TY - JOUR
T1 - Live-cell screening platform using human-induced pluripotent stem cells expressing fluorescence-tagged cytochrome P450 1A1
AU - Kim, Ji Woo
AU - Im, Ilkyun
AU - Kim, Hyemin
AU - Jeon, Jang Su
AU - Kang, Eun Hye
AU - Jo, Seongyea
AU - Chun, Hang Suk
AU - Yoon, Seokjoo
AU - Kim, Jong Hoon
AU - Kim, Sang Kyum
AU - Park, Han Jin
N1 - 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).
PY - 2020/7/1
Y1 - 2020/7/1
N2 - 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.
AB - 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.
KW - AHR
KW - CRISPR-Cas9
KW - CYP1A1
KW - high-content screening
KW - toxicology
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U2 - 10.1096/fj.201903110R
DO - 10.1096/fj.201903110R
M3 - Article
C2 - 32421247
AN - SCOPUS:85084827400
VL - 34
SP - 9141
EP - 9155
JO - The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
JF - The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
SN - 1530-6860
IS - 7
ER -