title: Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency.
abstract: The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.
authors: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA [4] Max Delbrück Center, Robert-Rössle Strasse 10, 13125 Berlin, Germany [5] Berlin Institute of Health, Kapelle-Ufer 2, 10117 Berlin, Germany.
authors: Sebastian
authors: S
authors: Diecke
authors: Departments of Pediatrics and Genetics; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Jiamiao
authors: J
authors: Lu
authors: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Jaecheol
authors: J
authors: Lee
authors: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Vittavat
authors: V
authors: Termglinchan
authors: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Nigel G
authors: NG
authors: Kooreman
authors: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Paul W
authors: PW
authors: Burridge
authors: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Antje D
authors: AD
authors: Ebert
authors: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Jared M
authors: JM
authors: Churko
authors: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Arun
authors: A
authors: Sharma
authors: Departments of Pediatrics and Genetics; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Mark A
authors: MA
authors: Kay
authors: 1] Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, California 94305, USA [2] Stanford Cardiovascular Institute; Stanford University School of Medicine, Stanford, California 94305, USA [3] Department of Medicine, Division of Cardiology; Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Joseph C
authors: JC
authors: Wu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D003062:Codon; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D007438:Introns; D007621:Karyotyping; D000090062:Kruppel-Like Factor 4; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D000071317:Nanog Homeobox Protein; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics
chemical_list: D003062:Codon; D019309:Epstein-Barr Virus Nuclear Antigens; D018398:Homeodomain Proteins; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; C000715187:Lin28A protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53
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