Matoba Nobumasa, Yamashita Tomoki, Takayama Kazuo, Sakurai Fuminori, Mizuguchi Hiroyuki
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan; Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.
Differentiation. 2018 Nov-Dec;104:13-21. doi: 10.1016/j.diff.2018.09.005. Epub 2018 Sep 24.
Hepatocyte-like cells differentiated from human iPS cells are expected to be utilized in pharmaceutical research and regenerative medicine. Recently, various culture methods for human iPS cell maintenance have been developed. However, it is not well known whether human iPS cell maintenance method affects hepatic differentiation potency. In this study, we cultured human iPS cells using four maintenance methods: ReproStem medium with feeder cells (mouse embryonic fibroblasts), AK02N medium with iMatrix-511 (E8 fragments of laminin511), Essential 8 medium with Vitronectin N (N-terminal domain of vitronectin), TeSR-E8 medium with Vitronectin XF (xeno-free vitronectin). Then, these human iPS cells were differentiated into the hepatocyte-like cells. Interestingly, the gene expression levels of definitive endoderm markers in the definitive endoderm cells generated from human iPS cells cultured with ReproStem or TeSR-E8 medium were higher than those in other groups. The gene expression level of foregut marker, HHEX, in the definitive endoderm cells generated from human iPS cells cultured with ReproStem medium was higher than that in other groups. Consistently, the expression levels of hepatocyte markers, albumin and urea secretion capacity, and CYP3A4 activity in the hepatocyte-like cells generated from human iPS cells cultured with ReproStem medium were higher than those in the other groups. Our data indicated that the most suitable human iPS cell maintenance method for efficient hepatic differentiation was the on-feeder method which uses mouse embryonic fibroblasts, but not feeder-free methods. In conclusion, human iPS cell maintenance method largely affects hepatic differentiation potency.
源自人诱导多能干细胞（iPS细胞）分化而来的类肝细胞有望应用于药物研究和再生医学。近来，已开发出多种用于人iPS细胞培养的方法。然而，人iPS细胞培养方法是否会影响肝脏分化潜能尚不清楚。在本研究中，我们使用四种培养方法培养人iPS细胞：含饲养层细胞（小鼠胚胎成纤维细胞）的ReproStem培养基、含iMatrix-511（层粘连蛋白511的E8片段）的AK02N培养基、含玻连蛋白N（玻连蛋白的N端结构域）的Essential 8培养基、含无动物成分玻连蛋白（XF）的TeSR-E8培养基。然后，将这些人iPS细胞分化为类肝细胞。有趣的是，用ReproStem或TeSR-E8培养基培养的人iPS细胞所产生的定形内胚层细胞中，定形内胚层标志物的基因表达水平高于其他组。用ReproStem培养基培养的人iPS细胞所产生的定形内胚层细胞中，前肠标志物HHEX的基因表达水平高于其他组。同样，用ReproStem培养基培养的人iPS细胞所产生的类肝细胞中，肝细胞标志物、白蛋白和尿素分泌能力以及CYP3A4活性的表达水平高于其他组。我们的数据表明，对于高效肝脏分化而言，最合适的人iPS细胞培养方法是使用小鼠胚胎成纤维细胞的饲养层培养法，而非无饲养层培养法。总之，人iPS细胞培养方法在很大程度上影响肝脏分化潜能。
