AR42J-B-13 cell: An expandable progenitor to generate an unlimited supply of functional hepatocytes

Wallace, Karen; Fairhall, Emma A.; Charlton, Keith A.; Wright, Matthew C.

doi:10.1016/j.tox.2010.05.008

Cited by 25 publications

(49 citation statements)

References 105 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is also important to keep the culture plates undisturbed for at least 4 h after plating to allow proper cell adherence. Figure 2 shows hepatocytes in culture as early as 5 h and out to 120 h. As seen by phase contrast microscopy, the morphology of the hepatocytes changes over time in culture consistent with documented morphological changes (Bhandari et al 2001;Godoy et al 2010;Li et al 2010;Talamini et al 1997: Wallace et al 2010. These cellular changes include cell polarization, de-differentiation as well as down regulation of gene expression (Bhandari et al 2001;Godoy et al 2010;Li et al 2010;Talamini et al 1997: Wallace et al 2010.…”

Section: Hepatocyte Culture and Cellular Changessupporting

confidence: 55%

“…Because of their central role in liver function, the use of freshly isolated primary hepatocytes has become an important tool to investigate various aspects of liver biology including metabolic disease, toxicology, oncology, and parasitology (Goncalves et al 2007;Qian et al 2007;Wallace et al 2010;Warner et al 2003;Wu et al 2002). Hepatocytes isolated using the standard two step perfusion method have been used in culture as well as in cell suspension assays for many years (Klaunig et al 1981;Li et al 2010;Warner et al 2003).…”

Section: Introductionmentioning

confidence: 99%

“…Hepatocytes isolated using the standard two step perfusion method have been used in culture as well as in cell suspension assays for many years (Klaunig et al 1981;Li et al 2010;Warner et al 2003). In these ex vivo assays, cells are used immediately after isolation to avoid changes in gene regulation, polarization and dedifferentiation (Bhandari et al 2001;Godoy et al 2010;Talamini et al 1997;Wallace et al 2010). The main downside of the current protocols is the requirement for cell purification through gradient centrifugation which extends the time that the cells are manipulated and can result in reduced cell number and viability.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development

et al. 2011

View full text Add to dashboard Cite

Primary mouse hepatocytes are an important tool in the biomedical research field for the assessment of hepatocyte function. Several methods for hepatocyte isolation have been published; however, many of these methods require extensive handling and can therefore compromise the viability and function of the isolated cells. Since one advantage of utilizing freshly isolated cells is to maintain an environment in which the cells are more comparable to their in vivo state, it is important to have robust methods that produce cells with high viability, good purity and that function in a similar manner to that in their in vivo state. Here we describe a modified twostep method for the rapid isolation and characterization of mouse primary hepatocytes that results in high yields of viable cells. The asialoglycoprotein receptor (ASGPR), which is one of the most abundant cell surface receptors on hepatocytes, was used to monitor the function of the isolated hepatocytes by demonstrating specific binding of its ligand using a newly developed flow cytometry based ligand-receptor binding assay. Also, an in vitro screening method for siRNA drug candidates was successfully developed utilizing freshly isolated hepatocytes with minimum culture time.

show abstract

Section: Hepatocyte Culture and Cellular Changessupporting

confidence: 55%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development

et al. 2011

View full text Add to dashboard Cite

show abstract

“…Hepatocytes are the primary defining cell of the liver, performing the vast majority of its functions (Wallace et al , 2010a). Isolation and/or culture of hepatocytes are therefore common experimental techniques employed in the study of liver functions (Wallace et al , 2010a).…”

mentioning

confidence: 99%

Utility of B-13 Progenitor-Derived Hepatocytes in Hepatotoxicity and Genotoxicity Studies

Probert¹,

Chung

Cockell

et al. 2013

Toxicological Sciences

Self Cite

View full text Add to dashboard Cite

AR42J-B-13 (B-13) cells form hepatocyte-like (B-13/H) cells in response to glucocorticoid treatment. To establish its utility in toxicity and genotoxicity screening, cytochrome P450 (CYP) induction, susceptibility to toxins, and transporter gene expression were examined. Conversion to B-13/H cells resulted in expression of male-specific CYP2C11 and sensitivity to methapyrilene. B-13/H cells constitutively expressed CYP1A, induced expression in response to an aryl hydrocarbon receptor agonist, and activated benzo[α]pyrene to a DNA-damaging species. Functional CYP1A2 was not expressed due to deletions in the Cyp1a2 gene. A B-13 cell line stably expressing the human CYP1A2 was therefore engineered (B-13−TR/h1A2) and the derived B-13/H cells expressed metabolically functional CYP1A2. Treatment with the cooked food mutagen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine resulted in a dose-dependent increase in DNA damage. B-13/H cells expressed constitutive androstane receptor (CAR) and induced CYP2B1 mRNA levels in response to classical CAR activators. However, translation to functional CYP2B1 protein was low and increased minimally by CAR activator treatment. B-13/H cells expressed high levels of pregnane X-receptor (PXR) and induced CYP3A1 in response to classical PXR activators. CYP3A genes were inducible, functional, and activated aflatoxin B1 to a DNA-damaging species. All 23 major hepatic transporters were induced when B-13 cells were converted to B-13/H cells, although in many cases, levels remained below those present in adult rat liver. However, bile salt export pump, Abcb1b, multidrug resistance-associated protein, and breast cancer resistance protein transporters were functional in B-13/H cells. These data demonstrate that the B-13 cell generates hepatocyte-like cells with functional drug metabolism and transporter activities, which can alone—or in a humanized form—be used to screen for hepatotoxic and genotoxic endpoints in vitro.

show abstract

“…Transdifferentiation is a form of metaplasia and describes the irreversible switching of cellular differentiation in fully differentiated cells (Burke and Tosh, 2005;Wallace et al, 2010a). Administration of glucocorticoid to rats results in the appearance of hepatocytes in the pancreas ).…”

Section: Introductionmentioning

confidence: 99%

Serine/threonine protein kinase SGK1 in glucocorticoid-dependent transdifferentiation of pancreatic acinar cells to hepatocytes

Wallace

Long

Fairhall

et al. 2011

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

Elevated glucocorticoid levels result in the transdifferentiation of pancreatic acinar cells into hepatocytes through a process that requires a transient repression of WNT signalling upstream of the induction of C/EBP-β. However, the mechanism by which glucocorticoid interacts with WNT signalling is unknown. A screen of microarray data showed that the serine/threonine protein kinase SGK1 (serum- and glucocorticoid-regulated kinase 1) was markedly induced in the model B-13 pancreatic rat acinar cell line after glucocorticoid treatment (which converts them into hepatocyte-like ‘B-13/H’ cells) and this was confirmed at the level of mRNA (notably an alternatively transcribed SGK1C form) and protein. Knockdown of SGK1 using an siRNA designed to target all variant transcripts inhibited glucocorticoid-dependent transdifferentiation, whereas overexpression of the human C isoform (and also the human SGK1F isoform, for which no orthologue in the rat has been identified) alone – but not the wild-type A form – inhibited distal WNT signalling Tcf/Lef transcription factor activity, and converted B-13 cells into B-13/H cells. These effects were lost when the kinase functions of SGK1C and SGK1F were mutated. Inhibition of SGK1 kinase activity also inhibited glucocorticoid-dependent transdifferentiation. Expression of SGK1C and SGK1F resulted in the appearance of phosphorylated β-catenin, and recombinant SGK1 was shown to directly phosphorylate purified β-catenin in vitro in an ATP-dependent reaction. These data therefore demonstrate a crucial role for SGK1 induction in B-13 cell transdifferentiation to B-13/H hepatocytes and suggest that direct phosphorylation of β-catenin by SGK1C represents the mechanism of crosstalk between glucocorticoid and WNT signalling pathways.

show abstract

AR42J-B-13 cell: An expandable progenitor to generate an unlimited supply of functional hepatocytes

Cited by 25 publications

References 105 publications

A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development

A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development

Utility of B-13 Progenitor-Derived Hepatocytes in Hepatotoxicity and Genotoxicity Studies

Serine/threonine protein kinase SGK1 in glucocorticoid-dependent transdifferentiation of pancreatic acinar cells to hepatocytes

Contact Info

Product

Resources

About