Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors.
Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors.
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DOI：
10.1186/1471-213x-10-81
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发表时间：
2010-08-03
影响因子：
--
通讯作者：
Duncan SA
中科院分区：
文献类型：
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作者：
Si-Tayeb K;Noto FK;Sepac A;Sedlic F;Bosnjak ZJ;Lough JW;Duncan SA
使用慢病毒将人体细胞重编程为诱导多能干细胞（iPS），由于病毒DNA序列整合到受体细胞的基因组中，可能会限制其治疗作用。最近的研究表明，人类iPS细胞可以使用外泌体质粒、可切除转座子、腺病毒或仙台病毒、mRNA或重组蛋白来产生。虽然这些方法提供了一种进步，但协议也有一些缺点。通常，这些程序需要亚克隆来鉴定没有外源DNA的人类iPS细胞，需要病毒学知识和安全处理程序，或者需要详细了解蛋白质生物化学。在这里，我们报告了一种简单的方法，使用标准技术转染编码OCT4、NANOG、SOX2和LIN28的表达质粒，从而促进了人类体细胞的重编程，而不需要额外的稳定性或选择。由此产生的人类iPS细胞没有DNA整合，表达多能性标记，并在免疫缺陷动物中形成畸胎瘤。这些iPS细胞也能够在培养中定向分化为肝细胞样细胞和心肌细胞样细胞。简单的瞬时转染编码重编程因子的质粒DNA足以从无外源DNA整合的原代成纤维细胞中产生人类iPS细胞。这种方法非常容易获得，并且可以扩大iPS细胞在人类疾病和发育研究中的应用。
The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.