Nonparenchymal Cell Fraction Research Articles

Epithelial colonies cultured from human explanted liver in subacute hepatic failure exhibit hepatocyte, biliary epithelial, and stem cell phenotypic markers.

The liver in subacute hepatic failure may become enriched for hepatic progenitor cells. Liver tissue from such a patient was collagenase digested and, from the nonparenchymal cell fraction, epithelioid colonies were developed. Albumin and alpha-1-antitrypsin (AAT) were secreted for greater than 120 days from these colonies. Reverse transcription-polymerase chain reaction showed expression of markers of both hepatocyte and biliary epithelial phenotypes (cytokeratins 7, 18, and 19, albumin and AAT, hepatocyte growth factor receptor, transforming growth factor beta receptor type II, gamma-glutamyl transpeptidase, biliary glycoprotein). The cell cycle regulator p21 was also expressed. The POU domain transcription factor octamer-binding protein 4 was present in these cells, but not in RNA or cDNA prepared from adult human liver. These markers were maintained even after 165 days culture. Proliferating epithelial-like cells with combined hepatocyte- and biliary-epithelial-specific functional markers and a stem cell marker can be isolated from the nonparenchymal fraction of liver cells in subacute hepatic failure.

Purification of Fetal Mouse Hepatoblasts by Magnetic Beads Coated with Monoclonal Anti-E-Cadherin Antibodies and Their In Vitro Culture

A simple, rapid, and reproducible method of fetal hepatoblast purification was established to investigate mechanisms controlling interactions between hepatoblasts and nonparenchymal cells during liver development. Because E-cadherin is exclusively expressed on the cell membrane of hepatoblasts, magnetic beads coated with monoclonal antibodies to an extracellular epitope of its molecule were used to purify hepatoblasts from a cell suspension prepared from 12.5-day fetal mouse livers. The purity and yield in the hepatoblast fraction prepared in our protocol were more than 90% and approximately 30%, respectively. The nonparenchymal fraction rarely contained hepatoblasts; the rate of hepatoblast contamination in this fraction was less than 1%. Separate cultures of these two fractions were compared with cocultures of both fractions. In culture of the hepatoblast fraction, hepatoblasts formed aggregates similar to a bunch of grapes via their loose adhesion, floating in the medium after 24 h, and dissociated into single cells from the aggregates after 120 h of culture. By contrast, in the mixed culture, the majority of hepatoblasts formed multicellular spheroids after 24 h, and these spheroids changed into monolayer cell sheets after 120 h of culture. The cells comprising these monolayer sheets abundantly expressed albumin and carbamoylphosphate synthase I. In the mixed culture, fibroblastic cells also proliferated extensively with spreading on glass slides and surrounded the hepatoblast or hepatocyte colonies. On the other hand, fibroblastic cells spreading on glass slides decreased gradually in cultures of the nonparenchymal cell fraction alone. These findings indicated that the coexistence of hepatoblasts and nonparenchymal cells may be essential for their mutual survival, proliferation, differentiation, and morphogenesis. The conditioned medium of fetal liver cell cultures could partially replace the effects of the nonparenchymal cells on hepatoblasts in vitro. Our isolation protocol for fetal mouse hepatoblasts using immunobeads can greatly facilitate studies on mechanisms of cell–cell interactions during liver development.

Colony-forming capacity of porcine liver epithelial cells in culture.

Previously, we reported the presence of certain nonparenchymal epithelial cells (NPECs) in adult porcine livers that demonstrate differentiation patterns including an emergence of duct-like structures (DLSs) in the colonies. In the present study, we examined the effect of supplements to the NAIR-1 medium (Dulbecco modified Eagle medium [DMEM]-F12 containing 5% fetal bovine serum [FBS] and 11 supplements) used in these cultures on formation of DLSs-emerged colonies (type I colonies). No type I colonies were observed in the cultures of the nonparenchymal cell fraction when Roswell Park Memorial Institute-1640 medium or DMEM-F12 (1:1) supplemented with 5% FBS was used as the culture medium. NAIR-1 medium without each component did not produce any significant results. No type I colonies were formed when epidermal growth factor, and hydrocortisone and insulin mixture (A) or nicotinamide and l-ascorbic acid phosphate magnesium salt (Asc2P) mixture (B) was added to the DMEM-F12 medium supplemented with 5% FBS. However, when a combination of A and B was added, colonies were formed at a significant level. Together, the number of type I colonies was increased in the combination of A and B containing a higher concentration of Asc2P. We conclude that NPECs need a mixture of Asc2P and other components as supplements for type 1 colony formation.

Evidence for a Sustained Inflammatory Response of the Hepatic Microcirculation after Hemorrhagic Shock

Defined microcirculatory disturbances have been demonstrated during the first hours after hemorrhagic shock involving complex pathophysiological mechanisms. Since clinical complications associated with the inflammatory reaction that accompanies shock and resuscitation become apparent within days, our aim was to establish an experimental model of hemorrhagic shock which simulates this clinical situation. Therefore, a nonlethal rat model was developed allowing for the investigation of the hepatic microcirculation by intravital microscopy up to 4 days after hermorrhagic shock. Anesthetized Sprague-Dawley rats were subjected to hemorrhagic hyptension by blood withdrawal reducing MAP (mean arterial blood pressure) to 40 mm Hg. After 60 min of hypovolemic hypotension, the animals were resuscitated. After 1, 2, 3, or 4 days, sinusoidal perfusion pattern and leukocyte adhesion characteristics were investigated using intravital fluorescence microscopy. In separate, but parallel experiments, the time course of TNFα (tumor necrosis factor α) and ICAM-1 (intercellular adhesion molecule-1) mRNA expressio was examined in enriched parenchymal and non-parenchymal liver cell fractions by Northern blot hybridization. At day 1, sinusoidal narrowing as seen in previous experiments a few hours after shock was not observed but occurred at days 2 and 3 after shock. Permanent adhesion of leukocytes to the sinusoidal endothelium reached a maximum at day 2 after shock and remained significantly increased until day 4 compared with controls. An early TNFα mRNA accumulation was observed in shock animals but not in sham operated controls. In contrast, a sustained expression of ICAM-1 and prolonged accumulation of white blood cells were observed. The data reflect an early transient TNFα mRNA response and a sustained inflammatory reaction associated with shock and resuscitation. The inflammatory pathological alteration of the hepatic microcirculation after hemorrhagic shock are paralleled by a sustained disturbance of the sinusoidal flow patterns.

Separation and characterization of the nonparenchymal cell fraction from the porcine liver

Separation and characterization of the nonparenchymal cell fraction from the porcine liver

Differential gene expression of CINC, NOS II, and ICAM-1 in endotoxemic liver cells by rG-CSF.

We have recently demonstrated that recombinant granulocyte colony-stimulating factor (rG-CSF) modulates lipopolysaccharide (LPS)-induced Kupffer cell activation with subsequent reduction in hepatic leukocyte-endothelial cell interaction, thereby achieving protection against microcirculatory perfusion failure and hepatic dysfunction. To further clarify the underlying mechanisms, rG-CSF treated liver cells were tested for the LPS-induced gene expression of cytokine-induced neutrophil chemoattractant (CINC) and intercellular adhesion molecule-1 (ICAM-1) as potential chemotactic and leukocyte-recruiting factors and for the gene expression of inducible nitric oxide synthase (NOS II) as potential modulator of leukocyte adherence. Using a collagenase, DNAse/pronase digestion technique, hepatic parenchymal and nonparenchymal cell fractions were obtained from livers of in vivo rG-CSF pretreated Sprague-Dawley rats 2 h after LPS exposure. mRNA transcripts were assessed using northern blot analysis. In control livers only ICAM-1 mRNA was found constitutively expressed in hepatic nonparenchymal cells. rG-CSF per se did not affect NOS II, CINC, or ICAM-1 expression in hepatic liver cells, while LPS induced a marked expression of NOS II, CINC, and ICAM-1 in nonparenchymal cells and, to a lesser extent, in hepatocytes. Administration of rG-CSF prior to LPS exposure tended to increase NOS II, CINC, and ICAM-1 mRNA transcripts in hepatocytes. In nonparenchymal cells, however, NOS II and CINC were found reduced in rG-CSF pretreated animals upon LPS exposure. The present data show a strikingly different cell type specific pattern of inflammatory response genes in rG-CSF-modulated hepatic endotoxemia. Reduced expression of NOS II, in particular of CINC, in the nonparenchymal cell fraction may contribute to the reduced leukocyte adherence and thus attenuation of cell-dependent tissue injury in rG-CSF pretreated endotoxemic animals.

Growth potential and differentiation capacity of adult rat hepatocytes in vitro.

We have previously reported a medium that supports the continuous growth of hepatocytes without their losing replicative potential and differentiation capacity for an extended period. The medium contains four key substances in addition to fetal bovine serum, that is, epidermal growth factor, nicotinamide, ascorbic acid 2-phosphate, and dimethyl sulfoxide. When a nonparenchymal cell fraction containing small hepatocytes and nonparenchymal cells was cultured in this medium, small hepatocytes grew clonally and differentiated into cells expressing either mature hepatocyte marker proteins or biliary cell marker proteins. The growth potential of small hepatocytes was variable among the cells, the highest case being that of a single cell that produced a colony containing over 100 cells in 10 days. When a hepatocyte was allowed to divide for 105 days, it produced a colony of approximately 0.2 mm2, which contained approximately 1,700 hepatocytes, indicating that the cell divided more than 10 times. Thus, for the first time, we showed the presence of a small compartment of bipotent and highly replicative clonogenic hepatocytes in the rat adult liver in vitro.

Activation of Proα2(I) Collagen Promoter during Hepatic Fibrogenesis in Transgenic Mice

We previously identified the promoter sequence that is essential for basal and TGF-β-stimulated transcription of α2(I) collagen gene (COL1A2). In the present study, we examined whether the promoter is activated during hepatic fibrogenesis by utilizing transgenic mice harboring the COL1A2 upstream sequence. Intraperitoneal CCl4 administration activated the −17 kb COL1A2 promoter more than 10-fold, whereas partial hepatectomy resulted in no significant change in the promoter activity. The non-parenchymal cell fraction, but not parenchymal hepatocytes, isolated from mice harboring the −313 COL1A2 promoter linked to a β-galactosidase reporter gene contained large amounts of β-galactosidase and endogenous COL1A2 mRNAs. β-galactosidase activity in the cells from CCl4-treated mice was significantly higher than in those from untreated animals. These results indicated that different molecular mechanisms control COL1A2 transcription in CCl4-induced liver injury/fibrosis and physiological regeneration after partial hepatectomy, and that the −313 COL1A2 promoter is activated in a cell type-specific manner during hepatic fibrogenesis.

Growth and differentiation of adult rat hepatocytes regulated by the interaction between parenchymal and non-parenchymal liver cells.

We have devised a medium which supports the continuous growth of hepatocytes without losing their replicative potential and differentiation capacity for a longer period. The medium HCGM, contains four key substances in addition to foetal bovine serum. They are epidermal growth factor, nicotinamide, ascorbic acid 2-phosphate and dimethylsulphoxide. When a non-parenchymal cell fraction containing small hepatocytes and non-parenchymal cells was cultured in HCGM, small hepatocytes grew clonally and differentiated into cells expressing either mature hepatocyte marker proteins or biliary cell marker proteins. Thus, for the first time, we showed the presence of a small compartment of bipotent and highly replicative clonogenic hepatocytes in the rat adult liver. HCGM also supported the growth of stellate cells (Ito cells) which were in the original preparation, suggesting the important role of stellate cells for the successful cultivation of hepatocytes. Together, these results suggest that a microenvironment is produced as a result of cooperative interactions between hepatocytes and stellate cells: one which stimulates the growth and differentiation of clonogenic hepatocytes.

Research workshop: Techniques for primary culture of liver cells

The aim of this study was to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells. Kupffer cells were isolated following sequential pronase/collagenase digestion of the liver and enrichment of a non-parenchymal cell fraction by a single-density gradient centrifugation step using 30% metrizamide. Kupffer cells were isolated and further purified from this cell fraction by centrifugal elutriation. Kupffer cells were isolated at 1017 g at 48-110 mL/min. All Kupffer cell fractions exhibited phagocytosis of 3 μm latex beads. Kupffer cell fractions isolated at 48 and 60 mL/min were predominantly ED2 negative while later fractions (80-110 mL/min) were ED2 positive. Kupffer cells were adherent in culture after 2 h. This method for Kupffer cell isolation resulted in a yield of 80-120 ×10 6 Kupffer cells per liver.

Isolation and primary culture of rat Kupffer cells.

The aim of this study was to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells. Kupffer cells were isolated following sequential pronase/collagenase digestion of the liver and enrichment of a non-parenchymal cell fraction by a single-density gradient centrifugation step using 30% metrizamide. Kupffer cells were isolated and further purified from this cell fraction by centrifugal elutriation. Kupffer cells were isolated at 1017 g at 48-110 mL/min. All Kupffer cell fractions exhibited phagocytosis of 3 microm latex beads. Kupffer cell fractions isolated at 48 and 60 mL/min were predominantly ED2 negative while later fractions (80-110 mL/min) were ED2 positive. Kupffer cells were adherent in culture after 2 h. This method for Kupffer cell isolation resulted in a yield of 80-120 x 10(6) Kupffer cells per liver.

A role for the liver nonparenchymal cell fraction in immunomodulation

A role for the liver nonparenchymal cell fraction in immunomodulation

Expression pattern of heme oxygenase isoenzymes 1 and 2 in normal and stress-exposed rat liver.

Heme oxygenase (HO) catalyzes the oxidative cleavage of the alpha-mesocarbon of Fe-protoporphyrin-IX yielding equimolar amounts of biliverdin-IXa, iron, and carbon monoxide. The HO-system consists of two isoenzymes, namely HO-2 and the inducible isoform HO-1, also referred to as heat shock protein (hsp) 32. Although both parenchymal and non-parenchymal liver cells participate in heme metabolism, the expression pattern of the isoenzymes in normal and stress exposed liver is unknown. To study this, rats underwent either endotoxin (lipopolysaccharide [LPS]) challenge, hemorrhagic hypotension, glutathione (GSH) depletion, or cobalt chloride injection, all known to provoke oxidative stress. HO-2 messenger RNA (mRNA) and protein were constitutively expressed in hepatocytes, Kupffer/endothelial-, and stellate (Ito-) cell enriched fractions. Although both non-parenchymal cell fractions expressed HO-1 transcripts, HO-1 immunoreactive protein was restricted to Kupffer cells in the normal liver. In contrast to HO-2, a significant increase in HO-1 on the whole organ level was noted by hemorrhagic hypotension, GSH depletion, and cobalt chloride injection. However, the distinct stress models led to a strikingly different cell-type specific and sublobular expression pattern of HO-1 gene expression. HO-1 was inducible in sinusoidal lining cells (hemorrhagic hypotension, LPS challenge), in periportal (cobalt chloride), or pericentral (GSH depletion, hemorrhagic hypotension) hepatocytes. The blockade of protein translation before hemorrhage by cycloheximide reduced upregulation of HO-1/hsp32 mRNA significantly (65.4% reduction, P < .05), whereas the inducibility of hsp70 transcript was maintained. In addition to transcriptional regulation, HO-1 seems to be subject to posttranscriptional control in particular in non-parenchymal cells.

Lecithin:Retinol Acyltransferase and Retinyl Ester Hydrolase Activities Are Differentially Regulated by Retinoids and Have Distinct Distributions between Hepatocyte and Nonparenchymal Cell Fractions of Rat Liver ,

The cellular distribution of enzymes that esterify retinol and hydrolyze retinyl esters (RE) was studied in liver of vitamin A–sufficient,–deficient, and deficient rats treated with retinoic acid or N-(4-hydroxyphenyl)-retinamide. Livers were perfused and cell fractions enriched in hepatocytes, and nonparenchymal cells were obtained for assays of RE and enzyme activity. The specific activity of lecithin:retinol acyltransferase (LRAT) was approximately 10-fold greater in the nonparenchymal cell than the hepatocyte fraction from both vitamin A–sufficient and retinoid-treated rats. Total RE mass, newly synthesized [3H]RE and LRAT activity were positively correlated in liver and isolated cells of both normal (P < 0.0001) and retinoid-treated rats (P < 0.0002). In nonparenchymal cells, these three constituents were nearly equally enriched as evaluated by their relative specific activity values (RSA, defined as the percentage of recovered activity divided by the percentage of recovered protein), which were each significantly greater than 1.0, with values of 4.3 for total RE mass (P < 0.05), 3.6 for newly synthesized [3H]RE (P < 0.01) and 3.8 for LRAT activity (P < 0.01). In contrast, the specific activities of neutral and acid bile salt–independent retinyl ester hydrolases (REH) did not vary with vitamin A status, and their RSA values were close to 1.0 in both hepatocytes and nonparenchymal cells. These data show that LRAT and REH are differentially regulated by retinoids and that these enzymes also differ in their spacial distribution between liver parenchymal and nonparenchymal cells.

2,3,7,8-Tetrachlorodibenzo- p-dioxin affects retinol esterification in rat hepatic stellate cells and kidney

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD), a highly toxic environmental contaminant, interferes with retinoid homeostasis. To elucidate the underlying mechanism, the activities of lecithin : retinol and acyl-CoA : retinol acyltransferase (LRAT and ARAT) were determined in liver, kidney, and hepatic parenchymal and nonparenchymal cell fractions from rats 7 days after a single oral dose of 10 μg TCDD/kg body weight (b.w). Severely depressed LRAT activity in hepatic stellate cells, and greatly increased LRAT activity in kidneys, as well as decreased ARAT activity in stellate cells, were seen in TCDD-treated rats. Although the relevance of decreased ARAT activity under physiological conditions is not clear, the changed LRAT activities most likely contributes significantly to the TCDD-induced effects on tissue retinyl ester levels. It is intriguing that TCDD affects LRAT activity in hepatic stellate cells and kidney in opposite directions. The results suggest that effects of TCDD on retinyl ester tissue levels could be due to a specific interaction with retinoid metabolism.

IL-6 stimulates vitronectin gene expression in vivo.

We tested the hypothesis that vitronectin (Vn) is regulated as an acute phase reactant in response to inflammatory stimuli. In initial experiments, Vn levels were measured during the surgically induced acute phase response in humans. The plasma concentration of Vn increased approximately twofold following elective orthopedic surgery and remained elevated up to 5 days. To examine the mechanism(s) of increased Vn synthesis, hepatic Vn mRNA expression and serum levels were examined in three rat models of acute inflammation: LPS (i.v.), CFA (i.p.), or turpentine (s.c.) injection. The serum concentration of Vn increased approximately twofold 24 h following treatment with turpentine. The expression of Vn mRNA in the liver increased markedly as early as 3 h after treatment in these models and remained elevated up to 18 h. Northern blot analysis of RNA isolated from fractionated liver cells derived from rats treated with LPS indicated that Vn was mainly expressed in hepatocytes, but not in the endothelial or nonparenchymal cell fractions. To analyze the individual effects of raised corticosterone and IL-6 levels on the expression of hepatic Vn mRNA, rats were injected (i.p.) with either dexamethasone or purified recombinant rat IL-6. Vn mRNA expression was elevated within 1 h after IL-6 injection, whereas dexamethasone-injected rats showed unchanged Vn expression. Vn mRNA also was increased in rats chronically injected with IL-6. These results indicate that the Vn gene is up-regulated in acute and chronic inflammation, and this induction is primarily mediated by IL-6.

Models for Hepatic Progenitor Cell Activation

Activation of liver progenitor cells was studied in rat liver induced to regenerate after carbon tetrachloride (CCl4) or D-galactosamine (GalN) injury. A change in the concentration of histone-3 mRNA was used as a marker for cell proliferation and the fetal form of alpha-fetoprotein (AFP) mRNA as a marker for fetal hepatoblasts. gamma-Glutamyltranspeptidase (GGT) and glutathione-S-transferase P were used as markers for activation of putative liver progenitor cells. After CCl4 administration, the proliferative response was high but confined primarily to parenchymal cells. No changes in the relative expression of albumin, glutathione-S-transferase P or insulin-like growth factor-II were observed. On the other hand, the level of AFP mRNA was increased modestly and predominantly in the nonparenchymal cell (NPC) fraction. After GalN administration, proliferation of NPC began within 24 hr, primarily in the portal area around the bile ducts. Activated cells were bile "duct-like" in appearance, had scant cytoplasm, and a pale, oval-shaped nucleus. On Day 2, they formed rows and clusters, expanding from the portal zone and invading the parenchyma, as well as proliferating in regions of focal necrosis. On Days 3 and 5, NPC expressing histone-3 mRNA expanded further, forming pseudoducts and islet-like structures (NPC structures) throughout the hepatic lobule. Proliferating NPC were positive for GGT. Some GGT-positive cells on Days 3 and 5 were also positive for fetal AFP mRNA. Expression of fetal AFP mRNA lagged behind that of GGT by 24 hr, was highest on Day 5, and then declined. Expression of albumin mRNA and glucose 6-phosphatase decreased during the first 48 hr after GalN administration and then resumed. These findings indicate that after GalN injury, the liver responds with activation of putative progenitor cells that proliferate and then differentiate through the hepatocyte lineage, whereas the regenerative response after CCl4 administration is primarily through proliferation of preexisting hepatocytes.

Arachidonate metabolism in D-galactosamine or carbon tetrachloride-induced acute and chronic liver injuries in rats

Arachidonate metabolism was examined in rats with experimentally induced acute and chronic liver injuries. Acute liver injury was induced by a single administration of D-galactosamine (D-Galn) and lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). Chronic liver injury was produced by several administrations of CCl4 for 5 weeks. Non-parenchymal liver cells from rats with D-Galn/LPS-induced acute liver injury produced prominently leukotriene B4 and 5-hydroxy-arachidonic acid which were hardly synthesized by the normal rat liver. No apparent changes were observed in the arachidonate metabolism of the non-parenchymal cells of the acute CCl4-injured liver. In chronic liver injury, the production of 6-ketoprostaglandin F1 alpha, a stable metabolite of prostaglandin I2, by the non-parenchymal cell fraction was significantly enhanced in contrast with the fixed amount of the other arachidonate metabolites. These results suggested the arachidonate metabolism by non-parenchymal liver cells might change according to the pathogenesis of the liver disease.

Vitamin A metabolism: new perspectives on absorption, transport, and storage

Vitamin A metabolism: new perspectives on absorption, transport, and storage

Presence of Liver CGRP/Amylin Receptors in Only Nonparenchymal Cells and Absence of Direct Regulation of Rat Liver Glucose Metabolism by CGRP/Amylin

Human calcitonin gene-related peptide (hCGRP-1) and human amylin (hA) have been reported to increase hepatic glucose output in vivo and to bind with high affinity to rat liver plasma membranes, resulting in increased cAMP production. These observations have led to the hypothesis that CGRP or amylin may be physiological regulators of liver glucose metabolism. Liver plasma membranes are derived from several cell types, including parenchymal (hepatocyte), Kupffer, endothelial, lipid storage, and smooth muscle cells. Because the parenchymal cell is responsible for the contribution of the liver to whole-body glucose homeostasis, it is important to verify the location and activity of the CGRP/amylin receptor to this cell. These studies separate liver cells prepared by collagenase digestion into parenchymal and nonparenchymal fractions by metrizamide gradient and differential centrifugation. 125I-labeled [Tyr-0]hCGRP-1 bound with high affinity to nonparenchymal cell fraction and was displaced by both hCGRP-1 and hA. hCGRP-1 bound with greater affinity than hA (Kd = 2.1 +/- 1.6 x 10(-11) vs. 2.6 +/- 1.2 x 10(-8) M) in a manner similar to the binding reported for liver plasma membrane fraction. Linear regression of receptor concentration against nonparenchymal cell count per milliliter was significant (r = 0.999, P = 0.026), leading to an estimate of 3000 receptors/cell. The parenchymal cell fraction bound very little 125I-[Tyr-0]hCGRP-1, and regression of receptor concentration against parenchymal cell count per milliliter was not significant (r = -0.708, P = 0.29), suggesting that binding was not due to parenchymal cells but instead to contamination by nonparenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)