Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.
Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.
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DOI：
10.1371/journal.pone.0014397
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发表时间：
2010-12-30
期刊：
影响因子：
3.7
通讯作者：
Na J
中科院分区：
文献类型：
--
作者：
Plews JR;Li J;Jones M;Moore HD;Mason C;Andrews PW;Na J
已使用多种方法诱导体细胞重新进入多能状态。重编程基因的病毒转导效率更高，但涉及将病毒序列随机插入人类基因组。虽然诱导多能干 (iPS) 细胞可以通过可拆卸的 PiggyBac 转座子系统或附加体系统获得，但这两种方法仍然使用 DNA 构建体，因此需要对所得细胞系进行彻底分析，以确认它们没有有害的基因修饰。因此，不使用 DNA 来改变细胞命运的方法将在再生医学中非常有用。在这项研究中，我们合成了编码OCT4、SOX2、cMYC、KLF4和SV40大T（LT）的mRNA，并将它们电穿孔到人成纤维细胞中。转染后，成纤维细胞表达这些因子的水平与人胚胎干（ES）细胞相当或更高。引入 mRNA 后 4 小时内，异位表达的 OCT4 定位于细胞核。用编码所有五种因子的 mRNA 混合物转染成纤维细胞显着增加了内源性 OCT4、NANOG、DNMT3β、REX1 和 SALL4 的表达。当这些转染的成纤维细胞也暴露于几种小分子（丙戊酸、BIX01294和5'-氮杂-2'-脱氧胞苷）并在人胚胎干细胞（ES）培养基中培养时，它们在30天内形成碱性磷酸酶活性和OCT4蛋白阳性的小聚集体。我们的结果表明，mRNA 转染是精确控制蛋白质表达水平的有用方法，并且重编程因子的短期表达足以激活分化细胞中的多能性基因。
Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS) cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine. In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT) and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES) cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5′-aza-2′-deoxycytidine) and cultured in human embryonic stem cell (ES) medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days. Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.
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克隆衍生的人类胚胎干细胞系在长期培养过程中保持多能性和增殖潜力
影响因子：
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作者：
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