Improvement of C3A cell metabolism for usage in bioartificial liver support systems

Filippi, Céline; Keatch, Steven A.; Rangar, D; Nelson, Leonard J.; Hayes, Peter; Plevris, John

doi:10.1016/j.jhep.2004.06.012

Cited by 40 publications

(28 citation statements)

References 36 publications

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“…This data shows that C3A cells have a similar phenotype to the parental HepG2 cell line as regards urea cycle expression (Mavri-Damelin et al, 2007). However, C3A cells are cited in the literature as being able to produce urea, and whilst this has been assumed to be reflective of a functional urea cycle (David et al, 2004;Ellis et al, 1996;Filippi et al, 2004;Stange et al, 1995;Wang et al, 1998), we have conclusively shown by stable isotope labelling, that ammonia cannot be specifically removed by this pathway and converted to urea. This has significant implications in the development and use of C3A cells in BAL devices, where they are currently used and considered to possess urea cycle function and therefore ammonia detoxificatory capability (Ellis et al, 1996); of course this study did not address their potential value in liver failure via provision of the other synthetic and detoxificatory functions that they possess.…”

Section: Discussionmentioning

confidence: 74%

“…Two such cell lines, HepG2 and the HepG2 subclone C3A, are commonly used (Kelly and Darlington, 1989). Previous studies have shown that C3A cells, in particular, are capable of producing urea, a finding which has been generally interpreted as indicating the presence of a fully functional urea cycle capable of ammonia detoxification via this specific pathway, and has led to their use in clinically trialled bioartificial devices (Chen et al, 2003;David et al, 2004;Ellis et al, 1996;Filippi et al, 2004;Wang et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

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Cells for bioartificial liver devices: The human hepatoma‐derived cell line C3A produces urea but does not detoxify ammonia

Mavri-Damelin

Damelin

Eaton³

et al. 2007

Biotech & Bioengineering

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Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function.

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Section: Discussionmentioning

confidence: 74%

Section: Introductionmentioning

confidence: 99%

Cells for bioartificial liver devices: The human hepatoma‐derived cell line C3A produces urea but does not detoxify ammonia

Mavri-Damelin

Damelin

Eaton³

et al. 2007

Biotech & Bioengineering

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“…HepG2, HEK293, and C3A cells (American Type Culture Collection, Rockville, MD) were cultured as recommended with 10% fetal bovine serum. HepG2 is derived from a hepatocellular carcinoma and C3A is a clonal derivative of HepG2 that exhibits a better differentiated hepatic phenotype (Filippi et al, 2004). The cloning of UGT2B7_v1, v5, and v7 cDNAs encoding UGT2B7 i1, i2, and i4 proteins and methodologies for transfection of HEK293 cells to produce stable clones have been described (Ménard et al, 2011).…”

Section: Methodsmentioning

confidence: 99%

Modulation of the UGT2B7 Enzyme Activity by C-Terminally Truncated Proteins Derived from Alternative Splicing

et al. 2013

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The enzyme UGT2B7 is one of the most active UDP-glucuronosyltransferases (UGTs) involved in drug metabolism and in maintaining homeostasis of endogenous compounds. We recently reported the existence of 22 UGT2B7 mRNAs, two with a classic 59 region but alternative 39 ends namely UGT2B7_v5 (containing a novel terminal exon 6b) and _v7 (exon 5 excluded) that encode enzymatically inactive isoforms 2 and 4 (i2 and i4), respectively. The v1 mRNA encoding the UGT2B7 enzyme (renamed isoform 1 or i1) is coexpressed with the splice variants v5 and v7 in human liver, kidney, and small intestine and the hepatic cell lines HepG2 and C3A. The presence of alternate v5 and v7 transcripts in isolated polysomes from these hepatic cells further supports endogenous protein translation. Cellular fractionation of clonal HEK293 cell lines overexpressing UGT2B7 isoforms demonstrates that i1, i2, and i4 proteins colocalize in the microsomal/Golgi fraction, whereas i2 and i4 can also be found in the cytosol; a finding sustained by immunofluorescence experiments using tagged proteins. By modifying splice variant abundance in overexpression in HEK293 and HepG2 cells as well as RNA interference experiments in HepG2 and C3A cells, we observe drug glucuronidation phenotypes compatible with variant-mediated repression of UGT2B7 activity without consequent alteration of the apparent enzyme affinity (K m ). Finally, coimmunoprecipitation experiments support a direct proteinprotein interaction of i2 and i4 proteins with the functional UGT2B7 enzyme as a potential causative mechanism. These findings point toward a novel autoregulatory mechanism of the UGT2B7 glucuronidation pathway by naturally occurring alternative i2 and i4 proteins.

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“…In addition to marker expression, we studied hESC-derived hepatocyte functionality. hESC-derived AW and AB HLCs (AWHLCs and ABHLCs, respectively) were matured using either L-15 (26) or Eagle's Minimal Essential Medium (MEME) in the presence or absence of a mixture known to induce hepatocyte function in vitro (27). After incubation, hepatocyte metabolism was measured by 2 assays, ureagenesis and gluconeogenesis.…”

Section: Aw-driven Hesc Differentiation Produces Hlcs That Express a mentioning

confidence: 99%

“…Hepatocyte metabolism was measured in the context of gluconeogenesis and ureagenesis as described (27). CYP1A2 activity was measured in 1 ϫ 10 5 HLCs or freshly isolated human hepatocytes (Johnson & Johnson) using 2 M 7-ethoxyresorufin in culture medium for 24 h at 37°C.…”

Section: Measurement Of Hepatocyte Metabolismmentioning

confidence: 99%

Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling

Hay

Fletcher

Payne

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

389

354

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Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function.definitive endoderm ͉ function ͉ hepatocyte ͉ drug metabolism ͉ high throughput H uman embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos and demonstrate pluripotency in vitro and in vivo (1, 2). Such attributes allow hESCs to be differentiated down all germ lineages in large numbers and offer significant advantages over their adult stem cell counterparts, which are generally limited in their capacity to differentiate and proliferate (3). Although these hESCs provide a valuable source of adult differentiated cells, homogeneous cellular differentiation to specific germ layers has proven difficult to achieve. One potential explanation for this failure is that cells do not receive sequential developmental cues that they do in vivo.Wnt signaling has been shown to play an important role in hESC self-renewal and differentiation and stimulates numerous intracellular signal transduction cascades, including the canonical pathway regulating gene expression in the nucleus and what seems to be a network of noncanonical pathways regulating many other aspects of cell biology [reviewed by Cadigan and Liu (4)]. In the absence of Wnt signaling, ␤-catenin is targeted for degradation; however, active Wnt signaling inhibits ␤-catenin destruction, resulting in its nuclear translocation (5, 6). After nuclear localization, ␤-catenin dimerizes with the nuclear proteins from the T cell factor/lymphoid enhancer factor (TCF/ LEF) family and transactivates gene expression. TCF/LEFs are not only present in transcriptional activator complexes; they also play a role in corepressor complex assembly (6).The important role played by Wnt signaling during gastrulation in vivo is evidenced by gene knockouts or dominant negatives (7, 8). Wnt3-mediated Brachyury expression is also important for migration of precursor cells through the anterior region of the primitive streak (PS). The subsequent specification of the anterior region of the PS to mesoderm or endoderm is likely to depend on the duration and magnitude of Nodal signaling (...

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Improvement of C3A cell metabolism for usage in bioartificial liver support systems

Cited by 40 publications

References 36 publications

Cells for bioartificial liver devices: The human hepatoma‐derived cell line C3A produces urea but does not detoxify ammonia

Cells for bioartificial liver devices: The human hepatoma‐derived cell line C3A produces urea but does not detoxify ammonia

Modulation of the UGT2B7 Enzyme Activity by C-Terminally Truncated Proteins Derived from Alternative Splicing

Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling

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