Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.
Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.
复制标题
通过附加型载体的最佳组合，增强从成人外周血单核细胞中生成无整合 iPSC。
DOI：
10.1016/j.stemcr.2016.04.005
复制
发表时间：
2016-06-14
影响因子：
5.9
通讯作者：
Zhang XB
中科院分区：
文献类型：
--
作者：
Wen W;Zhang JP;Xu J;Su RJ;Neises A;Ji GZ;Yuan W;Cheng T;Zhang XB
我们之前报道了使用改进的episomal载体（EV）系统从成人外周血（PB）中产生无整合诱导多能干细胞，该系统使用脾脏灶形成病毒U3启动子和一个额外的因子BCL-XL (B)。在这里，我们通过优化向量组合显示效率提高了约100倍。两个最关键的因素是：(1)OCT4 (O)和SOX2 (S)通过2A连接子等摩尔表达；(2)在重编程过程中，MYC (M)与KLF4 (K)的比值更高且逐渐增加，通过使用两个单独的载体来表达M和K，而不是一个。EV质粒组合（OS + M + K + B）在重编程效率上与仙台病毒相当，但成本只是后者的一小部分。生成的iPSCs在多能性和表型上与我们以前的方法没有区别。这一改进为情景向量在PB重编程中的广泛应用奠定了基础。利用两个episomal载体表达MYC和KLF4对PB重编程至关重要，优化的episomal载体组合显示重编程能力提高了~ 100倍，该系统在生成无整合iPSCs方面与仙台病毒相当。在这篇文章中，Zhang、Cheng和同事表明，使用两个episomal载体表达MYC和KLF4导致人类PB MNCs重编程能力提高了~ 100倍。这种改进的episal载体系统在产生不整合的多能干细胞方面与仙台病毒相当，但成本只是后者的一小部分。
We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B) is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming. Expression of MYC and KLF4 with two episomal vectors is critical for PB reprogramming Optimized episomal vector combination shows an ∼100-fold increase in reprogramming This system is comparable with Sendai virus in generating integration-free iPSCs In this article, Zhang, Cheng, and colleagues show that the use of two episomal vectors to express MYC and KLF4 leads to an ∼100-fold increase in reprogramming of human PB MNCs to pluripotency. This improved episomal vector system is comparable with Sendai virus in generating integration-free iPSCs but at a fraction of the cost.