Fibroblast-Derived Induced Pluripotent Stem Cells Show No Common Retroviral Vector Insertions

Abstract

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.

Epigenetic reprogramming and induced pluripotency

“…Similarly, one or several of the viral copies present in iPS cells might integrate into, and activate, a gene(s) that facilitates the reacquisition of a pluripotent, self-renewing state. However, the sequencing of viral insertion sites in iPS cells derived from fibroblasts (Varas et al, 2008), liver and stomach cells did not reveal any common integration sites, suggesting that insertional mutagenesis does not play an essential role in the induction of pluripotency. The possibility that retroviral insertion is required for the generation of iPS cells was finally excluded by two recent independent studies that produced mouse iPS cells by transiently introducing the four reprogramming factors into somatic cells using either nonintegrating adenoviruses (Stadtfeld et al, 2008c) or transient plasmid transfection .…”

<em>In vivo</em> Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

“…However, it contains the risks from the possibility of insertional mutagenesis, stable transduction and long-term proto-oncogene expression 21,22 . Nonviral gene transfer vectors such as plasmid DNA 6,7,23 or RNA 10 delivery using liposomes or electroporation have also been explored.…”

Generation of induced pluripotent stem cells using site-specific integration with phage integrase

“…The most efficient way to generate iPS cells is to introduce the transcription factor genes with integrating viral vectors. A concern with this method is that integration of the viral vector into random chromosomal locations may disturb vital gene functions (7). This complication is even more problematic if the iPS cells are to be used for a therapeutic purpose.…”