of integrating the wealth of information emerging from basic fibrosis research. In this Review, we focus on recent advances in liver fibrosis research as a paradigm for wound healing in solid organs and the role of the immune system in regulating and balancing this response.

Liver fibrosis and repair: immune regulation of wound healing in a solid organ.

Identifying molecular factors that contribute to resolution of liver fibrosis.

Following chronic liver injury or when hepatocyte proliferation is impaired, ductular reactions containing hepatic progenitor cells (HPCs) appear in the periportal regions and can regenerate the liver parenchyma. HPCs exist in a niche composed of myofibroblasts, macrophages and laminin matrix. Galectin-3 (Gal-3) is a β-galactoside-binding lectin that binds to laminin and is expressed in injured liver in mice and humans.

Galectin-3 regulates hepatic progenitor cell expansion during liver injury.

To investigate the potential of therapies which reduce glucocorticoid action in patients with type 2 diabetes we performed a randomised, double-blinded, placebo-controlled crossover study of acute glucocorticoid blockade, using the glucocorticoid receptor antagonist RU38486 (mifepristone) and cortisol biosynthesis inhibitor (metyrapone), in 14 men with type 2 diabetes. Stable isotope dilution methodologies were used to measure the rates of appearance (Ra) of glucose, glycerol and free fatty acids (FFAs), including during a low dose (10mU/m(2)/min) hyperinsulinaemic clamp, and sub-group analysis was conducted in patients with high or low liver fat content measured by magnetic resonance spectroscopy (n=7/group). Glucocorticoid blockade lowered fasting glucose and insulin levels, and improved insulin sensitivity of FFA and glycerol turnover and hepatic glucose production. Amongst this population with type 2 diabetes high liver fat was associated with hyperinsulinaemia, higher fasting glucose levels, peripheral and hepatic insulin resistance, and impaired suppression of FFA oxidation and FFA and glycerol turnover during hyperinsulinaemia. Glucocorticoid blockade had similar effects in those with and without high liver fat. Longer term treatments targeting glucocorticoid action may be useful in type 2 diabetes with and without fatty liver.

Article	Year
Reply to Elastin-based molecular MRI of liver fibrosis. Hepatology (Baltimore, Md.)\| PubMed ID: 23424101	2013
Twelve tips for teachers to encourage student engagement in academic medicine. Medical teacher\| PubMed ID: 23496123	2013
Bone marrow injection stimulates hepatic ductular reactions in the absence of injury via macrophage-mediated TWEAK signaling. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23576749	2013
Developing high-fidelity hepatotoxicity models from pluripotent stem cells. Stem cells translational medicine\| PubMed ID: 23757504	2013
Relaxin modulates human and rat hepatic myofibroblast function and ameliorates portal hypertension in vivo. Hepatology (Baltimore, Md.)\| PubMed ID: 23873655	2014
Origins of fibrosis: pericytes take centre stage. F1000prime reports\| PubMed ID: 24049641	2013
ECAT-V: where clinical and research training meet. The Veterinary record\| PubMed ID: 24141161	2013
Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nature medicine\| PubMed ID: 24216753	2013
Accurate prediction of drug-induced liver injury using stem cell-derived populations. Stem cells translational medicine\| PubMed ID: 24375539	2014
Proteinase activated receptor 1 mediated fibrosis in a mouse model of liver injury: a role for bone marrow derived macrophages. PloS one\| PubMed ID: 24475094	2014
Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nature reviews. Immunology\| PubMed ID: 24566915	2014
Identifying molecular factors that contribute to resolution of liver fibrosis. Gastroenterology\| PubMed ID: 24680971	2014
Galectin-3 regulates hepatic progenitor cell expansion during liver injury. Gut\| PubMed ID: 24837171	2014
Effects of acute glucocorticoid blockade on metabolic dysfunction in patients with type 2 diabetes with and without fatty liver. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 25104497	2014

John Iredale

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