Imamura Masanori, Okuno Hironobu, Tomioka Ikuo, Kawamura Yoshimi, Lin Zachary Yu-Ching, Nakajima Ryusuke, Akamatsu Wado, Okano Hirotaka James, Matsuzaki Yumi, Sasaki Erika, Okano Hideyuki
Department of Physiology, School of Medicine, Keio University, Tokyo, Japan.
Methods Mol Biol. 2012;925:21-48. doi: 10.1007/978-1-62703-011-3_2.
Pluripotent stem cells can provide us with an enormous cell source for in vitro model systems for development. In 2006, new methodology was designed to generate pluripotent stem cells directly from somatic cells, and these cells were named induced pluripotent stem cells (iPSCs). This method consists of technically simple procedures: donor cell preparation, gene transduction, and isolation of embryonic stem cell-like colonies. The iPSC technology enables cell biologists not only to obtain pluripotent stem cells easily but also to study the reprogramming events themselves. Here, we describe the protocols to generate iPSCs from somatic origins by using conventional viral vectors. Specifically, we state the usage of three mammalian species: mouse, common marmoset, and human. As mouse iPSC donors, fibroblasts are easily prepared, while mesenchymal stem cells are expected to give rise to highly reprogrammed iPSCs efficiently. Common marmoset (Callithrix jacchus), a nonhuman primate, represents an alternative model to the usual laboratory animals. Finally, patient-specific human iPSCs give us an opportunity to examine the pathology and mechanisms of dysregulated genomic imprinting. The iPSC technology will serve as a valuable method for studying genomic imprinting, and conversely, the insights from these studies will offer valuable criteria to assess the potential of iPSCs.
多能干细胞能够为我们提供用于发育的体外模型系统的巨大细胞来源。2006年,人们设计出了新方法,可直接从体细胞中产生多能干细胞,这些细胞被命名为诱导多能干细胞(iPSC)。该方法包含技术上简单的步骤:供体细胞制备、基因转导以及胚胎干细胞样集落的分离。iPSC技术不仅使细胞生物学家能够轻松获得多能干细胞,还能研究重编程事件本身。在此,我们描述使用传统病毒载体从体细胞来源产生iPSC的方案。具体而言,我们阐述了三种哺乳动物的使用方法:小鼠、普通狨猴和人类。作为小鼠iPSC供体,成纤维细胞易于制备,而间充质干细胞有望高效产生高度重编程的iPSC。普通狨猴(Callithrix jacchus),一种非人灵长类动物,是常用实验动物之外的另一种模型。最后,患者特异性的人类iPSC为我们提供了一个机会来研究基因组印记失调的病理学和机制。iPSC技术将成为研究基因组印记的一种有价值的方法,相反,这些研究的见解将为评估iPSC的潜力提供有价值的标准。