(A) study on drug metabolism in human pluripotent stem cell-derived hepatocyte-like cells인간 전분화능 줄기세포 유래 간세포에서의 약물대사에 관한 연구

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Hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) have been considered as a promising cell model for predicting drug safety and xenobiotic-induced hepatotoxicity. However, their application has been still restricted because of low expression and activity of drug metabolizing enzymes (DMEs), especially cytochrome P450 (CYP) enzymes. To determine the mechanism underlying limited DME expression in hPSC-HLCs, we investigated epigenetic and transcriptional regulatory factors involve in the regulation of DME expression. Firstly, we analyzed epigenetic regulation in terms of DNA methylation and histone modifications of CYPs in human embryonic stem cell (hESC)-HLCs. Repressive histone modification and DNA methylation was associated with diminished expression of CYP genes in hESC-HLCs. Inhibition of DNA methyltransferases (DNMTs) induced demethylation of the CpG sites of CYP1A1 and CYP1A2, leading to the up-regulation of their transcription. Moreover, combinatorial inhibition of DNMTs and histone deacetylases (HDACs) increased the transcript levels of CYP1A1, CYP1A2, CYP1B1, and CYP2D6. These results imply that limited expression of CYP genes in hESC-HLCs is associated with epigenetic regulatory factors such as DNMTs and HDACs. Next, we investigated the transcriptional activities of xenobiotic receptors, especially constitutive androstane receptor (CAR), pregnane X receptor (PXR), and aryl hydrocarbon receptor (AHR), that are capable of regulating transcription of a large number of DMEs and transporters. We found that low expression of xenobiotic receptors (CAR and PXR) contributes to low activity of DMEs in hPSC-HLCs. Most DMEs and transporters that are regulated by CAR and PXR were transcriptionally down-regulated in hPSC-HLCs. Transcriptional expression of CAR and PXR was highly repressed in hPSC-HLCs, whereas AHR mRNA level was comparable to that of adult liver. Also, ligand-induced transcriptional activation was observed only at AHR in hPSC-HLCs. Promoter hypermethylation of CAR and PXR was associated with diminished transcriptional activities in hPSC-HLCs. Treatment of AHR-selective ligands enhanced transcriptional level of AHR-dependent target genes (CYP1A1 and CYP1B1) by direct AHR-DNA binding at the xenobiotic response element. Thus, AHR seems intrinsically to function as xenosensor as well as ligand-dependent transcription factor in hPSC-HLCs. Furthermore, expression of AHR-dependent target genes was significantly inhibited by an antagonist. These results indicate that hPSC-HLCs are able to screen toxic substances related to the AHR signaling and to identify potential AHR-targeted therapeutics. Taken together, our findings indicate that transcription of DMEs and transporters in hPSC-HLCs is modulated by epigenetic factor as well as xenobiotic receptors. This implies that better understanding of DME regulation including epigenetic and transcriptional control may lead to generation of HLCs exhibiting functionally matured phenotypes in drug metabolism.
Advisors
Han, Yong-Mahnresearcher한용만researcher
Description
한국과학기술원 :생명과학과,
Publisher
한국과학기술원
Issue Date
2015
Identifier
325007
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명과학과, 2015.8,[viii, 104 p. :]

Keywords

Human pluripotent stem cell; Hepatocyte; Drug metabolizing enzyme; Epigenetics; Xenobiotic receptor; 인간 전분화능 줄기세포; 간세포; 약물대사효소; 후성유전학; 생체이물 수용체

URI
http://hdl.handle.net/10203/241831
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=669248&flag=dissertation
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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