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.

Figure 1

Experimental strategy for mapping retroviral insertion sites in iPS clones. DNA was extracted from individual iPS cell clones, and the fragments between the retroviral 5′ long terminal repeats and mouse genomic DNA were amplified by LM-PCR. The resulting amplicons were then sequenced either after cloning in bacterial plasmids or directly by high-throughput sequencing. Retroviral insertions were then determined by performing Ensembl BLAST searches against a mouse genome database. Abbreviations: iPS, induced pluripotent stem; LM-PCR, ligation-mediated polymerase chain reaction.

Figure 2

Distribution of retroviral insertion sites among mouse chromosomes. The horizontal lines across the chromosomes indicate sites of insertions.

Figure 3

Distribution of the number of gene functions included in the best gene networks of 50 groups of 65 randomly selected genes plus Oct4, Sox2, Klf4, and c-Myc.