Author:
Miyata Shoko,Saku Noriaki,Akiyama Saeko,Javaregowda Palaksha Kanive,Ite Kenta,Takashima Nagisa,Toyoda Masashi,Yura Kei,Kimura Tohru,Nishina Hiroshi,Nakazawa Atsuko,Kasahara Mureo,Nonaka Hidenori,Kiyono Tohru,Umezawa Akihiro
Abstract
Abstract
Background
Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI).
Methods
To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations.
Results
The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen.
Conclusions
iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.
Publisher
Springer Science and Business Media LLC
Subject
Cell Biology,Biochemistry, Genetics and Molecular Biology (miscellaneous),Molecular Medicine,Medicine (miscellaneous)
Reference48 articles.
1. In Vitro Drug Interaction Studies—Cytochrome P450 enzyme- and transporter-mediated drug interactions guidance for industry. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER); 2020. https://www.fda.gov/media/134582/download
2. Anthérieu S, Chesné C, Li R, Camus S, Lahoz A, Picazo L, et al. Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. Drug Metab Dispos. 2010;38:516–25.
3. De Assuncao TM, Sun Y, Jalan-Sakrikar N, Drinane MC, Huang BQ, Li Y, et al. Development and characterization of human-induced pluripotent stem cell-derived cholangiocytes. Lab Invest. 2015;95:1218.
4. Hankowski KE, Hamazaki T, Umezawa A, Terada N. Induced pluripotent stem cells as a next-generation biomedical interface. Lab Invest. 2011;91:972–7.
5. Santostefano KE, Hamazaki T, Biel NM, Jin S, Umezawa A, Terada N. A practical guide to induced pluripotent stem cell research using patient samples. Lab Invest. 2015;95:4–13.
Cited by
10 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献