Maintenance of rat hepatocytes under inflammation by coculture with human orbital fat-derived stem cells

Preservation of hepatocyte functions in vitro will undoubtedly help the management of acute liver failure. The coculture system may be able to prevent functional decline of hepatocytes. It has already been shown that hepatocytes, when cocultured with bone marrow mesenchymal stem cells, could undergo long-term culture in vitro without loss of functions. In this study, human orbital fat-derived stem cells were isolated and cocultured with rat hepatocytes. When treated with serum from an acute liver failure patient, rat hepatocyte monoculture showed reduction of cell viability and loss of liverspecific functions. However, rat hepatocytes in the coculture system were still able to secret albumin and synthesize urea. IL-6 was significantly elevated in the coculture of rat hepatocyte with orbital fat-derived stem cells, and it might be the key immunoregulator which protects rat hepatocytes against inflammation. Our data confirmed that orbital fat-derived stem cells, or other adipose tissue-derived stem cells, are an ideal candidate to support rat hepatocyte functions in vitro.     

Stimulation of hepatocyte survival and suppression of CCl4-induced liver injury by the adenovirally introduced C/EBPbeta gene

Gene therapy has attracted attention as a potentially effective alternative to liver transplantation for the treatment of hepatic failure. We chose the C/EBPbeta gene, which plays vital roles in liver regeneration, as a candidate for gene therapy, and examined its effect on hepatocyte survival and the suppression of liver inflammation. C/EBPbeta gene overexpression significantly maintained hepatocyte viability during 12 days of the culture. Urea synthesis ability, which is a liver-specific function, in Adv-C/EBPbeta-infected hepatocytes was stably maintained during the culture, but the activity per cell was significantly lower than that in non-infected cells. On the contrary, DNA synthesis activity in Adv-C/EBPbeta-infected hepatocytes was significantly higher than that in non-infected cells. COX-2 was induced in Adv-C/EBPbeta-infected hepatocytes, and the addition of NS398, a specific inhibitor of COX-2, suppressed the viability-maintenance effect. COX-2 was thus shown to be involved in the survival effect of C/EBPbeta gene. The introduction of the C/EBPbeta gene into liver-damaged mice significantly suppressed the serum AST and ALT activities. These results indicate that C/EBPbeta appears to be a survival factor under stressful conditions, and the introduction of the gene has therapeutic function against liver injury.     

Enhanced liver-specific functions of endothelial cell-covered hepatocyte hetero-spheroids

The performance of an extracorporeal bioartificial liver (BAL) support system depends on the functional activities of the hepatocytes immobilized in the system. One of the most promising techniques in retaining liver-specific functions is co-culturing hepatocytes with other cell types, such as epithelial cells, endothelial cells and dermal fibroblasts. Primary rat hepatocytes were suspension co-cultured with rat prostate endothelial cell line (RPEn) for 20 h in a spinner vessel to form hetero-spheroids, which contain the two types of the cells, i.e., hepatocytes and endothelial cells in the same spheroid. For the subsequent culture, the hetero-spheroids were entrapped in a Ca-alginate gel bead. From the results of incorporation efficiency test, it was found that RPEn cells have a significantly higher attachment affinity to hepatocytes than human dermal fibroblast and rat liver epithelial cells. We clearly found out that RPEn cells located on the surface of the hepatocyte spheroids from immunostained paraffin sections of the hetero-spheroids. Identical with in vivo liver tissue, laminin was stained at the surface of the hetero-spheroids. Ultrastructures of liver tissue, such as bile canaliculus-like and Disse’s space-like structures, were also found at the surface of the hetero-spheroids. In vivo liver tissue, in which hepatocytes were covered with sinusoidal endothelial cells, was partly mimicked by the endothelial cell-covered hepatocyte spheroids. And the hetero-spheroids showed significantly higher and stable albumin secretion and ammonia removal activities than pure spheroids for 12 days of observations.

Therefore, the endothelial cell-covered hepatocyte hetero-spheroids may offer a useful study model of epithelial–mesenchymal interactions and information about liver tissue engineering research as well as a substitute of a cell source of a BAL system.     

Spheroid formation and expression of liver specific functions of primary rat hepatocytes co-cultured with bone marrow cells

We have developed a new co-culture system consisting of adhesive bone marrow cells (A-BMCs), non-adhesive bone marrow cells (NA-BMCs) and hepatocytes with improved hepatocyte immobilization efficiency and better maintenance of liver specific functions. The composition of the inoculated cells affected the morphology of hepatocytes. Spheroids formed spontaneously when rat hepatocytes were co-cultured with bone marrow cells (BMCs). However, the addition of NA-BMCs to existing hepatocyte monolayers did not change their morphologies to spheroids. On the other hand, NA-BMCs dramatically increased hepatocyte immobilization efficiency. Hepatocytes co-cultured with either NA-BMCs or A-BMCs maintained their albumin production activities significantly better than hepatocyte culture alone. Interestingly, the two fractions of BMCs appear to have combination effects on hepatocytes to maintain albumin production activity. We conclude that co-culture of hepatocytes and BMCs is an effective strategy to enhance hepatocyte immobilization efficiency and functions in vitro.     

Cytosolic sulfotransferase 2B1b promotes hepatocyte proliferation gene expression in vivo and in vitro

Cytosolic sulfotransferase 2B1b (SULT2B1b) catalyzes the sulfation of 3β-hydroxysteroids and functions as a selective cholesterol and oxysterol sulfotransferase. Activation of liver X receptors (LXRs) by oxysterols has been known to be an antiproliferative factor. Overexpression of SULT2B1b impairs LXR's response to oxysterols, by which it regulates lipid metabolism. The aim of this study was to investigate in vivo and in vitro effects of SULT2B1b on liver proliferation and the underlying mechanisms. Primary rat hepatocytes and C57BL/6 mice were infected with adenovirus encoding SULT2B1b. Liver proliferation was determined by measuring the proliferating cell nuclear antigen (PCNA) immunostaining labeling index. The correlation between SULT2B1b and PCNA expression in mouse liver tissues was determined by double immunofluorescence. Gene expressions were evaluated by quantitative real-time PCR and Western blot analysis. SULT2B1b overexpression in mouse liver tissues increased PCNA-positive cells in a dose- and time-dependent manner. The increased expression of PCNA in mouse liver tissues was only observed in the SULT2B1b transgenic cells. Small interference RNA SULT2B1b significantly inhibited cell cycle regulatory gene expressions in primary rat hepatocytes. LXR activation by T0901317 effectively suppressed SULT2B1b-induced gene expression in vivo and in vitro. SULT2B1b may promote hepatocyte proliferation by inactivating oxysterol/LXR signaling.     

Development of hepatocyte spheroids immobilization technique using alternative encapsulation method

Primary hepatocytes of small animals such as rat and rabbit were often used for the study of extracorporeal liver support systems. Freshly isolated rat hepatocytes form spheroids within two days when cultivated as suspension in spinner vessels. These spheroids showed enhanced liver specific functions and more differentiated morphology compared to hepatocytes cultured as monolayers. However, shear stress caused by continuous agitation deteriorated spheroids gradually. In this work we immobilized spheroids to prolong liver specific activities. First, hepatocyte spheroids were suspended in collagen solution containing calcium chloride and then dropped into alginate solution. A thin layer of calcium alginate was formed around the droplet and then was removed after the inner collagen was gelled by treatment of sodium citrate buffer. Spheroids embedded in collagen-gel bead maintained liver specific functions such as albumin secretion rate longer than hepatocyte spheroids exposed to shear stress. Therefore, we suggest that this immobilization technique may offer an effective long-term hepatocyte cultivation and facilitate the development of a bioartificial liver support device.     

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.     

Hypoxic conditions restore lost sensitivity to the growth inhibitory effect of transforming growth factor beta-1 (TGF beta-1) in proliferating rat hepatocytes in vitro

TGF beta-1 is known to be a growth inhibitor of regenerating liver, and an inducer of hepatocyte apoptosis in primary culture. However, hepatocytes can proliferate after partial hepatectomy even at high serum TGF beta-1 concentrations. In this study we used the primary cultures of rat hepatocytes for 10 days to investigate how TGF beta-1 affects proliferating hepatocytes. DNA synthesis peaked on day 8 of culture, and TGF beta-1-induced apoptosis was significantly suppressed on day 8 compared to days 2, 5, and 10. Flow-cytometric analysis revealed that hepatocytes that had incorporated BrdU were resistant to the apoptotic effect of TGF beta-1, and Northern blot analysis showed that TGF beta receptor mRNA was down-regulated on day 8. Hypoxic conditions restores TGF beta receptor mRNA expression and the lost sensitivity of proliferating hepatocyte to TGF beta-1.     

Fractionation of Rat Hepatocyte Subpopulations with Varying Metabolic Potential, Proliferative Capacity, and Retroviral Gene Transfer Efficiency

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated thatsmalldiploid andlargepolyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.     

Analysis of the role of the integrin signaling pathway in hepatocytes during rat liver regeneration

To explore the role of the integrin signaling pathway in hepatocytes during rat liver regeneration, the integrin signaling pathway-related gene expression profile in hepatocytes of regenerative liver was detected using Rat Genome 230 2.0 array. The chip data showed that 265 genes of the integrin signaling pathway were included by Rat Genome 230 2.0 array and 132 genes showed significant expression changes in hepatocytes of regenerative liver. The numbers of up-, down- and up/down-regulated genes were 110, 15 and 7 respectively. In addition, bioinformatics and systems biology methods were used to analyze the role of the integrin signaling pathway in hepatocytes. The analysis of gene synergy value indicated that paths 1, 8, 12, and 15 promoted hepatocyte proliferation at the priming phase of liver regeneration; paths 1, 3, 8, and 12–15 enhanced hepatocyte proliferation at the progressing phase; paths 11 and 14 promoted hepatocyte proliferation, while paths 12 and 13 reduced hepatocyte proliferation at the terminal phase. Additionally, the other 8 paths (2, 4, 5–7, 9–10, and 16) were not found to be related to liver regeneration. In conclusion, 132 genes and 8 cascades of the integrin signaling pathway participated in regulating hepatocyte proliferation during rat liver regeneration.     

High levels of Myc expression are required for the robust proliferation of hepatocytes,but not for the sustained weak proliferation

In contrast to the robust proliferation exhibited following acute liver injury, hepatocytes exhibit long-lasting proliferative activity in chronic liver injury. The mechanistic differences between these distinct modes of proliferation are unclear. Hepatocytes exhibited robust proliferation that peaked at 2 days following partial hepatectomy in mice, but this proliferation was completely inhibited by hepatocyte-specific expression of MadMyc, a Myc-suppressing chimeric protein. However, Myc suppression induced weak but continuous hepatocyte proliferation, thereby resulting in full restoration of liver mass despite an initial delay. Late-occurring proliferation was accompanied by prolonged suppression of proline dehydrogenase (PRODH) expression, and forced PRODH overexpression inhibited hepatocyte proliferation. In hepatocytes in chronic liver injury, Myc was not activated but PRODH expression was suppressed in regenerating hepatocytes. In liver tumors, PRODH expression was often suppressed, especially in the highly proliferative tumors with distinct Myc expression. Our results indicate that the robust proliferation of hepatocytes following acute liver injury requires high levels Myc expression and that there is a compensatory Myc-independent mode of hepatocyte proliferation with the regulation of proline metabolism, which might be relevant to liver regeneration in chronic injury.     

Effect of apoE on triglyceride emulsion interaction with hepatocyte and hepatoma G2 cells

The uptake and internalization of a triglyceride emulsion by rat hepatocytes in culture less than 24 hr was either inhibited or uninfluenced by apoE. ApoE significantly increased the uptake of these emulsions in later cultures. Specific low density lipoprotein (LDL) binding was similar for hepatocyte monolayers prior to and after 24 hr. Rat hepatocytes in culture for 2 days, which were treated with collagenase, detached and then replated within 1 hr and were apoE-responsive in 2 hr. Heparin inhibited the apoE stimulation in both hepatocytes and hepatoma monolayers. Heparin wash of hepatocytes or hepatoma cells incubated with apoE-[14C]triolein emulsions at 4 degrees C resulted in a considerable loss in radiolabeled cell lipid. A similar wash after 37 degrees C incubations produced little loss suggesting internalization. Hepatocytes had lower affinity but similar apoE-emulsion binding capacity compared to hepatoma cells. Triolein emulsions with apoE were significantly more rapidly metabolized by the hepatocyte than unsupplemented emulsions. The apoE-mediated hepatocyte lipid uptake was inhibited by apoC proteins. High molar ratios of free fatty acid/albumin also suppressed hepatocyte apoE-mediated lipid uptake. Both rat high density lipoprotein (HDL) and LDL inhibited with a potency directly related to their content of apoE. Human LDL and HDL without apoE also inhibited the interaction with less potency than the rat lipoproteins. Human HDL inhibition was diminished after removal of apoC proteins.     

Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycopmtein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The results showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfection efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatocytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.     

Species-specific toxicity of diclofenac and troglitazone in primary human and rat hepatocytes

Troglitazone was withdrawn from the market shortly after approval for diabetes type II therapy because of strong hepatotoxic effects in man that could not be predicted from regulatory animal or in vitro studies. Another pharmaceutical that is regularly associated with adverse effects on the liver, sometimes leading to acute liver failure, is the widely used non-steroidal anti-inflammatory drug (NSAID) diclofenac. Since the underlying molecular mechanisms are not yet fully known, we treated primary rat and human hepatocyte monolayer cultures for 24 h with different doses of troglitazone and diclofenac to analyze species differences related to toxicity in vitro. Metformin an antidiabetic drug which does not cause severe adverse reactions served as negative control. Human hepatocytes showed a higher sensitivity to troglitazone than rat hepatocytes, while diclofenac-induced cytotoxicity at fairly similar concentrations. By co-treatment with specific inhibitors for cytochrome P450 (CYP) 2C and CYP3A - the major phase I enzymes involved in liver xenobiotic metabolism - we could confirm the prominent role of CYP3A in the bioactivation of troglitazone as well as the role of CYP3A and CYP2C in the activation of diclofenac. Inhibition of these enzymes increased the viability of treated cells in both species. Furthermore, we were able to demonstrate marked species differences in gene expression patterns of troglitazone treated rat and human hepatocytes. In contrast to rat hepatocytes, human cells showed distinct upregulation of various CYPs, regulators of xenobiotic metabolism and marker genes for oxidative stress. In contrast, gene expression alterations in rat and human hepatocytes treated with Diclofenac were rather similar. Altogether our study showed that species-specific effects as well as indications for the mode of action of compounds can be addressed by the use of primary hepatocyte cultures from various species in combination with gene expression profiling.     

Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.     

Effect of 2,5-anhydro-D-mannitol on membrane potential in rat hepatocyte couplets and hepatocyte monolayer cultures

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM), which depletes liver cells of ATP, has been shown to alter liver cell membrane potential (V(m)) in situ and in superfused liver slices. To study this effect of 2,5-AM on hepatocytes in more detail, patch-clamp experiments in the current-clamp mode were performed using two established models, rat hepatocyte couplets and confluent rat hepatocytes in primary culture. 2,5-AM, which has previously been shown to hyperpolarize hepatocytes in superfused liver slices and in vivo, failed to alter V(m) of hepatocyte couplets. Increasing intracellular Ca(2+) by addition of thapsigargin or ionomycin also did not evoke a change of V(m). This is most likely due to a lack of Ca(2+)-dependent K(+) channels in rat hepatocyte couplets. In contrast, 2,5-AM depolarized the cells in confluent hepatocyte monolayers. This depolarization was mimicked after inhibition of Na(+)/K(+) ATPase by ouabain. Ouabain was also able to block 2, 5-AM's effect on monolayer V(m). Thus, 2,5-AM affects the membrane potential of isolated and cultured hepatocytes in a way not comparable with cells integrated in the liver.     

Preservation of hepatocyte plasma membrane domains during cell division in situ in regenerating rat liver

We have utilized antibodies against five domain-specific integral proteins of the rat hepatocyte plasma membrane to examine the fates of the plasma membrane domains during hepatocyte division in the regenerating rat liver. The proteins were quantified on immunoblots of liver homogenates prepared during the peak of hepatocyte mitotic activity, 28-30 hr after two-thirds hepatectomy. Two sinusoidal/lateral proteins, CE 9 and the asialoglycoprotein receptor, and one bile canalicular protein, dipeptidylpeptidase IV, were not changed significantly in amount; whereas one sinusoidal/lateral protein, the epidermal growth factor receptor, and one bile canalicular protein, HA 4, were reduced to less than or equal to 50% of control levels. Light microscopic examination of plastic sections of regenerating liver tissue revealed that the mitotic hepatocytes generally appeared to retain normal contacts with neighboring interphase hepatocytes. Immunofluorescence was used to localize the domain-specific proteins on mitotic hepatocytes identified in 0.5-micron frozen sections of 28- to 30-hr regenerating liver tissue. Independent of mitotic stage, the hepatocytes retained mutually exclusive bile canalicular and sinusoidal/lateral domains, as defined at the molecular level by the distributions of specific proteins, such as HA 4 and CE 9, respectively.