Morphologic and gene expression criteria for identifying human induced pluripotent stem cells.
Morphologic and gene expression criteria for identifying human induced pluripotent stem cells.
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DOI：
10.1371/journal.pone.0048677
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Dezawa M
中科院分区：
文献类型：
--
作者：
Wakao S;Kitada M;Kuroda Y;Ogura F;Murakami T;Niwa A;Dezawa M
诱导多能干细胞（iPS）可以通过四种因子Oct 3/4、Sox 2、Klf 4和c-Myc的强制表达从体细胞产生。虽然在诱导过程中有各种各样的集落生长，但只有少数集落发育成iPS细胞。研究人员目前使用视觉观察来识别iPS细胞并选择类似胚胎干细胞（ES细胞）的集落，并且没有建立客观标准。因此，我们详尽地分析了用编码单个因子的四种逆转录病毒载体（两个实验中的192和203个菌落）和用编码所有四种因子的单个多顺反子逆转录病毒载体（两个实验中的199和192个菌落）转染后从人成纤维细胞产生的所有菌落的形态和基因表达。在这里，我们证明了出现的菌落的形态特征可以根据六个参数进行分类，并且所有生成的可以传代的菌落在用四种逆转录病毒载体转染的菌落中被分类为七种亚型，在用单个多顺反子逆转录病毒载体转染的菌落中被分类为六种亚型，两者都包括iPS细胞菌落。iPS细胞的基本条件是：具有单个核仁的细胞;核与核仁（N/Nls）比为2.19;细胞大小为43.5 µm2;核与细胞质（N/C）比为0.87;集落中的细胞密度为5900个细胞/mm 2;细胞层的数量为单个。最重要的是，基因表达分析首次揭示了内源性Sox 2和Cdx 2在iPS细胞中特异性表达，而通常用于鉴定iPS细胞的标志物Oct 3/4和Nanog在iPS细胞以外的集落中表达，这表明Sox 2和Cdx 2是鉴定iPS细胞的可靠标志物。我们的研究结果表明，形态学参数和内源性Sox 2和Cdx 2的表达可以用来准确地识别iPS细胞。
Induced pluripotent stem (iPS) cells can be generated from somatic cells by the forced expression of four factors, Oct3/4, Sox2, Klf4, and c-Myc. While a great variety of colonies grow during induction, only a few of them develop into iPS cells. Researchers currently use visual observation to identify iPS cells and select colonies resembling embryonic stem (ES) cells, and there are no established objective criteria. Therefore, we exhaustively analyzed the morphology and gene expression of all the colonies generated from human fibroblasts after transfection with four retroviral vectors encoding individual factors (192 and 203 colonies in two experiments) and with a single polycistronic retroviral vector encoding all four factors (199 and 192 colonies in two experiments). Here we demonstrate that the morphologic features of emerged colonies can be categorized based on six parameters, and all generated colonies that could be passaged were classified into seven subtypes in colonies transfected with four retroviral vectors and six subtypes with a single polycistronic retroviral vector, both including iPS cell colonies. The essential qualifications for iPS cells were: cells with a single nucleolus; nucleus to nucleolus (N/Nls) ratio ∼2.19: cell size ∼43.5 µm2: a nucleus to cytoplasm (N/C) ratio ∼0.87: cell density in a colony ∼5900 cells/mm2: and number of cell layer single. Most importantly, gene expression analysis revealed for the first time that endogenous Sox2 and Cdx2 were expressed specifically in iPS cells, whereas Oct3/4 and Nanog, popularly used markers for identifying iPS cells, are expressed in colonies other than iPS cells, suggesting that Sox2 and Cdx2 are reliable markers for identifying iPS cells. Our findings indicate that morphologic parameters and the expression of endogenous Sox2 and Cdx2 can be used to accurately identify iPS cells.
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2001 年 8 月 9 日之前提取的三种人类胚胎干细胞系的核型稳定性、基因分型、分化、无饲养维持和基因表达取样
影响因子：
4
作者：
Brimble, SN;Zeng, XM;Schulz, TC
通讯作者：
Schulz, TC
影响因子：
5.2
作者：
Ware, Carol B.;Nelson, Angelique M.;Blau, C. Anthony
通讯作者：
Blau, C. Anthony
影响因子：
56.9
作者：
Thomson, JA;Itskovitz-Eldor, J;Jones, JM
通讯作者：
Jones, JM
DOI：
10.1073/pnas.0911647107
发表时间：
2010-05-11
影响因子：
11.1
作者：
Kuroda, Yasumasa;Kitada, Masaaki;Dezawa, Mari
通讯作者：
Dezawa, Mari
影响因子：
64.5
作者：
Takahashi, Kazutoshi;Tanabe, Koji;Yamanaka, Shinya
通讯作者：
Yamanaka, Shinya