Efficient transfection of embryonic and adult stem cells
Efficient transfection of embryonic and adult stem cells
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DOI：
10.1634/stemcells.22-4-531
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发表时间：
2004-01-01
期刊：
影响因子：
5.2
通讯作者：
Verfaillie, CM
中科院分区：
文献类型：
--
作者：
Lakshmipathy, U;Pelacho, B;Verfaillie, CM
胚胎干细胞和成体干细胞分化为特定细胞类型的能力在细胞和基因治疗中具有巨大的治疗潜力。实现这一潜力取决于高效和优化的干细胞遗传操作方案。在这里报道的研究中，我们证明了使用核转染作为一种将质粒DNA引入胚胎和成体干细胞的方法，其效率明显高于电穿孔或基于脂质转染方法。利用增长型绿色荧光蛋白（eGFP）作为报告基因，瞬时且稳定地转染小鼠胚胎干细胞，其转染速率比传统方法高近10倍。转染后的细胞保留了其干细胞特性，包括干细胞标记物SSEA1、Oct4和Rex1的持续表达；胚状体的形成；在适当的诱导剂存在下分化为心肌细胞；当注射到发育中的囊胚中时，会产生嵌合体。转染人胚胎癌细胞和人胚胎干细胞也获得了较高的转染水平。特别是难以转染的成体干细胞，包括骨髓和多能成体祖细胞，也可以通过核转染的方法有效地转染。基于我们的研究结果，我们得出结论，在将质粒DNA引入多种胚胎和成体干细胞方面，核转染优于目前可用的方法。通过核转染实现的高水平转染将使其成为一种快速筛选工具，用于评估异位表达的转录因子对干细胞组织特异性分化的影响。
The ability of embryonic stem cells and adult stem cells to differentiate into specific cell types holds immense potential for therapeutic use in cell and gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. In the study reported here, we demonstrate the use of nucleofection as a method to introduce plasmid DNA into embryonic and adult stem cells with significantly greater efficiency than electroporation or lipid-based transfection methods have. Using enhanced green fluorescent protein (eGFP) as a reporter gene, mouse embryonic stem cells were transfected both transiently and stably at a rate nearly 10-fold higher than conventional methods. The transfected cells retained their stem cell properties, including continued expression of the stem cell markers SSEA1, Oct4, and Rex1; formation of embryoid bodies; differentiation into cardiomyocytes in the presence of appropriate inducers; and, when injected into developing blastocysts, contribution to chimeras. Higher levels of transfection were also obtained with human embryonic carcinoma and human embryonic stem cells. Particularly hard-to-transfect adult stem cells, including bone marrow and multipotent adult progenitor cells, were also transfected efficiently by the method of nucleofection. Based on our results, we conclude that nucleofection is superior to currently available methods for introducing plasmid DNA into a variety of embryonic and adult stem cells. The high levels of transfection achieved by nucleofection will enable its use as a rapid screening tool to evaluate the effect of ectopically expressed transcription factors on tissue-specific differentiation of stem cells.