Sekine K, Takebe T, Suzuki Y, Kamiya A, Nakauchi H, Taniguchi H
Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Kanagawa, Japan.
Transplant Proc. 2012 May;44(4):1127-9. doi: 10.1016/j.transproceed.2012.03.001.
Although hepatocytes can be an option for liver transplantation, the shortage of donor organs continues to worsen. Since the development of induced pluripotent stem (iPS) cell technology, it is eagerly anticipated to produce functional elements from pluripotent stem cells. These functional cells differentiated from iPS cells could be used for transplantation, drug screening, and in vitro toxicology.
Human iPS cells are maintained on Mitomycin C-treated mouse embryonic fibroblast layers in DMEM-Ham F12-based medium supplemented with Knockout Serum Replacement, nonessential amino acids, 2-mercaptoethanol, and Glutamax. Differentiation of human iPS cells into a definitive endodermal lineage was induced with PRMI 1640 medium supplemented with B27 and 100 ng/mL human activin A. Two B27 supplements were examined with and without insulin. Furthermore, the PI3 kinase inhibitor LY294002 was used to examine the effect of inhibiting insulin signaling.
We established efficient induction of definitive endodermal differentiation from iPS cells. Quantitative analysis revealed efficient (93.03 ± 2.74%) differentiation of human iPS cells into definitive endoderm cells using B27 minus insulin. This protocol may contribute as a fundamental technique to promote human iPS studies to develop cellular sources for transplantation.
尽管肝细胞可作为肝移植的一种选择，但供体器官短缺问题仍在不断恶化。自诱导多能干细胞（iPS细胞）技术发展以来，人们热切期待从多能干细胞中产生功能性细胞成分。这些由iPS细胞分化而来的功能性细胞可用于移植、药物筛选和体外毒理学研究。
人iPS细胞培养于经丝裂霉素C处理的小鼠胚胎成纤维细胞层上，培养基为基于DMEM-Ham F12的培养基，并添加了敲除血清替代品、非必需氨基酸、2-巯基乙醇和谷氨酰胺。用人PRMI 1640培养基添加B27和100 ng/mL人激活素A诱导人iPS细胞分化为确定的内胚层谱系。研究了添加和不添加胰岛素的两种B27补充剂。此外，使用PI3激酶抑制剂LY294002研究抑制胰岛素信号的作用。
我们建立了从iPS细胞高效诱导确定的内胚层分化的方法。定量分析显示，使用不含胰岛素的B27时，人iPS细胞向确定的内胚层细胞的分化效率很高（93.03±2.74%）。该方案可能作为一项基础技术，有助于推动人iPS研究以开发用于移植的细胞来源。
