Since the pioneering work from Takahashi and Yamanaka [1
] first demonstrated the possibility of converting differentiated murine somatic cells into iPS cells with similar characteristics and functional properties as embryonic stem cells via the introduction of a set of transcription factors via viral gene transfer, several groups have confirmed the general principle of reprogramming by applying modified methods to cells of different origins and species, including human and non-human primate cells [2
]. Even though the reprogramming strategies are rapidly evolving and there are recent reports documenting generation of iPS cells without the use of integrating viral vectors [13
], the transfer and expression of the crucial transcription factors via retro- or lentiviral gene transfer remains the most commonly-utilized reprogramming strategy in the preclinical and laboratory setting, due to its robustness and reproducibility. Moreover, the reprogramming efficiency in the episomal and protein transduction studies was substantially lower in comparison to the viral gene delivery methods, raising the possibility that a selection of de novo
mutations in the parental cell population contributed to the induction of the iPS phenotype [13
Whether activation or dysregulation of cellular genes caused by genomic vector insertions contributes to or modifies the process of iPS cell generation or properties are important to fully investigate, even if non-integrating delivery systems are eventually utilized for future clinical applications. Are specific integration sites found repeatedly in iPS cell lines, indicating that dysregulation of one or more genes greatly enhance the chance of iPS generation? Or specific gene classes? Are genes near integration sites reproducibly up-or down-regulated?
Our study is the first to investigate the vector insertion profile in human iPS- cell lines. Prior studies reported on integration sites found in murine iPS cells generated utilizing standard MLV retroviral vectors, and did not reveal any recurrent integration sites shared by different iPS clones [33
]. Given differences in the ease of transformation of murine versus human cells of all types, and the lower efficiency of iPS generation from human versus murine cells, it is important to carry out insertion studies specifically in human iPS cell lines. Human iPS cells were initially created utilizing either standard MLV or lentiviral HIV vectors [1
]. Because MLV vectors have been associated more directly with genotoxicity and insertional proto-oncogene activation in various clinical and experimental systems than lentiviral vectors, we felt iPS cells created utilizing MLV vectors were unlikely to move forward into clinical applications or be utilized by most laboratory investigators [35
We therefore focused on analysis of IS in 8 human iPS cell lines created via transfer of four transcription factors utilizing safety-modified HIV-based lentiviral vectors [2
]. These vectors drive gene expression with an internal promoter and have the LTR enhancer regions deleted. However, this type of vector has still been shown to perturb the expression of adjacent genes [39
]. Even if iPS cells shut down expression of the vector transgenes during the derivation process, it is possible that dysregulation of adjacent genes might persist and impact on iPS properties.
In order to circumvent possible restriction enzyme bias and to increase the probability of identifying all IS present in these clones, we performed independent LAM-PCR reactions using two restriction enzymes. Using this approach, we independently validated 66% of the IS; for the remaining IS the alternative restriction site was either too far or too close from the LTR-genome junction to allow amplification or an unambiguous mapping. The number of IS detected by LAM-PCR was also in very close approximation to the number of IS predicted by Southern blotting ( and ), providing reassurance that virtually all IS were identified. It is critical with LAM-PCR to use stringent criteria for identification of IS, including presence of the appropriate LTR junctions and at least 95% matching to the human genome for the entire length of the cloned fragment [41
]. We found no IS shared between the 8 examined cell lines. Furthermore, mRNA expression analysis did not reveal any overlapping dysregulation of genes near insertion sites. Pathway and network studies showed a wide functional diversity of the lentivirally tagged genes, all indicating that insertional mutagenesis is unlikely to systematically contribute to the reprogramming process.
Proto-oncogenes were over-represented in the list of insertion sites in iPS cells, similar to analysis of the murine dataset obtained by Aoi et al. [33
]. The target cells were highly proliferative embryonic or foreskin fibroblasts, and proto-oncogenes may be highly expressed in these target cells and therefore more likely targets. HIV vectors are known to preferentially integrate in genes that are highly expressed [42
Although we did not detect any IS shared between independent iPS clones, 6 of the 8 clones were found to harbour unusual “double” insertions, with two proviral insertions located very close to each other on the same chromosomal allele (). To our knowledge, this phenomenon has not been previously reported in any insertion site survey, neither in our own published analysis of 702 MLV IS and 501 SIV IS in rhesus macaques [43
], nor in insertion site analyses of cells transduced with HIV vectors similar to those used to create iPS clones [45
]. The loci with double insertions did not fall into any particular class of genes, nor were they shared between clones or differentially expressed between clones. This suggests no direct functional link with the success of reprogramming and achieving pluripotency, but instead an epiphenomenon of the lentiviral reprogramming procedures. In order to obtain iPS clones, there is strong positive selection for cells that were able to be simultaneously transduced with four different vectors, unlike previous MLV and HIV experiments, where integration profiles were carried out on cell populations harbouring fewer copies, and without selection pressure for a high copy number per cell. The target cells were exposed to high titer stocks of all vectors, and only those that successfully integrated and expressed proviruses carrying all four transgenes were able to grow out as iPS cells. Most of the cells had more than one copy of each vector. Possibly in cells successfully transduced simultaneously with so many vector copies events occur during integration process that somehow favor nearby dual integrants, for instance more than one provirus utilizing the same molecular site as part of the integrase complex, resulting in adjacent integration after tethering to chromatin. Successful reprogramming to fully functional iPS cells not only requires the correct stoichiometrical expression of the factors, but also the subsequent silencing of the viral vectors [46
]. It is possible that one or both requirements are better achieved with proviruses integrated close together [47