Transplantation of Highly Differentiated Immortalized Human Hepatocytes to Treat Acute Liver Failure

Kobayashi, N.; Miyazaki, M.; Tanaka, Nobukazu; Namba, M.

doi:10.1097/00007890-199905150-00051

Cited by 29 publications

(39 citation statements)

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“…For all experiments, we used a fetus-derived immortalized hepatocyte cell line, OUMS-29, which has been established by Kobayashi et al. 16 and has been confirmed to have the activities of several metabolic enzymes, such as cytochrome P450 (CYP) 1A1, CYP1A2, CYP2E1, bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase, and the gene expression of CYP3A. 21 The hepatoma cell lines HLF and HepG2 were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan) and the Cancer Cell Repository, Tohoku University (Sendai, Japan), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…16 Furthermore, a high functionality in the protein production in OUMS-29 cells has enabled ex vivo experiments for hepatocyte transplantation. 16,17 An in vitro model for PPAR␥-mediated terminal differentiation of adipocytes has been analyzed in detail from the aspect of cell cycle. 18 Those cells, treated with PPAR␥ ligands arrested at the G1 phase, expressed several cyclindependent kinase inhibitors, such as p21 WAF1/Cip1 , p27 Kip1 , and p18 INK4c .…”

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confidence: 99%

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Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes

Shishido

2003

Hepatology

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Troglitazone has been withdrawn from therapeutic options for diabetes mellitus because of its severe hepatocyte toxicity of unknown pathogenesis. The aim of the present study was to assess both morphologic and functional alterations in the mitochondria of troglitazonetreated hepatocytes. A polarized human hepatocyte cell line, OUMS-29, was used in this study. The mitochondrial volume and the mitochondrial transmembrane potential (⌬⌿ m ) were examined using flow cytometry with nonylacridine orange (NAO) and rhodamine-123, respectively. An ultrastructural examination of the troglitazone-treated OUMS-29 cells was performed using transmission electron microscopy (TEM). Reactive oxygen species (ROS) production was assessed using flow cytometry with dihydroethidium and 2 ,7 -dichlorodihydrofluorescein diacetate. A significant increase in the mitochondrial volume of the troglitazone-treated cells was found by the NAO analysis, in comparison with pioglitazone-treated and ciglitazone-treated cells. The increase in volume was due to a marked enlargement in the mitochondria. The markedly enlarged mitochondria with intramitochondrial electrondense deposits were confirmed on TEM, which showed myelin-like structures, indicating degraded membrane constituents. The troglitazone-treated cells showed a significant decline in the ⌬⌿ m per unit mitochondrial volume but resulted in no clear cell death. ROS analysis revealed a significant production of hydrogen peroxide in the troglitazone-treated hepatocytes. This production was attenuated using an antioxidant, N-acetyl-L-cysteine. In conclusion, troglitazone caused overproduction of hydrogen peroxide, which deteriorated both mitochondrial membrane structures and mitochondrial function, leading to a possible priming for the severe hepatocyte toxicity. (HEPATOLOGY 2003;37:136-147.) T roglitazone, a ligand for peroxisome proliferatoractivated receptor ␥ (PPAR␥), used to be administered as an antidiabetic drug until it was discovered to be associated with hepatotoxicity and withdrawn from the market after reports of several dozens of deaths or cases of severe hepatic failure requiring liver transplantation. 1,2 In contrast to troglitazone, 2 other glitazones, pioglitazone and rosiglitazone, are still on the market; however, several cautionary reports have suggested that the latter 2 drugs show hepatotoxicity as a class effect of glitazones. [2][3][4][5][6][7][8][9] Although the troglitazone-associated hepatotoxicity has been considered idiosyncratic direct hepatocyte toxicity, 10 the precise mechanism has remained unclear. In our recent study, mitochondrial damage was ultrastructurally demonstrated in troglitazone-treated human hepatoma cells, 11 allowing us to first focus on alterations in the mitochondrial structure and the function in the troglitazone-treated human hepatocytes. Troglitazone is known

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes

Shishido

2003

Hepatology

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“…17 The 2 basic types of human hepatocyte lines are those derived from human tumors 18,19 and those created by immortalizing human hepatocytes. [20][21][22] Tumor-derived line. One of the most developed BAL systems currently under trial, the ELAD system 23,24 (VITAGEN Inc, La Jolla, CA), uses the C3A cell line, a clonal derivative of the hepatoblastoma-based HepG2 cell line, which is reported to have a more highly differentiated hepatic phenotype than the HepG2 line.…”

Section: Human Hepatocyte Cell Linesmentioning

confidence: 99%

“…Human hepatocyte lines derived from normal liver and immortalized by coculture with rat epithelial cells, such as HH01, HH02, HH09, and HH25 21 cells, show viability in continuous culture for several years and express a spectrum of differentiated hepatocyte functions. This admixture of nonhuman 20 and HepZ. 28 The OUMS-29 is derived from human fetal hepatocytes transfected with the pSV3neo plasmid and contains the genes encoding simian virus 40 large T antigen (SV40T) and neomycin phosphotransferase.…”

Section: Human Hepatocyte Cell Linesmentioning

confidence: 99%

“…The OUMS-29 cells express genes of many liver-specific functions, including albumin, asialoglycoprotein receptor, glutamine synthetase, glutathione-S-transferase-p, and human blood coagulation factor X. 20 Injected into 90% hepatectomy rats, these cells resulted in significant clinical improvement in terms of blood ammonia level, hepatic encephalopathy grade, and survival. 20 Furthermore, preliminary data with the OUMS-29 line did not show tumorigenic features when transplanted into athymic nude mice 29 compared with the HepG2 line, which caused multiple tumor nodules within 2 weeks of infusion into similar mice.…”

Section: Human Hepatocyte Cell Linesmentioning

confidence: 99%

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Which hepatocyte will it be? Hepatocyte choice for bioartificial liver support systems

Tsiaoussis

Newsome

Nelson

et al. 2001

Liver Transplantation

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Liver failure, notwithstanding advances in medical management, remains a cause of considerable morbidity and mortality in the developed world. Although bioartificial liver (BAL) support systems offer the potential of significant therapeutic benefit for such patients, many issues relating to their use are still to be resolved. In this review, these issues are examined in terms of the functions required, the cells of choice in such a system, and the most appropriate environment to optimize the function of such cells T he development of nonbiological systems to support critically ill patients with acute liver failure (ALF) that incorporate plasmapheresis, 1 charcoal hemoperfusion, 2 and ultrafiltration techniques 3 has not had a significant impact on survival. Consequently, hepatocytebased biological systems have attracted intense interest worldwide but have also generated most of the controversy. Uncertainty exists about the most suitable biological component and the risk for zoonoses and immune rejection in the host. In addition, the optimization of culture conditions and thus metabolic function of the biological component have not been systematically studied, although continuing improvements in these areas have rendered this therapeutic avenue in ALF more promising. To compound matters, the question of which aspects of hepatocyte metabolism in an artificial liver are required to sustain life and promote recovery remains largely unanswered, further limiting development. In this review, the biological substrate of such systems is critically examined and insights are provided into the ideal system.

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Heterologous Over-expression ofα-1,6-Fucosyltransferase fromRhizobium sp.: Application to the Synthesis of the Trisaccharideβ-D-GlcNAc(1→4)- [α-L-Fuc-(1→6)]-D-GlcNAc, Study of the Acceptor Specificity and Evaluation of Polyhydroxylated Indolizidines as Inhibitors

et al. 2001

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An efficient heterologous expression system for overproduction of the enzyme alpha-1,6-Fucosyltransferase (alpha-1,6-FucT) from Rhizobium sp. has been developed. The gene codifying for the alpha-1,6-FucT was amplified by PCR using specific primers. After purification, the gene was cloned in the plasmid pKK223-3. The resulting plasmid, pKK1,6FucT, was transformed into the E. coli strain XL1-Blue MRF'. The protein was expressed both as inclusion bodies and in soluble form. Changing the induction time a five-fold increase of enzyme expressed in soluble form was obtained. In this way five units of enzyme alpha-1,6-FucT can be obtained per liter of culture. A crude preparation of the recombinant enzyme was used for the synthesis of the branched trisaccharide alpha-D-GlcNAc-(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GlcNAc (3), from chitobiose (2) and GDP-Fucose (1). After purification, the trisaccharide 3 was obtained in a 84% overall yield. In order to elucidate the structural requirements for the acceptors, the specificity of the enzyme was studied towards mono-, di- and trisaccharides, which are structurally related to chitobiose. The enzyme uses, among others, the disaccharide N-acetyl lactosamine as a good substrate; the monosaccharide GlcNAc is a weak acceptor. Finally, several racemic polyhydroxylated indolizidines have been tested as potential inhibitors of the enzyme. Indolizidine 21 was the best inhibitor with an IC50 of 4.5 x 10(-5) M. Interestingly, this compound turned out to be the best mimic for the structural features of the fucose moiety in the presumed transition state.

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Transplantation of Highly Differentiated Immortalized Human Hepatocytes to Treat Acute Liver Failure

Cited by 29 publications

References 0 publications

Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes

Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes

Which hepatocyte will it be? Hepatocyte choice for bioartificial liver support systems

Heterologous Over-expression ofα-1,6-Fucosyltransferase fromRhizobium sp.: Application to the Synthesis of the Trisaccharideβ-D-GlcNAc(1→4)- [α-L-Fuc-(1→6)]-D-GlcNAc, Study of the Acceptor Specificity and Evaluation of Polyhydroxylated Indolizidines as Inhibitors

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