title: No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells.
abstract: Derivation of induced pluripotent stem (iPS) cells requires the expression of defined transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28) in the targeted cells. Lentiviral or standard retroviral gene transfer remains the most robust and commonly used approach. Low reprogramming frequency overall, and the higher efficiency of derivation utilizing integrating vectors compared to more recent nonviral approaches, suggests that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. We provide for the first time an extensive analysis of the lentiviral integration profile in human iPS cells. We identified a total of 78 independent IS in eight recently established iPS cell lines derived from either human fetal fibroblasts or newborn foreskin fibroblasts after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28. The number of IS ranged from 5 to 15 IS per individual iPS clone, and 75 IS could be assigned to a unique chromosomal location. The different iPS clones had no IS in common. Expression analysis as well as extensive bioinformatic analysis did not reveal functional concordance of the lentiviral targeted genes between the different clones. Interestingly, in six of the eight iPS clones, some of the IS were found in pairs, integrated into the same chromosomal location within six base pairs of each other or in very close proximity. Our study supports recent reports that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes.
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Winkler
authors.forename: Amy
authors.initials: A
authors.lastname: Cantilena
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Métais
authors.forename: Xiuli
authors.initials: X
authors.lastname: Xu
authors.forename: Anh-Dao
authors.initials: AD
authors.lastname: Nguyen
authors.forename: Bhavesh
authors.initials: B
authors.lastname: Borate
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Antosiewicz-Bourget
authors.forename: Tyra G
authors.initials: TG
authors.lastname: Wolfsberg
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D065150:Cellular Reprogramming; D019295:Computational Biology; D005786:Gene Expression Regulation; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000821:virology*; D000090062:Kruppel-Like Factor 4; D016086:Lentivirus / Q000502:physiology*; D016662:Virus Integration