Liver Cell Function Research Articles

Simulation and experimental research on micro-channel for detecting cell status in bio-artificial liver.

Bioartificial liver support system (BALSS) based on culturing hepatocytes is an important research field for the treatment of acute liver failure. It is necessary to monitor the state of liver cell functions during the treatment of BALSS in order to guide clinical treatment. To design a micro-channel chip to achieve flash mixing for timely detection of liver cell status in bioreactors and improving liver cells growth environment to ensure the efficacy of the bio-artificial liver support system. Alanine aminotransferase (ALT) and Urea are chosen as detection indicators to reflect the degree of liver cell injury and the detoxification function. A diamond tandem structure micro-channel is designed and optimized to achieve the efficient mixing of serum and ALT or Urea reagent. The simulation and experimental results show that the diamond tandem structure micro-channel can significantly improve the mixing efficiency and meet the online detecting requirements. The easily controllable diamond tandem structure micro-channel combines the advantages of active and passive mixer and can effectively mix the serum and ALT or Urea reagent. It lays the foundation for online monitoring of liver cells and will help to improve the viability of liver cell in the bioreactor.

180 Evaluation of leptin and ghrelin levels in the blood serum of patients diagnosed with cystic fibrosis

Objectives Leptin and ghrelin are peptide hormones which, working antagonistically, regulate the energy balance of the body. The aim of the study was to evaluate the levels of leptin and ghrelin in the blood serum of patients diagnosed with cystic fibrosis (CF), in relation to their nutritional status and function of liver cells. Methods The study group consisted of 34 patients, aged from 3 months to 18 years (mean age 4.5 years) who were diagnosed with CF on the basis of screening and/or diagnostic tests. The analysis included the nutritional status, CFTR gene mutation, assessment of the exocrine pancreas function, abnormal lipid metabolism, as well as liver cell function parameters. In all children, serum leptin and ghrelin levels were measured by means of an immunoenzymatic test, using reagents from DRG Instruments. The results were statistically analyzed. Results Serum ghrelin levels were significantly lower in the youngest patients ( Conclusion Ghrelin and leptin levels in children with cystic fibrosis correlate with the nutritional status and can be an early marker of exocrine pancreatic insufficiency.

Characterization of Liver-Specific Functions of Human Fetal Hepatocytes in Culture

This study was designed to assess liver-specific functions of human fetal liver cells proposed as a potential source for hepatocyte transplantation. Fetal liver cells were isolated from livers of different gestational ages (16-22 weeks), and the functions of cell preparations were evaluated by establishing primary cultures. We observed that 20- to 22-week-gestation fetal liver cell cultures contained a predominance of cells with hepatocytic traits that did not divide in vitro but were functionally competent. Fetal hepatocytes performed liver-specific functions at levels comparable to those of their adult counterpart. Moreover, exposure to dexamethasone in combination with oncostatin M promptly induced further maturation of the cells through the acquisition of additional functions (i.e., ability to store glycogen and uptake of indocyanine green). In some cases, particularly in cultures obtained from fetuses of earlier gestational ages (16-18 weeks gestation), cells with mature hepatocytic traits proved to be sporadic, and the primary cultures were mainly populated by clusters of proliferating cells. Consequently, the values of liver-specific functions detected in these cultures were low. We observed that a low cell density culture system rapidly prompted loss of the mature hepatocytic phenotype with downregulations of all the liver-specific functions. We found that human fetal liver cells can be cryopreserved without significant loss of viability and function and evaluated up to 1 year in storage in liquid nitrogen. They might, therefore, be suitable for cell banking and allow for the transplantation of large numbers of cells, thus improving clinical outcomes. Overall, our results indicate that fetal hepatocytes could be used as a cell source for hepatocyte transplantation. Fetal liver cells have been used so far to treat end-stage liver disease. Additional studies are needed to include these cells in cell-based therapies aimed to treat liver failure and inborn errors of metabolism.

Pharmacokinetics, Safety and Tolerability of Melissa officinalis Extract which Contained Rosmarinic Acid in Healthy Individuals: A Randomized Controlled Trial

The aim of this study was to evaluate the safety, tolerability and pharmacokinetics of single dose of Melissa officinalis extract which contained rosmarinic acid, including food-effects in healthy individuals. A total of eleven healthy individuals were randomly assigned to treatment arms in the two studies [Study 1 (fasted state) and Study 2 (fed state)]. Rosmarinic acid in serum was measured by a coulometric detection method using High-Performance Liquid Chromatography electrochemical detector. The serum concentration of total rosmarinic acid peaked at 1 hour after administration of Melissa officinalis extract containing 500mg rosmarinic acid in fasted state, with a maximum serum concentration 162.20 nmol/ L. The area under the curve for intact rosmarinic acid was calculated from the serum concentration-time profile to be 832.13 nmol • hour/ L. Food intake increases area under the curve and delayed time at which the maximum serum concentration. Rosmarinic acid supplementation did not affect liver, kidney, or blood cell function parameters. No adverse event was reported by any of the participants due to the study treatment. Single dose of Melissa officinalis extract containing 500 mg rosmarinic acid appears to be safe and tolerable in healthy individuals. Food intake increased the exposure of rosmarinic acid and delayed absorption of rosmarinic acid in healthy individuals.Trial RegistrationTrial Registration: UMIN-CTR UMIN000004997

Effects of chronic exposure to microcystin-LR on hepatocyte mitochondrial DNA replication in mice.

Microcystins (MCs) are produced by cyanobacterial blooms, and microcystin-LR (MC-LR) is the most toxic among the 80 MC variants. Data have shown that the liver is one of the specific target organs for MC-LR, which can cause mitochondrial DNA (mtDNA) damage, resulting in mitochondrial dysfunction. However, the underlying mechanism is still unclear. In the present study, we evaluated the genetic toxicity of MC-LR in mice drinking water at different concentrations (1, 5, 10, 20, and 40 μg/L) for 12 months. Our results showed that long-term and persistent exposure to MC-LR increased the 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels of DNA in liver cells, damaged the integrity of mtDNA and nuclear DNA (nDNA), and altered the mtDNA content. Notably, MC-LR exposure can change the expression of mitochondrial genes and nuclear genes that are critical for regulating mtDNA replication and repairing oxidized DNA. They also further impaired the function of mitochondria and liver cells.

Modification of nanocellulose by poly-lysine can inhibit the effect of fumonisin B1 on mouse liver cells

Fumonisin B1 is an important mycotoxin, mainly produced by Fusarium verticillioides. It has toxic effects on liver, brain, and kidney cells. The first aim of this study was to synthesize nanocellulose modified with poly-lysine (NMPL), and the second aim was to evaluate the adsorption of fumonisin B1 by NMPL. As third aim, the function of mouse liver cells was investigated after exposure to fumonisin B1, and fumonisin B1+ NMPL. In this study, NMPL was prepared using cross-linker, and then incubated with fumonisin B1 at controlled conditions. After incubation, the adsorption and release of fumonisin B1 were evaluated in each condition. Next, mouse liver cells were separately exposed to fumonisin B1, NMPL, and (fumonisin B1+NMPL). Then, the level of aniline aminotransferase (ALT) and aspartate aminotransferase (AST) was evaluated. It was found that both adsorption and release of fumonisin B1 were not affected by temperature and incubation time, but affected by pH and concentration of NMPL. Also, this study showed NMPL could adsorb fumonisin B1 in different foodstuffs. Importantly, although the levels of ALT and AST were increased when the cells were treated with fumonisin B1 alone, they were not affected when exposed to NMPL or (fumonisin B1+NMPL). The authors suggest that NMPL is a good adsorbent to remove and inhibit fumonisin B1.

Concentrations of Ghrelin and Leptin in Children with Cystic Fibrosis

Ghrelin and leptin are peptide hormones which, working antagonistically, regulate the energy balance of the body. Abnormal concentrations of these hormones are observed in the course of gastrointestinal diseases, where the clinical picture is characterized by nutritional disorders. Approximately 70% of patients with cystic fibrosis have body weight deficiency due to exocrine pancreatic insufficiency, respiratory failure, and secondary circulatory disorders as well as eating disorders. The aim of the study was to evaluate the levels of ghrelin and leptin in the blood serum of patients diagnosed with cystic fibrosis, in relation to their nutritional status and function of liver cells. Patients and methods: The study group consisted of 34 patients, 17 girls (50%) and 17 boys (50%), aged from 3 months to 18 years (mean age 4.5 years) who were diagnosed with cystic fibrosis on the basis of screening and/ or diagnostic tests. The analysis included the nutritional status (body weight and height, BMI), CFTR gene mutation, assessment of the exocrine pancreas function (albumin and glucose concentration in the blood serum, acid steatocrit in stool), abnormal lipid metabolism (cholesterol and triglyceride levels in blood serum), as well as liver cell function parameters and cholestasis (serum activity amino-transpherase, gamma-glutamyl transpeptidase, concentration of bile acids, coagulation parameters). In all children, serum ghrelin and leptin levels were measured by means of an immunoenzymatic test, using reagents from DRG Instruments. The results were statistically analyzed. Results: Serum ghrelin levels were significantly lower in the youngest patients (<1 year of age) compared with older age groups. Ghrelin concentration was significantly lower in patients with salt wasting syndrome (p<0.05). The statistically lower serum leptin levels were observed in patients with growth deficiency and increased parameters of cholestasis, particularly in the youngest age group. Conclusion: Ghrelin and leptin levels in children with cystic fibrosis correlate with the nutritional status and can be an early marker of exocrine pancreatic insufficiency.

Adipose tissue-derived mesenchymal stem cells differentiated into hepatocyte-like cells in vivo and in vitro.

Cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vivo and in vitro hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs) and to explore its therapeutic use. AT-MSCs were isolated and cultured with hepatic differentiation medium. Bioactivity assays were used to study the properties of AT-MSCs. The morphology of differentiated AT-MSCs in serum-free hepatic differentiation medium changed into polygonal epithelial cells, while the morphology of AT-MSCs in a similar medium containing 2% fetal bovine serum remained unchanged. The differentiated cells cultured without serum showed hepatocyte-like cell morphology and hepatocyte-specific markers, including albumin (ALB) and α-fetoprotein. The bioactivity assays revealed that hepatocyte-like cells could take up low-density lipoprotein (LDL) and store glycogen. Furthermore, trichostatin A (TSA) enhanced ALB production and LDL uptake by the hepatocyte-like cells, analogous to the functions of human liver cells. ALB was detected in the livers of the CCl4-injured mice one month post-transplantation. This suggested that transplantation of the human AT-MSCs could relieve the impairment of acute CCl4-injured livers in nude mice. This therefore implied that adipose tissue was a source of multipotent stem cells which had the potential to differentiate into mature, transplantable hepatocyte-like cells in vivo and in vitro. In addition, the present study determined that TSA was essential to promoting differentiation of human MSC towards functional hepatocyte-like cells. The relief of liver injury following treatment with AT-MSCs suggested their potential as a novel therapeutic method for liver disorders or injury.

Adult human liver contains intermediate-type proteasomes with different enzymatic properties

Background. The 20S proteasome is the proteolytic core of the major intracellular protein degradative system, the ubiquitin-proteasome system. Since little is known about proteasomes of human liver, we have investigated the proteasome spectrum in adult human liver.Material and methods. 20S proteasomes were chromatographically purified from adult human liver and from HuH7 cells. They were divided into subpopulations and subtypes and characterized with regard to their proteolytic activities using short fluorogenic oligo- and long poly-peptide substrates. Their subunit composition was studied by immunoblotting.Results. Proteasomes from adult human liver tissue can be separated into three subpopulations (I, II, III), each of which is composed of several subtypes, which total to a spectrum of 14 different subtypes. Two minor subtypes contain only the immuno-subunits β1i and β5i but not their standard counterparts; all others are intermediate subtypes containing β1 and β5 standard- and β1i and β5i immuno-subunits in various compositions. With regard to the proteolytic activities we observed that a decreasing content of subunit β1i in the subtypes goes along with a decreasing ratio of chymotrypsin-like/caspase-like activity, whereas the degradation rate of a 30 mer polypeptide substrate increased with decreasing β1i content. By comparison, 20S proteasomes from HuH7 cells do not contain immuno-subunits but are pure standard proteasomes, which can be separated into three subtypes.Conclusion. These findings suggest that adult human liver contains a spectrum of 14 different 20S proteasome subtypes with different enzymatic properties reflecting most probably an adaptive response of liver cell functions to challenging factors during lifetime.

Surface wettability of model microporous membranes enhances rat liver cell functions in sub-confluent adherent culture in a continuous-flow recycle bioreactor depending on the ammonia concentration challenge

Polymeric membranes used in bioreactors for bioartificial livers are generally chosen for their transport and separation properties to provide liver cells with adequate nutrients supply and avoid rejection. Possible effects of membrane surface properties on cell metabolism are generally given little consideration. The reported effects of membrane surface wettability on adherent liver cells are qualitative and inconsistent, possibly due to the variation of other surface properties and the culture in Petri dishes, often at confluence, under uncontrolled time- and space-varying metabolite concentrations.In this investigation, rat liver cells were cultured in sub-confluent adhesion on model membranes hydrophilized by physical treatment featuring varying surface wettability in a continuous-flow recycle bioreactor. Bioreactor optimization permitted to culture cells at uniform and measurable pericellular concentrations of metabolic substrates, and to challenge them with controlled increasing ammonia concentrations. Membrane surface wettability was characterized in terms of water sorption, dynamic contact angle, and oxygen content by XPS. The kinetics of oxygen consumption, ammonia elimination and urea synthesis of cells adherent on membranes with increasing wettability was characterized at increasing ammonia concentrations. Cells exhibited increasingly better metabolic functions on membranes with increasing surface wettability. Metabolic reaction rate differences were increasingly more evident at increasing ammonia concentrations. Membrane surface wettability appeared to mainly affect cell capacity to respond to the ammonia challenge.

English

BACKGROUND: The involvement of liver in dengue fever is not uncommon as reported in literature since 1970. Liver and nervous system involvement simultaneously predicts poor outcome in dengue fever. Atypical manifestations include liver involvement with elevation of enzymes, central nervous involvement (encephalopathy) and cardiac alterations (myocarditis). Liver involvement in dengue fever is manifested by the elevation of transaminases representing reactive hepatitis, due to direct attack of virus itself or the use of hepatotoxic drugs. OBJECTIVE OF THE STUDY: Study of serum aminotransferase levels in dengue fever. METHODOLOGY: In this descriptive, cross sectional study, all patients who presented to the Department of medicine with dengue IgM positive were included. Study period of 24 months from July 2010-June 2012, patients attending to M.S. Ramaiah medical college were included (n=166). RESULTS: 166 patients reactive for dengue virus specific IgM antibody were studied. As per WHO classification, 137 (82.5%) patients were classified as dengue fever, 20 (12%) as dengue hemorrhagic fever, and 9 (5.4%) as dengue shock syndrome. Mean age of dengue infection patients was 35.71 ±12.9 (SD) years, with male to female ratio nearly equal. Hepatic dysfunction is very common in all forms of dengue infection, with AST rising significantly more than ALT. Serum aminotransferase levels appear to have a directly proportional correlation with grading of dengue infection. Hyperbilirubinemia, elevated transaminases, hypoproteinemia, hypoalbuminemia and deranged coagulation profile were seen in higher frequency in DHF and DSS group as compared to classical DF group. AST and ALT were significantly higher in patients with secondary infection (IgM & IgG positive) as compared to primary infection (IgM positive). CONCLUSION: Serum aminotransferase levels are significantly raised in all forms of dengue infection and it directly correlates with severity of infection. Serum aspartate aminotransferase was significantly raised compared to alanine aminotransferase levels in all forms of dengue infection. The degree of affection of serum albumin and prothrombin time which are absolute indicators of liver cell function correlated with severity of dengue infection.

Effects of different types of fluid resuscitation on hepatic mitochondria and apoptosis

The aim of this study was to observe the effects of different types of fluid resuscitation on hepatic mitochondria and apoptosis in hemorrhagic shock, and the corresponding mechanisms. Forty rats were divided into five groups: Sham surgery (Sham group), shock (Shock group), Ringer’s lactate resuscitation (RL group), hydroxyethyl starch resuscitation (HES group) and autologous blood resuscitation (BL group). A model of hemorrhagic shock was successfully induced in the latter four groups. The recovery objective was to maintain the mean arterial pressure (MAP) of the rats at 80 mmHg. Two hours after the end of the recovery experiment, fresh liver samples were examined in order to observe the changes in the morphology and mitochondrial membrane potential (ΔΨm). In addition, the levels of succinate dehydrogenase (SDH) activity were assessed, and a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was conducted to evaluate the level of apoptosis in the liver cells. In the Shock, RL, HES and BL groups, mitochondrial ultrastructural damage in the liver cells, significant reductions in liver cell function, liver ΔΨm and SDH activity, and the apoptosis of hepatocytes were more apparent compared with those in the Sham group. In the BL group, compared with the RL and HES groups, the injuries to the mitochondrial ultrastructure and liver cell function were significantly reduced, the hepatic ΔΨm and SDH activity were significantly increased and the hepatocyte apoptosis index (AI) was significantly reduced (P<0.05). In conclusion, in a rat model of hemorrhagic shock, different methods of fluid resuscitation may improve the liver cells with regard to mitochondrial ultrastructure and function, the stability of liver ΔΨm, the activity of SDH and the inhibition of liver cell apoptosis. The results indicate that infusion with autologous blood followed by RL solution is a preferable method of fluid resuscitation compared with HES.

Rainbow trout peptidoglycan recognition protein has an anti-inflammatory function in liver cells

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are structurally conserved through evolution in both invertebrate and vertebrate animals. PGRPs exert diverse host-defense functions both through direct antibacterial activity and through indirect effects, including the induction of antimicrobial peptides and the modulation of inflammation and immune responses. In this study, we identified the gene encoding a long form of PGRP (OmPGRP-L1) from the rainbow trout, Oncorhynchus mykiss, and investigated whether it has immunomodulating activity in a rainbow trout hepatoma cell line RTH-149 challenged with fish pathogenic bacteria. OmPGRP-L1 contains the conserved PGRP domain and the four Zn2+-binding amino acid residues required for amidase activity. In RTH-149 cells, OmPGRP-L1 expression was increased by bacterial stimulation. Loss-of-function and gain-of-function experiments indicated that OmPGRP-L1 is involved in the expression of pro-inflammatory cytokines. Silencing of OmPGRP-L1 in RTH-149 cells challenged with Edwardsiella tarda dramatically increased the expression of IL-1β and TNF-α. In contrast, overexpression of OmPGRP-L1 or its amidase-inactive mutant OmPGRP-L1(C472S) resulted in down-regulation of IL-1β and TNF-α expression. When overexpressed in RTH-149 cells, OmPGRP-L1 inhibited NF-κB activity with or without bacterial stimulation. Collectively, these findings suggest that OmPGRP-L1 has an anti-inflammatory function, independent of its amidase activity, possibly via NF-κB inhibition in liver cells.

Ethoxybenzyl diethylenetriamine pentaacetic acid Relationship between liver function and relative signal intensity of MR cholangiography using gadolinium

Objective To evaluate the effect of liver function on the biliary visualization of MR cholangiography (CE-MRC) using gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA). Methods Thirty-nine patients were divided into three groups based on Child-Pugh score (23 patients in group A,11 in B,and 5 in C).CE-MRC was scanned 50 minutes after Gd-EOBDTPA injection.Biliary visualization of CE-MRC at that time was qualitatively assessed by 5-point scale.The relative signal intensity (RSI) of the common bile duct (CBD) was quantitatively measured.The serum bilirubin level and the model of end-stage liver disease (MELD) score were recorded.Kruskal-Wallis test was used to compare the rating scores among three groups.Bonferroni method was used to further pairwise comparison.The one-way ANOVA was used to compare the RSI of CBD among three groups.And regression analysis was used to evaluate the relationship between RSI and the influenced factors. Results Visualization rating scores of the CBD,left hepatic duct (LHD),right hepatic duct (RHD),subbranches of intrahepatic bile ducts (SIBD),cystic duct (CD) and gallbladder (GB) were 4 (3,4),3 (2,4),3 (2,4),1 (1,2),3 (2,4),3 (2,4) of group A;3 (0,3),1 (0,2),1 (0,2),0 (0,1),2 (0,2),2 (0,2) of group B; and 1 (1,2),1 (1,1),1 (1,1),0 (0,0),0 (0,1),0 (0,1) of group C.Statistically significant differences were found in visualization rating scores of the CBD,LHD,RHD,SIBD,CD and GB among the 3 groups (χ2=17.881,18.707,23.328,13.016,20.170,19.938,P<0.01).Statistically pairwise comparison differences were found between group A and B,either between group A and C.The RSI of the CBD of these three groups was 17.12±0.41,3.95±0.63 and 3.33±0.30 respectively.There were statistically significant difference between 3 groups. (F=235.843,P<0.01).Statistically significant differences of the RSI were shown between group A and B,also between group A and C.The total serum bilirubin level and the MELD score predicted the RSI of the CBD (β=-0.834,-1.042,P<0.01). Conclusion Liver function could affect CE-MRC with Gd-EOB-DTPA and RSI of the CBD.The RSI may reflect liver cell function. Key words: Liver diseases; Contrast media; Magnetic resonance imaging

Protective effect of Tanreqing injection on acute hepatic injury induced by CCl4 in rats

To observe the protective effect of Tanreqing injection(TRQ) on carbon tetrachloride-induced acute hepatic injury in rats. Rats were randomly divided into the normal group and the model group, and injected subcutaneously with 100% CCl4 5 mL x kg(-1) to establish the single CCl4 infection model, in order to observe the changes in rat liver injury after 3 h and 6 h. Subsequently, the multiple CCl4 infection liver injury model was reproduced by subcutaneously injecting 100% CCl4 (5 mL x kg(-1)), 50% CCl4 olive oil solution (2 mL x kg(-1)) and then 20% CCl4 olive oil solution (2 mL x kg(-1)). At 6 h after the first CCl4 injection, the rats were divided into six groups: the model group, the control group, the diammonium glycyrrhizinate-treated group, and TRQ high, middle and low dose groups. They were injected through caudal veins, while a normal control group was set up. Their weight and liver-body ratio were observed. Hepatic inflammation was observed with HE staining. Assay kits were adopted to detect ALT, AST, T. Bil, D. Bil, CHE, TBA, gamma-GT and Alb. According to the single injection model, serum AST and T. Bil of model rats were obviously increased at 6 h after single subcutaneous injection of CCl4, with disordered lobular structure in liver tissues, notable swollen liver cells and remarkable liver injury. According to the results of the multiple injection pharmacological experiment, compared with the normal group, the model group had higher serum ALT, AST, and gamma-GT activities (P < 0. 05), TBA and T. Bil contents (P < 0.05) and lower CHE activity (P < 0.05). HE staining showed disorganized lobular structure in liver tissues and notable ballooning degeneration in liver cells. Compared with the model group, TRQ high and middle dose groups and the diammonium glycyrrhizinate-treated group showed significant charges in serum liver function and inflammation in liver cells. Specifically, TRQ high and middle dose groups were superior to the diammonium glycyrrhizinate-treated group. Tanreqing injection has significant protective effect on CCl4-induced acute hepatic injury in rats.

Editorial to accompany “Effects of statins on liver cell function and inflammation in septic rats”

DOI of original article: 10.1016/j.jss.2012.0 * Corresponding author. Department of Sur Tel.: þ1 253 968 2361; fax: þ1 253 968 5900. E-mail address: matthew.martin1@amed 0022-4804/$ e see front matter Published by http://dx.doi.org/10.1016/j.jss.2013.03.055 “All who drink of this remedy recover in a short time, except those whom it does not help, who all die. Therefore it’s obvious that it fails only in incurable cases”. Galen

Coactivator-Associated Arginine Methyltransferase 1 Regulates Fetal Hematopoiesis and Thymocyte Development

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). In this study, we find that reduced thymopoiesis in Carm1(-/-) mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver (FL) and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1(-/-) E14.5 or E18.5 FL. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1(-/-) FL cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1(-/-) FL cells in a CARM1-sufficient host showed that CARM1 is required cell autonomously in hematopoietic cells. Coculture of Carm1(-/-) FL cells on OP9-DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation.

Toxicity Study of a Novel Oral Iron Chelator: 1-(N-Acetyl-6-Aminohexyl)-3 Hydroxy-2-Methylpyridin-4-One (CM1) in Transgenic b-Thalassemia Mice

Deferiprone (DFP) (MW=139 Da, Kpart=0.11) is an effective iron chelator used for the treatment of iron overload in thalassemia patients, but the drug is not free from side effects. We have synthesized a novel oral bidentate iron chelator, 1-(N-acetyl-6-aminohexyl)-3-hydroxypyridin-4-one (CM1) (MW=256 Da, Kpart=0.53), which is an analogue of DFP. This compound is more lipophilic than DFP and can bind iron efficiently. Our current results have demonstrated that CM1 reduced iron-induced redox damage and decreased levels of the intracellular iron pool (LIP) in cultured hepatocytes, effectively. However, the toxicity of CM1 remains largely unknown. The aim of this study was to therefore examine the toxicity of CM1 treatment in an animal model under normal and iron overload conditions. To induce iron overload, transgenic s-thalassemia (BKO) mice were fed with a 0.2% (w/w) ferrocene-supplemented diet (Fe diet) for 240 days. The mice received three doses of CM1 orally (50,100 and 200 mg/kg), every day for 180 days. Blood was collected from the tail vein every 45 days during treatment for the measurement of hemoglobin (Hb) levels, white blood cells (WBC) and platelet numbers. We also determined the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), which are markers of liver damage. Treatment with CM1 at the assigned doses did not markedly alter the numbers of WBC and the platelets, and the Hb level in BKO mice fed with either N diet or Fe diet. Importantly, all the treatments slightly increased the activities of plasma AST, ALT and ALP in BKO mice after 150 days. Nonetheless, hematoxylin and eosin staining results did not show abnormal morphological changes of the spleen, liver and heart tissues. The results imply that CM1 may not be toxic to bone marrow cells and liver cell function in BKO mice under normal and iron overload conditions.

Humoral Mechanisms Regulating the Functions of Progenitor Cells in Chronic Hepatitis

We studied the secretory function of the liver and bone marrow microenvironment cells under conditions of chronic CCl4-induced hepatitis. Enhanced production of substances stimulating parenchymal tissue-specific precursors and stem cell homing factors by liver cells was observed. In situ binding of stem cells by bone marrow elements increased due to production of various cooperation and adhesion factors against the background of reduced SDF-1 secretion.

GROWTH AND DEVELOPMENT SYMPOSIUM: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-1912

Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent differentiation of the PICM-19 cells, enhance our ability to genetically modify the cells, and provide a better model system to investigate porcine hepatic metabolism.