Human induced pluripotent stem cell line transformed with fluorescent protein-labeled cytochrome P450 and AHR modulator screening method using same
The present invention relates to a human induced pluripotent stem cell line transformed with fluorescent protein-tagged CYP1A1 and an AHR modulator screening method using the same. Specifically, a human-induced pluripotent stem cell line (hiPSC line) in which a gene was edited to express a cytochrome P450 1A1 (CYP1A1) protein in a state of fusion with a fluorescent protein without inhibiting its unique function was prepared, and it was confirmed that an AHR modulator could be screened by screening cells in a living state using the cell line-derived liver cells better than in the case of using existing human primary hepatocytes (hPH) or HepG2 cells. Therefore, the CYP1A1-mCherry hiPSC cell line of the present invention can be effectively used for screening AHR modulating compounds.
1 . A human induced pluripotent stem cell (hiPSC) line for screening an AHR (aryl hydrocarbon receptor) modulator,
the cell line expressing a fluorescent protein-tagged CYP1A1 protein, wherein the fluorescent-tagged CYP1A1 protein is produced by CRISPR-Cas9-mediated knock-in at the CYP1A1 locus using:
(i) a single guide RNA selected from SEQ ID NOs: 1-3; and
(ii) a targeting vector comprising a fluorescent protein insertion cassette,
the cell line having been deposited under the accession number KCTC 14186BP.
2 . The human induced pluripotent stem cell line for screening an AHR modulator according to claim 1 , wherein the fluorescent protein is selected from the group consisting of green fluorescence protein (GFP), cyan fluorescence protein (CFP), yellow fluorescence protein (YFP) and red fluorescence protein (RFPD).
3 . The human induced pluripotent stem cell line for screening an AHR modulator according to claim 1 , wherein the fluorescent protein is mCherry.
4 . The human induced pluripotent stem cell line for screening an AHR modulator according to claim 1 , wherein the human induced pluripotent stem cell line is prepared by transfection using pMCDT-A vector as a targeting vector.
5 . The human induced pluripotent stem cell line for screening an AHR modulator according to claim 1 , wherein the human induced pluripotent stem cell line expresses the markers NANOG, OCT4, SOX2, TRA-1-60 and TRA-1-81.
6 . The human induced pluripotent stem cell line for screening an AHR modulator according to claim 1 , wherein the human induced pluripotent stem cell line expresses Pax6, Otx2, alpha-SMA, EOMES, Brachyury, FOXA2, GATA4 and Sox17 upon differentiation into three germ layers.
7 . An AHR modulator screening method comprising the following steps:
1) contacting a test substance to the human induced pluripotent stem cell line (hiPSC line) of claim 1 ;
2) measuring the signal intensity of the fluorescent protein in the human induced pluripotent stem cell line contacted with the test substance; and
3) selecting a test substance that changes the signal intensity of the fluorescent protein compared to a control sample.
8 . The AHR modulator screening method according to claim 7 , wherein the control sample of step 3) is BaP or TCDD.
9 . An AHR modulator screening method according to claim 7 , wherein the screening method is performed by a high content screening (HCS) system.
10 . The AHR modulator screening method according to claim 7 , wherein the screening method is performed while the cells are alive.
11 . The AHR modulator screening method according to claim 7 , wherein the AHR modulator is at least one selected from the group consisting of papaverine hydrochloride, nordihydroguaiaretic acid, glafenine, yangonin and desmethoxyyangonin.