Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog
Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog
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DOI：
10.1634/stemcells.2005-0475
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发表时间：
2006-06-01
期刊：
影响因子：
5.2
通讯作者：
Wiederholt, Kristin A.
中科院分区：
文献类型：
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作者：
Hough, Shelley R.;Clements, Ian;Wiederholt, Kristin A.
RNA干扰（RNAi）作为研究干细胞基础生物学或以特定方式指导分化的工具具有很大的前景。在RNAi实验中实现有效和特异性基因沉默的障碍包括转染效率和RNAi沉默效应子的功效和特异性的限制。在这里，我们结合联合收割机的方法，有效的脂质介导的交付与化学修饰的RNAi化合物沉默基因相关的多能性，以指导小鼠胚胎干细胞的分化。在用OCT 4或Nanog靶向RNAi化合物转染胚胎干细胞后，OCT 4或Nanog转录物和蛋白质的水平相应地降低。OCT 4表达的减少与滋养外胚层基因Cdx 2、Hand1和PL-1的诱导相关，6天后形成具有滋养层巨细胞表型的细胞。Nanog表达的减少与胚外内胚层基因GATA 4、GATA 6和层粘连蛋白B1的诱导相关，随后产生具有壁内胚层表型的细胞群。我们的结果表明，RNAi化合物对OCT 4或Nanog的瞬时抑制足以诱导向胚外谱系分化，这支持了这些转录因子以剂量依赖性方式影响细胞命运的模型。
RNA interference (RNAi) holds great promise as a tool to study the basic biology of stem cells or to direct differentiation in a specific manner. Barriers to achieving efficient and specific gene silencing in RNAi experiments include limitations in transfection efficiency and in the efficacy and specificity of RNAi silencing effectors. Here, we combine methods of efficient lipid-mediated delivery with chemically modified RNAi compounds to silence genes related to pluripotency, in order to direct differentiation of mouse embryonic stem cells. After transfection of embryonic stem cells with OCT4 or Nanog-targeted RNAi compounds, levels of OCT4 or Nanog transcript and protein were reduced accordingly. Reduction in OCT4 expression correlated with induction of trophectoderm genes Cdx2, Hand1, and PL-1, with formation of cells with trophoblast giant cell phenotype after 6 days. Reduction in Nanog expression correlated with induction of extraembryonic endoderm genes GATA4, GATA6, and laminin B1, with subsequent generation of groups of cells with parietal endoderm phenotype. Our results indicate that transient inhibition of OCT4 or Nanog by RNAi compounds is sufficient to induce differentiation toward extraembryonic lineages, which supports the model that these transcription factors function in a dose-dependent manner to influence cell fate.