Real‐time fluorometric evaluation of hepatoblast proliferation in vivo and in vitro using the expression of <i>CYP3A7</i> coding for human fetus‐specific P450

Okuyama, Shota; Kawamura, Fumihiko; Kubiura, Musashi; Tsuji, S.; Osaki, Mitsuhiko; Kugoh, Hiroyuki; Oshimura, Mitsuo; Kazuki, Yasuhiro; Tada, Masako

doi:10.1002/prp2.642

Cited by 5 publications

(6 citation statements)

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“…In contrast to mammalians, the avian liver has much less connective tissue than the mammalian liver and lacks a true lobular structure [15,40]. Hepatocytes exhibit a lower differentiation ability and relatively simpler function during the embryonic stage compared to that during the early postnatal period [41,42]. Therefore, a simple hepatocyte separation method for avians is possible.…”

Section: Discussionmentioning

confidence: 99%

Developmental Changes of Duckling Liver and Isolation of Primary Hepatocytes

Bao

Wang

et al. 2023

Animals

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The liver is the main site of fat synthesis and plays an important role in the study of fat deposition in poultry. In this study, we investigated the developmental changes of duckling livers and isolated primary duck hepatocytes. Firstly, we observed morphological changes in duckling livers from the embryonic period to the first week after hatching. Liver weight increased with age. Hematoxylin-eosin and Oil Red O staining analyses showed that hepatic lipids increased gradually during the embryonic period and declined post-hatching. Liver samples were collected from 21-day-old duck embryos for hepatocyte isolation. The hepatocytes showed limited self-renewal and proliferative ability and were maintained in culture for up to 7 days. Typical parenchymal morphology, with a characteristic polygonal shape, appeared after two days of culture. Periodic acid-Schiff (PAS) staining analysis confirmed the characteristics of duck embryo hepatocytes. PCR analysis showed that these cells from duck embryos expressed the liver cell markers ALB and CD36. Immunohistochemical staining and immunofluorescence analysis also confirmed ALB and CK18 expression. Our findings provide a novel insight regarding in vitro cell culture and the characteristics of hepatocytes from avian species, which could enable further studies concerning specific research on duck lipid metabolism.

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Section: Discussionmentioning

confidence: 99%

Developmental Changes of Duckling Liver and Isolation of Primary Hepatocytes

Bao

Wang

et al. 2023

Animals

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“…We report here results of mRNA expression of genes responsible for key hepatic functions versus culture duration in prolonged cultured human hepatocytes. The hepatocyte-specific genes evaluated include hepatic proteins ALB, TR, and TTR that are generally regarded as markers of mature hepatocytes (Lok and Loh, 1998;Ascoli et al, 2006;Buxbaum et al, 2008;Bal et al, 2013;Fujiwara and Amisaki, 2013;Lee and Wu, 2015;Alemi et al, 2016;Zorzi et al, 2019); the plasma membrane receptor asialoglycoprotein receptor1 (ASGR1) used routinely for the delivery of therapeutic agents specifically to hepatocytes (Merwin et al, 1994;Kim et al, 2005;Thapa et al, 2015;Huang et al, 2017); P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 that are considered the key isoforms responsible for drug metabolism (Rendic and Guengerich, 2010;Zanger and Schwab, 2013) as well as CYP3A7, a CYP3A isoform mainly expressed in fetal but also expressed in adult livers (Kamataki et al, 1995;Greuet et al, 1996;Okuyama et al, 2020); drug uptake transporters SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3 and SLCO2B1 (Fenner et al, 2012;Barton et al, 2013;Bi et al, 2019), and efflux transporters ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2 (Matsushima et al, 2005;Ishiguro et al, 2008;Pfeifer et al, 2014) that play key roles in the regulation of intracellular concentrations of drugs and their metabolites that are transporter substrates. Expression of the housekeeping gene HPRT1 (Nishimura et al, 2006) as a function of culture duration was also evaluated for comparison to the above-mentioned hepatocyte-specific genes.…”

Section: Discussionmentioning

confidence: 99%

“…CYP3A4 isoform highly expressed in human fetal liver but also in some adult populations (Kamataki et al, 1995;Greuet et al, 1996;Okuyama et al,…”

Section: Cyp3a7mentioning

confidence: 99%

Messenger RNA Expression of Albumin, Transferrin, Transthyretin, Asialoglycoprotein Receptor, Cytochrome P450 Isoform, Uptake Transporter, and Efflux Transporter Genes as a Function of Culture Duration in Prolonged Cultured Cryopreserved Human Hepatocytes as Collagen-Matrigel Sandwich Cultures: Evidence for Redifferentiation upon Prolonged Culturing

Yang

2021

Drug Metabolism and Disposition

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“…Fluorescence microscopic images were captured using a BZ‐9000 fluorescence microscope (Keyence) and an A1 confocal microscope (Nikon). Reagents, software, and methods used in this study have been described in detail previously 9 in METHODS. Primer sets used for Genomic PCR and RT‐qPCR are listed in Table .…”

Section: Methodsmentioning

confidence: 99%

Transgenic HepaRG cells expressing CYP2D6 as an improved model of primary human hepatocytes

Okuyama

Mine

Nakamura

et al. 2022

Pharmacology Res & Perspec

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CYP2D6 and CYP3A4, which are members of the cytochrome P450 superfamily of metabolic enzymes, play major roles in the metabolism of commonly available drugs. CYP3A4 is involved in the metabolism of 50% of drugs on the market, whereas CYP2D6 is involved in the metabolism of 25% of them. CYP2D6 exhibits a high degree of polymorphic nature in the human population, causing individual differences in CYP2D6 expression and enzymatic activity. Therefore, accurate prediction of drug metabolism and toxicity require a human adult hepatocyte cell model that mimics individual responses in the average population. HepaRG cells, a human hepatocellular carcinoma cell line, is the only cell line that can differentiate into hepatocyte‐like cells with high expression of CYP3A4 but poor expression of CYP2D6. To solve this problem, we developed transgenic HepaRG cell clones expressing either full‐length or spliced CYP2D6 at various levels with an easy monitoring system for CYP2D6 expression in living cells under a fluorescent microscope. As CYP2D6 mRNA, protein, and fluorescence intensity were closely correlated among transgenic HepaRG clones, fluorescence levels will provide a simple tool for quality assurance of CYP2D6‐expressing HepaRG cells. Thus, the package of transgenic HepaRG cell clones expressing CYP2D6 at various levels will provide an improved hepatocyte model that reflects the average or individual reactions in the human population for in vitro studies of drug metabolism and toxicity involving CYP2D6 and CYP3A4.

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Real‐time fluorometric evaluation of hepatoblast proliferation in vivo and in vitro using the expression of CYP3A7 coding for human fetus‐specific P450

Abstract: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 5 publications

References 48 publications

Developmental Changes of Duckling Liver and Isolation of Primary Hepatocytes

Developmental Changes of Duckling Liver and Isolation of Primary Hepatocytes

Transgenic HepaRG cells expressing CYP2D6 as an improved model of primary human hepatocytes

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