Kime Cody, Rand Tim A, Ivey Kathryn N, Srivastava Deepak, Yamanaka Shinya, Tomoda Kiichiro
Gladstone Institute of Cardiovascular Disease, San Francisco, California.
Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, California.
Curr Protoc Hum Genet. 2015 Oct 6;87:21.2.1-21.2.21. doi: 10.1002/0471142905.hg2102s87.
The advent of induced pluripotent stem (iPS) cell technology has revolutionized biomedicine and basic research by yielding cells with embryonic stem (ES) cell-like properties. The use of iPS-derived cells for cell-based therapies and modeling of human disease holds great potential. While the initial description of iPS cells involved overexpression of four transcription factors via viral vectors that integrated within genomic DNA, advances in recent years by our group and others have led to safer and higher quality iPS cells with greater efficiency. Here, we describe commonly practiced methods for non-integrating induced pluripotent stem cell generation using nucleofection of episomal reprogramming plasmids. These methods are adapted from recent studies that demonstrate increased hiPS cell reprogramming efficacy with the application of three powerful episomal hiPS cell reprogramming factor vectors and the inclusion of an accessory vector expressing EBNA1.
诱导多能干细胞（iPS）技术的出现彻底改变了生物医学和基础研究，它能产生具有胚胎干细胞（ES）样特性的细胞。将iPS衍生细胞用于基于细胞的治疗和人类疾病建模具有巨大潜力。虽然iPS细胞的最初描述涉及通过整合到基因组DNA中的病毒载体过表达四种转录因子，但近年来我们团队和其他团队的进展已产生了更安全、质量更高且效率更高的iPS细胞。在此，我们描述了使用游离重编程质粒核转染产生非整合诱导多能干细胞的常用方法。这些方法改编自最近的研究，这些研究表明，应用三种强大的游离型人iPS细胞重编程因子载体并加入表达EBNA1的辅助载体可提高人iPS细胞重编程效率。
