Exploring interactions between rat hepatocytes and nonparenchymal cells using gene expression profiling

Khetani, Salman R.; Szulgit, Greg; Rio, Jo A. Del; Barlow, Carrolee; Bhatia, Sangeeta N.

doi:10.1002/hep.20351

Cited by 115 publications

(131 citation statements)

References 46 publications

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“…4D demonstrates basal surface staining for HS4C3 in co-cultures of primary rat hepatocytes (26). Finally, the small leucine-rich proteoglycan, decorin, which was previously shown to be important in the long-term maintenance of hepatocyte function can be also detected in oxygenated cocultures (29). For additional images see Fig.…”

Section: Figmentioning

confidence: 86%

Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

Kidambi

Yarmush

Novik³

et al. 2009

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

193

147

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The liver is a major site for the metabolism of xenobiotic compounds due to its abundant level of phase I/II metabolic enzymes. With the cost of drug development escalating to over $400 million/ drug there is an urgent need for the development of rigorous models of hepatic metabolism for preclinical screening of drug clearance and hepatotoxicity. Here, we present a microenvironment in which primary human and rat hepatocytes maintain a high level of metabolic competence without a long adaptation period. We demonstrate that co-cultures of hepatocytes and endothelial cells in serum-free media seeded under 95% oxygen maintain functional apical and basal polarity, high levels of cytochrome P450 activity, and gene expression profiles on par with freshly isolated hepatocytes. These oxygenated co-cultures demonstrate a remarkable ability to predict in vivo drug clearance rates of both rapid and slow clearing drugs with an R 2 of 0.92. Moreover, as the metabolic function of oxygenated co-cultures stabilizes overnight, preclinical testing can be carried out days or even weeks before other culture methods, significantly reducing associated labor and cost. These results are readily extendable to other culture configurations including three-dimensional culture, bioreactor studies, as well as microfabricated co-cultures. drug discovery ͉ liver metabolism ͉ tissue engineering

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Section: Figmentioning

confidence: 86%

Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

Kidambi

Yarmush

Novik³

et al. 2009

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

193

147

View full text Add to dashboard Cite

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“…One explanation for this effect may be the presentation of cadherins, providing for homoadhesive cell to cell contacts. HSCs switch cadherin expression from the E-to N-type during in vivo and in vitro activation (Lim et al 2007) and both E-and N-cadherins as well as the truncated T-type have been identified as potent mediators of hepatocyte differentiation (Khetani et al 2004(Khetani et al , 2008.…”

Section: Discussionmentioning

confidence: 99%

Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells

Krause

Saghatolislam

Koenig

et al. 2009

In Vitro Cell.Dev.Biol.-Animal

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Primary hepatocytes lose their differentiated functions rapidly when in culture. Our aim was to maintain the differentiated status of hepatocytes in vitro by means of vital hepatic stellate cells (HSCs), their soluble and particulate factors and lipid extracts. Hepatocytes were placed into collagen-coated culture dishes in the presence of HSCs at different ages of pre-culture, with or without direct cell to cell contacts, at different cell ratios and in monoculture with cellular HSC components in place of vital cells. Changes in morphology and enhancement of phosphoenolpyruvate carboxykinase (PCK) activity by glucagon were used to determine the differentiated status of hepatocytes in 2d-short-term culture. HSCs proved able to maintain the differentiated function of hepatocytes in coculture either by direct cell contacts or via factors derived from HSC-conditioned medium. In comparison, however, without cellular contact to hepatocytes five to ten times as many HSCs were necessary to increase the PCK activity to the same degree as in the presence of intercellular contacts. Whereas stimulation in the presence of HSC/hepatocyte contacts was independent of HSC culture age only quiescent, resting HSCs (precultured for 1-2 d) were able to stimulate hepatocytes significantly via soluble factors. Culturing of hepatocytes with a lipid extract or a particulate fraction from HSCs clearly displayed a very strong beneficial effect on enzyme activity and morphology. HSCs maintain hepatocyte function and structure through preferentially cell-bound signalling and transfer of lipids.

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“…However, other studies have also reported that the activities of some enzymes and some hepatic functions are modified by certain in vitro conditions being used (Guillouzo and GuguenGuillouzo, 1992;Rogiers et al, 1995;Khetani et al, 2004). For example, cultured hepatocytes expressing parenchymal functions could be maintained only in monolayer cultures for a limited period and showed a rapid decay of many of their differentiated cell properties and functions, particularly of drug-metabolizing enzymes, in other cultures (Gó mez-Lechó n et al, 1990;Guillouzo, 1998).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Cytochrome P450 Expression in Murine Embryonic Stem Cell-Derived Hepatic Tissue System

Tsutsui

Ogawa²,

Inada³

et al. 2006

Drug Metab Dispos

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ABSTRACT:An in vitro system for liver organogenesis from murine embryonic stem (ES) cells has been recently established. This system is expected to be applied to the development of a new drug metabolism assay system that uses ES cells as a substitute for animal experiments. The objective of this study was to elucidate the drug metabolism profiles of the murine ES cell-derived hepatic tissue system compared with those of primary cultures of murine adult and fetal hepatocytes. The expression of the genes of the cytochrome P450 (P450) family, such as Cyp2a5, Cyp2b10, Cyp2c29, Cyp2d9, Cyp3a11, and Cyp7a1, was observed in the murine ES cell-derived hepatic tissue system at 16 days and 18 days after plating (A16 and A18). To investigate the activities of these P450 family enzymes in the murine ES cell-derived hepatic tissue system at A16 and A18, testosterone metabolism in this system was analyzed. Testosterone was hydroxylated to 6␤-hydroxytestosterone (6␤-OHT), 16␣-OHT, 2␣-OHT, and 2␤-OHT in this system, and was not hydroxylated to 15␣-OHT, 7␣-OHT, and 16␤-OHT. This metabolism profile was similar to that of fetal hepatocytes and different from that of adult hepatocytes. Furthermore, pretreatment with phenobarbital resulted in a 2.5-and 2.6-fold increase in the production of 6␤-OHT and 16␤-OHT. Thus, evidence for drug metabolic activities in relation to P450s has been demonstrated in this system. These results in this system would be a stepping stone of the research on the development and differentiation to adult liver.Embryonic stem (ES) cells are pluripotent and can differentiate in vitro and in vivo. There have been several reports on the differentiation of murine or human ES cells into hepatocyte-like or albuminproducing cells and their isolation (Chinzei et al., 2002;Jochheim et al., 2004;Shirahashi et al., 2004); these cells also differentiate into a variety of other cell lineages. Thus far, in all of the above researches, the ES cells were differentiated into a single cell lineage by the addition of specific growth factors and chemicals to the culture. Limiting these differentiation systems during in vivo liver development is considered difficult because of the multiple functions and complex structure of the liver. However, we recently succeeded in establishing an in vitro system of liver morphogenesis by using murine ES cells (Ogawa et al., 2005). This system consists of not only hepatocytes but also cell lineages such as cardiomyocytes and endothelial cells that support liver-specific functions and differentiations. The system is more efficient with respect to hepatic functions such as albumin production and ammonia degradation. Furthermore, the expression of the transthyretin, ␣-fetoprotein, ␣1-antitrypsin, and tyrosine aminotransferase genes is higher in this system than in the cultures of hepatic cell lines and murine primary cultures of adult hepatocytes. This system is expected to have many practical applications. It can be used in the development of new drugs and in drug metabolism assays as an alterna...

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Exploring interactions between rat hepatocytes and nonparenchymal cells using gene expression profiling

Cited by 115 publications

References 46 publications

Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells

Characterization of Cytochrome P450 Expression in Murine Embryonic Stem Cell-Derived Hepatic Tissue System

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