Abstract
Human-induced pluripotent stem cells (hiPSCs) are invaluable sources for drug screening and toxicity tests because of their differentiation potential and proliferative capacity. Recently, the CRISPR-Cas9-mediated homologous recombination system has enabled reporter knock-ins at desired loci in hiPSCs, and here, we generated a hiPSC reporter line expressing mCherry-tagged cytochrome P450 1A1 (CYP1A1), which can be utilized to screen for the modulators of aryl hydrocarbon receptor (AHR) in live cells. CYP1A1-mCherry hiPSCs exhibited typical characteristics of pluripotent stem cells such as marker expression, differentiation potential, and normal karyotype. After differentiation into hepatocyte-like cells (HLCs), CYP1A1-mCherry fusion protein was expressed and localized at the endoplasmic reticulum, and induced by AHR agonists. We obtained 23 hits modulating CYP1A1 expression from high-content screening with 241 hepatotoxicity chemicals and nuclear receptor ligands, and identified three upregulating chemicals and two downregulating compounds. Responses of hiPSC-HLCs against an AHR agonist were more similar to human primary hepatocytes than of HepG2 hepatocellular carcinoma cells. This platform has the advantages of live-cell screening without sacrificing cells (unlike previously available CYP1A1 reporter cell lines), as well as an indefinite supply of cells, and can be utilized in a wide range of screening related to AHR- and CYP1A1-associated diseases in desired cell types.
Original language | English |
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Pages (from-to) | 9141-9155 |
Number of pages | 15 |
Journal | FASEB Journal |
Volume | 34 |
Issue number | 7 |
DOIs | |
Publication status | Published - 2020 Jul 1 |
Bibliographical note
Funding Information:This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, Republic of Korea (No. NRF‐2017R1C1B2010444 and No. NRF‐2018M3A9H1021384).
Funding Information:
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, Republic of Korea (No. NRF-2017R1C1B2010444 and No. NRF-2018M3A9H1021384).
Publisher Copyright:
© 2020 Federation of American Societies for Experimental Biology
Keywords
- AHR
- CRISPR-Cas9
- CYP1A1
- high-content screening
- toxicology
ASJC Scopus subject areas
- Biotechnology
- Biochemistry
- Molecular Biology
- Genetics