title: Simple generation of human induced pluripotent stem cells using poly-beta-amino esters as the non-viral gene delivery system.
abstract: Reprogramming of somatic cells to induced pluripotent stem (iPS) cells can be achieved by the delivery of a combination of transcription factors, including Oct4, Sox2, Klf4, and c-Myc. Retroviral and lentiviral vectors are commonly used to express these four reprogramming factors separately and obtain reprogrammed iPS cells. Although efficient and reproducible, these approaches involve the time-consuming and labor-intensive production of retroviral or lentiviral particles together with a high risk of working with potentially harmful viruses overexpressing potent oncogenes, such as c-Myc. Here, we describe a simple method to produce bona fide iPS cells from human fibroblasts using poly-β-amino esters as the transfection reagent for the delivery of a single CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc, and a GFP reporter gene (OSKMG). We demonstrate for the first time that poly-β-amino esters can be used to deliver a single polycistronic reprogramming vector into human fibroblasts, achieving significantly higher transfection efficiency than with conventional transfection reagents. After a protocol of serial transfections using poly-β-amino esters, we report a simple methodology to generate human iPS cells from human fibroblasts avoiding the use of viral vectors.
authors.affiliation: Center of Regenerative Medicine of Barcelona, Doctor Aiguader 88, p7, 08003 Barcelona, Spain.
authors.forename: Núria
authors.initials: N
authors.lastname: Montserrat
authors.forename: Elena
authors.initials: E
authors.lastname: Garreta
authors.forename: Federico
authors.initials: F
authors.lastname: González
authors.forename: Jordán
authors.initials: J
authors.lastname: Gutiérrez
authors.forename: Cristina
authors.initials: C
authors.lastname: Eguizábal
authors.forename: Víctor
authors.initials: V
authors.lastname: Ramos
authors.forename: Salvador
authors.initials: S
authors.lastname: Borrós
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Izpisua Belmonte
mesh_terms: D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D007621:Karyotyping; D000090062:Kruppel-Like Factor 4; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000502:physiology; D011108:Polymers / Q000737:chemistry*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000502:physiology; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D050814:Octamer Transcription Factor-3; D011108:Polymers; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors; C507253:poly(beta-amino ester)