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 6β-hydroxytestosterone (6β-OHT), 16α-OHT, 2α-OHT, and 2β-OHT in this system, and was not hydroxylated to 15α-OHT, 7α-OHT, and 16β-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 6β-OHT and 16β-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 albumin-producing 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, α-fetoprotein, α1-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 alternative to animal experiments and in vitro experiments using primary hepatocytes. Furthermore, it can be used for the development of bioartificial liver systems.
In the present study, we investigated the drug metabolism capability of the ES cell-derived hepatic tissue system. First, to identify and quantify the expression of cytochrome P450 (P450) genes, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used. Second, the basal activities of selective P450 enzymes were determined by measuring their testosterone hydroxylation activity and comparing with those observed in both murine adult and fetal hepatocytes. Furthermore, the phenobarbital inducibility of selective P450 enzymes was analyzed. This is the first study that investigates both the expression of the P450 genes and the testosterone hydroxylation activities in a murine ES cell-derived hepatic tissue system.
Materials and Methods
Chemicals. Testosterone (4-androsten-17β-ol-3-on), 15α-hydroxytestosterone (15α-OHT), 6β-OHT, 2α-OHT, 2β-OHT, 11α-hydroxyprogesterone, and phenobarbital were purchased from Sigma (St. Louis, MO). 16α-OHT, 16β-OHT, and 7α-OHT were purchased from Daiichi Pure Chemicals Corp. (Tokyo, Japan). All other chemicals used in this study were of analytical grade.
Differentiation of Murine Embryonic Stem Cells. E14-1 ES cells that were derived from 129/Ola mice were grown on mitomycin C-treated mouse embryonic fibroblast feeder layers. The murine ES cell-derived hepatic tissue system, adult hepatocytes, and fetal hepatocytes were obtained as described previously (Ogawa et al., 2005). In brief, the E14-1 cells were used for all the following experiments as the parent ES cell line. These cells were dissociated with 0.25% trypsin, 1% chicken serum (Invitrogen Corp., Carlsbad, CA), and 1 mM EDTA in phosphate-buffered saline and resuspended in Iscove's modified Dulbecco's medium (Invitrogen Corp.) containing 20% fetal bovine serum, 1 mM sodium pyruvate, 100 μM nonessential amino acids, and 100 μM 2-mercaptoethanol without leukemia inhibitory factor. The cells were cultured in a hanging drop (1000 cells per 50-μl drop) in an atmosphere of 5% CO2 at 37°C for 5 days. Twenty 5-day-old embryoid bodies (EBs) were plated on a 6-cm dish coated with gelatin containing the differentiation medium. The day on which the 5-day-old EBs were plated on the dish was denoted as day 0 (A0).
Isolation and Culture of Murine Primary Hepatocytes. To obtain fetal hepatocytes, 129/sv mouse liver at E15 was minced and dissociated with collagenase II (Sigma) in Hanks' buffer (Invitrogen Corp.). The cells were seeded on a gelatin-coated dish (six-well plate) at a density of 2 × 105 cells/dish containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 μM nonessential amino acids, and a mixture of 100 U/ml penicillin, 1000 μg/ml streptomycin, and 292 μg/ml glutamine for a few hours and washed once with the same solution as the ES cell-derived hepatic tissue system cultured medium. The medium was changed daily.
Primary adult hepatocytes were isolated from 129/sv male mice (Japan SLC, Inc., Shizuoka, Japan; 6–10 weeks old) using the two-step collagenase perfusion method. The hepatocytes were separated from the resulting cell suspension by centrifugation and again centrifuged through a 50% Percoll gradient (Sigma). The isolated hepatocytes were plated onto gelatin-coated dishes (6-cm) at a density of 4 × 105 cells/dish. Culture medium was the same as above and was changed a few hours later to remove unattached hepatocytes. After 12 h of culture, these murine primary hepatocytes were used for the experiment.
The animal experiments were carried out at the Research Center for Human and Environmental Sciences, Shinshu University, and were conducted in accordance with the ethical guidelines of the Shinshu University.
Assay for Albumin Protein. To confirm albumin production, samples of the cultured medium were analyzed using the Lebis ELISA kit (Shibayagi, Gunma, Japan). After daily incubation with the murine ES cell-derived hepatic tissue system, the media samples were collected at 15, 16, 17, and 18 days after plating (A15, A16, A17, and A18).
Quantitative RT-PCR Analysis for Cytochrome P450 Gene Expression. Total RNA was isolated from the cell pellets using the SV Total RNA Isolation System (Promega, Tokyo, Japan) along with the DNase treatment step. The mRNA levels in the ES-derived hepatic tissue system at A16 and A18, and in 12-h-old and 60-h-old primary cultured adult and fetal hepatocytes were determined by quantitative RT-PCR using the SYBR Green and GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA).
The primers used for quantitative RT-PCR are listed in Table 1. All the primers were synthesized by Operon Technologies (Tokyo, Japan). Designed primers included Cyp2b10 (Jackson et al., 2004), Cyp2d9, Cyp3a11 (Pan et al., 2000), Cyp2a5, Cyp7a1 (Wang and Seed, 2003), 2c29 (Jackson et al., 2004), and albumin (Miyake et al., 2002). Hypoxanthine phosphoribosyltransferase, which is a housekeeping gene, was used as the internal standard (Ball et al., 2002).
Testosterone Metabolism Assay. The murine ES cell-derived hepatic tissue system at A16 and A18, and the 12-h-old and 60-h-old primary cultured adult and fetal hepatocytes were washed with Dulbecco's phosphate-buffered saline (Invitrogen Corp.) and incubated for 40 min at 37°C with the cultured medium containing 0.25 mM testosterone (Sigma). After incubation, the reaction was terminated by aspirating the medium from the plates. The amounts of testosterone metabolite products, i.e., 15α-OHT, 6β-OHT, 7α-OHT, 16α-OHT, 16β-OHT, 2α-OHT, and 2β-OHT, were measured according to the method described previously by Arlotto et al. (1991) with some modification. In brief, the cultured medium samples were placed in 5 ml of ethyl acetate, and 11α-hydroxyprogesterone was added as an internal standard. Subsequently, it was vigorously mixed in a vortex mixer and centrifuged at 3000 rpm for 5 min. The organic phase was transferred and evaporated to dryness under a stream of nitrogen gas. The residue was reconstituted in 100 μl of methanol/water (1:1 v/v) and analyzed by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: column, Cadenza CD-C18 (15 cm × 4.6 mm i.d.; Intakt, Kyoto, Japan); mobile phases, A: methanol/water/acetonitrile (39:60:1 v/v) and B: methanol/water/acetonitrile (80:18:2 v/v). The gradient elution system was as follows: 0 min, B = 18%; 12 min, B = 18%; 17 min, B = 80%; 20 min, B = 80%; 23 min, B = 18%; and 35 min, B = 18%. The metabolites were detected by UV absorbance at 254 nm. The retention times of the testosterone metabolites were as follows: 15α-OHT, 14.4 min; 6β-OHT, 14.8 min; 7α-OHT, 15.8 min; 16α-OHT, 20.6 min; 16β-OHT, 23.7 min; 2α-OHT, 25.0 min; 2β-OHT, 25.4 min; 11α-hydroxyprogesterone, 26.4 min; and testosterone, 28.8 min. The peak of each metabolite was compared with that of the internal standard to determine the amount of metabolites.
Phenobarbital Induction Assay. The murine ES cell-derived hepatic tissue system at A21, and 12-h-old primary cultured adult and fetal hepatocytes were exposed to 2 mM phenobarbital (PB) for 48 h. PB was prepared as an aqueous solution and added directly to the culture daily. The culture was then treated with 0.25 mM testosterone, and the amounts of 6β-OHT and 16β-OHT were measured by HPLC using the method described in the above section (testosterone metabolism).
Statistical Analysis. Each experiment was performed in three different cultures. Results are shown as means ± S.E. p < 0.05 was considered as statistically significant.
Results
Murine Embryonic Stem Cell-Derived Hepatic Tissue System. Twenty 5-day-old EBs were placed together on gelatin-coated plastic dishes containing the differentiation medium. Contracting cardiomyocytes appeared in the EB outgrowths. The EB outgrowths were cultured in the differentiation medium for 18 days (A18) and then were formed to hepatic tissue-like morphology having endothelial cell networks and albumin-producing cell area, as previously reported (Ogawa et al., 2005). The amount of albumin released from the ES cell-derived hepatic tissue system into the medium was measured in each medium at A15 to A18 by enzyme-linked immunosorbent assay. As seen in Fig. 1, the albumin level increased gradually from A15 to A18. This result suggests that the ES cell-derived hepatic tissue system had been differentiated from ES cells.
P450 Family Genes Were Expressed in the Murine Embryonic Stem Cell-Derived Hepatic Tissue System. The P450 enzymes catalyze the oxidative metabolism of endogenous and exogenous compounds and play a major role in the biotransformation of xenobiotics. To measure the constitutive expression of the P450 family genes in the murine ES cell-derived hepatic tissue system, quantitative RT-PCR was used. The expression of the P450 family genes (Cyp2a5, Cyp2b10, Cyp2c29, Cyp2d9, and Cyp3a11), with regard to the hydroxyl metabolism of testosterone, was detected in the murine ES cell-derived hepatic tissue system at A16 and A18, and in 12-h-old and 60-h-old primary cultured adult and fetal hepatocytes (Fig. 2). Furthermore, the expression of the liver-specific genes, Cyp7a1 and albumin, was also observed in the murine ES cell-derived hepatic tissue system (Fig. 2), and this system was shown to be actually ES cells differentiated into liver tissue.
Testosterone Metabolism Profiles. Testosterone is metabolized in a regioselective manner by different P450 enzymes and can be used as a multienzymatic substrate to simultaneously investigate the activities of multiple enzymes. Testosterone (0.25 mM) was added into the ES cell-derived hepatic tissue system at A16 and A18, and 12-h-old and 60-h-old primary cultured adult and fetal hepatocytes, and then the cells were exposed for 40 min (Fig. 3). The metabolized testosterone was analyzed as hydroxylated products (15α-OHT, 6β-OHT, 7α-OHT, 16α-OHT, 16β-OHT, 2α-OHT, and 2β-OHT). Specifically, the products (15α-OHT, 6β-OHT, 7α-OHT, 16α-OHT, 16β-OHT, and 2α-OHT) are the indexes of specific cytochrome P450 (Cyp2a4/5, Cyp3a, Cyp2a4/5 and 2d9, Cyp2d9 and 2b, Cyp2c29 and 2b, and Cyp2d) activities. In the murine ES cell-derived hepatic tissue system, 6β-OHT, 16β-OHT, 2α-OHT, and 2β-OHT were observed at A16 and A18; however, 15α-OHT, 7α-OHT, and 16α-OHT were not observed. This hydroxylation pattern of the murine ES cell-derived hepatic tissue system was similar to that of the fetal hepatocytes, and was different from that of adult hepatocytes. The P450 functionality in the murine ES cell-derived hepatic tissue system at A16 and A18 was assessed from these results, and it was demonstrated to be similar to that of the fetal hepatocytes.
Phenobarbital Induces P450 Expression in Embryonic Stem Cell-Derived Hepatic Tissue System. The induction of P450 expression is a common cellular defense mechanism against the toxicity of foreign compounds. The ES cell-derived hepatic tissue system, adult hepatocytes, and fetal hepatocytes were incubated for 48 h with 2 mM PB, and the induction potential of P450 expression was investigated by measuring the amounts of testosterone metabolite products (6β-OHT and 16β-OHT). In the ES cell-derived hepatic tissue system, the hydroxylation of testosterones (i.e., 6β-OHT, and 16β-OHT) was 2.5- and 2.6-fold higher in the presence of PB than in the absence of PB, as shown in Fig. 4A. Furthermore, PB increased the amounts of 6β-OHT and 16β-OHT in the murine adult hepatocytes (by 1.3- and 2.3-fold; Fig. 4B) and fetal hepatocytes (by 1.9- and 1.1-fold; Fig. 4C). These results revealed that the murine ES cell-derived hepatic tissue system shows the same P450 inducibility as the murine adult hepatocytes and fetal hepatocytes.
Discussion
The purpose of this study was to characterize the metabolic capability of the novel in vitro system for liver organogenesis from murine ES cells, namely, the murine ES cell-derived hepatic tissue system. We have detected the expression of not only the Cyp2b5, Cyp2b10, Cyp2c29, Cyp2d9, and Cyp3a11 genes but also that of the Cyp7a1 gene in the murine ES cell-derived hepatic tissue system. Cyp7a1 is known as a liver-specific gene; it is not expressed in the yolk sac and is induced in developing embryonic bodies (Asahina et al., 2004). Therefore, our result demonstrated that the murine ES cell-derived hepatic tissue system has differentiated ES cells into hepatic tissue. Furthermore, P450 enzyme (Cyp2b, Cyp2d, Cyp2c29, and Cyp3a) activities were identified in this system by measuring the testosterone metabolite products. Therefore, it has been demonstrated that drug-metabolizing P450 enzymes are truly present and are functionally active in the murine ES cell-derived hepatic tissue system.
The liver plays a major role in drug metabolism. Primarily, this can be attributed to the relatively high exposure of the liver to chemicals. Cultured hepatocytes are being widely used for drug metabolism and toxicity studies for new drug development (Guillouzo, 1998; Rodríguez-Antona et al., 2000; Tirona et al., 2003). The endpoints for in vitro hepatocyte functions such as albumin secretion, mitochondrial function, lactate dehydrogenase leakage, generation of reactive oxygen species, activities of alanine aminotransferase and aspartate aminotransferase, or intracellular glutathione levels have been generally evaluated in these cultured hepatocytes (Viluksela et al., 1996; Delraso and Channel, 1999; De Smet et al., 2000; Trohalaki S et al., 2002; Riss and Moravec, 2004). 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 Guguen-Guillouzo, 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 (Gómez-Lechón et al., 1990; Guillouzo, 1998). Furthermore, it has been reported that in vitro hepatic models using immortalized hepatocyte-like cell lines or hepatoma-derived cell lines are not considered to be fully representative of hepatic functions in the view of their drug-metabolizing enzymes (Grant et al., 1988; Donato et al., 1994, 1999, 2003; Courjault-Gautier et al., 1997; Rodriguez-Antona et al., 2002).
Our novel in vitro system consists of not only hepatocytes but also other cell lineages such as cardiomyocytes and endothelial cells that support liver-specific functions and differentiations; these cell lineages correspond to those involved in liver organogenesis in vivo (Ogawa et al., 2005). Furthermore, it also shows higher levels of hepatic functions, such as albumin production and ammonia degradation, as well as the expression of transthyretin, α-fetoprotein, α1-antitrypsin, and tyrosine aminotransferase genes than those observed in the cultures of other hepatic cell lines and murine primary culture of adult hepatocytes. In the present study, the expression of P450 genes and their activities in the murine ES cell-derived hepatic tissue system were characterized, and it is suggested that the murine ES cell-derived hepatic tissue system has the capability to maintain drug-metabolic functions. In addition, hepatocyte-endothelial cell interaction is very important for the generation of full hepatic function in hepatocytes, liver development, and proliferation of the embryo (Ogawa et al., 2005). Based on these findings, this system is expected to be able to mimic in vivo functions of the liver.
PB is the prototype for a large number of structurally diverse chemicals that induce P450 belonging to the Cyp2b and Cyp3a gene families (Waxman and Azaroff, 1992; Gonzalez et al., 1993; Denison and Whitlock, 1995) and other xenochemical-metabolizing enzymes (Waxman and Azaroff, 1992; Honkakoshi et al., 1998). In the present study, PB was used to confirm P450 inducibility in the murine ES cell-derived hepatic tissue system; in fact, PB increased the production of testosterone metabolites (namely, 6β-OHT and 16β-OHT) in this system. Therefore, it is confirmed that PB induction of P450, a specific metabolic property of primary hepatocytes, is also present in the murine ES cell-derived hepatic tissue system.
A part of testosterone hydroxylation (6β-OHT, 16β-OHT, 2α-OHT, and 2β-OHT) was detected and maintained in the ES cell-derived hepatic tissue system at A16 and A18. But 15α-OHT, 7α-OHT, and 16α-OHT were not detected. This phenomenon was similar to that of fetal hepatocytes and was different from that of adult hepatocytes in that all of the hydroxylated testosterone was detected and its activities were markedly decreased within 60 h.
In the future, detailed studies to investigate the mechanisms of this phenomenon would be helpful to characterize the detailed metabolic profile of this system for development and differentiation to intact liver.
Acknowledgments
We are grateful to Prof. Nobuaki Yoshida and Prof. Yoichiro Iwakura (Institute of Medical Science, University of Tokyo) for providing us the E14-1 ES cells and RIKEN Cell Bank for the STO cell line.
Footnotes
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This study was supported by grants from the Ministry of Education, Sports, Science and Technology of Japan (Tokyo, Japan) (15700314; 13470150, Grantin-Aid for 21st Century COE program), and the Hokuto Foundation of Bioscience (Nagano, Japan).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.007674.
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ABBREVIATIONS: ES, embryonic stem; RT-PCR, reverse transcription-polymerase chain reaction; OHT, hydroxytestosterone; EB, embryoid body; HPLC, high performance liquid chromatography; PB, phenobarbital.
- Received October 2, 2005.
- Accepted January 6, 2006.
- The American Society for Pharmacology and Experimental Therapeutics