Live-cell screening platform using human-induced pluripotent stem cells expressing fluorescence-tagged cytochrome P450 1A1.

FASEB J 2020 Jul 18;34(7):9141-9155. Epub 2020 May 18.

Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, Republic of Korea.

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.

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201903110RDOI Listing
July 2020

Publication Analysis

Top Keywords

stem cells
12
pluripotent stem
12
cells
8
differentiation potential
8
cytochrome p450
8
p450 1a1
8
human-induced pluripotent
8
live-cell screening
8
ahr agonists
4
agonists hits
4
expressed localized
4
localized endoplasmic
4
induced ahr
4
reticulum induced
4
endoplasmic reticulum
4
hits modulating
4
high-content screening
4
hepatotoxicity chemicals
4
chemicals nuclear
4
nuclear receptor
4

Similar Publications