Protocol for generating transgene-free naive human induced pluripotent stem cells from somatic cells using modified Sendai viral system.

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS
Kaho Washizu, Shinya Yamanaka, Akira Kunitomi
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引用次数: 0

Abstract

Sendai virus (SeV) vector represents a powerful tool for generating naive and primed human induced pluripotent stem cells (iPSCs) from somatic cells. Here, we present a protocol for the generation of transgene-free naive human iPSCs from human dermal fibroblasts (HDFs) and human peripheral mononuclear cells (PBMCs) using a modified SeV vector system. We describe steps for thawing the HDFs or PBMCs, reseeding HDFs, SeV vector infection, reseeding the SeV-infected HDFs or PBMCs on an irradiated mouse embryonic fibroblast (iMEF) plate, switching to t2iLGö+Y medium, passaging the generated naive iPSCs, removing the SeV vectors, and cryopreserving the naive iPSCs. For complete details on the use and execution of this protocol, please refer to Kunitomi et al.1,2.

利用改进的仙台病毒系统从体细胞中产生无转基因的幼稚人诱导多能干细胞的方案。
仙台病毒(SeV)载体是一种从体细胞生成初始和引物的人诱导多能干细胞(iPSCs)的强大工具。在这里,我们提出了一种使用改良的SeV载体系统从人真皮成纤维细胞(HDFs)和人外周单核细胞(PBMCs)中生成无转基因的初始人iPSCs的方案。我们描述了以下步骤:解冻HDFs或PBMCs,重新播种HDFs, SeV载体感染,在辐照的小鼠胚胎成纤维细胞(iMEF)板上重新播种SeV感染的HDFs或PBMCs,切换到t2iLGö+Y培养基,传代生成的初始iPSCs,去除SeV载体,并冷冻保存初始iPSCs。有关本协议使用和执行的完整细节,请参考Kunitomi等人1,2。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
STAR Protocols
STAR Protocols Biochemistry, Genetics and Molecular Biology-General Biochemistry, Genetics and Molecular Biology
CiteScore
2.00
自引率
0.00%
发文量
789
审稿时长
10 weeks
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