Generation of Induced Pluripotent Stem Cell Lines from Adult Rat Cells
Generation of Induced Pluripotent Stem Cell Lines from Adult Rat Cells
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从成年大鼠细胞产生诱导多能干细胞系
DOI：
10.1016/j.stem.2008.11.013
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发表时间：
2009-01-09
期刊：
影响因子：
23.9
通讯作者：
Xiao, Lei
中科院分区：
文献类型：
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作者：
Liao, Jing;Cui, Chun;Xiao, Lei
实验室大鼠（Rattus norvegicus）是第一个驯化用于科学研究的哺乳动物物种，150多年来一直被用作生理学、药理学、毒理学、营养学、行为学、免疫学和肿瘤形成的动物模型（Jacob，1999）。尽管有这样的历史，大鼠在功能遗传学研究和人类疾病模型的产生方面远远落后于小鼠，因为缺乏功能性生殖系能力大鼠胚胎干细胞（ESC），这在反向遗传学方法中至关重要。在这里，我们报告了诱导多能干细胞（iPSC）从成年大鼠细胞的产生，并证明了iPSC技术提供了一种可行的方法来建立多能干细胞的一个物种中，胚胎干细胞先前已被证明是难以建立从早期胚胎。在27年前衍生出第一个小鼠ESC系之后（Evans和考夫曼，1981;马丁，1981），进行了许多努力来建立大鼠ESC，迄今为止没有成功（Brenin等人，1997; Demers等人，2007; Iannaccone等人，1994; Mashimo等人，2008年; Schulze等人，2006;上田等人，2008; Vassilieva等人，2000）。所有已报道的细胞系都只能被称为ESC样细胞，因为它们在以下一个或多个方面不符合ESC的标准：（1）很少有细胞系能长时间增殖而保持未分化和保持正常核型;（2）当将细胞注射到免疫缺陷小鼠中时，没有报告包含所有三个生殖层的晚期畸胎瘤，这表明这些大鼠ESC样细胞缺乏分化成所有三个胚层的衍生物的能力;和（3）当大鼠ESC样细胞被注射到胚泡中时，细胞仅贡献于胚外组织。没有令人信服的证据表明细胞可以促进其他组织（Brenin等人，1997年; Demers等人，2007;上田等人，2008年）。这些不成功的尝试表明，使用传统方法从胚泡建立大鼠ESCs可能不可行。另一种称为诱导多能干细胞（iPSC）的多能干细胞可以通过使用确定的转录因子重编程成体细胞来产生（Liao等人，2008年; Park等人，2008; Takahashi等人，2007; Takahashi和Yamanaka，2006; Yu等人，2007年）。尽管首次报道的小鼠iPSC没有显示种系传递（Takahashi和Yamanaka，2006），但最近证实了种系传递和同源重组（Hanna et al.，2007; Okita等人，2007; Wernig等人，2007年）。这些报道表明，iPSC可以在体内取代ESC以产生基因敲除和敲入株。因此，iPSCs可能是非常有价值的，特别是对于大鼠和其他物种，其中ESCs是不可用的，或难以分离。本报告中使用的大鼠原代耳成纤维细胞（PEF）或原代骨髓（BMC）细胞来自10周龄SD大鼠。重编程方案的示意图如图1A所示，详细的实验程序包括在在线补充数据中。使用含有重编程因子混合物的病毒来培养成体细胞。转导后两天，通过胰蛋白酶消化收获细胞，并以5 × 104个细胞/孔的六孔板接种到MEF（鼠胚胎成纤维细胞）上。第二天，重新培养培养基（含有10%FBS的DMEM）。
The laboratory rat (Rattus norvegicus) was the first mammalian species domesticated for scientific research, and it has been used as an animal model for physiology, pharmacology, toxicology, nutrition, behavior, immunology, and neoplasia for over 150 years (Jacob, 1999). Despite this history, the rat lags far behind the mouse in functional genetic studies and generation of human disease models because of the absence of functional germline-competent rat embryonic stem cells (ESCs), which are vital in reverse genetics approach. Here, we report the generation of induced pluripotent stem cells (iPSCs) from adult rat cells and demonstrate that the iPSC technique provides a feasible approach to establish pluripotent stem cells for a species in which ESCs have previously proven to be difficult to establish from the early embryo. After the first mouse ESC lines were derived 27 years ago (Evans and Kaufman, 1981; Martin, 1981), many efforts were made to establish rat ESCs, without success to date (Brenin et al., 1997; Demers et al., 2007; Iannaccone et al., 1994; Mashimo et al., 2008; Schulze et al., 2006; Ueda et al., 2008; Vassilieva et al., 2000). All of the reported cell lines could only be termed ESC-like cells because they did not meet the criteria of ESCs in one or more of the following aspects:(1) few cell lines could proliferate for a long period of time while remaining undifferentiated and maintaining normal karyotypes;(2) an advanced teratoma containing all three germ layers was not reported when the cells were injected into immune-deficient mice, suggesting that these rat ESC-like cells lacked the ability to differentiate into derivatives of all three germ layers; and (3) when rat ESC-like cells were injected into a blastocyst, the cells only contributed to extraembryonic tissue. No convincing evidence was reported that the cells could contribute to other tissues (Brenin et al., 1997; Demers et al., 2007; Ueda et al., 2008). These unsuccessful attempts suggest that establishing rat ESCs from a blastocyst might not be feasible using traditional methods. Another type of pluripotent stem cells called induced pluripotent stem cells (iPSCs) can be generated by reprogramming adult cells using defined transcription factors (Liao et al., 2008; Park et al., 2008; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Yu et al., 2007). Although the first reported mouse iPSCs did not show germline transmission (Takahashi and Yamanaka, 2006), germline transmission and homologous recombination were recently demonstrated (Hanna et al., 2007; Okita et al., 2007; Wernig et al., 2007). These reports suggest that iPSCs can replace ESCs in vivo to generate gene knockout and knockin strains. Thus, iPSCs could be extremely valuable, especially for rat and other species in which ESCs are not available, or difficult to isolate.The rat primary ear fibroblast (PEF) or primary bone marrow (BMC) cells used in this report were derived from 10-week-old SD rats. A schematic diagram of the reprogramming protocol is shown in Figure 1 A, and detailed Experimental Procedures are included in the Supplemental Data online. Virus containing a cocktail of reprogramming factors was used to transduce the adult cells. Two days after transduction, the cells were harvested by trypsinization and plated onto MEF (murine embryonic fibroblast cells) at 5 3 104 cells per well of a sixwell plate. The next day, the medium (DMEM containing 10% FBS) was re-