George Yeoh

A requirement for DNA synthesis in foetal hepatocyte differentiation: Effect of cytosine arabinoside on the appearance of the liver isoenzyme of pyruvate kinase

Publisher: Elsevier BV

Date: 11-1984

DOI: 10.1111/J.1432-0436.1984.TB00265.X

Abstract: In hepatocyte cultures derived from 15-day-old foetal rats, the appearance of the liver (L) form of pyruvate kinase is blocked when cytosine arabinoside is added on the 2nd day of culture. When added on the 3rd day of culture, the inhibitor of DNA synthesis does not prevent the appearance of the enzyme. If cytosine arabinoside is added on the 2nd day of culture and removed on the 4th day, the enzyme is detected by the 6th day of culture. The specificity of the action of cytosine arabinoside for the L form of pyruvate kinase is in contrast with the lack of effect observed on total protein synthesis and the activity of the embryonic (M2) form of the enzyme.

Glucagon stimulation of DNA-synthesis in neonatal rat liver - evidence for a dnase-mediated effect

Publisher: Wiley

Date: 05-1973

DOI: 10.1111/J.1432-1033.1973.TB02782.X

Abstract: Kabuki syndrome (KS) is a complex multisystem developmental disorder associated with mutation of genes encoding histone-modifying proteins. In addition to craniofacial, intellectual, and cardiac defects, KS is also characterized by humoral immune deficiency and autoimmune disease, yet no detailed molecular characterization of the KS-associated immune phenotype has been reported. We sought to characterize the humoral immune defects found in patients with KS with lysine methyltransferase 2D (KMT2D) mutations. We comprehensively characterized B-cell function in a cohort (n = 13) of patients with KS (age, 4 months to 27 years). Three quarters (77%) of the cohort had a detectable heterozygous KMT2D mutation (50% nonsense, 20% splice site, and 30% missense mutations), and 70% of the reported mutations are novel. Among the patients with KMT2D mutations (KMT2D(Mut/+)), hypogammaglobulinemia was detected in all but 1 patient, with IgA deficiency affecting 90% of patients and a deficiency in at least 1 other isoform seen in 40% of patients. Numbers of total memory (CD27(+)) and class-switched memory B cells (IgM(-)) were significantly reduced in patients with KMT2D(Mut/+) mutations compared with numbers in control subjects (P < .001). Patients with KMT2D(Mut/+) mutations also had significantly reduced rates of somatic hypermutation in IgG (P = .003) but not IgA or IgM heavy chain sequences. Impaired terminal differentiation was noted in primary B cells from patients with KMT2D(Mut/+) mutations. Autoimmune pathology was observed in patients with missense mutations affecting the SET domain and its adjacent domains. In patients with KS, autosomal dominant KMT2D mutations are associated with dysregulation of terminal B-cell differentiation, leading to humoral immune deficiency and, in some cases, autoimmunity. All patients with KS should undergo serial clinical immune evaluations.

Differential regulation of rodent hepatocyte and oval cell proliferation by interferon gamma

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 2005

DOI: 10.1002/HEP.20645

Abstract: Hepatocytes and intrahepatic progenitor cells (oval cells) have similar responses to most growth factors but rarely proliferate together. Oval cells constitute a reserve compartment that is activated when hepatocyte proliferation is inhibited. Interferon gamma (IFN-gamma) increases in liver injury that involves oval cell responses, but it is not upregulated during liver regeneration after partial hepatectomy. Based on these observations, we used well-characterized lines of hepatocytes (AML-12 cells) and oval cells (LE-6 cells) to investigate the potential mechanisms that regulate differential growth responses in hepatocytes and oval cells. We show that IFN-gamma blocks hepatocyte proliferation in vivo, and that in combination with either tumor necrosis factor (TNF) or lipopolysaccharide (LPS), it causes cell cycle arrest in hepatocytes but stimulates oval cell proliferation in cultured cells. The hepatocyte cell cycle arrest is reversible, is p53-independent, and is not associated with apoptosis. Treatment of AML-12 hepatocytes with IFN-gamma/LPS or IFN-gamma/TNF, but not with in idual cytokines, induced NO synthase and generated NO, while similarly treated oval cells produced little if any NO. Generation of NO by an NO donor reproduced the inhibitory effect of the cytokine combinations on AML-12 cell replication, while NO inhibitors abolish the replication deficiency. In conclusion, we propose that IFN-gamma, in conjunction with TNF or LPS, can both inhibit hepatocyte proliferation through the generation of NO and stimulate oval cell replication. The response of hepatocytes and oval cells to cytokine combinations may contribute to the differential proliferation of these cells in hepatic growth processes.

DIFFERENTIATION OF OVAL CELLS INTO DUCT-LIKE CELLS IN PRENEOPLASTIC LIVER OF RATS PLACED ON A CHOLINE-DEFICIENT DIET SUPPLEMENTED WITH ETHIONINE

Publisher: Oxford University Press (OUP)

Date: 1994

DOI: 10.1093/CARCIN/15.12.2747

Abstract: Feeding male Wistar rats a choline-deficient diet containing 0.07% DL-ethionine (CDE diet) for up to 5 weeks results in the production of two distinct non-parenchymal cell populations, oval and duct-like cells. These cells can undergo replication and display different patterns of expression of glutathione S-transferases (GSTs) and pyruvate kinases (PKs). Oval cells were first detected around the periportal region after 1 week of CDE treatment and infiltrated the parenchyma after 2 weeks. Duct-like structures first appeared as isolated ducts in the parenchymal region at 2 weeks and were easily detected after 2.5 weeks. These duct-like structures differed from the bile ducts which reside in the portal region. Large concentrations of duct-like structures in cyst-like clusters were detected after 5 weeks. Enlargement of these structures from single ducts to clusters of up to 20 ducts was observed over 3-5 weeks of CDE treatment. The number of cells forming a duct increased from 5 to 30 cells. We established a double immunocytochemical staining technique to characterize the oval and duct-like cells for their expression of GSTs and PKs. pi GST and M2-PK, which are fetal hepatocytes isoenzymes, are present in virtually all the oval and duct-like cells. Most of the oval cells are devoid of the adult hepatocytes markers, alpha GST, mu GST and L-PK. There are two sub-populations of duct-like cells, one which expresses only fetal markers and the other which co-expresses the adult and fetal isoenzymes. Hence, oval cells display characteristics of fetal hepatocytes and some duct-like cells appear more mature than oval cells. Using a combination of double immunocytochemical and [3H]thymidine labelling techniques we have established that oval cells differentiate into duct-like cells.

ROLE OF MYONUCLEI IN MUSCLE REGENERATION - INVITRO STUDY

Publisher: Wiley

Date: 08-1978

DOI: 10.1002/JCP.1040960213

Abstract: It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell ision to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or "freed" as a result of the trauma, play any role in muscle regeneration remains unresolved. In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub-cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H3-thymidine and their ultimate fate, either as "free" myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub-cultured. In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re-enter the mitotic cycle.

ENZYMIC DIFFERENTIATION IN CULTURED FETAL HEPATOCYTES OF THE RAT - INDUCTION OF SERINE DEHYDRATASE ACTIVITY BY DEXAMETHASONE AND DIBUTYRYL-CYCLIC-AMP

Publisher: Elsevier BV

Date: 07-1983

DOI: 10.1111/J.1432-0436.1983.TB01325.X

Abstract: Serine dehydratase activity is absent from the rat foetal liver and normally appears in the immediate postnatal period. In foetal hepatocytes cultured from livers of various gestational ages, enzyme activity can be induced only in the simultaneous presence of dexamethasone and dibutyryl cyclic AMP in the culture medium. Adrenalin and glucagon can replace dibutyryl cyclic AMP. Actinomycin D and cordycepin both repress the response, a result that suggests the induction of enzyme synthesis involves the initial transcription of the enzyme gene(s). Inducibility is assessed in cultures prepared from foetuses aged between 15 and 19 days of gestation after 48 h of culture. No induction is obtained in cells from 15 day foetuses, only a marginal induction from 16 day foetuses, and a substantial induction from older foetuses. In cultures from older foetuses, 6-18 fold inductions are already demonstrable after 24 h of culture. While hepatocytes from more mature foetuses are able to acquire inductibility during culture, cells taken from 15 day foetuses do not develop in the same manner in spite of being maintained under identical conditions. These results suggest that a differentiation event occurs in vivo at about day 16 of foetal development which renders the hepatocyte inducible when cultured. Cells taken prior to this stage do not appear to acquire inducibility. This system represents a case of enzymic differentiation and requires the simultaneous presence of two inducer molecules. The mechanism of induction may represent a unique system in cellular differentiation.

Differential lymphotoxin-β and interferon gamma signalling during mouse liver regeneration induced by chronic and acute injury

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 2005

DOI: 10.1002/HEP.20520

Abstract: The liver regenerates after acute injury via hepatocyte cell ision during chronic injury, when hepatocyte replication is impaired or blocked, liver progenitor oval cells mediate liver regeneration. If both regeneration options are blocked in animal models, then liver failure and death ensues. The mechanisms underlying oval cell induction, proliferation, and subsequent liver regeneration remain poorly characterized. In particular, cell-signaling pathways that distinguish the alternative pathways are unknown. This study shows that in a mouse model, hepatic expression of lymphotoxin-beta (LTbeta) and interferon gamma (IFNgamma) transcripts is increased in response to the choline-deficient, ethionine-supplemented (CDE) diet, which induces oval cell-mediated liver regeneration. Oval cells express LTbeta and IFNgamma transcripts, contributing to the increased expression in the liver of mice fed the CDE diet. An attenuated oval cell response to such a diet was observed in LTbeta receptor-, LTbeta-, and IFNgamma-gene targeted mice. Loss of LTbeta and LTbeta receptor signaling reduced the number of oval cells expressing A6 and muscle pyruvate kinase. The lack of IFNgamma signaling reduced muscle pyruvate kinase(+), but not A6(+), oval cells. In contrast, partial hepatectomy suppressed LTbeta and IFNgamma transcripts. We also show that IFNgamma induces STAT-3 phosphorylation in an oval cell line. In conclusion, LTbeta, LTbeta receptor, and IFNgamma are involved in oval cell-mediated, but not hepatocyte-mediated, liver regeneration, and the absence of these pathways impairs the oval cell-dependent regenerative response.

Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.JHEP.2006.08.015

Abstract: Liver regeneration following chronic injury is associated with inflammation, the proliferation of liver progenitor (oval) cells and fibrosis. Previous studies identified interferon-gamma as a key mediator of oval cell proliferation. Interferon-gamma is known to regulate Th1 cell activities during immune challenge. Therefore, we hypothesised that progenitor cell-mediated regeneration is associated with a Th1 immune response. C57Bl/6 (normal Th1 response) and BALB/c mice (deficient in Th1 signalling) were placed on a carcinogenic diet to induce liver injury, progenitor cell proliferation and fibrosis. Serum transaminases and mortality were elevated in BALB/c mice fed the diet. Proliferation of liver progenitor cells was significantly attenuated in BALB/c animals. The pattern of cytokine expression and inflammation differed between strains. Liver fibrosis and hepatic stellate cell activation were significantly inhibited in BALB/c mice compared to C57Bl/6. In addition, interferon-gamma knockout mice also showed reduced fibrosis compared to wild type. These findings are in contrast to published results, in which interferon-gamma is shown to be anti-fibrogenic. Our data demonstrate that the hepatic progenitor cell response to a CDE diet is inhibited in mice lacking Th1 immune signalling and further show that this inhibition is associated with reduced liver fibrosis.

Antiproliferative effects of interferon alpha on hepatic progenitor cells in vitro and in vivo

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 05-2006

DOI: 10.1002/HEP.21170

Abstract: Hepatic progenitor cells (called oval cells in rodents) proliferate during chronic liver injury. They have been suggested as targets of malignant transformation in chronic liver diseases, including chronic hepatitis C. Interferon alpha therapy reduces the risk of hepatocellular carcinoma (HCC) in chronic hepatitis C regardless of viral clearance. The aim of this study was to determine whether interferon alpha could reduce the risk of HCC by modifying preneoplastic events in the hepatic progenitor cell population. Pre- and post-treatment liver biopsies were evaluated for changes in t he hepaticprogenitor cell population in 16 patients with non-responding chronic hepatitis C Interferon alpha-based treatment significantly reduced the numbers of c-kit-positive hepatic progenitor cells by 50%. To determine the mechanism of cell number reduction, the effects of interferon alpha on murinehepatic progenitor cells were studied in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay and proliferating cell nuclear antigen staining showed that interferon alpha had a dose-dependent, anti-proliferative effect Interferon alpha stimulated hepatocytic and biliary differentiation of the oval cell lines reflected by increased expression of albumin and cytokeratin19 accompanied by decreased expression of alphafetoprotein and Thy-1. To validatethese results in vivo, mice were placed on the choline-deficient, ethionine-supplemented diet to induce liver injury and oval cell proliferation and treated with pegylated interferon alpha 2b for 2 weeks. This resulted in a significant four-fold reduction in the number of oval cells (P < .05). In conclusion, interferon alpha-based treatment reduced the number of hepatic progenitor cells in chronic liver injury by modulating apoptosis, proliferation, and differentiation. Supplementay material for this article can

Transferrin endocytosis and iron uptake in developing myogenic cells in culture: effects of microtubular and metabolic inhibitors, sulphydryl reagents and lysosomotrophic agents.

Publisher: Wiley

Date: 12-1988

DOI: 10.1002/JCP.1041370313

Abstract: The experiments described in this study were designed to investigate receptor-mediated endocytosis of transferrin and its role in iron uptake by cultured chick presumptive myoblasts ( iding and non- iding) and myotubes. The effects of a variety of inhibitors on the internalization of transferrin and iron were investigated and three main effects were found: (i) sulphydryl reagents and microtubular inhibitors reduced the rate of transferrin and iron internalization to similar degrees, (ii) metabolic inhibitors reduced the rate of iron uptake more than that of transferrin endocytosis, and (iii) lysosomotrophic agents almost completely abolished iron accumulation by the cells without any effect on the rate of transferrin internalization. The results suggest that metabolic energy is required not only for the endocytosis of transferrin but also for subsequent steps in the iron uptake process, and that iron release from transferrin occurs in acidified endosomes. Overall, these experiments show that all or virtually all of the iron taken up by developing muscle cells from transferrin occurs as a consequence of receptor-mediated endocytosis of the protein.

GLUCAGON STIMULATION OF DNA-SYNTHESIS IN NEONATAL RAT-LIVER - STUDIES ON ENZYMES OF DNA-SYNTHESIS

Publisher: Elsevier BV

Date: 02-1972

DOI: 10.1016/0020-711X(72)90020-1

The vascularised chamber device significantly enhances the survival of transplanted liver organoids

Publisher: Cold Spring Harbor Laboratory

Date: 25-04-2023

DOI: 10.1101/2023.04.24.538062

Abstract: Organoid transplantation has a promising future in the treatment of liver disease, but a major limitation is the lack of guidance on the most appropriate method for transplantation that maximises organoid survival. Human induced pluripotent stem cell (hiPSC)-derived liver progenitor cell organoids were transplanted into four different transplantation sites in a mouse model of liver disease, using five organoid delivery methods. Organoids were transplanted into the vascularised chamber device established in the groin, or into the liver, spleen, and subcutaneous fat. For organoid transplantations into the liver, organoids were delivered either in Matrigel alone, or in Matrigel and a polyurethane scaffold. At 2 weeks post-transplantation, the vascularised chamber had the highest organoid survival, which was 5.1x higher than the site with second highest survival ( p =0.0002), being the intra-hepatic scaffold approach. No organoid survival was observed when delivered into the liver without a scaffold, or when injected into the spleen. Very low survival occurred in transplantations into subcutaneous fat. Animals with the vascularised chamber also had the highest levels of human albumin (0.33 ± 0.09 ng/mL). This study provides strong evidence supporting the use of the vascularised chamber for future liver organoid transplantation studies, including its translation into clinical therapy.

The association between murine cytomegalovirus induced hepatitis and the accummulation of oval cells

Publisher: Wiley

Date: 04-01-1998

DOI: 10.1046/J.1365-2613.1998.00089.X

Abstract: The accumulation of oval cells is an early event in the development of hepatocellular carcinoma induced by certain experimental regimes involving hepatocarcinogens. Oval cells have also been observed during chronic hepatitis induced by alcohol and iron overload. In this study, livers of murine cytomegalovirus (MCMV) infected mice were examined to determine whether hepatitis induced by this virus could initiate oval cell proliferation. BALB/c and C57BL/6 mice were infected with MCMV and studied 4, 8, 10 and 12 months later, alongside control (uninfected) mice. The livers were examined histochemically, immunocytochemically and by in situ hybridization to identify oval cells, inflammatory cells and proliferating cells. Oval cells were seen in the periportal regions of livers from MCMV infected BALB/c mice. These increased in number from 4 to 12 months after infection in parallel with increases in the numbers of inflammatory cells, even though cells expressing MCMV antigens were no longer evident in these s les. Proliferating oval cells and hepatocytes were identified by PCNA staining, indicating an increased level of liver regeneration in the infected livers. C57BL/6 mice are less susceptible to persistent MCMV hepatitis and had fewer oval cells than BALB/c mice. Thus the study demonstrates an association between MCMV induced hepatitis, inflammation, and presence of oval cells.

GLUTATHIONE-S-TRANSFERASE (MU CLASS) AS AN EARLY MARKER OF AZASERINE-INDUCED FOCI IN THE RAT PANCREAS

Publisher: Oxford University Press (OUP)

Date: 1991

DOI: 10.1093/CARCIN/12.7.1237

Abstract: The alpha, mu and pi classes of glutathione S-transferase (GST) were evaluated as early immunocytochemical markers for the development of atypical foci within the pancreases of azaserine treated rats. Changes detected with haematoxylin and eosin (H&E) were compared with those detected by immunocytochemistry using antibodies raised against each class of GST. All foci detected with H&E staining were classified as acidophilic atypical acinar cell nodules (AACN), which have previously been reported in this model. All of these AACN overexpressed GST mu. However, 64% of foci detected with GST mu staining had not been identified as AACN during a prior examination with H&E. Re-evaluation of the H&E sections revealed that some of these foci showed subtle morphological changes which are indicative of AACN. In many cases, however, no morphological difference could be seen with H&E staining. We conclude that immunocytochemical staining for GST mu is a more reliable and sensitive method than H&E for detecting the early stages of azaserine-induced foci. Furthermore, we suggest that studies on the incidence and growth of these foci can be shortened considerably if GST mu staining is used in conjunction with H&E.

Detection of histidine kinases via a filter-based assay and reverse-phase thin-layer chromatographic phosphoamino acid analysis

Publisher: Elsevier BV

Date: 12-2003

DOI: 10.1016/J.AB.2003.08.035

Abstract: The methods that detect histidine phosphorylation have largely been either laborious or difficult to apply quantitatively. The major difficulty in assessing for its presence is its alkali-stable, acid-labile nature. While an assay that detects alkali-stable phosphorylation has been developed, it does not distinguish phosphohistidine from other alkali-stable phosphoamino acids. Using this established method, we extend the assay to facilitate the specific detection of phosphohistidine. We use the acid-lability of phosphohistidine as a defining feature in our approach for its detection. In addition, reverse-phase thin-layer chromatography was utilized to conclusively demonstrate the viability of the conditions that we implement in the assay for the selective detection of phosphohistidine. In summary, this report describes a rapid filter-based kinase assay that quantitatively measures histidine kinase activity, even in the presence of tyrosine kinase activity.

DIMETHYL-SULFOXIDE INDUCTION OF TRANSFERRIN RECEPTORS ON FRIEND-ERYTHROLEUKEMIA CELLS

Publisher: Elsevier BV

Date: 08-1979

DOI: 10.1016/0045-6039(79)90008-3

Abstract: The genetic heterogeneity of the spirochete Borrelia burgdorferi within single adult black-legged ticks from Shelter Island, N.Y., was determined by cold, single-strand conformation polymorphism (SSCP) analysis. The central region of the ospA gene of B. burgdorferi from infected ticks was lified by nested PCR. Amplified product of the correct size was obtained from 20 to 45 ticks (44%). This is the fraction of ticks that is expected to be infected with B. burgdorferi. Four variant classes were determined by SSCP analysis. Eight ticks were infected with a single variant, nine ticks were infected with two variants, two ticks were infected with three variants, and one tick was infected with all four variants. DNA from each variant was sequenced. Five different sequences were found. The sequence of each variant was different from that of another variant by a single base. SSCP analysis could distinguish three of the four single-base changes found in the region.

PHOSPHOENOLPYRUVATE CARBOXYKINASE IN CULTURED FETAL HEPATOCYTES FROM THE RAT - ONTOGENY OF HORMONE INDUCIBILITY AND ROLE OF GLUCOCORTICOIDS AND INSULIN IN ENZYME-INDUCTION

Publisher: Wiley

Date: 02-1980

DOI: 10.1111/J.1432-1033.1980.TB04404.X

Abstract: Fasting and especially intermittent fasting have been shown to be an effective intervention in many diseases, such as obesity and diabetes. The fasting-mimicking diet (FMD) has recently been found to ameliorate metabolic disorders. To investigate the effect of a new type of low-protein low-carbohydrate FMD on diabetes, we tested an FMD in

THE DEVELOPMENT OF PHENYLALANINE-HYDROXYLASE IN RAT-LIVER - INVIVO, AND INVITRO STUDIES UTILIZING FETAL HEPATOCYTE CULTURES

Publisher: Elsevier BV

Date: 06-1988

DOI: 10.1111/J.1432-0436.1988.TB00590.X

Abstract: Phenylalanine hydroxylase (PAH) is first detected in the liver of 21-day-gestation rats. Activity increases after birth, and in 10-day-postnatal rats it is about equal to that observed in the adult. The developmental pattern for the enzyme is reflected in the level of its mRNA determined by hybridization to 32P-cDNA, which is specific for PAH. Studies with cultured adult hepatocytes reveal that the addition of dexamethasone and dibutyryl cAMP to the medium maximizes the yield of enzyme. Hepatocytes derived from 21-day-gestation rats will produce enzyme in cultures maintained in medium supplemented with dexamethasone and dibutyryl cAMP. However, less mature cells, taken from 19-day-gestation rats do not produce measurable levels of enzyme activity. The relative amounts of PAH mRNA in the respective cultures reflect the level of PAH activity. Interestingly, after 3 days of culture, 19-day-gestation hepatocytes can be shown to express PAH mRNA. Therefore, with respect to the expression of PAH, we conclude that 19-day-gestation liver cells will differentiate during culture.

Human Amnion Epithelial Cell Therapy for Chronic Liver Disease

Publisher: Hindawi Limited

Date: 07-08-2019

DOI: 10.1155/2019/8106482

Abstract: Liver fibrosis is a common consequence of chronic liver disease. Over time, liver fibrosis can develop into liver cirrhosis. Current therapies for liver fibrosis are limited, and liver transplant is the only curative therapy for patients who progress to end-stage disease. A potential approach to treat chronic liver disease with increasing interest is cell-based therapy. Among the multiple cell types which have been proposed for therapeutic uses, human amnion epithelial cells and amniotic fluid-derived mesenchymal cells are promising. These cells are highly abundant, and their use poses no ethical concern. Furthermore, they exert potent anti-inflammatory and antifibrotic effects in animal models of liver injury. This review highlights the therapeutic characteristics and discusses how human amnion epithelial cells can be utilised as a therapeutic tool for chronic liver disease.

FACTORS AFFECTING INCORPORATION OF C-14 LEUCINE INTO ALBUMIN AND TRANSFERRIN BY LIVER IN POSTNATAL RAT

Publisher: Elsevier BV

Date: 1975

DOI: 10.1016/0005-2787(75)90144-6

Abstract: The mechanisms responsible for the increase in incorporation of radioactive amino acid into albumin, transferrin and total soluble liver protein which occurs in the immediate postnatal period in the rat was investigated in rats delivered surgically in the last 2 days of gestation. The in vivo incorporation of [14C]leucine into the proteins in the liver was low at birth, but increased rapidly during the first half hour after delivery and then more gradually during the subsequent 4.5 h. Neonatal adrenalectomy had no effect on this pattern of results. Intraperitoneal administration of an amino acid supplement had little effect on [14C]leucine incorporation immediately after birth but increased incorporation at 0.5 h and eliminated the second phase of rising incorporation values between 0.5 and 5 h. The in vitro incorporation of 14C into albumin, transferrin and total protein by slices of the liver from animals immediately after delivery was as great as with slices from animals 5 h after delivery. It is concluded that the initial increase in synthesis of proteins in the liver in the first 0.5 h after delivery is probably due to an increase in the supply of metabolic energy due to improved oxygenation of the rats and that the slower increase in protein synthesis between 0.5-5.0 h results from an improved supply of amino acids to liver cells. It is unlikely that changes in the secretion of adrenal hormones in involved.

Oval cell-mediated liver regeneration: Role of cytokines and growth factors

Publisher: Wiley

Date: 20-12-2003

DOI: 10.1046/J.1440-1746.2003.02906.X

Abstract: In experimental models, which induce liver damage and simultaneously block hepatocyte proliferation, the recruitment of a hepatic progenitor cell population comprised of oval cells is invariably observed. There is a substantial body of evidence to suggest that oval cells are involved in liver regeneration, as they differentiate into hepatocytes and biliary cells. Recently, bone marrow cells were shown to be a source of a stem cells with the capacity to repopulate the liver. Presently, the relationship between bone marrow cells and oval cells is unclear. Investigations will be greatly assisted by the availability of in vitro models based on a knowledge of cytokines that affect oval cells. While the cytokines, which regulate the different hematopoietic lineages, are well characterized, there is relatively little information regarding those that influence oval cells. This review outlines recent developments in the field of oval cell research and focuses on cytokines and growth factors that have been implicated in regulating oval cell proliferation and differentiation.

AZASERINE-INDUCED PANCREATIC FOCI - DETECTION, GROWTH, LABELING INDEX AND RESPONSE TO RAW SOYA FLOUR

Publisher: Oxford University Press (OUP)

Date: 1992

DOI: 10.1093/CARCIN/13.9.1519

Abstract: Atypical acinar cell foci were induced in the pancreases of rats by injection of azaserine. An incubation period of 6 weeks was sufficient for the detection of all glutathione S-transferase mu positive foci. In chow-fed rats, the labelling index of foci was 12-fold higher than normal pancreatic tissue. Feeding rats raw soya flour (RSF) for up to 20 weeks did not increase the number of foci per pancreas but did produce significant increases in labelling index and growth rate. In normal pancreatic tissue, the trophic response was complete after 4 weeks of RSF feeding. In foci, however, the trophic response to RSF was prolonged. Involution of normal pancreatic tissue was seen in rats fed RSF for 19 weeks and then switched to chow 1 week prior to death. No evidence for involution was seen in the foci of these animals, although a 40-fold reduction was seen in labelling index. The labelling index of these foci was reduced to the level seen in normal tissue of chow-fed rats. These results are consistent with increased cholecystokinin (CCK) responsiveness and CCK dependence in azaserine-induced pancreatic foci.

Uptake and cytotoxicity of chitosan nanoparticles in human liver cells

Publisher: Elsevier BV

Date: 12-2010

DOI: 10.1016/J.TAAP.2010.08.029

Abstract: Despite extensive research into the biomedical and pharmaceutical applications of nanoparticles, and the liver being the main detoxifying organ in the human body, there are limited studies which delineate the hepatotoxicity of nanoparticles. This paper reports on the biological interactions between liver cells and chitosan nanoparticles, which have been widely recognised as biocompatible. Using the MTT assay, human liver cells were shown to tolerate up to 4h of exposure to 0.5% w/v of chitosan nanoparticles (18±1 nm, 7.5±1.0 mV in culture medium). At nanoparticle concentrations above 0.5% w/v, cell membrane integrity was compromised as evidenced by leakage of alanine transaminase into the extracellular milieu, and there was a dose-dependent increase in CYP3A4 enzyme activity. Uptake of chitosan nanoparticles into the cell nucleus was observed by confocal microscopic analysis after 4h exposure with 1% w/v of chitosan nanoparticles. Electron micrographs further suggest necrotic or autophagic cell death, possibly caused by cell membrane damage and resultant enzyme leakage.

Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma

Publisher: Oxford University Press (OUP)

Date: 03-2002

DOI: 10.1093/CARCIN/23.3.435

Abstract: Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.

EFFECT OF ONCOGENIC VIRUS ON MUSCLE DIFFERENTIATION

Publisher: Proceedings of the National Academy of Sciences

Date: 10-1975

DOI: 10.1073/PNAS.72.10.4051

Abstract: Chick muscle cultures infected with wild-type Rous sarcoma virus form myotubes, but these myotubes vacuolate and by day 6 most have degenerated, leaving only large numbers of transformed mononucleated, replicating cells. Muscle cultures infected with a temperature-sensitive mutant (TS) at permissive temperatures behave as cells infected with wild-type Rous sarcoma virus. TS-infected cells reared for 8 days at nonpermissive temperature form contracting myotubes, plus large numbers of fibroblastic cells. If these cultures are lowered to permissive temperature, within 72 hr the myotubes vacuolate and degenerate, whereas the mononucleated cells transform. If replicating TS-transformed cells after 8 days at permissive temperature are shifted to nonpermissive temperature, within 72 hr many cells fuse and form contracting, post-mitotic myotubes. Creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) levels parallel the formation and degeneration of myotubes during these temperature shifts. If the viral transforming gene is expressed in the post-mitotic myotubes it is lethal, whereas it is not lethal if expressed in replicating percursor myogenic cells. The viral gene expression at permissive temperature blocks further myogenesis depending on the position of the cells in the myogenic program. The virus does not cancel the replicating, transformed myogenic cells' commitment to, or position in, the myogenic lineage. When the transforming action of the virus is suppressed, the normal myogenic program resumes.

A modified choline-deficient, ethionine-supplemented diet protocol effectively induces oval cells in mouse liver

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 09-2001

DOI: 10.1053/JHEP.2001.26751

Abstract: Several reliable and reproducible methods are available to induce oval cells in rat liver. Effective methods often involve inhibiting proliferation in hepatocytes using an alkylating agent, then subjecting the rat to partial hepatectomy (PH). The surgery is difficult to perform reproducibly in mice. Approaches that do not include partial hepatectomy, such as administration of D-galactosamine, are ineffective in mice. We found that a choline-deficient, ethionine-supplemented (CDE) diet, which is very effective in rats, leads to high morbidity and mortality when administered to mice. This article outlines an alternative protocol by which a CDE diet can be administered to mice. This diet is shown to be highly effective for oval cell induction, without causing high mortality. It takes less time and is at least as effective as other commonly used protocols for inducing oval cells in mice.

Invading macrophages play a major role in the liver progenitor cell response to chronic liver injury

Publisher: Elsevier BV

Date: 09-2010

DOI: 10.1016/J.JHEP.2010.04.010

Abstract: Although a strong association between liver progenitor cells (LPCs) and inflammation exists in many chronic liver diseases, the exact role of the immune system in LPC-mediated hepatic regeneration remains unclear. A number of pro-inflammatory factors were identified in cytokine knockout mice in which the LPC response was attenuated but neither the mechanism nor the producing cells are known. To identify the critical immune cells and cytokines required in the LPC response, we compared two diet-induced models of liver injury with two recently established transgenic models of immune-mediated hepatitis. Despite severe inflammation being observed in all models, the generation of LPCs was highly dependent on the cause and kinetics of liver damage. The LPC response was associated with an increase of macrophages and CD8(+) T cells but not natural killer cells. T cell-deficient mice were able to mount a LPC response, albeit delayed, suggesting that T cells are not essential. Mice mounting an LPC response showed elevated numbers of Kupffer cells and invading CX(3)CR1(high)CCR2(high) macrophages secreting persistent high levels of tumour necrosis factor alpha (TNFalpha), a major cytokine involved in the LPC response. Liver macrophages are an important determinant of LPC expansion during liver regeneration in models of diet- and immune-mediated liver injury. Invading macrophages in particular provide pro-mitogenic cytokines such as TNFalpha that underpin the process. LPC themselves are a source of chemokines (CCL2, CX(3)CL1) that attract infiltrating macrophages.

TRANSFERRIN RECEPTORS AND IRON UPTAKE DURING ERYTHROID CELL-DEVELOPMENT

Publisher: Elsevier BV

Date: 05-1982

DOI: 10.1016/0005-2736(82)90547-8

Abstract: Experiments were performed to determine the level of transferrin receptors and rate of transferrin-bound iron uptake by various immature erythroid cell populations. Developing erythroid cells from the rat and mouse foetal liver at various stages of gestation were studied. In addition Friend leukaemic cells grown in culture were examined. The transferrin receptor level of Friend cells was similar to that of erythroid cells from the mouse foetal liver. During erythroid cell development the transferrin receptor level increased from about 300,000 per cell at the early normoblast stage to reach a maximum of about 8000,000 per cell on intermediate normoblasts. Further maturation of intermediate normoblasts was accompanied by a decline in the number of transferrin receptors, reaching a level of 105,000 in the circulating reticulocyte. The rate of iron uptake from transferrin during erythroid cell development was found to correlate closely with the number of transferrin receptors. In each of the immature erythroid cell populations studied the rate of iron uptake was about 36 iron atoms per receptor per hour. These results indicate that the level of transferrin receptors may be the major factor which determines the rate of iron uptake during erythroid cell development.

Oval cell numbers in human chronic liver diseases are directly related to disease severity

Publisher: Elsevier BV

Date: 02-1999

DOI: 10.1016/S0002-9440(10)65299-6

HORMONAL-REGULATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE IN CULTURED FETAL HEPATOCYTES FROM THE RAT

Publisher: Wiley

Date: 12-1979

DOI: 10.1111/J.1432-1033.1979.TB06266.X

Abstract: In this study, we evaluated a rapid whole-blood test, BM-test Helicobacter pylori, for detection of H. pylori infection in 144 and 48 patients with other gastrointestinal symptoms and with gastric cancer, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the test correlated well with the standards used for the calculation, i.e., serology by enzyme-linked immunosorbent assay or culture and histology.

EFFECT OF CORYNETOXIN ISOLATED FROM PARASITIZED ANNUAL RYEGRASS ON ALBUMIN AND TRANSFERRIN SYNTHESIS AND SECRETION BY CULTURED FETAL-RAT HEPATOCYTES

Publisher: Elsevier BV

Date: 04-1984

DOI: 10.1016/0014-4827(84)90392-6

Abstract: A group of glycolipid toxins, corynetoxin (CT), isolated from parasitized annual ryegrass, was shown to suppress the synthesis of both albumin and transferrin by cultured fetal rat hepatocytes. Based on [3H]leucine incorporation, inhibition of transferrin synthesis was greater than that of both albumin and total protein synthesis. As a result, the secretion of albumin and transferrin was decreased. The incorporation of [3H]N-AcGlc into cellular glycoproteins was only marginally affected by CT, although a dramatic reduction was observed with respect to the secreted proteins. Transferrin secreted into the culture medium was substantially non-glycosylated, judging by the absence of [3H]N-AcGlc. These studies suggested that the toxin preferentially affects the synthesis, and hence the secretion of glycoproteins, although it did not block the secretion of the proteins albumin and transferrin, as these did not accumulate intercellularly. Since transferrin labelled with [3H]leucine but not [3H]N-AcGlc is detected in the culture medium of hepatocytes exposed to CT, it was concluded that glycosylation of the protein is not required for secretion. This study shows that the effects of CT on protein synthesis and secretion in cultured hepatocytes are similar to those reported for tunicamycin (TM).

DIRECTED ISSUE: LIVER GROWTH, DEVELOPMENT, AND DISEASE DEDICATED TO ROBERT HENRY (ROB) COSTA Preface

Publisher: Elsevier BV

Date: 02-2011

DOI: 10.1016/J.BIOCEL.2010.09.004

An Akt/Hypoxia-Inducible Factor-1 alpha/Platelet-Derived Growth Factor-BB Autocrine Loop Mediates Hypoxia-Induced Chemoresistance in Liver Cancer Cells and Tumorigenic Hepatic Progenitor Cells

Publisher: American Association for Cancer Research (AACR)

Date: 15-05-2009

DOI: 10.1158/1078-0432.CCR-08-2127

Abstract: Purpose: The goals of the present study were to investigate the mechanism of hypoxia-mediated chemoresistance in liver cancer cells and tumorigenic hepatic progenitor (oval) cells and to determine whether disrupting an Akt/hypoxia-inducible factor-1α (HIF-1α) latelet-derived growth factor (PDGF)-BB autocrine loop can enhance chemotherapeutic efficacy in hypoxia. Experimental Design: Five hepatocellular carcinoma (HCC) cell lines and two hepatic progenitor cell lines were treated in vitro with cisplatin under both normoxic and hypoxic conditions. To generate ischemic hypoxia for tumor cells in vivo, hepatic artery ligation was applied to an orthotopic HCC model. Cisplatin and YC1, which is a HIF-1α inhibitor, were administered by portal vein and intratumoral injections, respectively. Results: Cell viability was higher under hypoxic than normoxic conditions. HIF-1α and Akt were up-regulated under hypoxic conditions, forming an autocrine signaling loop with PDGF-BB. Akt/HIF-1α/PDGF-BB signaling regulated Akt to confer cisplatin resistance to HCC cell lines in vitro. This autocrine signaling loop also contributed to chemoresistance in the tumorigenic hepatic progenitor cell line PIL2 under hypoxic conditions but not in the nontumorigenic cell line PIL4. In an orthotopic HCC model, combining blockade of HIF-1α activity with ischemic hypoxia significantly enhanced the efficacy of chemotherapy, leading to suppression of tumor growth and prolongation of animal survival. Conclusion: Blockade of Akt/HIF-1α/PDGF-BB autocrine signaling could enhance the chemosensitivity of liver cancer cells and tumorigenic hepatic progenitor cells under hypoxic conditions and thus provide an effective therapeutic strategy for HCC.

Insulin antagonism of dexamethasone induction of tyrosine aminotransferase in cultured fetal hepatocytes. A correlation between enzyme activity, synthesis, level of messenger RNA and transcription.

Publisher: Wiley

Date: 06-1989

DOI: 10.1111/J.1432-1033.1989.TB14849.X

Abstract: Previous studies have shown that insulin depresses the induction of tyrosine aminotransferase by glucocorticoids in cultured fetal rat hepatocytes. However, the site at which this inhibitory effect is exerted was not elucidated, since only enzyme activity was determined in such studies. Therefore, the effect of insulin on tyrosine aminotransferase synthesis, the level of its mRNA as well as the rate of transcription of the gene in isolated nuclei have been determined. The results obtained indicate that in cultures exposed to dexamethasone, Bt2cAMP, insulin and combinations of these additives, there is an excellent correlation between the enzyme activity, enzyme synthesis and the level of mRNA. Run-on transcription experiments indicate that the reduction in the level of mRNA by insulin in dexamethasone-supplemented cultures is the result of a diminished rate of gene transcription.

Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma

Publisher: Springer Science and Business Media LLC

Date: 29-04-2023

DOI: 10.1186/S13148-023-01482-0

Abstract: Epigenetic silencing of tumor suppressor genes (TSGs) is a key feature of oncogenesis in hepatocellular carcinoma (HCC). Liver-targeted delivery of CRISPR-activation (CRISPRa) systems makes it possible to exploit chromatin plasticity, by reprogramming transcriptional dysregulation. Using The Cancer Genome Atlas HCC data, we identify 12 putative TSGs with negative associations between promoter DNA methylation and transcript abundance, with limited genetic alterations. All HCC s les harbor at least one silenced TSG, suggesting that combining a specific panel of genomic targets could maximize efficacy, and potentially improve outcomes as a personalized treatment strategy for HCC patients. Unlike epigenetic modifying drugs lacking locus selectivity, CRISPRa systems enable potent and precise reactivation of at least 4 TSGs tailored to representative HCC lines. Concerted reactivation of HHIP , MT1M , PZP , and TTC36 in Hep3B cells inhibits multiple facets of HCC pathogenesis, such as cell viability, proliferation, and migration. By combining multiple effector domains, we demonstrate the utility of a CRISPRa toolbox of epigenetic effectors and gRNAs for patient-specific treatment of aggressive HCC.

THE DEVELOPMENT OF RAT ALPHA-2-MACROGLOBULIN - STUDIES INVIVO AND IN CULTURED FETAL-RAT HEPATOCYTES

Publisher: Wiley

Date: 02-1988

DOI: 10.1111/J.1432-1033.1988.TB13842.X

Abstract: During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.

EFFECT OF GLUCAGON ON DNA-SYNTHESIS IN RAT SPLEEN AND BONE-MARROW

Publisher: Wiley

Date: 15-04-1972

DOI: 10.1016/0014-5793(72)80230-8

Liver progenitor cell interactions with the extracellular matrix

Publisher: Hindawi Limited

Date: 03-2013

DOI: 10.1002/TERM.1470

Abstract: Liver progenitor cells (LPCs) are a promising source of cells to treat liver disease by cell therapy, due to their capability for self-replication and bipotentiality. In order to establish useful culture systems of LPCs and apply them to future clinical therapies, it is necessary to understand their interactions with their microenvironment and especially with the extracellular matrix (ECM). There is considerable evidence from in vivo studies that matrix proteins affect the activation, expansion, migration and differentiation of LPCs, but the information on the role that specific ECMs play in regulating LPCs in vitro is more limited. Nevertheless, current studies suggest that laminin, collagen type III, collagen type IV and hyaluronic acid help to maintain the undifferentiated phenotype of LPCs and promote their proliferation when cultured in media supplemented with growth factors chosen for LPC expansion, whereas collagen type I and fibronectin are generally associated with a differentiated phenotype under the same conditions. Experimental evidence suggests that α6β1 and α5β1 integrins as well as CD44 on the surface of LPCs, and their related downstream signals, are important mediators of interactions between LPCs and the ECM. The interactions of LPCs with the ECM form the focus of this review and the contribution of ECM molecules to strategies for optimizing in vitro LPC cultures for therapeutic applications is discussed.

Human Liver Progenitor Cell Lines Are Readily Established From Non-Tumorous Tissue Adjacent to Hepatocellular Carcinoma

Publisher: Mary Ann Liebert Inc

Date: 08-2010

DOI: 10.1089/SCD.2009.0304

Abstract: Non-tumorous liver tissue removed during surgery to resect hepatocellular carcinoma (HCC) is potentially a useful source of material from which cells, particularly liver progenitor/stem cells (LPCs), can be isolated to establish cell lines. The purpose of this study was to evaluate the applicability of the "plate-and-wait" method to derive LPCs from resections to remove HCC. Three independent non-tumorous liver s les from HCC resection and 3 s les from liver donors were used for LPC isolation. Staining for LPC markers, OV6, CK19, and EpCAM, in the above liver s les demonstrated staining in only 2 of the non-tumorous s les. We isolated 2 human liver epithelial cell lines (HLECs) from these 2 s les. These HLECs were positive for general stem cell markers CD133, EpCAM, and Oct4. They expressed the liver progenitor cell markers OV6, CK14, and M2PK but not CK19. They also expressed the hepatocellular markers albumin, CK8, CK18, HNF4-alpha, and the drug-metabolizing gene CYP3A4. These cells accumulated glycogen, indocyanine green, and synthesized urea. They produced colonies in soft agar that showed anchorage-independent growth and their tumorigenic status was confirmed when they produced tumors following transfer to athymic nude mice. In contrast, the third non-tumorous tissue and 3 normal liver s les did not produce cell lines. This study establishes a correlation between the presence of LPCs in the source liver tissue and the ability to derive cell lines from these tissues. The phenotypic similarities between the LPCs and the HLECs suggest that a precursor-product relationship may exist between the 2 cell types.

Direct effects of interleukin-6 on liver progenitor oval cells in culture

Publisher: Wiley

Date: 11-2004

DOI: 10.1111/J.1067-1927.2004.12605.X

Abstract: Following acute injury, liver is usually regenerated from hepatocytes by a process that is dependent on interleukin (IL)-6. If this pathway is impaired, restoration of the liver mass and ultimately the survival of the animal are dependent on recruitment of cells from a precursor cell population, either a stem cell or an oval cell. Importantly, oval cells are also implicated in tumorigenesis. A carcinogenic choline-deficient ethionine supplemented (CDE) diet is capable of inducing substantial numbers of oval cells that we can isolate and utilize to identify cytokines, which affect oval cell proliferation and differentiation. Currently, a putative role of IL-6 in oval cell biology is suggested by the elevation of IL-6 in liver and serum of mice treated with a CDE diet and knockout mouse studies. Also, when IL-6 is injected into the peritoneal cavity of mice on the CDE diet, oval cell numbers are increased compared to mice on the CDE diet alone. We investigated the role of human IL-6 on p53 null immortalized murine oval cell lines (PIL), finding that they express transcripts for the IL-6 receptor and gp 130, STAT-3 is phosphorylated upon IL-6 stimulation, IL-6 induces IL-6 production, and proliferation is induced by IL-6. In addition, we show that mouse primary oval cells also express IL-6 receptor and gp 130 mRNA. These findings suggest that IL-6 directly stimulates oval cells and an autocrine mechanism may sustain oval cell proliferation.

Assessing health systems for type 1 diabetes in sub-Saharan Africa: developing a 'Rapid Assessment Protocol for Insulin Access'

Publisher: Springer Science and Business Media LLC

Date: 12-2006

DOI: 10.1186/1472-6963-6-17

Abstract: In order to improve the health of people with Type 1 diabetes in developing countries, a clear analysis of the constraints to insulin access and diabetes care is needed. We developed a Rapid Assessment Protocol for Insulin Access, comprising a series of questionnaires as well as a protocol for the gathering of other data through site visits, discussions, and document reviews. The Rapid Assessment Protocol for Insulin Access draws on the principles of Rapid Assessment Protocols which have been developed and implemented in several different areas. This protocol was adapted through a thorough literature review on diabetes, chronic condition management and medicine supply in developing countries. A visit to three countries in sub-Saharan Africa and meetings with different experts in the field of diabetes helped refine the questionnaires. Following the development of the questionnaires these were tested with various people familiar with diabetes and/or healthcare in developing countries. The Protocol was piloted in Mozambique then refined and had two further iterations in Zambia and Mali. Translations of questionnaires were made into local languages when necessary, with back translation to ensure precision. In each country the protocol was implemented in 3 areas – the capital city, a large urban centre and a predominantly rural area and their respective surroundings. Interviews were carried out by local teams trained on how to use the tool. Data was then collected and entered into a database for analysis. The Rapid Assessment Protocol for Insulin Access was developed to provide a situational analysis of Type 1 diabetes, in order to make recommendations to the national Ministries of Health and Diabetes Associations. It provided valuable information on patients' access to insulin, syringes, monitoring and care. It was thus able to sketch a picture of the health care system with regards to its ability to care for people with diabetes. In all countries where this tool was used the involvement of local stakeholders resulted in the process acting as a catalyst in bringing diabetes to the attention of the health authorities.

Histone H4 histidine kinase displays the expression pattern of a liver oncodevelopmental marker.

Publisher: Oxford University Press (OUP)

Date: 03-06-2004

DOI: 10.1093/CARCIN/BGH222

Transdifferentiation of pancreatic progenitor cells to hepatocyte-like cells is not serum-dependent when facilitated by extracellular matrix proteins.

Publisher: Springer Science and Business Media LLC

Date: 12-03-2018

DOI: 10.1038/S41598-018-22596-Z

Abstract: The rising prevalence of chronic liver disease, coupled with a permanent shortage of organs for liver transplantation, has sparked enormous interest in alternative treatment strategies. Previous protocols to generate hepatocyte-like cells (HLCs) via pancreas-to-liver transdifferentiation have utilised fetal bovine serum, introducing unknown variables and severely limiting study reproducibility. Therefore, the main goal of this study was to develop a protocol for transdifferentiation of pancreatic progenitor cells to HLCs in a chemically defined, serum-free culture medium. The clonal pancreatic progenitor cell line AR42J-B13 was cultured in basal growth medium on uncoated plastic culture dishes in the absence or presence of Dexamethasone on uncoated, laminin- or fibronectin-coated culture substrata, with or without serum supplementation. The hepatocytic differentiation potential was evaluated: (i) morphologically through bright-field and scanning electron microscopy, (ii) by assessing pancreatic and hepatic marker expression and (iii) by determining the function of HLCs through their ability to synthesise glycogen or take up and release indocyanine green. Here we demonstrate for the first time that transdifferentiation of pancreatic cells to HLCs is not dependent on serum. These results will assist in converting current differentiation protocols into procedures that are compliant with clinical use in future cell-based therapies to treat liver-related metabolic disorders.

Liver stem cells: A scientific and clinical perspective

Publisher: Wiley

Date: 05-2008

DOI: 10.1111/J.1440-1746.2008.05383.X

Abstract: The promise of liver stem cells lie in their potential to provide a continual and readily available source of liver cells that can be used for gene therapy, cellular transplant, bioartificial liver-assisted devices, drug toxicology testing and use as an in vitro model to understand the developmental biology of the liver. Both the rodent and human embryonic stem cell, bone marrow hematopoietic stem cell, mesenchymal stem cell, umbilical cord blood cell, fetal liver progenitor cell, adult liver progenitor cell as well as the mature hepatocyte have been reported to be capable of self-renewal, giving rise to daughter hepatocytes both in vivo and in vitro. These cells can repopulate livers in animal models of liver injury and seemingly improve liver function. However, significant challenges still exist before these cells can be used in humans. These include lack of consensus in immunophenotype of liver progenitor cells, uncertainty of the physiological role of reported candidate stem rogenitor cell, practicality in obtaining sufficient quantity of cells for clinical use and concerns over ethics, long-term efficacy and safety. Current molecular techniques of stem cell identification are confounded by cell fusion, horizontal gene transfer, incomplete differentiation and fetal microchimerism. Reports of stem cell transplantation and phase 1 trials of bone marrow transplantation in humans for liver diseases are exciting but require more robust verification. We review the evidence for various candidate stem cells, human clinical trials reported to date and highlight the challenges facing clinicians in their quest to use liver stem cells to save lives.

Hepatic expression of the tumor necrosis factor family member lymphotoxin‐β is regulated by interleukin (IL)‐6 and IL‐1β: transcriptional control mechanisms in oval cells and hepatoma cell lines

Publisher: Wiley

Date: 20-05-2005

DOI: 10.1111/J.1478-3231.2005.01080.X

Abstract: Lymphotoxin-beta (LT-beta) plays an important role in inflammation and its promoter contains a functional nuclear factor-kappaB (NF-kappaB) element, rendering it a likely target of pro-inflammatory cytokines. Inflammatory cytokines play a central role in liver regeneration resulting from acute or chronic liver injury, with interleukin (IL)-6 signaling essential for liver regeneration induced by partial hepatectomy. In hepatic oval cells observed following chronic liver injury, LT-beta levels are upregulated, suggesting a link between LT-beta and liver regeneration. The expression of LT-beta in hepatic oval cell and hepatocellular carcinoma cell lines was further investigated, along with its responsiveness to IL-6 and IL-1beta. Key regulatory cis-acting elements of the LT-beta promoter that mediate IL-6 responsiveness (Sp/BKLF, Ets, NF-kappaB and Egr-1/Sp1) and IL-1beta responsiveness (NF-kappaB and Ets) of hepatic LT-beta expression were identified. The novel binding of basic Kruppel-like factor (BKLF) proteins to an apparent composite Sp/BKLF site of the LT-beta promoter was shown to mediate IL-6 responsiveness. Binding of NF-kappaB p65 50 heterodimers and Ets-related transcription factors to their respective sites mediates responsiveness to IL-1beta. The identification of IL-6 and IL-1beta as activators of LT-beta supports their involvement in LT-beta signaling in liver regeneration associated with chronic liver damage.

Liver inflammation and cytokine production,but not acute phase protein synthesis, accompany the adult liverprogenitor (oval) cell response to chronic liver injury

Publisher: Wiley

Date: 08-2005

DOI: 10.1111/J.1440-1711.2005.01346.X

Abstract: Oval cells are facultative liver progenitor cells, which are invoked during chronic liver injury in order to replenish damaged hepatocytes and bile duct cells. Previous studies have observed inflammation and cytokine production in the liver during chronic injury. Further, it has been proposed that inflammatory growth factors may mediate the proliferation of oval cells during disease progression. We have undertaken a detailed examination of inflammation and cytokine production during a time course of liver injury and repair, invoked by feeding mice a choline-deficient, ethionine-supplemented (CDE) diet. We show that immediately following initial liver injury, B220-expressing leucocytes transiently infiltrate the liver. This inflammatory response occurred immediately before oval cell numbers began to expand in the liver, suggesting that the two events may be linked. Two waves of liver cytokine production were observed during the CDE time course. The first occurred shortly following commencement of the diet, suggesting that it may represent a hepatic acute phase response. However, examination of acute phase marker expression in CDE-fed mice did not support this hypothesis. The second wave of cytokine expression correlated with the expansion of oval cell numbers in the liver, suggesting that these factors may mediate oval cell proliferation. No inflammatory signalling was detected following withdrawal of the injury stimulus. In summary, our results document a close correlation between inflammation, cytokine production and the expansion of oval cells in the liver during experimental chronic injury.

Appearance of oval cells in the liver of rats after long-term exposure to ethanol

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 1996

DOI: 10.1002/HEP.510230120

Abstract: Epidemiological studies show an increased risk of developing liver cancer among alcoholics. There is some agreement that ethanol itself is not carcinogenic, but it may enhance the tumorigenic process by inducing drug-metabolizing enzymes, suppression of the immune system or by affecting DNA repair enzymes. Precisely how ethanol predisposes or promotes the development of hepatoma is unknown. Hepatocarcinogenesis induced by a choline-deficient, ethionine-supplemented (CDE) diet produces extensive alteration of the liver architecture with the emergence and rapid proliferation of oval cells. This study examines whether chronic alcohol consumption induces the proliferation of oval cells. Oval cells induced in rats maintained on a 5% ethanol liquid diet (ELD) for up to 24 months, or fed a CDE diet for up to 4 weeks, are compared using a panel of liver-specific markers. In CDE-treated rats, oval cells staining positively for alpha-fetoprotein (AFP), pi-class glutathione S-transferase (pi GST), and the embryonic form of pyruvate kinase (M2-PK) are observed after 1 week. Similar cells are seen in ELD-treated rats after 2 months. Their numbers increase with time, and incorporation of [3H]thymidine confirms they are a iding population. Acute damage induced by partial hepatectomy and CCI4 poisoning did not induce the appearance of oval cells. We conclude that chronic ethanol consumption induces oval cell proliferation. We suggest that, in addition to other proposed mechanisms, an alteration in cellular composition of the liver be considered as an explanation for the increased incidence of liver cancer among alcoholics.

DNA-POLYMERASE ACTIVITY IN MUSCLE CULTURES

Publisher: Rockefeller University Press

Date: 04-1978

DOI: 10.1083/JCB.77.1.99

Abstract: Nuclei within myotubes do not synthesize DNA for replication. Accordingly, cultures of myotubes display low levels of DNA polymerase activity. The coincidental decline in DNA polymerase activity and increased formation of multinucleated myotubes during culture does not prove that the loss of capacity to synthesize DNA is a consequence of fusion. Tne experiments described demonstrate that myogenic cells prevented from fusing have low levels of DNA polymerase activity. This is consistent with the notion that, in myogenic cultures, there is a population of mononucleated cells, the myoblasts, which have withdrawn from the mitotic cycle before fusion.

THE DEVELOPMENT OF MIXED-FUNCTION AMINE OXIDASE IN CULTURED FETAL-RAT HEPATOCYTES AND ITS RELATION TO 3'-METHYL-4-N,N-DIMETHYLAMINOAZOBENZENE EFFECTS ON TYROSINE AMINOTRANSFERASE ACCUMULATION

Publisher: Oxford University Press (OUP)

Date: 1983

DOI: 10.1093/CARCIN/4.11.1499

Abstract: The ability of cultured foetal rat hepatocytes to metabolize the carcinogen 3'-methyl-4-dimethylaminoazobenzene (MDAB) is shown to correlate with the effectiveness of the carcinogen in suppressing the accumulation of tyrosine aminotransferase (TAT). MDAB is ineffective in cultures of 15-day gestation liver which are unable to carry out oxidation of MDAB as judged by the conversion of [3H]MDAB to a non-ether extractable form. In contrast, 19-day gestation hepatocytes can perform this function, and correspondingly the levels of TAT are suppressed in these cultures in the presence of MDAB. When 15-day gestation hepatocytes are maintained for beyond 3 days in culture, they acquire the ability to oxidize MDAB and accordingly become susceptible to the carcinogen.

Ethanol interactions with a choline-deficient, ethionine-supplemented feeding regime potentiate pre-neoplastic cellular alterations in rat liver

Publisher: Oxford University Press (OUP)

Date: 10-2002

DOI: 10.1093/CARCIN/23.10.1685

Abstract: To examine the effect of ethanol on hepatocarcinogenesis induced by a choline-deficient, ethionine-supplemented (CDE) diet, rats were fed either an ethanol-supplemented diet or ethanol-free, isocaloric diet for 2 months, followed by a CDE diet or control diet for up to 8 months. Changes to cellular composition and pattern of gene expression in the liver were determined at 0 and 3 days, and 1, 2 and 3 weeks after commencing the CDE diet, using histological/immunochemical techniques and northern analysis. Oval cells in the liver were identified morphologically and by expression of pi-glutathione S-transferase (pi-GST), alpha-fetoprotein (AFP) and the embryonic isoform of pyruvate kinase (M2-PK). Oval cell numbers and changes in the pattern of gene expression induced by the CDE diet were accelerated by pre-treatment with ethanol. At all stages, the proportion of oval cells in the test group exceeded that in controls. After 1 week, oval cells had spread sufficiently from the periportal region to be observed pericentrally in test animals and by 3 weeks, extensive formation of ductal structures was apparent, which were absent in controls. Additionally, M2-PK and AFP mRNA were detected earlier, and in greater abundance in animals pre-treated with ethanol. After 8 months of CDE treatment, one or two small hepatic foci ( 40 hepatocytes) and numbers of foci, correlating with the extent of liver damage and varying from 5 to 50% of the liver section. Our data suggest that ethanol pre-treatment potentiates the short-term effects of the CDE diet by enhancing oval cell proliferation, while chronic CDE administration enhances the appearance of pre-malignant hepatic foci that are observed with ethanol pre-treatment alone.

Expansion and hepatocytic differentiation of liver progenitor cells in vivo using a vascularized tissue engineering chamber in mice.

Publisher: Mary Ann Liebert Inc

Date: 03-2010

DOI: 10.1089/TEN.TEC.2009.0519

HEPATOCYTE DIFFERENTIATION INVITRO - INITIATION OF TYROSINE AMINOTRANSFERASE EXPRESSION IN CULTURED FETAL-RAT HEPATOCYTES

Publisher: Rockefeller University Press

Date: 12-1989

DOI: 10.1083/JCB.109.6.3403

Abstract: A fetal rat hepatocyte culture system has been used to study the molecular mechanisms of tyrosine aminotransferase (TAT) gene expression during development. It has previously been shown that TAT activity can be detected in 19-d, but not 15-d, gestation hepatocytes on the first day of culture (Yeoh, G. C. T., F. A. Bennett, and I. T. Oliver. 1979. Biochem. J. 180:153-160). In this study enzyme activity, synthesis, and mRNA levels were determined in hepatocytes isolated from 13-, 15-, and 19-d gestation rats maintained in culture for 1, 2, or 3 d and exposed to dexamethasone. TAT expression is barely detectable in 13-d gestation hepatocytes even after 3 d in culture. Hepatocytes isolated from 15-d gestation fetuses have undetectable levels of enzyme activity and synthesis on the first day of culture both can be assayed by days 2 and 3. TAT mRNA levels in these hepatocytes, measured by hybridization with a specific cDNA, increase substantially during culture. TAT activity, synthesis, and mRNA are evident on the first and subsequent days of culture in 19-d gestation hepatocytes. Transcription measurements in isolated nuclei indicate that the increase in TAT mRNA in 15- and 19-d gestation hepatocytes is associated with an increase in transcription of the gene. Immunocytochemical studies demonstrated that the increase in TAT expression correlated with an increase in the proportion of hepatocytes expressing the enzyme, rather than a simultaneous increase in all hepatocytes. These results support the proposal that a subpopulation of 15-d fetal hepatocytes undergo differentiation in culture with respect to TAT.

TNF/LT alpha double knockout mice display abnormal inflammatory and regenerative responses to acute and chronic liver injury

Publisher: Springer Science and Business Media LLC

Date: 03-11-2005

DOI: 10.1007/S00441-004-1003-6

Abstract: Following acute liver injury, hepatocytes ide to facilitate regeneration. However, during chronic injury, hepatocyte proliferation is typically blocked and repair is mediated through liver progenitor (oval) cells. Signalling of the p55 tumour necrosis factor (TNF) receptor is central to these processes. Two ligands for p55 are known: TNF and lymphotoxin-alpha (LTalpha). However, one study suggests that another exists that mediates liver injury following viral challenge. We have therefore investigated whether ligands other than TNF and LTalpha are required for liver regeneration following either acute or chronic injury. Wild-type and double TNF/LTalpha knockout (TNF-/-LTalpha-/-) mice were subjected to either partial hepatectomy (PHx) or a choline-deficient ethionine-supplemented (CDE) diet. Proliferating hepatocytes, oval cells and inflammatory cells were identified and quantified in liver sections by immunohistochemistry. Liver inflammatory cells were characterised by cell surface antigen expression. Liver damage and mortality were monitored. Both hepatocyte and oval cell proliferation was reduced in TNF-/-LTalpha-/- mice. Lymphocyte clusters were evident in all TNF-/-LTalpha-/- livers and were heterogeneous, comprising B and T lymphocytes. PHx evoked liver inflammation in TNF-/-LTalpha-/- but not wild-type mice, whereas no difference was apparent between genotypes in CDE experiments. Thus, TNF/LTalpha signalling mediates liver regeneration involving both hepatocytes and progenitor cells. The hyper-inflammatory response following PHx in TNF-/-LTalpha-/- animals, which is absent following CDE-induced injury, demonstrates that the two forms of liver injury evoke discrete inflammatory responses and provides a model in which such differences can be examined further.

What fires prometheus? The link between inflammation and regeneration following chronic liver injury

Publisher: Elsevier BV

Date: 2008

DOI: 10.1016/J.BIOCEL.2007.11.025

Abstract: Liver progenitor cells (LPCs) play a major role in the regeneration process after chronic liver damage, giving rise to hepatocytes and cholangiocytes. Thus, they provide a cell-based therapeutic alternative to organ transplant, the current treatment of choice for end-stage liver disease. In recent years, much attention has focused on unravelling the cytokines and growth factors that underlie this response. Liver regeneration following acute damage is achieved by proliferation of mature hepatocytes yet similar cytokines, most related to the inflammatory process, are implicated in both acute and chronic liver regeneration. Thus, many recent studies represent attempts to identify LPC-specific factors. This review summarises our current understanding of LPC biology with a particular focus on the liver inflammatory response being associated with the induction of LPCs in the liver. We will describe: (i) the pathways of liver regeneration following acute and chronic damage (ii) the similarities and differences between the two pathways (iii) the liver inflammatory environment (iv) the unique features of liver immunology as well as (v) the interactions between liver immune cells and LPCs. Combining data from studies on the LPC-driven regeneration process with the knowledge in the field of liver immunology will improve our understanding of the LPC response and allow us to regulate these cells in vivo and in vitro for future therapeutic strategies to treat chronic liver disease.

Upregulation of lymphotoxin beta expression in liver progenitor (oval) cells in chronic hepatitis C

Publisher: BMJ

Date: 09-2003

DOI: 10.1136/GUT.52.9.1327

Abstract: Bipotent liver progenitor (oval) cells with the ability to differentiate into hepatocytes and biliary epithelium have recently been identified in human subjects with hepatitis C. Animal studies suggest that members of the tumour necrosis factor family, including lymphotoxin beta (LT-beta), regulate oval cell proliferation in liver disease, but its role in human liver disease is unclear. This study seeks to establish a role for LT-beta in hepatitis C related liver injury and to provide evidence that its increased expression is related to the presence of oval cells. Liver biopsy specimens were obtained from patients with chronic hepatitis C virus (HCV) infection (n=20). Control liver s les (n=5) were obtained from liver resection or transplant surgery. LT-beta expression in liver biopsy specimens was studied using quantitative real time polymerase chain reaction and immunohistochemistry. LT-beta mRNA levels were similar in control and HCV liver in the absence of fibrosis. In subjects with portal fibrosis, LT-beta mRNA levels were elevated 2.2-fold over control liver levels (p=0.04). In subjects with bridging fibrosis, LT-beta mRNA levels increased 4.4-fold over control liver levels (p=0.02). LT-beta mRNA levels in subjects with established cirrhosis were increased 3.3-fold compared with controls and 2.6-fold compared with mild liver damage (p=0.02). Immunohistochemical analysis established that LT-beta was expressed by oval cells, inflammatory cells, and small portal hepatocytes. In chronic HCV infection, LT-beta expression is observed in multiple hepatic cell types, including oval cells. LT-beta expression is significantly increased when fibrosis or cirrhosis is present, suggesting a role for LT-beta in the pathogenesis of chronic hepatitis C and a possible role in oval cell mediated liver regeneration.

InForm software: a semi-automated research tool to identify presumptive human hepatic progenitor cells, and other histological features of pathological significance

Publisher: Springer Science and Business Media LLC

Date: 21-02-2018

DOI: 10.1038/S41598-018-21757-4

Abstract: Hepatic progenitor cells (HPCs) play an important regenerative role in acute and chronic liver pathologies. Liver disease research often necessitates the grading of disease severity, and pathologists’ reports are the current gold-standard for assessment. However, it is often impractical to recruit pathologists in large cohort studies. In this study we utilise PerkinElmer’s “InForm” software package to semi-automate the scoring of patient liver biopsies, and compare outputs to a pathologist’s assessment. We examined a cohort of eleven acute hepatitis s les and three non-alcoholic fatty liver disease (NAFLD) s les, stained with HPC markers (GCTM-5 and Pan Cytokeratin), an inflammatory marker (CD45), Sirius Red to detect collagen and haematoxylin/eosin for general histology. InForm was configured to identify presumptive HPCs, CD45 +ve inflammatory cells, areas of necrosis, fat and collagen deposition (p 0.0001). Hepatitis s les were then evaluated both by a pathologist using the Ishak-Knodell scoring system, and by InForm through customised algorithms. Necroinflammation as evaluated by a pathologist, correlated with InForm outputs (r 2 = 0.8192, p 0.05). This study demonstrates that the InForm software package provides a useful tool for liver disease research, allowing rapid, and objective quantification of the presumptive HPCs and identifies histological features that assist with assessing liver disease severity, and potentially can facilitate diagnosis.

Opposing roles of gp130-mediated STAT-3 and ERK-1/2 signaling in liver progenitor cell migration and proliferation

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 2007

DOI: 10.1002/HEP.21535

Abstract: Gp130-mediated IL-6 signaling may play a role in oval cell proliferation in vivo. Levels of IL-6 are elevated in livers of mice treated with a choline-deficient ethionine-supplemented (CDE) diet that induces oval cells, and there is a reduction of oval cells in IL-6 knockout mice. The CDE diet recapitulates characteristics of chronic liver injury in humans. In this study, we determined the impact of IL-6 signaling on oval cell-mediated liver regeneration in vivo. Signaling pathways downstream of gp130 activation were also dissected. Numbers of A6(+ve) liver progenitor oval cells (LPCs) in CDE-treated murine liver were detected by immunohistochemistry and quantified. Levels of oval cell migration and proliferation were compared in CDE-treated mouse strains that depict models of gp130-mediated hyperactive ERK-1/2 signaling (gp130(deltaSTAT)), hyperactive STAT-3 signaling (gp130(Y757F) and Socs-3(-/deltaAlb)) or active ERK-1/2 as well as active STAT-3 signaling (wild-type). The A6(+ve) LPC numbers were increased with IL-6 treatment in vivo. The gp130(Y757F) mice displayed increased A6(+ve) LPCs numbers compared with wild-type and gp130(deltaSTAT) mice. Numbers of A6(+ve) LPCs were also increased in the livers of CDE treated Socs-3(-/deltaAlb) mice compared with their control counterparts. Lastly, inhibition of ERK-1/2 activation in cultured oval cells increased hyper IL-6-induced cell growth. For the first time, we have dissected the gp130-mediated signaling pathways, which influence liver progenitor oval cell proliferation. Hyperactive STAT-3 signaling results in enhanced oval cell numbers, whereas ERK-1/2 activation suppresses oval cell proliferation.

Liver stem cells

Publisher: Wiley

Date: 08-2005

DOI: 10.1080/15216540500215606

Abstract: The concept of a liver stem cell or progenitor cell has not been widely accepted until the last decade. Studies investigating liver regeneration under conditions which totally or substantially preclude hepatocyte proliferation report the proliferation of a subpopulation of small, oval-shaped cells, which are first observed in the portal triad, adjacent to the terminal ducts. These cells, termed liver progenitor oval cells (LPCs) are shown to participate in liver regeneration in a variety of rodent models of chronic liver damage. They express markers common to hepatocytes and cholangiocytes suggesting they are a common precursor of both liver cell lineages. Supporting evidence for liver stem cells has also come from cell tracing studies which show transdifferentiation of bone marrow cells into hepatocytes in both human and animal models. Another important issue is the link between LPCs and hepatocellular carcinoma (HCC). The widening liver donor-recipient gap a consequence of poor donation rates coupled with increasing incidence of liver disease highlights the importance of establishing the utility of cell transplant as an alternative to treat liver disease. In this regard, liver stem cells and progenitor cells may have a significant role to play. To successfully utilize liver stem cells or LPCs for cell therapy, we have to first develop methods for maintaining and differentiating them in culture. This technology must be based on a thorough understanding of conditions which regulate their behaviour in vitro. In particular, we need to know which growth factors and cytokines affect them and their mechanism of action. Since they are a potential source of HCC, it is also necessary to understand the mechanisms which underlie their transformation to cancer.

Liver-specification of human iPSC-derived endothelial cells transplanted into mouse liver

Publisher: Cold Spring Harbor Laboratory

Date: 29-06-2023

DOI: 10.1101/2023.06.28.546913

Abstract: Liver sinusoidal endothelial cells (LSECs) play an important role in liver development, regeneration and pathophysiology, but the differentiation process that generates their unique tissue-specific phenotype is poorly understood and difficult to study as primary cells are only available in limited quantities. To address this, we hypothesised that human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) can produce hiPSC-derived LSECs upon transplantation into the livers of Fah −/− /Rag2 −/− /Il2rg −/− mice, and serve as a model to study LSEC specification. Progressive and long-term repopulation of the liver vasculature was observed, as iECs expanded along the sinusoids that run between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptome of transplanted cells at time-points between 1 and 12 weeks were compared against primary human adult LSECs, which demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1 , GATA4 , and FOS play a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process include EMCN and CLEC14A . Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contain zonal subpopulations with a region-specific phenotype. Collectively, this study confirms that hiPSC can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs.

TNF-inducible expression of lymphotoxin-β in hepatic cells: an essential role for NF-κB and Ets1 transcription factors.

Publisher: Elsevier BV

Date: 11-2012

DOI: 10.1016/J.CYTO.2012.05.029

Abstract: As TNF is one of the earliest signals that can be detected in the leukocyte-derived inflammatory cascade which drives subsequent cytokine production, we are interested in determining whether TNF is one of the initiating factors controlling liver remodeling and regeneration following chronic liver damage. One of the early responses is the expression of lymphotoxin-β by hepatic progenitor oval cells. The aim of this study was to determine whether hepatic expression of LT-β was controlled by TNF and to understand the basis of this regulation. We previously showed that LT-β expression is transcriptionally controlled via the TNF-induced, inflammatory NF-κB pathway in T lymphocytes. Here we show that TNF is able to upregulate LT-β expression in hepatic cells at the transcriptional level by the binding of NF-κB p50 65 heterodimers and Ets1 to their respective sites in the LT-β promoter.

A Transcriptomic Signature of Mouse Liver Progenitor Cells

Publisher: Hindawi Limited

Date: 2016

DOI: 10.1155/2016/5702873

Abstract: Liver progenitor cells (LPCs) can proliferate extensively, are able to differentiate into hepatocytes and cholangiocytes, and contribute to liver regeneration. The presence of LPCs, however, often accompanies liver disease and hepatocellular carcinoma (HCC), indicating that they may be a cancer stem cell. Understanding LPC biology and establishing a sensitive, rapid, and reliable method to detect their presence in the liver will assist diagnosis and facilitate monitoring of treatment outcomes in patients with liver pathologies. A transcriptomic meta-analysis of over 400 microarrays was undertaken to compare LPC lines against datasets of muscle and embryonic stem cell lines, embryonic and developed liver (DL), and HCC. Three gene clusters distinguishing LPCs from other liver cell types were identified. Pathways overrepresented in these clusters denote the proliferative nature of LPCs and their association with HCC. Our analysis also revealed 26 novel markers, LPC markers, including Mcm2 and Ltbp3 , and eight known LPC markers, including M2pk and Ncam . These markers specified the presence of LPCs in pathological liver tissue by qPCR and correlated with LPC abundance determined using immunohistochemistry. These results showcase the value of global transcript profiling to identify pathways and markers that may be used to detect LPCs in injured or diseased liver.

EXPRESSION OF ALPHA-CLASS, MU-CLASS, AND PI-CLASS GLUTATHIONE S-TRANSFERASES IN OVAL AND DUCTAL CELLS IN LIVER OF RATS PLACED ON A CHOLINE-DEFICIENT, ETHIONINE-SUPPLEMENTED DIET

Publisher: Oxford University Press (OUP)

Date: 1992

DOI: 10.1093/CARCIN/13.10.1879

Abstract: Expression of the alpha, mu and pi class glutathione S-transferases (GSTs) in hepatocytes, oval cells and ductal cells derived from the livers of rats placed on a choline-deficient, ethionine-supplemented (CDE) diet for 5 weeks was investigated. An overall decrease in the expression of alpha and mu class GSTs and an over-expression of pi class GST was observed in the liver after CDE treatment as indicated by Northern blotting analysis. Massive disruption of the liver with oval cell infiltration in the sinusoids throughout the lobule occurred after 5 weeks CDE treatment. 'Duct-like' structures consisting of oval-like cells (ductal cells) with rounder nuclei and more cytoplasm than oval cells within the sinusoids were also apparent. Immunocytochemical analysis revealed that the altered expression of GST in the whole liver is attributed to a differential expression of alpha, mu and pi class GSTs in the different cell types in the liver, including hepatocytes, oval cells around the portal region and among the sinusoids, and oval-like cells (ductal cells) in the 'duct-like' structures. In vitro studies using purified oval-ductal cells and hepatocyte populations confirmed the differential expression of GSTs in the varying cell populations in situ. The expression of the alpha and mu class GSTs in hepatocytes does not appear to be altered by the CDE diet. Heterogeneity in distribution of pi class GST was observed in the hepatocyte population, some hepatocytes were stained strongly while no staining was observed in others. Oval and ductal cells represent two distinct populations displaying different expression of GSTs. Pi class GST was detected in the majority of oval and ductal cells. Alpha class GST was detected in 50% of the ductal cell population. In contrast, mu class GST was absent in ductal cells and was present in 24% of oval cells around the portal region. This supports the view that ductal cells are not of bile ductal origin since mu GST is present in normal bile duct epithelial cells. Furthermore the change in expression of GSTs in the liver after CDE treatment is attributed to the large increase in oval and ductal cell populations.

Dual phenotypic expression of hepatocytes and bile ductular markers in developing and preneoplastic rat liver

Publisher: Oxford University Press (OUP)

Date: 1996

DOI: 10.1093/CARCIN/17.2.251

Abstract: This study supports the existence of a pluripotent liver stem cell population which has the potential to differentiate into hepatocytes and bile ductular cells. We compared the expression of hepatocyte-specific and bile ductular-specific markers in fetal and preneoplastic rat liver. L-pyruvate kinase (L-PK) and alpha glutathione S-transferase (GST) were used as adult hepatocyte-specific markers, while cytokeratin 19 (CK19) was used as a bile ductular-specific marker. pi GST and M2-pyruvate kinase (M2-PK), which are fetal hepatocyte-specific and expressed at high levels in the oval and duct-like cells, were also used. We characterized fetal liver derived from 13-21 days of gestation (E13-E21). pi GST was detected in the E18 hepatoblasts, which form the intrahepatic bile ducts, while CK19 was detected at E19. Some of these cells express alpha GST and L-PK from E19 to E21. Oval, duct-like and bile ductular cells in rats treated with a choline-deficient diet containing 0.07% ethionine (CDE diet) for up to 8 weeks were characterized by double immunocytochemistry. L-PK and alpha GST are absent from bile ductular cells in the normal adult liver and up to 3 weeks of CDE treatment. After 4-5 weeks on CDE treatment, the majority of bile ductular cells express L-PK, while at 6 weeks some co-express L-PK and alpha GST. There are two populations of oval cells, a major population expressing only the fetal hepatocyte markers, while a minor population expresses the fetal hepatocyte, adult hepatocyte and bile ductular markers. There are at least three different duct-like cell populations which co-express different markers and have characteristics of fetal hepatocytes at sequential stages of differentiation. One population co-expresses pi GST and M2-PK and is similar to fetal hepatocytes derived from E13-E14 fetuses. The second expresses the two fetal markers and L-PK, and this reflects characteristics of E15 hepatocytes. The third expresses pi GST, M2-PK, L-PK and alpha GST which is characteristic of E16-E19 hepatocytes. Upon withdrawal of the CDE diet, autoradiography using tritiated thymidine shows that oval and duct-like cells differentiate into hepatocytes. This study demonstrates that oval and duct-like cells express both hepatocytic and bile ductular markers, and have the capacity to differentiate into hepatocytes, characteristics similar to hepatoblasts in the developing rat liver.

Lymphotoxin-beta production following bile duct ligation: Possible role for Kupffer cells

Publisher: Wiley

Date: 12-10-2005

DOI: 10.1111/J.1440-1746.2005.04065.X

Abstract: Lymphotoxin-beta (LT-beta) may play a role in the pathogenesis of chronic liver injury. The aim of this study was to determine in an animal model of bile duct ligation liver injury whether LT-beta expression is induced and whether Kupffer cells are an intrahepatic source of LT-beta. Sprague-Dawley rats were ided into two groups: one group received a single dose of GdCl (a Kupffer cell-blocking agent, 10 mg/kg i.v.), whereas the other group received saline. One day later, the groups underwent bile duct ligation or a sham operation. Liver tissue was obtained on days 1, 3, 5, and 8 for assessment of Kupffer cell numbers, early fibrogenic events and LT-beta gene expression. Kupffer cells were isolated using pronase/collagenase perfusion and centrifugal elutriation. Hepatic LT-beta mRNA expression increased early following bile duct ligation. Pretreatment of bile duct-ligated animals with GdCl significantly reduced the number of Kupffer cells, delayed the rise in LT-beta expression, but had no effect on fibrogenesis. Recovery of the Kupffer cell population in these animals was accompanied by increased hepatic LT-beta expression. The LT-beta ligand and receptor were expressed by isolated normal Kupffer cells. Hepatic LT-beta expression is induced early following bile duct ligation. Kupffer cells may be an intrahepatic source of LT-beta.

EFFECTS OF DEXAMETHASONE AND CAMP ON TYROSINE AMINOTRANSFERASE EXPRESSION IN CULTURED FETAL-RAT HEPATOCYTES

Publisher: Wiley

Date: 07-1991

DOI: 10.1111/J.1432-1033.1991.TB16146.X

Abstract: Fetal hepatocyte cultures were used to investigate tyrosine aminotransferase (TyrAT) expression during development. Previous studies showed that TyrAT is synthesized by hepatocytes isolated from 15-day-gestation fetuses maintained in culture for two or more day, then exposed to dexamethasone. TyrAT expression was essentially undetectable on the first day of culture of hepatocytes derived from 15-day-gestation, or less mature, fetuses. Dexamethasone and cAMP are potent inducers of TyrAT and they synergistically induce TyrAT to extremely high levels when added simultaneously to cultured fetal hepatocytes. The effects of dibutyryl-cAMP (Bt2cAMP) alone and in combination with dexamethasone on TyrAT expression are investigated. Hepatocytes isolated from 15-day-gestation fetuses exposed to both inducers possessed detectable levels of TyrAT activity and mRNA on day 1 of culture, and this increased by day 3. In contrast, hepatocytes exposed to either inducing agent alone were essentially negative on day 1, but positive on day 3. This was shown to be a consequence of transcription. When 13-day-gestation hepatocytes were maintained in culture under identical conditions detectable levels of TyrAT mRNA were evident on day 1, and this increased by day 3. Immunocytochemical studies revealed that the appearance and subsequent increase in TyrAT production elicited by dexamethasone and Bt2cAMP were due to changes in the proportion of hepatocytes expressing the enzyme. Therefore, in the presence of both dexamethasone and Bt2cAMP, TyrAT expression can be detected in some cells at an earlier stage of liver development than reported previously.

Differential effects of gadolinium chloride on Kupffer cells in vivo and in vitro

Publisher: Elsevier BV

Date: 03-2004

DOI: 10.1016/J.BIOCEL.2003.08.004

Abstract: Gadolinium chloride (GdCl) is commonly used to study the role of Kupffer cells in liver disease in vivo. The in vitro effects of GdCl on cultured Kupffer cells are poorly characterised. The aim of this study was to characterise rat Kupffer cell TNFalpha production, phagocytic function, and ED1 and ED2 antigen expression following the administration of GdCl. For in vivo experiments, rats received 10mg/kg GdCl IV or sterile saline. Lipopolysaccharide 3mg/kg IP (LPS) was administered 4h prior to sacrifice on Days 1-3, 5 or 8 following GdCl injection. Hepatic ED1 and ED2 positive macrophage numbers and TNFalpha mRNA levels were determined. For in vitro experiments, Kupffer cells were cultured in the presence of 0-270 microM GdCl for 24h following which viability, TNFalpha protein production in response to LPS (10 ng/ml), phagocytosis, and ED1 and ED2 staining were evaluated. In vivo, the proportion of ED1 positive cells which were ED2 positive was reduced from 87 to 3% and hepatic TNFalpha mRNA levels following LPS declined by 60% over Days 1-5 after injection of GdCl (P<0.01). In vitro, phagocytosis declined with increasing concentrations of GdCl. GdCl (0-27 microM) did not effect cultured Kupffer cell viability, TNFalpha production, ED1 or ED2 staining. We conclude that GdCl significantly reduces ED2 expression by Kupffer cells in vivo. In vitro, GdCl has a dose dependent effect on phagocytosis but only effects viability and TNFalpha production at high concentrations. ED2 expression of cultured Kupffer cells is not affected by GdCl.

EFFECT OF CELL DENSITY, CONDITIONED MEDIUM AND CYTOSINE-ARABINOSIDE ON MYOGENESIS IN PRIMARY AND SECONDARY CULTURES

Publisher: Elsevier BV

Date: 1977

DOI: 10.1016/0014-4827(77)90069-6

ALBUMIN AND TRANSFERRIN SYNTHESIS IN LIVER OF RATS IN IMMEDIATE POSTNATAL-PERIOD

Publisher: Elsevier BV

Date: 12-1973

DOI: 10.1016/0005-2787(73)90028-2

Abstract: Direct gene delivery to the neurons of interest, without affecting other neuron populations in the cerebral cortex, represent a challenge owing to the heterogeneity and cellular complexity of the brain. Genetic modulation of corticospinal motor neurons (CSMN) is required for developing effective and long-term treatment strategies for motor neuron diseases, in which voluntary movement is impaired. Adeno-associated viruses (AAV) have been widely used for neuronal transduction studies owing to long-term and stable gene expression as well as low immunoreactivity in humans. Here we report that AAV2-2 transduces CSMN with high efficiency upon direct cortex injection and that transduction efficiencies are similar during presymptomatic and symptomatic stages in hSOD1(G93A) transgenic amyotrophic lateral sclerosis (ALS) mice. Our findings reveal that choice of promoter improves selectivity as AAV2-2 chicken β-actin promoter injection results in about 70% CSMN transduction, the highest percentage reported to date. CSMN transduction in both wild-type and transgenic ALS mice allows detailed analysis of single axon fibers within the corticospinal tract in both cervical and lumbar spinal cord and reveals circuitry defects, which mainly occur between CSMN and spinal motor neurons in hSOD1(G93A) transgenic ALS mice. Our findings set the stage for CSMN gene therapy in ALS and related motor neuron diseases.

PYRUVATE-KINASE ISOENZYME TRANSITIONS IN CULTURES OF FETAL-RAT HEPATOCYTES

Publisher: Elsevier BV

Date: 11-1988

DOI: 10.1016/0922-3371(88)90004-4

Abstract: Changes in the expression of two isoenzymic forms of pyruvate kinase in fetal hepatocyte cultures derived from 15- and 19-day gestation rats are studied by immunocytochemical localization of the respective antigens. Initially, in cultures established from 15-day gestation rats only the 'embryonic' form of the enzyme (M2-PK) is detected in all cells. Cells which stain positively for the liver specific form of the enzyme (L-PK) are not observed. After 2 days' culture, a significant number of cells have become positive for L-PK. All the positive cells have a morphology which is typical of liver parenchymal cells. However, the majority of parenchymal cells remain negative for L-PK while retaining M2-PK. In contrast, all cells which display a fibroblastic morphology, as well as clear epithelial cells are M2-PK positive, but L-PK negative. In 5-day-old cultures, all hepatocytes have become L-PK positive. Hepatocytes derived from 19-day gestation rat liver stain positively for L-PK on day 1 of culture in agreement with previously published biochemical data. A minor population of negative cells is non-parenchymal in appearance. All parenchymal cells are negative when the culture is stained with M2-PK specific antibody. Five days after the culture is established, many non-parenchymal cells are present. Such cells are L-PK negative and M2-PK positive and their presence in cultures derived from both 15- and 19-day gestation rats explains the persistence of M2-PK. This study reveals that during enzymic differentiation of fetal hepatocytes, all immature hepatocytes are initially capable of expressing M2-PK while they do not produce L-PK. During culture, a sub-population of these cells initiates synthesis of L-PK, indicating that only a fraction of the cells differentiate. At the same time, hepatocytes which do not stain for M2-PK appear, which suggests that cells which initiate L-PK synthesis have ceased to make M2-PK. Eventually all hepatocytes are L-PK positive and M2-PK negative, indicating that a switchover in expression of the pyruvate kinase isoenzymes has occurred.

Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 11-2003

DOI: 10.1053/JHEP.2003.50448

Regulation of microRNAs and their role in liver development, regeneration and disease

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BIOCEL.2014.04.002

Abstract: Since their discovery more than a decade ago microRNAs have been demonstrated to have profound effects on almost every aspect of biology. Numerous studies in recent years have shown that microRNAs have important roles in development and in the etiology and progression of disease. This review is focused on microRNAs and the roles they play in liver development, regeneration and liver disease particularly chronic liver diseases such as alcoholic liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, viral hepatitis and primary liver cancer. The key microRNAs identified in liver development and chronic liver disease will be discussed together with, where possible, the target messenger RNAs that these microRNAs regulate to profoundly alter these processes. This article is part of a Directed Issue entitled: The Non-coding RNA Revolution.

Inhibition of adult liver progenitor (oval) cell growth and viability by an agonist of the peroxisome proliferator activated receptor (PPAR) family member gamma, but not alpha or delta

Publisher: Oxford University Press (OUP)

Date: 25-05-2005

DOI: 10.1093/CARCIN/BGI138

Abstract: Multifaceted evidence links the development of liver tumours to the activation and proliferation of adult liver progenitor (oval) cells during the early stages of chronic liver injury. The aim of this study was to examine the role of the peroxisome proliferator activated receptors (PPARs): PPARalpha, delta and gamma, in mediating the behaviour of liver progenitor cells during pre-neoplastic disease and to investigate their potential as therapeutic targets for the treatment of chronic liver injury. We observed increased liver expression of PPARalpha and gamma in concert with expanding oval cell numbers during the first 21 days following commencement of the choline deficient, ethionine supplemented (CDE) dietary model of carcinogenic liver injury in mice. Both primary and immortalized liver progenitor cells were found to express PPARalpha, delta and gamma, but not gamma2, the alternate splice form of PPARgamma. WY14643 (PPARalpha agonist), GW501516 (PPARdelta agonist) and ciglitazone (PPARgamma agonist) were tested for their ability to modulate the behaviour of p53-immortalized liver (PIL) progenitor cell lines in vitro. Both PPARdelta and gamma agonists induced dose-dependent growth inhibition and apoptosis of PIL cells. In contrast, the PPARalpha agonist had no effect on PIL cell growth. None of the drugs affected the maturation of PIL cells along either the hepatocytic or biliary lineages, as judged by their patterns of hepatic gene expression prior to and following treatment. Administration of the PPARgamma agonist ciglitazone to mice fed with the CDE diet for 14 days resulted in a significantly diminished oval cell response and decreased fibrosis compared with those receiving placebo. In contrast, GW501516 did not affect oval cell numbers or liver fibrosis, but inhibited CDE-induced hepatic steatosis. In summary, PPARgamma agonists reduce oval cell proliferation and fibrosis during chronic liver injury and may be useful in the prevention of hepatocellular carcinoma.

THE EFFECT OF CARCINOGENS ON THE ACCUMULATION OF TYROSINE AMINOTRANSFERASE BY FETAL-RAT HEPATOCYTES IN CULTURE

Publisher: Elsevier BV

Date: 11-1982

DOI: 10.1016/0277-5379(82)90098-0

Abstract: The hepatocarcinogen 3'-methyl-4-dimethyl-aminoazobenzene (MDAB) suppresses the accumulation of tyrosine aminotransferase in cultured foetal hepatocytes. Experiments involving liver derived from foetuses of various ages reveals that a response is only obtained with rats older than 16-day gestation. It has been proposed that the lack of an effect in less mature hepatocytes is due to their inability to activate the carcinogen. Chemically synthesized analogues of MDAB which are considered likely to be activated forms of the procarcinogen are shown to be effective in the less mature cells. This supports the proposal that these cells may be unresponsive because they are unable to activate MDAB. Tests with other carcinogens reveal that the hepatocarcinogen dimethylbenzanthracene is also effective in 19-day gestation hepatocytes. However, the non-hepatocarcinogens azaserine and benz(a)pyrene are ineffective. Treatment with MDAB is shown not to alter the level of steroid receptor and reduce its translocation into the nucleus, suggesting that this is not the mechanism by which TAT is suppressed. The effect of the tumour promoter phorbol-myristate acetate (PMA) administered together with MDAB was shown not to modify the response to the carcinogen alone.

SYNTHESIS AND SECRETION OF ALBUMIN AND TRANSFERRIN BY FETAL RAT HEPATOCYTE CULTURES

Publisher: Elsevier BV

Date: 12-1979

DOI: 10.1016/0005-2787(79)90210-7

Abstract: Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.

Genetic manipulations utilizing albumin and alpha-fetoprotein promoter/enhancers affect both hepatocytes and oval cells

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 2004

DOI: 10.1002/HEP.20396

Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet.

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.BIOCEL.2007.06.008

Abstract: Oval cells have great potential for use in cell therapy to treat liver disease, however this cannot be achieved until the factors which govern their proliferation and differentiation are better understood. We describe a method to establish primary cultures of murine oval cells, and the derivation of two novel lines from these. Primary cultures from the livers of wildtype or TAT-GRE lacZ transgenic mice subjected to a choline-deficient, ethionine-supplemented diet comprised up to 80% oval cells at day 7 based on A6 or CK19 staining. Cell lines were clonally derived, which underwent spontaneous immortalisation following prolonged maintenance in culture. Immunostaining and RT-PCR demonstrated they express hepatocytic and biliary markers and they were therefore termed "bipotential murine oval liver" (BMOL) cells. Under proliferating culture conditions, BMOL or BMOL-TAT cells abundantly expressed oval cell and biliary markers, whereas mature hepatocytic markers were upregulated when the growth conditions were changed to facilitate differentiation. Hepatic differentiation of BMOL-TAT cells could be traced by measuring the expression of their lacZ transgene, which is driven by a promoter element from tyrosine aminotransferase (TAT), a marker of adult hepatocytes. Interestingly, haematopoietic markers were upregulated in superconfluent cultures, indicating a possible multipotentiality. None of the cell lines grew in semi-solid agar, nor did they form tumours in nude mice, suggesting they are non-tumourigenic. These novel murine oval cell lines, together with a reliable method for isolation and culture of primary oval cells, will provide a useful tool for investigating the contribution of oval cells to liver regeneration.

Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells.

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 15-03-2010

DOI: 10.1002/HEP.23663

Abstract: Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte-mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemochromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is promitotic for LPCs whether it acts directly is not known. In our murine choline-deficient, ethionine-supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor-inducible 14 (Fn14)] expression in the whole liver is massively upregulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE-induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2-week-CDE-treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin-positive cells. Stimulation of an Fn14-positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NFkappaB) activation and dose-dependent proliferation, which was diminished after targeting of the p50 NFkappaB subunit by RNA interference. TWEAK acts directly and stimulates LPC mitosis in an Fn14-dependent and NFkappaB-dependent fashion, and signaling via this pathway mediates the LPC response to CDE-induced injury and regeneration.

Enhanced liver progenitor cell survival and differentiation in vivo by spheroid implantation in a vascularized tissue engineering chamber

Publisher: Elsevier BV

Date: 05-2013

DOI: 10.1016/J.BIOMATERIALS.2013.02.011

Abstract: Liver tissue engineering is h ered by poor implanted cell survival due to inadequate vascularization and cell-cell/cell-matrix interactions. Here, we use liver progenitor cell (LPC) spheroids to enhance cell-cell/cell-matrix interactions, with implantation into an angiogenic in vivo mouse chamber. Spheroids were generated in vitro in methylcellulose medium. Day 2 spheroids were optimal for implantation (22,407 +/-645 cells/spheroid), demonstrating maximal proliferation (Ki67 immunolabeling) and minimal apoptosis (caspase-3 immunolabelling). In vivo chambers established bilaterally on epigastric vessels of immunodeficient mice were implanted with equivalent numbers of LPCs as a cell suspension (200,000 cells), or spheroids (9 spheroids). At day 14, a trend of increased LPC survival was observed in spheroid-implanted chambers [pan-cytokeratin (panCK+) cells, p = 0.38, 2.4 fold increase)], with significantly increased differentiation [cytokeratin 18 (CK18+) cells, p < 0.002, 5.1 fold increase)] compared to cell suspension-implanted chambers. At day 45, both measures were significantly increased in spheroid-implanted chambers (panCK, p < 0.006, 16 fold increase) (CK18, p < 0.019, 6 fold increase). Hepatic acini lates of CK18 + cells expressed hepatocyte nuclear factor 4-α and β-catenin, indicating ongoing hepatic differentiation. Spheroid cell-delivery significantly increased LPC survival and differentiation compared to conventional cell suspensions. This LPC spheroid/vascularized chamber model has clinical potential to generate three-dimensional vascularized liver tissue for liver replacement.

M6P/IGF2R modulates the invasiveness of liver cells via its capacity to bind mannose 6-phosphate residues

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1016/J.JHEP.2012.03.026

Trial-to-trial similarity and distinctness of muscle synergy activation coefficients increases during learning and with a higher level of movement proficiency

Publisher: Cold Spring Harbor Laboratory

Date: 22-09-2023

DOI: 10.1101/2023.09.19.558460

DEVELOPMENT OF UREA SYNTHESIS IN RAT LIVER

Publisher: Elsevier BV

Date: 12-1970

DOI: 10.1016/0020-711X(70)90036-4

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFα

Publisher: Rockefeller University Press

Date: 07-07-2008

DOI: 10.1083/JCB.200801010

Abstract: Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor κB (NF-κB) signaling, and sensitize cells to tumor necrosis factor α (TNFα). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1–Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1–TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-κB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFα-induced death occurs. TWEAK-induced loss of the cIAP1–TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFα-induced death, whereas primary cells remain resistant. Conversely, cIAP1–TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFα sensitization. Lysosomal degradation of cIAP1–TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

Single Nucleus RNA Sequencing of Pre-Malignant Liver Reveals Disease-Associated Hepatocyte State with HCC Prognostic Potential

Publisher: Cold Spring Harbor Laboratory

Date: 27-03-2022

DOI: 10.1101/2022.03.25.485695

Abstract: Current approaches to stage chronic liver diseases have limited utility to directly predict liver cancer risk. Here, we employed single nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and chronically injured pre-malignant livers using two distinct mouse models. Analysis of 40,748 hepatic nuclei unraveled a previously uncharacterized disease-associated hepatocyte transcriptional state (daHep). These cells were absent in healthy livers, but were increasingly prevalent as chronic liver disease progressed towards hepatocarcinogenesis. Gene expression deconvolution of 1,439 human liver transcriptomes from publicly available datasets revealed that daHep frequencies highly correlate with current histopathological liver disease staging systems. Importantly, we show that high daHep levels precede carcinogenesis in mice and humans and predict a higher risk of hepatocellular carcinoma (HCC) development. This novel transcriptional signature with diagnostic and, more importantly, prognostic significance has the potential to change the way chronic liver disease patients are staged, surveilled and risk-stratified.

TRANSFORMATION OF CULTURED FETAL-RAT LIVER-CELLS BY MDAB AND PHENOBARBITAL - MORPHOLOGICAL, BIOCHEMICAL AND IMMUNOCYTOCHEMICAL CHARACTERIZATION OF CELL-LINES

Publisher: Oxford University Press (OUP)

Date: 1989

DOI: 10.1093/CARCIN/10.6.1015

Abstract: Fetal rat liver cells derived from 19-day gestation rats were exposed in culture to the carcinogen, 3'-methyl-4-dimethyl-aminoazobenzene (MDAB) for 3 days and then maintained in medium supplemented with the tumor promoter, phenobarbital (PB). Tumors developed in immunodeficient mice inoculated with cells derived from cultures which had been maintained for more than 8 weeks. Histologically, three types of tumors could be distinguished. One contained epithelial-like cells, which resembled what has previously been described as 'clear' epithelial cells. The second contained cells which were more basophilic, with prominent nuclei and closely resembled the hepatoma cell line Mc-A-R-777. The third group of tumors possessed cells of both varieties. Cell lines derived from these tumors were then characterized by determining their capacity to synthesize and secrete alpha-fetoprotein, albumin and transferrin by measuring the incorporation of 35S-methionine into immunoprecipitates obtained by reaction with the respective specific antibodies and the content of the respective mRNAs were determined by hybridization to cDNAs. The activity of gamma-glutamyl-transpeptidase (GGT) and the liver specific enzyme tyrosine aminotransferase (TAT), as well as the induction of TAT by dexamethasone was also evaluated. The presence of these markers in some of the cell lines strongly suggests that they are derived from parenchymal cells. In contrast, other cell lines which morphologically resemble 'clear' epithelial cells are negative, suggesting that they may be derived from non-parenchymal epithelial cells which exist in the original culture. However, some epithelial-like cell lines derived from tumors of mixed morphology appear different to those established from tumors which contained only epithelial-like cells. These express low levels of transferrin and tyrosine aminotransferase suggesting that they may be more closely related to hepatocytes than those cells which are derived from tumors which originally comprised only epithelial cells. The absence or presence of liver markers correlates with the morphology of the respective cell lines since transferrin and TAT are only present in significant levels in those lines which comprise cells with a morphology resembling hepatoma cell lines. In cell lines which show mixed morphology, immunocytochemistry reveals that significant amounts of transferrin are only present in the parenchymal-like population. Growth rate measurements show that the faster growing cell lines generally possessed lower levels of transferrin and TAT expression. It can be concluded from these studies that it is possible to transform cells derived from fetal rat liver in culture using a hepatocarcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of iron status on glyceraldehyde 3-phosphate dehydrogenase expression in rat liver

Publisher: Wiley

Date: 13-02-1995

DOI: 10.1016/0014-5793(95)00023-3

Abstract: The influence of iron status on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene transcription, mRNA levels and distribution was determined in whole liver s les from adult Wistar rats. While iron loading did not alter GAPDH expression, iron deficiency evoked a 2.3-fold increase in the steady-state level of liver GADPH mRNA, but did not affect gene transcription or intracellular localisation of the message. Therefore, the over-expression of GAPDH mRNA in iron deficiency is probably due to increased message stability.

Sub-Cellular Localisation Studies May Spuriously Detect the Yes-Associated Protein, YAP, in Nucleoli Leading to Potentially Invalid Conclusions of Its Function

Publisher: Public Library of Science (PLoS)

Date: 06-02-2015

DOI: 10.1371/JOURNAL.PONE.0114813

THE CULTURE OF 12-DAY AND 13-DAY RAT EMBRYOS USING CONTINUOUS AND NONCONTINUOUS GASSING OF ROTATING BOTTLES

Publisher: Wiley

Date: 05-1984

DOI: 10.1002/JEZ.1402300210

Abstract: A technique for the culture of 12- and 13-day rat embryos is presented. The culture method described utilizes the opening of the extraembryonic membranes together with a simple bottle rotator during incubation to facilitate tissue oxygenation. This method was compared with a more elaborate device that enabled constant gassing during incubation. Best results were obtained with 12-day embryos cultured for 24 hr in closed bottles. Thereafter, there was a marked falloff in embryonic development in culture. Optimal medium conditions were 25% rat serum in tissue culture medium with a gas phase of 60% O2, 5% CO2, 35% N2. The culture method described allows for larger numbers of embryos to be cultured more simply than previous methods and should be valuable to workers wishing to study embryos in the more advanced stages of organogenesis.

STIMULATORY EFFECT OF GLUCAGON ON DNA SYNTHESIS IN NEONATAL RAT LIVER

Publisher: Elsevier BV

Date: 08-1971

DOI: 10.1016/0300-9629(71)90195-2

Abstract: Alcohol consumption is known to be associated with risky sexual behaviours, but this relationship may be complex and bidirectional. We explored whether alcohol consumption leads to the consumer being rated as more attractive than sober in iduals. Heterosexual social alcohol consumers completed an attractiveness-rating task, in which they were presented with pairs of photographs depicting the same in idual, photographed while sober and after having consumed alcohol (either 0.4 or 0.8 g/kg), and required to decide which image was more attractive. Photographs of in iduals who had consumed a low dose of alcohol (equivalent to 250 ml of wine at 14% alcohol by volume for a 70 kg in idual) were rated as more attractive than photographs of sober in iduals. This was not observed for photographs of in iduals who had consumed a high dose of alcohol. In addition to perceiving others as more attractive, a mildly intoxicated alcohol consumer may also be perceived as more attractive by others. This in turn may play a role in the relationship between alcohol consumption and risky sexual behaviour.

Evaluation of the Cellscreen system for proliferation studies on liver progenitor cells.

Publisher: Elsevier BV

Date: 12-2006

DOI: 10.1016/J.EJCB.2006.08.006

Abstract: Proliferation studies on mammalian cells have been disadvantaged by the limited availability of non-invasive assays as the majority of approaches are based on chemical treatment, s ling or staining of cells removed from culture. In this study, we utilised the Cellscreen system (Innovatis AG, Bielefeld, Germany), a non-invasive automated technique for measuring proliferation of adherent and suspension cells over time. We have evaluated the ability of the Cellscreen system to monitor and quantify growth of adherent liver progenitor cells over time and tested several applications, (i) serum reduction or (ii) treatment with a cytokine. Our results demonstrate that the Cellscreen system reproducibly documents pro- and anti-proliferative effects of cytokines and growth factors and quantifies changes by providing cell-doubling times for control and test cultures. However, we found that for the conversion of cell density values into absolute cell numbers different conversion factors, which better suit the in idual growth phases, need to be established. Collectively, these findings reveal that the Cellscreen system is applicable for the determination of cell proliferation of adherent and suspension cells in response to a variety of (growth) factors. It minimises operator participation and thus enables more rapid and larger screens and, being non-invasive, permits multiple assays on the same culture of cells. Hence, this technique proves superior to the common proliferation assays opening up new dimensions of proliferation studies in cell biology.

INSULIN ANTAGONISM OF GLUCOCORTICOID INDUCTION OF TYROSINE AMINOTRANSFERASE IN CULTURED FETAL HEPATOCYTES

Publisher: Wiley

Date: 08-1981

DOI: 10.1111/J.1432-1033.1981.TB05496.X

Abstract: Whereas dexamethasone is unable to induce the premature formation of hepatic tyrosine aminotransferase when administered to foetal rats in utero, the steroid can induce the enzyme in foetal rat liver if the liver is first removed from the environment in utero and grown in culture. Dexamethasone produced a significant induction of the enzyme at a concentration of 0.1 nM in cultured foetal hepatocytes, but for optimal induction the cells were exposed to 10 nM for 15 h. Growing the hepatocytes in the presence of physiological concentrations of insulin had no effect on the enzyme activity in control cells. However, the induction of the enzyme by dexamethasone was markedly diminished in the presence of insulin. This effect of insulin is both time-dependent and dose-dependent with significant inhibition being obtained with 1 nM insulin. Growing foetal hepatocytes in the presence of insulin has no effect on either the cellular level of glucocorticoid receptor or on the stability of dexamethasone-receptor complexes to undergo nuclear translocation suggesting that insulin inhibits some event subsequent to translocation. The results are discussed in relation to the postnatal appearance of tyrosine aminotransferase and suggest that the marked decline in the plasma concentration of insulin, that is known to occur at birth, is a major contributor to the postnatal induction of the enzyme.

Jekyll and Hyde: evolving perspectives on the function and potential of the adult liver progenitor (oval) cell

Publisher: Wiley

Date: 2005

DOI: 10.1002/BIES.20311

Abstract: The liver progenitor cell (LPC) has enormous potential for use in cell therapy to treat liver disease. Since liver regenerates readily from pre-existing hepatocytes, a role for LPCs and, indeed, their existence have been questioned. Research during the last decade has established that LPCs are an important alternative source of cells for liver regeneration. Their utility for cell therapy lies in their ability to generate both hepatocytes and cholangiocytes. However, they are observed in liver diseases that often lead to cancer and there is experimental evidence that implicates LPCs as the source of tumours. This article provides a brief history of the studies that established the functional importance of LPCs in liver disease. It focuses on mouse models that have led to the identification of factors that regulate LPC growth and differentiation and discusses LPCs derived from different sources. Recent promising results from both in vitro and vivo studies suggest that LPCs could be useful for cell therapy. In the context of liver disease, LPCs may indeed be the cell of the future and understandably "our favourite cell".

Bioenergetic differences selectively sensitize tumorigenic liver progenitor cells to a new gold(I) compound

Publisher: Oxford University Press (OUP)

Date: 15-04-2008

DOI: 10.1093/CARCIN/BGN093

Abstract: A hallmark of cancer cells is their ability to evade apoptosis and mitochondria play a critical role in this process. Delineating mitochondrial differences between normal and cancer cells has proven challenging due to the lack of matched cell lines. Here, we compare two matched liver progenitor cell (LPC) lines, one non-tumorigenic [p53-immortalized liver (PIL) 4] and the other tumorigenic (PIL2). Analysis of these cell lines and a p53 wild-type non-tumorigenic cell line [bipotential murine oval liver (BMOL)] revealed an increase in expression of genes encoding the antiapoptotic proteins cellular inhibitor of apoptosis protein (cIAP) 1 and yes associate protein in the PIL2 cells, which resulted in an increase in the protein encoded by these genes. PIL2 cells have higher mitochondrial membrane potential (Deltapsi(m)) compared with PIL4 and BMOL and had greater levels of reactive oxygen species, despite the fact that the mitochondrial antioxidant enzyme, manganese superoxide disumutase, was elevated at transcript and protein levels. Taken together, these results may account for the observed resistance of PIL2 cells to apoptotic stimuli compared with PIL4. We tested a new gold compound to show that hyperpolarized Deltapsi(m) led to its increased accumulation in mitochondria of PIL2 cells. This compound selectively induces apoptosis in PIL2 cells but not in PIL4 or BMOL. The gold compound depolarized the Deltapsi(m), depleted the adenosine triphosphate pool and activated caspase-3 and caspase-9, suggesting that apoptosis was mediated via mitochondria. This investigation shows that the non-tumorigenic and tumorigenic LPCs are useful models to delineate the role of mitochondrial dysfunction in tumorigenesis and for the future development of mitochondria-targeted chemotherapeutics that selectively target tumor cells.

Transferrin receptor numbers and transferrin and iron uptake in cultured chick muscle cells at different stages of development.

Publisher: Wiley

Date: 06-1987

DOI: 10.1002/JCP.1041310306

Abstract: The mechanism of iron uptake and the changes which occur during cellular development of muscle cells were investigated using primary cultures of chick embryo breast muscle. Replicating presumptive myoblasts were examined in exponential growth and after growth had plateaued. These were compared to the terminally differentiated cell type, the myotube. All cells, regardless of the state of growth or differentiation, had specific receptors for transferrin. Presumptive myoblasts in exponential growth had more transferrin receptors (3.78 +/- 0.24 X 10(10) receptors/micrograms DNA) than when ision had ceased (1.70 +/- 0.14 X 10(10) receptors/micrograms DNA), while myotubes had 3.80 +/- 0.26 X 10(10) receptors/micrograms DNA. Iron uptake occurred by receptor-mediated endocytosis of transferrin. While iron was accumulated by the cells, apotransferrin was released in an undegraded form. There was a close correlation between the molar rates of endocytosis of transferrin and iron. Maximum rates of iron uptake were significantly higher in myotubes than in presumptive myoblasts in either exponential growth or after growth had plateaued. There were two rates of exocytosis of transferrin, implying the existence of two intracellular pathways for transferrin. These experiments demonstrate that iron uptake by muscle cells in culture occurs by receptor-mediated endocytosis of transferrin and that transferrin receptor numbers and the kinetics of transferrin and iron uptake vary with development of the cells.

Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice

Publisher: Rockefeller University Press

Date: 18-12-2000

DOI: 10.1084/JEM.192.12.1809

Abstract: Hepatic stem cells (oval cells) proliferate within the liver after exposure to a variety of hepatic carcinogens and can generate both hepatocytes and bile duct cells. Oval cell proliferation is commonly seen in the preneoplastic stages of liver carcinogenesis, often accompanied by an inflammatory response. Tumor necrosis factor (TNF), an inflammatory cytokine, is also important in liver regeneration and hepatocellular growth. The experiments reported here explore the relationship among the TNF inflammatory pathway, liver stem cell activation, and tumorigenesis. We demonstrate that TNF is upregulated during oval cell proliferation induced by a choline-deficient, ethionine-supplemented diet and that it is expressed by oval cells. In TNF receptor type 1 knockout mice, oval cell proliferation is substantially impaired and tumorigenesis is reduced. Oval cell proliferation is impaired to a lesser extent in interleukin 6 knockout mice and is unchanged in TNF receptor type 2 knockout mice. These findings demonstrate that TNF signaling participates in the proliferation of oval cells during the preneoplastic phase of liver carcinogenesis and that loss of signaling through the TNF receptor type 1 reduces the incidence of tumor formation. The TNF inflammatory pathway may be a target for therapeutic intervention during the early stages of liver carcinogenesis.

Bone marrow cells play only a very minor role in chronic liver regeneration induced by a choline-deficient, ethionine-supplemented diet

Publisher: Elsevier BV

Date: 09-2008

DOI: 10.1016/J.SCR.2008.05.004

Abstract: Liver progenitor (oval) cells have enormous potential in the treatment of patients with liver disease using a cell therapy approach, but their use is limited by their scarcity and the number of donor livers from which they can be derived. Bone marrow may be a suitable source. Previously the derivation of oval cells from bone marrow was examined in rodents using hepatotoxins and partial hepatectomy to create liver damage. These protocols induce oval cell proliferation however, they do not produce the disease conditions that occur in humans. In this study we have used the choline-deficient, ethionine-supplemented (CDE) diet (which causes fatty liver) and viral hepatitis as models of chronic injury to evaluate the contribution of bone marrow cells to oval cells under conditions that closely mimic human liver disease pathophysiology. Following transplantation of lacZ-transgenic bone marrow cells into congenic mice, liver injury was induced and the movement of bone marrow cells to the liver monitored. Bone marrow-derived oval cells were observed in response to the CDE diet and viral injury but represented a minor fraction (0-1.6%) of the oval cell compartment, regardless of injury severity. In all situations only rare, in idual bone marrow-derived oval cells were observed. We hypothesized that the bone marrow cells may replenish oval cells that are expended by protracted liver injury and regeneration however, experiments involving a subsequent episode of chronic liver injury failed to induce proliferation of the bone marrow-derived oval cells that appeared as a result of the first episode. Bone marrow-derived hepatocytes were also observed in all injury models and controls at a frequency unrelated to that of oval cells. We conclude that during viral-and steatosis-induced liver disease the contribution of bone marrow cells to hepatocytes, either via oval cells or by independent mechanisms, is minimal and that the majority of oval cells responding to this injury are sourced from the liver.

LEVELS OF 2,3-DIPHOSPHOGLYCERATE IN FRIEND LEUKEMIC-CELLS

Publisher: Springer Science and Business Media LLC

Date: 05-1980

DOI: 10.1038/285108A0

Abstract: Most cells are thought to contain trace amounts of 2,3-diphosphoglycerate (DPG), as it acts as a cofactor in the interconversion of 2-phosphoglycerate and 3-phosphoglycerate by the glycolytic enzyme phosphoglyceromutase. DPG is synthesized from 1,3-diphosphoglycerate by the action of diphosphoglycerate mutase. Lowry et al. reported levels of 29 mumol DPG per kg wet weight brain tissue which is approximately 3 pmol per 10(8) cells, assuming that 1 g of brain tissue contains 10(9) cells. In contrast, erythroid cells contain 50-100 nmol DPG per 10(8) cells, depending on the species and the stage of development. This is of the order of a 1,000-fold more DPG compared with non-erythroid cells. In red cells DPG concentration modulates the binding of oxygen to haemoglobin. I show here that erythroid precurser cells also contain markedly raised levels of DPG.

The oval-shaped cell as a candidate for a liver stem cell in embryonic, neonatal and precancerous liver: Identification based on morphology and immunohistochemical staining for albumin and pyruvate kinase isoenzyme expression

Publisher: Springer Science and Business Media LLC

Date: 12-03-1997

DOI: 10.1007/S004180050109

Abstract: Oval cells observed in some experimental models of hepatocarcinogenesis can function as stem cells capable of differentiating into hepatocytes and bile ductular cells. Using markers which characterise embryonic hepatocytes, we showed that oval cells display different patterns of gene expression, suggesting some are more mature than others. In this study we looked for oval cells in developing liver, predicting that they are abundant in embryonic liver and decline in number during development. Albumin (ALB) serves as a liver-specific marker, and the isoenzymes of pyruvate kinase, M2-PK and L-PK, are used to identify immature and mature hepatocytes, respectively. Small oval-shaped cells expressing ALB, M2-PK and L-PK are found near the vascular spaces and portal areas in 20-day gestation (E20), E21, newborn, 3-day and 1-week-old rat liver. Similar cells expressing ALB and M2-PK, but not L-PK are seen only periportally in adult liver. These are abundant in early embryonic liver and decrease in number during development until only a few, located periportally, persist in the adult. Oval cells, located periportally a few days after commencing a choline-deficient, ethionine-supplemented diet, co-express ALB and M2-PK. Their similarity with respect to markers, morphology and location suggests that oval-shaped cells may be the progenitors of oval cells.

Kupffer cell cytokines interleukin-1 beta and interleukin-10 combine to inhibit phosphoenolpyruvate carboxykinase and gluconeogenesis in cultured hepatocytes

Publisher: Elsevier BV

Date: 08-2004

DOI: 10.1016/J.BIOCEL.2003.10.022

Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 2006

DOI: 10.1002/HEP.21466

Abstract: Oval cells are hepatocytic precursors that proliferate in late-stage cirrhosis and that give rise to a subset of human hepatocellular carcinomas. Although liver regeneration typically occurs through replication of existing hepatocytes, oval cells proliferate only when hepatocyte proliferation is inhibited. Transforming growth factor-beta (TGF-beta) is a key inhibitory cytokine for hepatocytes, both in vitro and in vivo. Because TGF-beta levels are elevated in chronic liver injury when oval cells arise, we hypothesized that oval cells may be less responsive to the growth inhibitory effects of this cytokine. To examine TGF-beta signaling in vivo in oval cells, we analyzed livers of rats fed a choline-deficient, ethionine-supplemented (CDE) diet for phospho-Smad2. Phospho-Smad2 was detected in more than 80% of hepatocytes, but staining was substantially reduced in oval cells. Ki67 staining, in contrast, was significantly more common in oval cells than hepatocytes. To understand the inverse relationship between TGF-beta signaling and proliferation in oval cells and hepatocytes, we examined TGF-beta signaling in vitro. TGF-beta caused marked growth inhibition in primary hepatocytes and the AML12 hepatocyte cell line. Two oval cell lines, LE/2 and LE/6, were less responsive. The greater sensitivity of the hepatocytes to TGF-beta-induced growth inhibition may result from the absence of Smad6 in these cells. Our results indicate that oval cells, both in vivo and in vitro, are less sensitive to TGF-beta-induced growth inhibition than hepatocytes. These findings further suggest an underlying mechanism for the proliferation of oval cells in an environment inhibitory to hepatocytic proliferation.

PHORBOL-MYRISTATE ACETATE (PMA) FAILS TO PREVENT THE APPEARANCE OF MARKERS ASSOCIATED WITH MUSCLE AND LIVER DIFFERENTIATION IN CULTURE

Publisher: Wiley

Date: 06-1981

DOI: 10.1016/S0309-1651(81)80013-6

Abstract: The tumour promoter PMA has been shown to both prevent and induce differentiation of a variety of cell types in culture. The reason for its paradoxical effects is not known. However, it is clear that PMA alters the cell membrane and therefore it is possible that PMA may only be effective in instances where differentiation is accompanied by changes to the cell membrane e.g. myoblast fusion during myogenesis. In this study, its effects on myoblast fusion as well as the appearance of the muscle specific isoenzyme of creatine phosphokinase (M-CPK) which is not fusion dependent is examined. It is shown that M-CPK accumulates in myogenic cultures exposed to PMA although fusion is prevented. PMA is also tested in foetal rat hepatocytes which differentiate and acquire the enzyme tyrosine aminotransferase during culture. There is no evidence which suggests that this change is membrane dependent. The tumour promoter does not prevent the accumulation of tyrosine aminotransferase in cultured foetal rat hepatocytes.

Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue

Publisher: The Optical Society

Date: 21-04-2015

DOI: 10.1364/BOE.6.001767

Interferon-gamma exacerbates liver damage, the hepatic progenitor cell response and fibrosis in a mouse model of chronic liver injury

Publisher: Elsevier BV

Date: 12-2007

DOI: 10.1016/J.JHEP.2007.06.022

Abstract: Several previous studies have suggested that interferon gamma (IFNgamma) may play a key role during hepatic progenitor cell (HPC) mediated liver regeneration. However to date, no studies have directly tested the ability of IFNgamma to mediate the HPC response in an in vivo model. Administration of IFNgamma to mice receiving a choline deficient, ethionine (CDE) supplemented diet to induce chronic injury resulted in an augmented HPC response. This was accompanied by increased inflammation, altered cytokine expression and hepatic fibrosis. Serum alanine aminotransferase activity, hepatocyte apoptosis and Bak staining were significantly increased in IFNgamma-treated, CDE-fed mice, demonstrating that liver damage was exacerbated in these animals. Administration of IFNgamma to control diet fed mice did not induce liver damage, however it did stimulate hepatic inflammation. Our results suggest that IFNgamma increases the HPC response to injury by stimulating hepatic inflammation and aggravating liver damage. This is accompanied by an increase in hepatic fibrogenesis, supporting previous reports which suggest that the HPC response may drive fibrogenesis during chronic liver injury.

THE EFFECT OF 3'-METHYL-4-DIMETHYL-AMINOAZOBEN ZENE ON FETAL-RAT HEPATOCYTES IN CULTURE

Publisher: Elsevier BV

Date: 07-1981

DOI: 10.1016/0014-2964(81)90229-2

Abstract: Acquired hemophilia A (AHA) is a hemorrhagic disorder. Whether or not severe thrombotic events can develop without the use of bypassing agents in AHA patients is unclear. An 80-year-old woman with AHA underwent immunosuppressive therapy with prednisolone at 1 mg/kg daily. After achieving remission, she suddenly developed multiple organ failure due to acute systemic thrombosis and died within a few hours of the diagnosis. Patients with AHA, especially those with risk factors for thrombosis, have a considerable risk of developing thrombosis during the recovery phase of factor VIII activity and should be carefully monitored by coagulation testing.

TRANSCRIPTIONAL-DEPENDENT AND POST-TRANSCRIPTIONAL-DEPENDENT REGULATION OF GLUTATHIONE-S-TRANSFERASE EXPRESSION IN RAT HEPATOCYTES AS A FUNCTION OF CULTURE CONDITIONS

Publisher: Wiley

Date: 23-11-1992

DOI: 10.1016/0014-5793(92)81434-N

Abstract: Transcriptional activity of the glutathione S-transferase (GST) alpha (subunits 1 and 2), mu (subunits 3 and 4) and pi (subunit 7) gene families has been analyzed using the nuclear 'run-on' technique on adult rat hepatocytes maintained for 4 days in conventional culture and for 4 and 12 days in co-culture with rat liver epithelial cells. Several medium conditions are included in this study, namely with or without fetal calf serum and with nicotinamide or dimethylsulphoxide. Hepatocytes co-cultured for 4 days maintain approximately 30-70% of the alpha gene family transcriptional activity, whatever the medium conditions, when compared to freshly isolated hepatocytes. A marked decrease is observed after 12 days of co-culture or when hepatocytes are maintained in conventional culture. The transcriptional activity of the mu gene family is maintained at 40-160% when hepatocytes are cultured with or without fetal calf serum, and is inducible by nicotinamide (approximately 4-fold) and dimethylsulphoxide (approximately 2-fold) in conventional culture and/or in co-culture. In contrast to freshly isolated hepatocytes, GST pi gene transcriptional activity is observed in conventional and co-cultured hepatocytes, irrespective of the medium conditions. Dimethylsulphoxide treatment however, represses the expression of GST 7 in vitro. These results demonstrate that the expression of GST alpha, mu and pi genes in conventional and co-cultured rat hepatocytes is controlled primarily at the level of transcription. It cannot be excluded, however, that dimethylsulphoxide stabilizes the GST mRNA levels in vitro.

Rapid Evaluation of Antrodia camphorata Natural Products and Derivatives in Tumourigenic Liver Progenitor Cells with a Novel Cell Proliferation Assay

Publisher: Wiley

Date: 28-09-2009

DOI: 10.1002/CMDC.200900238

Abstract: We report the syntheses of five natural product maleimide and maleic anhydrides from the mushroom Antrodia c horata. The ability of these compounds to affect proliferation in non-tumourigenic and tumourigenic liver progenitor cell lines was monitored by the Cellscreen system, a novel and nondestructive rapid-screening instrument. Additionally, a range of new aryl-functionalised differentiated derivatives were prepared through a Suzuki cross-coupling reaction to influence cell-growth effects. Several derivatives radically slowed the proliferation of liver progenitor cells however, of particular interest were two maleic anhydride derivatives containing aryl tethers. These analogues demonstrated selectivity for limiting the proliferation of tumourigenic progenitor cells in comparison with their non-tumourigenic counterparts. Also highlighted is the application of the Cellscreen system in medicinal chemistry to rapidly measure the effect of compound libraries on cell proliferation.

5' sequences direct developmental expression and hormone responsiveness of tyrosine aminotransferase in primary cultures of fetal rat hepatocytes

Publisher: Wiley

Date: 11-1997

DOI: 10.1111/J.1432-1033.1997.T01-1-00675.X

Abstract: Tyrosine aminotransferase (TyrAT) is one of several gluconeogenic enzymes which appear postnatally in humans and rodents in response to increased glucocorticoid and glucagon levels and decreased insulin. Primary cultured fetal rat hepatocytes older than day 15 of gestation (>E15) transcribe the TyrAT gene in response to the synergistic effect of dexamethasone and N6,2'-O-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP), whereas less mature hepatocytes ( E15 hepatocytes, and not <E15 hepatocytes, to be determined. This study reports that 11.1 kb of sequences upstream of the TyrAT transcription start site, which include a cAMP-responsive element (CRE) and a glucocorticoid-responsive element (GRE), are required for correct developmental regulation of gene expression in determined fetal hepatocytes. In contrast, the TyrAT CRE alone does not have this capability. Dexamethasone augments basal and Bt2cAMP-stimulated activity of the TyrAT CRE alone, suggesting that synergism may be due to interaction between the glucocorticoid and cAMP-signaling pathways. However, Bt2cAMP does not further increase dexamethasone-induced activity of the 11.1 kb 5' sequences when the TyrAT CRE is removed, thus excluding interaction of Bt2cAMP with the glucocorticoid pathway. Finally, insulin inhibition of dexamethasone-induced gene transcription is shown to be conferred by TyrAT 5' sequences. This study shows that cellular components, other than those which mediate hormonal regulation of genes, are required for determination of hepatocytes with respect to TyrAT. Since this phenomenon is observed with transient transfections, it is unlikely to involve higher-order chromatin structure.

Exploiting the unique regenerative capacity of the liver to underpin cell and gene therapy strategies for genetic and acquired liver disease

Publisher: Elsevier BV

Date: 11-2014

DOI: 10.1016/J.BIOCEL.2014.10.023

Abstract: The number of genetic or acquired diseases of the liver treatable by organ transplantation is ever-increasing as transplantation techniques improve placing additional demands on an already limited organ supply. While cell and gene therapies are distinctly different modalities, they offer a synergistic alternative to organ transplant due to distinct architectural and physiological properties of the liver. The hepatic blood supply and fenestrated endothelial system affords relatively facile accessibility for cell and/or gene delivery. More importantly, however, the remarkable capacity of hepatocytes to proliferate and repopulate the liver creates opportunities for new treatments based on emerging technologies. This review will summarise current understanding of liver regeneration, describe clinical and experimental cell and gene therapeutic modalities and discuss critical challenges to translate these new technologies to wider clinical utility. This article is part of a Directed Issue entitled: "Regenerative Medicine: the challenge of translation".

GLYCOLIPID TOXINS FROM PARASITIZED ANNUAL RYEGRASS - A COMPARISON WITH TUNICAMYCIN

Publisher: Elsevier BV

Date: 04-1982

DOI: 10.1016/0006-291X(82)91045-2

Abstract: An interesting question posed by the current evidence that severe acute respiratory syndrome coronavirus may be originated from an animal coronavirus is how such an animal coronavirus breaks the host species barrier and becomes zoonotic. In this report, we study the chronological order of genotypic changes in the spike protein of avian coronavirus infectious bronchitis virus (IBV) during its adaptation to a primate cell line. Adaptation of the Beaudette strain of IBV from chicken embryo to Vero cells showed the accumulation of 49 amino acid mutations. Among them, 26 (53.06%) substitutions were located in the S protein. Sequencing analysis and comparison of the S gene demonstrated that the majority of the mutations were accumulated and fixed at passage 7 on Vero cells and minor variants were isolated in several passages. Evidence present suggests that the dominant Vero cell-adapted IBV strain may be derived from the chicken embryo passages by selection of and potential recombination between the minor variants. This may explain why adaptation is a rapid process and the dominant strain, once adapted to a new host cell, becomes relatively stable.

Towards gene and cell therapy to treat OTC deficiency: a strategy based on utilising liver progenitor cells

Publisher: Springer Science and Business Media LLC

Date: 02-1976

DOI: 10.1038/259417A0

Abstract: A total of 118 trauma patients were prospectively randomized to receive either open or closed peritoneal lavage. In addition, comparisons were made between using an infraumbilical versus a supraumbilical approach in patients with pelvic fractures. No statistical difference was noted between the open and closed groups, despite the overall accuracy rate of 96.6%. The supraumbilical approach was vastly superior to the infraumbilical approach in patients with pelvic fracture with an accuracy rate of 90.9% versus 57%. In this regard, use of the infraumbilical approach in this setting is to be highly discouraged. Finally, patient body habitus, technique, and physician experience all play a role in the outcome of the lavage.

REGULATION OF GLUTATHIONE-S-TRANSFERASE GENE-EXPRESSION BY PHENOBARBITAL IN CULTURED ADULT-RAT HEPATOCYTES

Publisher: Wiley

Date: 17-06-1991

DOI: 10.1016/0014-5793(91)80772-U

Abstract: Previous studies, by using Northern blotting analyses, showed that phenobarbital (PB) affects the steady-state mRNA levels of glutathione S-transferase (GST) subunits 1/2, 3/4 and 7 in both conventional cultures of adult rat hepatocytes and co-cultures, with rat liver epithelial cells [Vandenberghe et al., 1989, FEBS Lett. 251, 59-64 Morel et al., 1989, FEBS Lett. 258, 99-102]. To determine whether PB acts at the transcriptional level, nuclear 'run on' experiments using cDNA probes hybridizing to GST subunits 1/2, 3/4 and 7 mRNA were performed on purified nuclei isolated from control and PB treated hepatocytes seeded under conventional and co-culture conditions. Data from this study demonstrate that the increase in steady-state mRNA levels observed in both conventional culture and co-culture after 4 days PB exposure results from an increased transcriptional activity of the GST genes. However, a substantial increase in steady-state mRNA levels in the absence of a commensurate increase in transcriptional activity at 12 days of co-culture, indicates that the barbiturate has also a stabilizing effect in vitro on the GST mRNAs.

Hepatic oval cell response to the choline-deficient, ethionine supplemented model of murine liver injury is attenuated by the administration of a cyclo-oxygenase 2 inhibitor

Publisher: Oxford University Press (OUP)

Date: 12-12-2006

DOI: 10.1093/CARCIN/BGI365

Abstract: Oval cell proliferation precedes neoplasia in many rodent models of hepatocellular carcinoma and prevention of this proliferative response can reduce the risk of subsequent carcinoma. This study aimed to determine whether a selective cyclo-oxygenase-2 (COX-2) inhibitor, SC-236, affects (i) the oval cell response to liver injury in a mouse model of hepatocarcinogenesis and (ii) an oval cell line. Four-week-old mice were fed either normal chow or a choline deficient, ethionine supplemented (CDE) diet in the presence or absence of SC-236. Liver histology and oval cell numbers were determined after 2, 4, 12 and 52 weeks of treatment. Oval cells were scored using morphological criteria and positive immuno-staining for the M(2)-isozyme of pyruvate kinase (M2PK) or A6. An immortalized oval cell line (PIL-2) was used to study the in vitro effects of SC-236 on oval cell proliferation, apoptosis and Akt phosphorylation. The percentage of M2PK-positive oval cells and COX-2-positive cells was reduced by 80% and 45%, respectively, in CDE-fed mice receiving SC-236 compared with CDE-fed animals not receiving SC-236. Some M2PK-positive oval cells were also COX-2 positive. The percentage of A6-positive cells was not affected by SC-236 administration to CDE-fed mice. Administration of SC-236 increased apoptosis as evidenced by a 73% increase in the number of TUNEL-positive cells at 2 weeks in CDE-fed mice. Primary oval cells and PIL-2 cells expressed COX-2. In vitro treatment of PIL-2 cells with SC-236 resulted in a dose-dependent preferential death of A6-negative cells. Administration of 25 and 50 microM Prostaglandin E(2) partially attenuated SC-236 induced cell death by 25%. In vitro oval cell death was associated with apoptosis and a 70% reduction in Akt phosphorylation. These results suggest that the SC-236 induced reduction of M2PK-positive oval cell numbers may be due to COX-2 dependent inhibition of Akt phosphorylation and induction of apoptosis.

Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes

Publisher: Elsevier BV

Date: 06-1996

DOI: 10.1016/1357-2725(96)00007-6

Abstract: In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5' long terminal repeat controlling transcription of the beta-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of beta-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to beta-galactosidase activity and cell survival. For maximum transfection of cells, 10 micrograms/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling beta-galactosidase and chlor henicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.

The Harry Perkins Institute Of Medical Research

Location: Australia

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University Of Western Australia

Location: Australia

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Human Amnion Epithelial Cells And Liver Repair

Start Date: 2014

End Date: 2016

Funder: National Health and Medical Research Council

Understanding And Applying White Blood Cell-mediated Effects On Liver Stem Cells To Treat Liver Disease.

Start Date: 2013

End Date: 2015

Funder: National Health and Medical Research Council

How Do Liver Stem Cells Grow And Develop Into Functional Liver Cells?

Start Date: 2008

End Date: 2010

Funder: National Health and Medical Research Council

Development Of Microscope-in-a-needle Devices For Improved Clinical Diagnostics

Start Date: 2014

End Date: 2016

Funder: National Health and Medical Research Council

Discovery Projects - Grant ID: DP0208385

Start Date: 10-2002

End Date: 02-2005

Amount: $141,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882866

Start Date: 12-2008

End Date: 12-2009

Amount: $470,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0668413

Start Date: 12-2006

End Date: 12-2007

Amount: $723,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989788

Start Date: 01-2009

End Date: 01-2010

Amount: $108,481.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882634

Start Date: 2008

End Date: 12-2009

Amount: $220,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100217

Start Date: 2010

End Date: 05-2011

Amount: $450,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989782

Start Date: 10-2009

End Date: 10-2010

Amount: $524,000.00

Funder: Australian Research Council