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Stem Cell Reports. 2016 Jun 14;6(6):873-884. doi: 10.1016/j.stemcr.2016.04.005. Epub 2016 May 5.

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

Author information

1
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Collaborative Innovation Center for Cancer Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China.
2
Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA 92350, USA.
3
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Collaborative Innovation Center for Cancer Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China. Electronic address: chengtao@ihcams.ac.cn.
4
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Collaborative Innovation Center for Cancer Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China; Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA 92350, USA. Electronic address: xzhang@llu.edu.

Abstract

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.

KEYWORDS:

episomal vectors; human induced pluripotent stem cells; peripheral blood mononuclear cells; sendai viral vectors

PMID:
27161365
PMCID:
PMC4911493
DOI:
10.1016/j.stemcr.2016.04.005
[Indexed for MEDLINE]
Free PMC Article

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