Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells☆
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).
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For information on hESC please visit http://www.cellartis.com/.
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These authors contributed equally to this study.