Differences in the Epigenetic Regulation of Cytochrome P450 Genes between Human Embryonic Stem Cell-Derived Hepatocytes and Primary Hepatocytes

Abstract

Human pluripotent stem cell-derived hepatocytes have the potential to provide in vitro model systems for drug discovery and hepatotoxicity testing. However, these cells are currently unsuitable for drug toxicity and efficacy testing because of their limited expression of genes encoding drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes. Transcript levels of major CYP genes were much lower in human embryonic stem cell-derived hepatocytes (hESC-Hep) than in human primary hepatocytes (hPH). To verify the mechanism underlying this reduced expression of CYP genes, including CYP1A1, CYP1A2, CYP1B1, CYP2D6, and CYP2E1, we investigated their epigenetic regulation in terms of DNA methylation and histone modifications in hESC-Hep and hPH. CpG islands of CYP genes were hypermethylated in hESC-Hep, whereas they had an open chromatin structure, as represented by hypomethylation of CpG sites and permissive histone modifications, in hPH. Inhibition of DNA methyltransferases (DNMTs) during hepatic maturation induced demethylation of the CpG sites of CYP1A1 and CYP1A2, leading to the up-regulation of their transcription. Combinatorial inhibition of DNMTs and histone deacetylases (HDACs) increased the transcript levels of CYP1A1, CYP1A2, CYP1B1, and CYP2D6. Our findings suggest that limited expression of CYP genes in hESC-Hep is modulated by epigenetic regulatory factors such as DNMTs and HDACs.

Comparative transcriptomics of hepatic differentiation of human pluripotent stem cells and adult human liver tissue

“…AOX1 is an oxidase that plays a key role in the metabolism of xenobiotics and drugs containing aromatic azaheterocyclic substituents (12). Finally, CYP2E1, a member of the cytochrome P450, is a phase I drug-metabolizing enzyme (33), and differences in epigenetic regulation of CYP2E1 between hPSC-HEP and primary hepatocytes have been determined (36), which may account for the impaired activity of this enzyme. As expected, AOX1 and CYP2E1 were not expressed in hPSC-HEP.…”

“…Finally, we have identified a module of genes expressed at lower levels in hPSC-HEP comprising CYP4A11, CYP2A6, CYP2B6, CYP2C9, CYP2E1, UGT2B4, and AOX1. Several studies suggest that epigenetic factors may underlie the impaired activity of hepatocyte maturity genes (19,36). Thus, investigating the dynamics of these factors during the differentiation, in addition to the pathways and transcription factors that regulate this module, may reveal several leads.…”

Cell sources forin vitrohuman liver cell culture models

“…However, Matrigel, which is of biological origin with batch-to-batch variations, is still commonly used for the coating of culture vessels. 117,134,137 To enable better defined culture conditions, Matrigel was replaced in a number of studies by defined matrix components such as E-cadherin, 111,141 vitronectin, 142 or laminin. 107 A further possibility to increase reproducibility is to substitute recombinant growth factors with small molecules during the hepatic differentiation of pluripotent stem cells.…”

The Uses of Stem Cells in Toxicology Studies

“…hiPSC'ler birçok sitokrom (CYP) P450 genini eksprese etmektedir. hiPSCkaynaklı hepatosit benzeri hücrelerin, ilaçların metabolizmasında görev alan Faz I ve Faz II enzimlerinin indüksiyon ve inhibisyon profillerinin değerlendirilmesinde kullanılabileceği düşünülmektedir [72,73]. Ayrıca bu hücrelerin ilaca bağlı hepatotoksik immünolojik olayları değerlendirmek için kullanılabileceği bildirilmiştir.…”