title: Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.
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.
authors.affiliation: 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.
authors.forename: Wei
authors.initials: W
authors.lastname: Wen
authors.affiliation: 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.
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA 92350, USA.
authors.forename: Ruijun Jeanna
authors.initials: RJ
authors.lastname: Su
authors.affiliation: Division of Regenerative Medicine MC1528B, Department of Medicine, Loma Linda University, 11234 Anderson Street, Loma Linda, CA 92350, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Neises
authors.affiliation: 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.
authors.forename: Guang-Zhen
authors.initials: GZ
authors.lastname: Ji
authors.affiliation: 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.
authors.forename: Weiping
authors.initials: W
authors.lastname: Yuan
authors.affiliation: 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.
authors.forename: Tao
authors.initials: T
authors.lastname: Cheng
authors.affiliation: 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.
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Zhang
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D015870:Gene Expression; D005818:Genetic Engineering / Q000191:economics / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D013155:Spleen Focus-Forming Viruses / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C494811:BCL2L1 protein, human; D015415:Biomarkers; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D051020:bcl-X Protein
keywords: episomal vectors; human induced pluripotent stem cells; peripheral blood mononuclear cells; sendai viral vectors