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Biol. Pharm. Bull. 39(12): 2015-2021 (2016)

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Biol. Pharm. Bull. 39(12): 2015-2021 (2016) Vol. 39, No. 12 Biol. Pharm. Bull. 39, 2015–2021 (2016) 2015 Regular Article Role of the Drug-Metabolizing Enzyme CYP during Mouse Liver Development Wataru Ochiai,# Akiyo Hirose,# Taisuke Kawamura, Kyoko Komachi, Yuka Yamamoto, Satoshi Kitaoka, Jo Hatogai, Yoshiki Kusunoki, Risako Kon, Nobutomo Ikarashi, and Kiyoshi Sugiyama* Department of Clinical Pharmacokinetics, School of Pharmacy and Pharmaceutical Sciences, Hoshi University; Tokyo 142–8501, Japan. Received June 15, 2016; accepted August 23, 2016 The drug-metabolizing enzyme CYP is mainly involved in the metabolism of various substances in the liver, such as drugs, endogenous substances, and carcinogens. Recent reports have also revealed that CYP1B1 plays a major role in the developmental process. Because the level of CYP expression is markedly high in the liver, we hypothesize that CYP plays a role in the developmental process of the liver. To verify this hypoth- esis, we analyzed the expression patterns of various CYP molecular species and their functions during the differentiation of embryonic stem cells (ES cells) into hepatocytes and the developmental process in mice. The results demonstrated that CYP2R1 and CYP26A1 are expressed at an earlier stage of the differentiation of ES cells into hepatocytes than hepatoblast-specific markers. Additionally, during the development of the mouse liver, CYP2R1 and CYP26A1 were mostly up-regulated during the stage when hepatoblasts appeared. In addition, when CYP2R1 and CYP26A1 expressions were forced in ES cells and liver of adult mice, they differentiated into hepatoblast marker positive cells. These results suggest that CYP2R1 and CYP26A1 may play a major role in hepatoblast cell differentiation during the development of the liver. Key words embryonic stem cell; liver; CYP; hepatoblast The mature liver contains a variety of cell types, such as ing in embryonic lethal.7) Mutation of the CYP1B1 gene is hepatocytes, sinusoidal endothelial cells, bile duct epithelial also known to cause primary congenital glaucoma in human cells, stellate cells and Kupffer cells. The hepatocyte account beings.8) The same trabecular defect in humans that causes for 80% of the volume of the liver and play a major role in primary congenital glaucoma was also observed in CYP1B1 liver functions, such as metabolism and excretion of drugs. and tyrosinase double-knockout mice.9) These reports indicate Additionally, the fetal liver is known to act as a hematopoietic that CYP not only works as a drug-metabolizing enzyme but organ. also plays a major role in development. We focused our atten- The expression of CYP is significantly up-regulated in the tion on the fact that the expression of CYP is much higher in liver, and CYP converts fat-soluble drugs into a water-soluble the liver than other organs10)and hypothesized that CYP may state so that they can be easily excreted. In addition to drug play a role in hepatocyte differentiation and proliferation dur- metabolism, CYP is also known to be involved in the oxida- ing liver development. In this study, we attempted to validate tive metabolism of endogenous substances, including steroids, this hypothesis by using the differentiation induction system bile acid, hormones, and eicosanoids. To date, 58 types of to differentiate embryonic stem (ES) cells into hepatocytes.11) CYP molecular species have been identified in humans, and ES cells are multipotent stem cells that can differentiate 108 types have been identified in mice.1–3) As the CYP mo- into various cell types through induction by humoral factors, lecular species share high amino acid sequence homology in such as cytokines.12) ES cells also provide a useful means for several substrate-recognition sites, in addition to the above- analyzing the differentiation mechanism of different cell types mentioned functions of CYP, it may also play a major role in by inducing ES cells to differentiate into target cell types. development and differentiation in the body. Because CYP begins to be expressed close to the time of hematopoiesis MATERIALS AND METHODS initiation during the fetal stage and CYP requires heme for its structure, CYP is considered to be involved in the develop- Animal Handling Pregnancy ICR mice (E11, 14) were ment of the liver during the fetal stage. Moreover, it has been purchased from Japan SLC, Inc. (Tokyo Laboratory Animals reported that the metabolites of endogenous substances and Science Co., Ltd., Tokyo, Japan). The mice were kept at room the intermediate metabolites of chemical substances have an temperature (r.t.) (24±1°C) and 55±5% humidity with 12 h of effect on the development of individuals and on homeosta- light (artificial illumination; 8:00–20:00). Food and water were sis.4–6) Therefore, CYP, which transiently appears during the available ad libitum. Each animal was used only once. The process of development, is thought to possibly play an impor- present study was conducted in accordance with the Guid- tant role in the development of the liver. ing Principles for the Care and use of Laboratory Animals, It has been reported that the knockout of either the as adopted by the Committee on Animal Research at Hoshi CYP26A1 gene in mice causes abnormal embryogeny result- University. Induction of Differentiation of ES Cells into Hepatocytes # These authors contributed equally to this work. Mouse ES cells (AES0125, Lot. 001) were purchased from the * To whom correspondence should be addressed. e-mail: [email protected] © 2016 The Pharmaceutical Society of Japan 2016 Biol. Pharm. Bull. Vol. 39, No. 12 (2016) Fig. 1. Schematic Diagram of the Protocol for Induction of Differentiation of ES Cells into Hepatocytes RIKEN BRC CELL BANK. Culture of the mouse ES cells MO, U.S.A.). was performed using a known methodology11) (Fig. 1). Frozen cDNA was synthesized from 1 µg purified total RNA using cells were thawed, dispersed at a density of 1×106 cells/mL in a High Capacity cDNA synthesis Kit (Applied Biosystems, culture medium, and seeded at 10 mL per 100 mm in a gelatin- Foster City, CA, U.S.A.). The concentration of the total RNA coated dish. (µg/mL) was calculated, and the purity of the total RNA was The induction of the differentiation of ES cells into hepa- evaluated by measuring its absorbance at 260 and 280 nm. tocytes was performed using a known methodology. The ES For each sample, 2.0 µL of 10×RT buffer, 0.8 µL of 25×de- cells were seeded at 2×105 cells per 35 mm gelatin-coated dish oxynucleotide triphosphate (dNTP) Mix, 2.0 µL of 10×RT or at 4×104 cells per well on a Lab-Tek™ II Chamber Slide Random Primer, 1.0 µL MultiScribe™ Reverse Transcriptase, (Nunc, CA, U.S.A.) and were incubated at 37°C in 5% CO2 1.0 µL ribonuclease (RNase) Inhibitor, and 3.2 µL ultrapure for 72 h in stem medium (DS farmabiomedical, Osaka, Japan) water, which were all included in the High Capacity cDNA (final conc. LIF: 1000 units/mL). The culture medium was synthesis Kit, were mixed gently on ice to prepare the 2×Re- then changed to stem medium containing LIF and 10−8 mol/L verse Transcription (RT) Master Mix. all-trans-retinoic acid (RA) (Wako Pure Chemical Industries, RT-PCR The following reagents were added to each well Ltd., Osaka, Japan), and the cells were incubated for 3 d. of the PCR 8 Strip Tube: 0.1 µL TaKaRa Ex Taq (TaKaRa-Bio, After 3 d, the cells were cultured with LIF (−) culture Shiga, Japan), 2.5 µL of 10×Ex Taq buffer, 2.0 µL dNTP mix- medium for 5 d in the presence of fibroblast growth factor ture, 1.25 µL dimethyl sulfoxide (DMSO), 1 µL cDNA solu- (FGF)-1, 100 ng/mL; FGF4, 20 ng/mL; hepatocyte growth fac- tion, 2.5 µL forward primer (20 pmol/µL), 2.5 µL reverse prim- tor (HGF), 50 ng/mL (Wako) at 106 cells per 100 mm gelatin- er (20 pmol/µL) and 13.15 µL ultrapure water. Using an iQ™ coated dishes. Thermal Cycler (Bio-Rad, Hercules, CA, U.S.A.), samples After 5 d, the cells were subcultured from the gelatin-coated were first denatured at 94°C for 2 min and then at 98°C for dishes to collagen-coated dishes. At this point, the cells were 10 s; then primer annealing was performed at 55 to 58°C for washed gently three times with ES Cell Qualified Dulbecco’s 30 s, followed by elongation at 72°C for 30 s. These steps con- phosphate buffered saline (D-PBS) and incubated with 1 mL stituted one cycle. After 30 to 35 cycles, an extension step was of PBS for subculture at 37°C in 5% CO2 for 2 min. The re- performed at 72°C for 1 min and 30 s to amplify the cDNA. action was stopped by adding serum-free ES Cell Qualified The forward and reverse primers for the hepatocellular Dulbecco’s modified Eagle’s medium (DMEM) (DS farma- differentiation markers, hepatocyte nuclear factor 3-beta biomedical), and the cells were collected and centrifuged at (HNF-3β), α-fetoprotein (AFP), delta-like 1 (DLK1), albumin 100×g for 3 min. After removing the supernatant, the cells (ALB), and tryptophan dioxygenase (TDO), and glyceralde- were resuspended in ES Cell Qualified DMEM and centri- hyde-3-phosphate dehydrogenase (GAPDH) as the housekeep- fuged at 100×g for 3 min, and the supernatant was removed. ing gene are listed in Table 1. After the PCR was completed, The cells were then resuspended in stem medium, seeded at 2.5 µL of 10× loading buffer was added to the 25 µL PCR 2×105 cells per 35 mm collagen-coated dish and incubated in products, and the solution was mixed well.
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