by Stuart P. Atkinson

Traditional techniques for the generation of induced pluripotent stem cells (iPSCs) require the use of integrating viral particles to deliver the required factors for efficient reprogramming. Reprogramming efficiency, however, lies in the region of only 0.02 to 1% of target cells, and this falls to much lower levels when non-integrational means of reprogramming factor delivery or expression are used. The low efficiency observed with integrational delivery suggests that other factors are involved in the reprogramming process, whilst the reduction of efficiency with non-integrational methods suggests that integration of the viral vectors into the host genome may be important. This may be due to prolonged expression of integrated vectors or the lack of dilution of the vectors upon cell proliferation. Another theory is that insertion of the viral vectors into the host genome somehow enhances the efficiency of reprogramming by perturbation of nearby gene expression. This would suggest that within each target cell type, iPSCs may have common viral integration sites.

In the April edition of Stem Cells, Winkler et al report the distribution of lentivirus integration sites in eight human iPSC lines generated from foetal and newborn fibroblasts with an aim to elucidate whether any common sites of integration are apparent and if these affect nearby gene expression. Previous studies in various mouse iPSC clones generated using retroviral vectors suggest that there are no common integration sites (Aoi et al and Varas et al), but Stem Cells Winkler et al are the first to report the investigation of lentiviral integration in human iPSC. Integration of lentiviruses has also been shown to cause perturbation of nearby gene expression (Hawley et al, and Hargrove et al). In the current study, however, no common integration sites were discovered in the eight iPSC lines studied and no overlap of dysregulated genes near integration sites was observed, suggesting that perturbation of gene expression by viral integration does not play a major role in human iPSC generation by lentiviral means.

Specifically, the authors used the so-called “Thompson factors” (OCT4, NANOG, SOX2 and LIN28) to create four iPSC lines from IMR90 foetal fibroblasts and four iPSC lines from newborn foreskin fibroblasts. Each of the eight lines showed appropriate human embryonic stem cell (hESC)-like characteristics and were able to differentiate into cells of the three primordial germ layers. Integration sites were identified using linear amplification-mediated PCR (LAM-PCR) followed by shotgun cloning and sequencing. This revealed 78 integration sites across the eight iPSC lines, with 75 assigned to a unique chromosomal location with 53 mapped within transcriptional units of RefSeq mRNAs. Compared to randomly in silico generated integration site controls, there was an over-representation of integration sites within 5kb of CpG islands and, separately, within a 30kb window of proto-oncogenes, although no common integration sites were found. Indeed the closest distance between two sites (across different clones) was over 500kb. mRNA analysis of genes associated with integration sites was carried out for 45 of the 53 mapped genes and it was found that five of these genes were dysregulated, although this was observed only once for each gene and in separate clones.

Altogether, this study strongly suggests that the integration site of lentiviral particles does not affect iPSC generation in human cells.

References

No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells.

Winkler T, Cantilena A, Métais JY, Xu X, Nguyen AD, Borate B, Antosiewicz-Bourget JE, Wolfsberg TG, Thomson JA, Dunbar CE.

Stem Cells. 2010 Apr;28(4):687-94.

Generation of pluripotent stem cells from adult mouse liver and stomach cells.

Aoi T, Yae K, Nakagawa M, Ichisaka T, Okita K, Takahashi K, Chiba T, Yamanaka S.

Science. 2008 Aug 1;321(5889):699-702.

Fibroblast-derived induced pluripotent stem cells show no common retroviral vector insertions.

Varas F, Stadtfeld M, de Andres-Aguayo L, Maherali N, di Tullio A, Pantano L, Notredame C, Hochedlinger K, Graf T.

Stem Cells. 2009 Feb;27(2):300-6.

Does retroviral insertional mutagenesis play a role in the generation of induced pluripotent stem cells?

Hawley RG.

Mol Ther. 2008 Aug;16(8):1354-5.

Globin lentiviral vector insertions can perturb the expression of endogenous genes in beta-thalassemic hematopoietic cells.

Hargrove PW, Kepes S, Hanawa H, Obenauer JC, Pei D, Cheng C, Gray JT, Neale G, Persons DA.

Mol Ther. 2008 Mar;16(3):525-33. Epub 2008 Jan 15.

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