| Summary: | Summary: 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. : 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. Keywords: human induced pluripotent stem cells, peripheral blood mononuclear cells, episomal vectors, sendai viral vectors
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