Elsevier

Biochemical Pharmacology

Volume 74, Issue 3, 1 August 2007, Pages 496-503
Biochemical Pharmacology

Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells

https://doi.org/10.1016/j.bcp.2007.05.009Get rights and content

Abstract

Human embryonic stem cells (hESC) offer a potential unlimited source for functional human hepatocytes, since they can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing several hepatic markers. Such cells could be used for, e.g. studies of drug metabolism and hepatotoxicity, which however would require a significant expression of drug metabolising enzymes. Thus, we have investigated the expression of cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs), drug transporters, transcription factors and other liver specific genes in hepatocyte-like cells derived from hESC using a simple direct differentiation protocol. The mRNA and protein expression of several important CYPs were determined using low density arrays, real time PCR and Western blotting. Significant CYP expression on the mRNA level was detected in hepatocyte-like cells derived from one out of two different hESC lines tested, which was much higher than in undifferentiated hESC and generally higher than in HepG2 cells. CYP1A2, CYP3A4/7 and low levels of CYP1A1 and CYP2C8/9/19 protein were detected in both lines. The mRNAs for a variety of CYPs and liver specific factors were shown to be inducible in both cell lines, and this was reflected in induced levels of CYP1A2 and CYP3A4/7 protein. This first report on expression of all major CYPs in hepatocyte-like cells derived from hESC represents an important step towards functional hepatocytes, but efforts to further differentiate the cells using optimized protocols are needed before they exhibit similar levels of drug metabolizing enzymes as primary human hepatocytes and liver.

Introduction

Human embryonic stem cells (hESC) are pluripotent and can give rise to cells of all three embryonic germ layers: endoderm, ectoderm, and mesoderm, and further on, to all somatic and germ cells [1], [2]. Thus, in the future, differentiated cells derived from hESC can potentially be used for regenerative therapies that may cure various diseases. HESC derived cells with functional characteristics of hepatic cells do not only have the potential of being used in bioreactors for extra corporal liver support in patients with liver failure [3], but also as a test system for studying hepatic metabolism of xenobiotics and hepatotoxicity. Since hESC have the ability to self-renew, the use of hESC derived hepatocytes can potentially provide an unlimited source of functional human hepatocytes, from the same genetic donor if desired, and thereby improve the predictability of toxicity tests and reduce the need for animal experimentation. However, the toxicity of xenobiotics is often dependent on their biotransformation into toxic and reactive metabolites and, therefore, the presence and distribution of biotransforming systems is required. At present, primary human hepatocytes constitute a common model for in vitro drug metabolism and toxicity testing. Nevertheless, the activity of drug metabolizing enzymes and many transporter functions are rapidly lost and/or changed when primary hepatocytes are cultured [4], [5], [6]. Moreover, many of the hepatoma cell lines, e.g. HepG2, which are used for in vitro studies, lack expression of many important drug metabolizing enzymes [7], [8].

Cytochrome P450s (CYPs) are mixed function monooxygenases and the major enzymes in phase I metabolism of xenobiotics. This oxidative metabolism results in, depending on the nature of the xenobiotic, inactivation and facilitated elimination, activation of pro-drugs or metabolic activation. The major site of CYP expression is the liver and CYP3A4 is the most abundant CYP isozyme in human adult liver. The enzymes of greatest importance for drug metabolism belong to the families 1–3, responsible for 70–80% of all phase I dependent metabolism of clinically used drugs [9], [10]. CYP expression and activity present large interindividual variations due to polymorphisms. Moreover, CYPs can be induced several fold or inhibited by specific drugs, resulting in additional, although transient, variability of metabolic activity [11].

Many reports on the differentiation of hESC have been published, mainly considering derivation of ectodermal or mesodermal lineages. Directed differentiation into endoderm has been more difficult to achieve, most likely due to a lack of early lineage-specific markers, and there are only few reports on hepatocyte-like cells derived from hESC [12], [13], [14], [15], [16]. In these reports, only limited studies of CYP expression and induction in hESC derived hepatocyte-like cells have been included. Schwartz et al. detected phenobarbital-inducible CYP expression as measured by quantitative RT-PCR and pentoxyresorufin-O-deethylase (PROD) activity in hepatocyte-like cells [12], whereas Rambhatla et al. reported inducible CYP1A2 activity as detected by ethoxyresorufin-O-deethylase (EROD) activity in hESC derived cells with hepatocyte-like characteristics [15].

We have previously shown that hepatocyte-like cells derived from hESC analyzed in the present study, display characteristic hepatic morphology and express liver markers such as HNF3β (Foxa2), liver fatty acid binding protein (LFABP), α-1-antitrypsin (α-1-AT), albumin, and cytokeratin 18. Furthermore, the cells accumulate glycogen, a feature typical for hepatocytes, and exhibit glutathione transferase protein expression and activity that closely resembles that of human hepatocytes [17]. In this study, we have evaluated the CYP mRNA and protein expression in hepatocyte-like cells derived from two different hESC lines using real time PCR based methods and Western blotting. We could detect mRNA from most of the CYP enzymes tested in one of the hepatocyte-like cells, as well as CYP1A2 and CYP3A4/7 protein. Inducibility of CYP1A2 and 3A4/7 was also demonstrated. These results are discussed in terms of the maturity of the hepatocytes-like cells and the need for further differentiation towards a suitable phenotype.

Section snippets

Cell and tissue material

In this study, we have used hepatocyte-like cells derived from hESC lines SA002, SA002.5 and SA167. These cell lines were established, characterized and cultured as previously described [1], [18] at Cellartis AB, after approval from the local ethics committee at Gothenburg University. Hepatocyte-like cells were derived using a direct 2D-differentiation protocol as previously described [17]. Briefly, hESC were allowed to differentiate for 18–30 days in VitroHES™ supplemented with 4 ng/ml human

Gene expression analysis using low density array

The mRNA expression of liver specific genes was analyzed in hepatocyte-like cells derived from hESC lines SA002 and SA167, undifferentiated hESC, MEFs, HepG2 cells, primary human hepatocytes, and human liver samples using TaqMan low density arrays (LDAs). The genes selected were in the categories cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs), transporters, transcription factors and other liver specific genes. Two genes, D-site albumin promoter binding protein (DBP) and

Discussion

Here, we show that hepatocyte-like cells derived from hESC express mRNA and protein for several hepatic CYPs. We also show that there is a clear difference in expression patterns between hepatocyte-like cells from the two hESC lines investigated. Thus, many of the liver specific genes, including most CYPs, were only detected in hepatocyte-like cells from line SA167, and not in cells from SA002, which indicates a difference in the degree of differentiation. It could be speculated whether the

Acknowledgements

We wish to thank Emma Wincent at Stockholm University for technical assistance. We are also thankful to Marie Rehnström, Gunilla Caisander, Anders Aspegren, and Markus Nordberg at Cellartis AB for their skillful assistance with harvesting the hepatocyte-like cells. This study was financed by the Swedish Animal Welfare Agency (grant number 2005-2291, 34-2962/04), EU (Vitrocellomics, contract number 018940) and the Swedish Research Council (project number K2005-03X-05949-25A).

References (32)

  • A.P. Li et al.

    Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug–drug interaction potential

    Chem Biol Interact

    (1999)
  • N. Heins et al.

    Derivation, characterization, and differentiation of human embryonic stem cells

    Stem Cells

    (2004)
  • N. Geijsen et al.

    Derivation of embryonic germ cells and male gametes from embryonic stem cells

    Nature

    (2004)
  • J.C. Gerlach et al.

    Use of primary human liver cells originating from discarded grafts in a bioreactor for liver support therapy and the prospects of culturing adult liver stem cells in bioreactors: a morphologic study

    Transplantation

    (2003)
  • T.K. Baker et al.

    Temporal gene expression analysis of monolayer cultured rat hepatocytes

    Chem Res Toxicol

    (2001)
  • C. Rodriguez-Antona et al.

    Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells

    Xenobiotica

    (2002)
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    For information on hESC please visit http://www.cellartis.com/.

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