Fibroblast-derived induced pluripotent stem cells show no common retroviral vector insertions.
Several laboratories have reported the reprogramming of mouse and human fibroblasts into pluripotent cells, using retroviruses carrying the Oct4, Sox2, Klf4, and c-Myc transcription factor genes. In these experiments the frequency of reprogramming was lower than 0.1% of the infected cells, raising the possibility that additional events are required to induce reprogramming, such as activation of genes triggered by retroviral insertions. We have therefore determined by ligation-mediated polymerase chain reaction (LM-PCR) the retroviral insertion sites in six induced pluripotent stem (iPS) cell clones derived from mouse fibroblasts. Seventy-nine insertion sites were assigned to a single mouse genome location. Thirty-five of these mapped to gene transcription units, whereas 29 insertions landed within 10 kilobases of transcription start sites. No common insertion site was detected among the iPS clones studied. Moreover, bioinformatics analyses revealed no enrichment of a specific gene function, network, or pathway among genes targeted by retroviral insertions. We conclude that Oct4, Sox2, Klf4, and c-Myc are sufficient to promote fibroblast-to-iPS cell reprogramming and propose that the observed low reprogramming frequencies may have alternative explanations.
Varas
Florencio
F
Differentiation and Cancer, Center for Genomic Regulation and Pompeu Fabra University, Barcelona, Spain.
Stadtfeld
Matthias
M
de Andres-Aguayo
Luisa
L
Maherali
Nimet
N
di Tullio
Alessandro
A
Pantano
Lorena
L
Notredame
Cedric
C
Hochedlinger
Konrad
K
Graf
Thomas
T
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D019295:Computational Biology; D005347:Fibroblasts / Q000166:cytology* / Q000821:virology; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism* / Q000821:virology; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D055748:SOXB1 Transcription Factors / Q000235:genetics
C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors