title: Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.
abstract: Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies are: 1) the use of a 2A "self-cleavage" peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College.
authors.forename: Wei
authors.initials: W
authors.lastname: Wen
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College.
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Zhang
authors.affiliation: Division of Regenerative Medicine, Department of Medicine, Loma Linda University.
authors.forename: Wanqiu
authors.initials: W
authors.lastname: Chen
authors.affiliation: Division of Regenerative Medicine, Department of Medicine, Loma Linda University.
authors.forename: Cameron
authors.initials: C
authors.lastname: Arakaki
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; Division of Regenerative Medicine, Department of Medicine, Loma Linda University.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Li
authors.affiliation: Division of Regenerative Medicine, Department of Medicine, Loma Linda University.
authors.forename: David
authors.initials: D
authors.lastname: Baylink
authors.affiliation: Department of Orthopaedic Surgery, Loma Linda University.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Botimer
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College.
authors.forename: Weiping
authors.initials: W
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; Center for Stem Cell Medicine, Chinese Academy of Medical Sciences; Department of Stem Cell & Regenerative Medicine, Peking Union Medical College; Collaborative Innovation Center for Cancer Medicine; Tianjin Key Laboratory of Blood Cell Therapy and Technology; 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 Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; Division of Regenerative Medicine, Department of Medicine, Loma Linda University; xzhang@llu.edu.
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Zhang
mesh_terms: D002454:Cell Differentiation / Q000235:genetics; D065150:Cellular Reprogramming; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D010957:Plasmids / Q000378:metabolism*
chemical_list: D019309:Epstein-Barr Virus Nuclear Antigens; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; C417029:EBV-encoded nuclear antigen 1