title: Enhanced Metabolizing Activity of Human ES Cell-Derived Hepatocytes Using a 3D Culture System with Repeated Exposures to Xenobiotics.
abstract: Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the cell-based therapy of liver disease and the development of effective in vitro toxicity screening tools. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized hepatic differentiation protocol, which produces >90% (BGO1 and CHA15) albumin-positive HLCs with no purification process from human embryonic stem cell lines. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating 3D spheroidal aggregate of HLCs, compared with 2D HLCs. The 3D differentiation method increased expression of nuclear receptors (NRs) that regulate the proper expression of key hepatic enzymes. Furthermore, significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids, compared with 2D HLCs. HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated further functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic metabolizing ability of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.
authors.affiliation: *Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences & Biotechnology, Science Campus, Korea University, Seoul, 136-713, Republic of Korea;
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Kim
authors.affiliation: *Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences & Biotechnology, Science Campus, Korea University, Seoul, 136-713, Republic of Korea;
authors.forename: Yu Jin
authors.initials: YJ
authors.lastname: Jang
authors.affiliation: *Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences & Biotechnology, Science Campus, Korea University, Seoul, 136-713, Republic of Korea;
authors.forename: Su Yeon
authors.initials: SY
authors.lastname: An
authors.affiliation: *Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences & Biotechnology, Science Campus, Korea University, Seoul, 136-713, Republic of Korea;
authors.forename: Jeongsang
authors.initials: J
authors.lastname: Son
authors.affiliation: *Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences & Biotechnology, Science Campus, Korea University, Seoul, 136-713, Republic of Korea;
authors.forename: Jaehun
authors.initials: J
authors.lastname: Lee
authors.affiliation: *Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences & Biotechnology, Science Campus, Korea University, Seoul, 136-713, Republic of Korea;
authors.forename: Gyunggyu
authors.initials: G
authors.lastname: Lee
authors.affiliation: *Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences & Biotechnology, Science Campus, Korea University, Seoul, 136-713, Republic of Korea;
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Park
authors.affiliation: Alternative Toxicological Methods Research Center, Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, 305-343, Republic of Korea;
authors.forename: Han-Jin
authors.initials: HJ
authors.lastname: Park
authors.affiliation: Department of Biomedical Science, CHA University, Seongnam, Kyeonggido, 463-400, Republic of Korea.
authors.forename: Dong-Youn
authors.initials: DY
authors.lastname: Hwang
authors.affiliation: *Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences & Biotechnology, Science Campus, Korea University, Seoul, 136-713, Republic of Korea; hanjiyou12@hanmail.net.
authors.forename: Jong-Hoon
authors.initials: JH
authors.lastname: Kim
authors.affiliation: *Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences & Biotechnology, Science Campus, Korea University, Seoul, 136-713, Republic of Korea; hanjiyou12@hanmail.net.
authors.forename: Jiyou
authors.initials: J
authors.lastname: Han
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003580:Cytochromes / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D000066449:Human Embryonic Stem Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D008111:Liver Function Tests; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D015262:Xenobiotics / Q000633:toxicity*
chemical_list: D003580:Cytochromes; D014157:Transcription Factors; D015262:Xenobiotics; D006899:Mixed Function Oxygenases
keywords: 3D culture; CYP450 enzymes; drug metabolism; hepatic maturation; human embryonic stem cell; repeated exposure