Generation of Mouse Induced Pluripotent Stem Cells by Protein Transduction
Generation of Mouse Induced Pluripotent Stem Cells by Protein Transduction
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DOI：
10.1089/ten.tec.2013.0026
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发表时间：
2014-05-01
影响因子：
3
通讯作者：
Dinnyes, Andras
中科院分区：
文献类型：
--
作者：
Nemes, Csilla;Varga, Eszter;Dinnyes, Andras
2006年Yamanaka和他的同事首次报道了从小鼠成纤维细胞中产生诱导多能干细胞（iPSC）后，体细胞重编程引起了极大的兴趣。在这里，我们报告了通过重组蛋白转导（Klf 4，Oct 4，Sox 2和c-Myc）从小鼠成纤维细胞中产生稳定的iPSC，这是一种旨在规避与基因递送系统相关的靶细胞基因组中外源序列整合所引起的风险的程序。将重组蛋白融合到谷胱甘肽-S-转移酶标签的框架中进行亲和纯化，并融合到反式激活因子转录-核定位信号多肽中以促进膜穿透和核定位。我们对来自近交系（C57 BL 6）和远交系（ICR）小鼠品系的胚胎成纤维细胞进行了重编程程序。以48小时的间隔用纯化的蛋白质处理细胞四次，并在完全胚胎干细胞（ESC）培养基中在丝裂霉素C处理的小鼠胚胎成纤维细胞（MEF）细胞上培养，直到形成集落。由远系繁殖的成纤维细胞产生的iPSC表现出与ESC相似的形态和生长特性，并且在未分化状态下持续超过20代。通过免疫细胞化学和逆转录-聚合酶链反应检查细胞的多能性相关标志物（Oct 4、Sox 2、Klf 4、cMyc、Nanog）。蛋白质iPSC（piPSC）形成胚状体，随后向所有三个胚层谱系分化。重要的是，piPSC可以整合到胚泡中，并在新生小鼠中导致不同程度的嵌合体。这些数据显示重组纯化的细胞穿透蛋白能够将MEF重编程为iPSC。我们还证明，所产生的细胞系的细胞满足真正的小鼠胚胎干细胞的所有要求：形成具有限定边界的圆形集落;具有以高核/质比附着在一起的趋势;表达关键的多能性标志物;并且能够在体外分化成外胚层、内胚层和中胚层，以及体内嵌合体形成。
Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2, and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in the frame to the glutathione-S-transferase tag for affinity purification and to the transactivator transcription-nuclear localization signal polypeptide to facilitate membrane penetration and nuclear localization. We performed the reprogramming procedure on embryonic fibroblasts from inbred (C57BL6) and outbred (ICR) mouse strains. The cells were treated with purified proteins four times, at 48-h intervals, and cultured on mitomycin C treated mouse embryonic fibroblast (MEF) cells in complete embryonic stem cell (ESC) medium until colonies formed. The iPSCs generated from the outbred fibroblasts exhibited similar morphology and growth properties to ESCs and were sustained in an undifferentiated state for more than 20 passages. The cells were checked for pluripotency-related markers (Oct4, Sox2, Klf4, cMyc, Nanog) by immunocytochemistry and by reverse transcription-polymerase chain reaction. The protein iPSCs (piPSCs) formed embryoid bodies and subsequently differentiated towards all three germ layer lineages. Importantly, the piPSCs could incorporate into the blastocyst and led to variable degrees of chimerism in newborn mice. These data show that recombinant purified cell-penetrating proteins are capable of reprogramming MEFs to iPSCs. We also demonstrated that the cells of the generated cell line satisfied all the requirements of bona fide mouse ESCs: form round colonies with defined boundaries; have a tendency to attach together with high nuclear/cytoplasmic ratio; express key pluripotency markers; and are capable of in vitro differentiation into ecto-, endo-, and mesoderm, and in vivo chimera formation.