Efficient Generation of Integration-Free iPS Cells from Human Adult Peripheral Blood Using BCL-XL Together with Yamanaka Factors

Rui-Jun Su,Daila S Gridley,Yuyou Duan,Nancy Appleby,David J Baylink,Jason B Kiroyan,Mary Kearns-Jonker,Kimberly J Payne,Amanda Neises,Xiao-Bing Zhang,K-H William Lau,Jun Wang,Xianmei Meng,Benjamin Tschudy-Seney,Zoran Ivanovic

doi:10.1371/journal.pone.0064496

Abstract

The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source—peripheral blood—has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34+ cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3−/CD19− cells or T/B cell-depleted MNCs for 4–6 days led to the generation of 20–30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Highlights

The successful generation of induced pluripotent stem cells from human somatic cells has revolutionized our understanding of the development and regeneration of cells, tissues and organs, igniting new hope for replacement therapies.[1,2,3] This breakthrough has been recently recognized by the Nobel Committee for Physiology or Medicine.[4,5,6] The development of novel approaches for generating integration-free iPSCs has eliminated the concern of integrating virus-associated genotoxicity in clinical applications
Building on our previous finding that our improved episomal vectors (EV) vector design leads to efficient reprogramming of cord blood (CB) CD34+ cells, [40] here we further develop this approach for the generation of integrationfree iPSCs from adult peripheral blood mononuclear cells (PBMNCs)
We have found that balanced expression of OCT4 and SOX2 (OS) driven by a single strong promoter efficiently induces CB CD34+ cells into pluripotency

Summary

Introduction

The successful generation of induced pluripotent stem cells (iPSCs) from human somatic cells has revolutionized our understanding of the development and regeneration of cells, tissues and organs, igniting new hope for replacement therapies.[1,2,3] This breakthrough has been recently recognized by the Nobel Committee for Physiology or Medicine.[4,5,6] The development of novel approaches for generating integration-free iPSCs has eliminated the concern of integrating virus-associated genotoxicity in clinical applications. Many one-step approaches such as adenovirus vectors, [15,16] plasmids, [17,18,19] minicircle DNAs, [20,21] artificial chromosome vectors [22] and protein transduction [23,24] are very inefficient in generating integration-free iPSCs. Relatively efficient approaches that have been readily reproduced in different labs include Sendai virus vector, [25,26,27,28,29,30] modified mRNA, [31,32,33] and oriP/EBNA1-based episomal vectors (EV). EV yields expression of reprogramming factors at sufficiently high levels for several cell divisions, allowing for successful reprogramming after only one infection, while the gradual depletion of plasmids during each cell division leads to the generation of integration-free iPSCs after approximately 2 months of culture

Methods

Results

Conclusion