Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.

Wei Wen,Ruijun Jeanna Su,Jian-Ping Zhang,Xiao-Bing Zhang,Amanda Neises,Jing Xu,Tao Cheng,Weiping Yuan,Guang-Zhen Ji

doi:10.1016/j.stemcr.2016.04.005

Abstract

SummaryWe 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.

Highlights

One decade ago, Takahashi and Yamanaka (2006) made a stunning discovery that mouse somatic cells can be reprogrammed into a pluripotent state after forced expression of defined factors composed of OCT4, SOX2, KLF4, and MYC
Expression of MYC and KLF4 in Two Individual Episomal Vectors instead of One Dramatically Increases Reprogramming Efficiency We have reported that the use of three episomal vector (EV) plasmids to express Yamanaka factors and BCL-XL (OS + B + MK) leads to efficient generation of integration-free induced pluripotent stem cells (iPSCs) from peripheral blood (PB) mononuclear cells (MNCs) (Su et al, 2013a; Zhang, 2013)
After nucleofection with different combinations of EV plasmids, cells were cultured in hypoxia with Stemline-based serum-free erythroid medium, which was gradually changed to iPSC induction medium (Figure 1B)

Summary

Introduction

One decade ago, Takahashi and Yamanaka (2006) made a stunning discovery that mouse somatic cells can be reprogrammed into a pluripotent state after forced expression of defined factors composed of OCT4 ( known as POU5F1), SOX2, KLF4, and MYC ( termed c-MYC). The finding in mouse cells was soon reproduced with human fibroblasts (Takahashi et al, 2007; Yu et al, 2007). This breakthrough has changed the landscape of personalized cell therapy, disease modeling, and drug screening. Fibroblasts are the widely used cellular source for many reprogramming studies reported in the last decade but with noticeable limitations (Zhang, 2013). Human fibroblasts are often obtained by skin biopsy, which is an invasive and non-sterile procedure. Skin cells bear more mutations due to environmental insults than cells from inside the body (Abyzov et al, 2012)

Results

Discussion

Conclusion