Wen Wei, Zhang Jian-Ping, Chen Wanqiu, Arakaki Cameron, Li Xiaolan, Baylink David, Botimer Gary D, Xu Jing, Yuan Weiping, Cheng Tao, Zhang Xiao-Bing
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College.
Division of Regenerative Medicine, Department of Medicine, Loma Linda University.
J Vis Exp. 2017 Jan 1(119):55091. doi: 10.3791/55091.
Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies are: 1) the use of a 2A "self-cleavage" peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.
诱导多能干细胞（iPSC）在疾病建模和再生治疗方面具有巨大潜力。我们之前报道了使用游离型载体（EV）从外周血单个核细胞（PB MNC）中生成无整合的iPSC。所使用的游离型载体是整合了来自爱泼斯坦 - 巴尔（EB）病毒的oriP和EBNA1元件的DNA质粒，它们分别允许质粒在哺乳动物细胞中复制和长期保留。通过进一步优化，从1 mL外周血中可获得数千个iPSC集落。实现高重编程效率的两个关键因素是：1）使用2A“自切割”肽连接OCT4和SOX2，从而实现这两个因子的等摩尔表达；2）使用两个载体分别表达MYC和KLF4。在此，我们描述了一种从成人外周血样本中生成无整合iPSC的分步方案。所生成的iPSC是无整合的，因为在传代五次后无法检测到残留的游离型质粒。尽管重编程效率与仙台病毒（SV）载体相当，但EV质粒比市售的SV载体经济得多。这种经济实惠的EV重编程系统在再生医学的临床应用中具有潜力，并为将PB MNC直接重编程为无整合的间充质干细胞、神经干细胞等提供了一种方法。
