Does Retroviral Insertional Mutagenesis Play a Role in the Generation of Induced Pluripotent Stem Cells? (2008)

To the Editor: In a recent report, Aoi et al.1Aoi T Yae K Nakagawa M Ichisaka T Okita K Takahashi K et al.Generation of pluripotent stem cells from adult mouse liver and stomach cells.Science. 2008; (e-pub ahead of print 14 February 2008)Google Scholar described the establishment of induced pluripotent stem (iPS) cells from adult mouse hepatocytes and gastric epithelial cells (designated iPS-Hep and iPS-Stm cells, respectively) by retroviral transduction of complementary DNAs encoding the Oct 3/4, Sox2, Klf4, and c-Myc transcription factors. The authors noted that no common retroviral integration sites (CISs) were detected among the iPS cells analyzed and therefore concluded that retroviral integration into specific sites was not required for iPS-cell generation. Examination of their data (Figures S7–11 in Aoi et al.1Aoi T Yae K Nakagawa M Ichisaka T Okita K Takahashi K et al.Generation of pluripotent stem cells from adult mouse liver and stomach cells.Science. 2008; (e-pub ahead of print 14 February 2008)Google Scholar) indicates that the iPS-cell clones contain integrations into CISs previously identified by retroviral-mediated tumor induction in mice, as listed in the Mouse Retrovirus Tagged Cancer Gene Database (RTCGD),2Akagi K Suzuki T Stephens RM Jenkins NA Copeland NG RTCGD: Retroviral Tagged Cancer Gene Database.Nucleic Acids Res. 2004; 32: D523-D527Crossref PubMed Google Scholar summarized in Supplementary Table S1 online. This is particularly noteworthy in the cases of iPS-Stm-cell clones 99-1 and 99-3. In iPS-Stm-cell clone 99-1, the c-Myc retroviral vector was found to have integrated downstream of the Zfp36l2 gene (originally designated the Evi134 CIS) associated with oncogenesis in several tumor models and required for the progression of mouse embryos beyond the two-cell stage of development.3Ramos SB Stumpo DJ Kennington EA Phillips RS Bock CB Ribeiro-Neto F et al.The CCCH tandem zinc-finger protein Zfp36l2 is crucial for female fertility and early embryonic development.Development. 2004; 131: 4883-4893Crossref PubMed Scopus (113) Google Scholar Among the retroviral integration sites in iPS-Stm-cell clone 99-3, the c-Myc retroviral vector was demonstrated to have integrated into the Sgms1 locus, a CIS implicated in several B-lymphoid and myeloid tumor models, whereas the Klf4 retroviral vector was shown to have integrated downstream of the Snai1 gene (originally designated the Evi154 CIS) that encodes a zinc-finger transcriptional repressor associated with B- and T-cell lymphomagenesis, and which plays a critical role in the establishment of left–right asymmetry during early embryonic development.4Murray SA Gridley T Snail family genes are required for left–right asymmetry determination, but not neural crest formation, in mice.Proc Natl Acad Sci USA. 2006; 103: 10300-10304Crossref PubMed Scopus (94) Google Scholar It is of interest that the iPS-Hep and iPS-Stm cells were observed to have fewer vector integrations on average than did iPS cells induced from mouse fibroblasts (Figure 3 in Aoi et al.1Aoi T Yae K Nakagawa M Ichisaka T Okita K Takahashi K et al.Generation of pluripotent stem cells from adult mouse liver and stomach cells.Science. 2008; (e-pub ahead of print 14 February 2008)Google Scholar). Two other recent publications have reported the reprogramming of mouse fibroblasts using a strategy in which the same four transcription-factor genes were expressed from doxycycline-inducible self-inactivating lentiviral vectors.5Brambrink T Foreman R Welstead GG Lengner CJ Wernig M Suh H et al.Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell. 2008; 2: 151-159Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar,6Stadtfeld M Maherali N Breault DT Hochedlinger K Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse.Cell Stem Cell. 2008; 2: 230-240Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar Because lentiviral vectors tend to preferentially integrate into transcriptional units,7Wu X Li Y Crise B Burgess SM Transcription start regions in the human genome are favored targets for MLV integration.Science. 2003; 300: 1749-1751Crossref PubMed Scopus (1143) Google Scholar in contrast to the gene-activating tendency shown by the gammaretroviral vectors used by Aoi et al.,1Aoi T Yae K Nakagawa M Ichisaka T Okita K Takahashi K et al.Generation of pluripotent stem cells from adult mouse liver and stomach cells.Science. 2008; (e-pub ahead of print 14 February 2008)Google Scholar they have generally been assumed to be more likely to inactivate endogenous gene expression upon integration. However, another recent study showed that an uninsulated lentiviral vector with an internal promoter caused significant insertional dysregulation of genes within 600-kilobase regions surrounding the vector integration sites, with documented instances of both increases and decreases in gene expression.8Hargrove PW Kepes S Hanawa H Obenauer JC Pei D Cheng C et al.Globin lentiviral vector insertions can perturb the expression of endogenous genes in β-thalassemic hematopoietic cells.Mol Ther. 2008; 16: 525-533Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar No integration-site analyses were performed in either of the fibroblast reprogramming reports that used the lentiviral systems,5Brambrink T Foreman R Welstead GG Lengner CJ Wernig M Suh H et al.Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell. 2008; 2: 151-159Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar,6Stadtfeld M Maherali N Breault DT Hochedlinger K Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse.Cell Stem Cell. 2008; 2: 230-240Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar but previous investigations involving various cell types have revealed that lentiviral gene delivery frequently results in high copy numbers of vector integrations per transduced target cell.9Hamaguchi I Woods NB Panagopoulos I Andersson E Mikkola H Fahlman C et al.Lentivirus vector gene expression during ES cell–derived hematopoietic development in vitro.J Virol. 2000; 74: 10778-10784Crossref PubMed Scopus (91) Google Scholar,10Woods NB Muessig A Schmidt M Flygare J Olsson K Salmon P et al.Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis.Blood. 2003; 101: 1284-1289Crossref PubMed Scopus (174) Google Scholar Of relevance in this context, we (A. Ramezani, T.S. Hawley, and R.G.H., manuscript submitted) and others11Modlich U Navarro S Mishra A Zychlinski D Maetzig T Brugman M et al.Importance of vector content and vector backbone to reduce the risk of insertional mutagenesis.Mol Ther. 2008; 16: S360Google Scholar have recently described growth-stimulating effects on mouse hematopoietic stem/progenitor cells associated with integration of self-inactivating lentiviral vector backbones, albeit at lower frequencies than conventional long terminal repeat–driven gammaretroviral vectors. For iPS-cell reprogramming to become clinically relevant, approaches that can replace high-copy retroviral transduction are clearly desirable.12Shi Y Do JT Desponts C Hahm HS Scholer HR Ding S A combined chemical and genetic approach for the generation of induced pluripotent stem cells.Cell Stem Cell. 2008; 2: 525-528Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar In the interim, in view of the intriguing coincidences between the vector integration sites elaborated in the iPS-Hep and iPS-Stm-cell clones characterized by Aoi et al.,1 and the above-mentioned RTCGD CIS genes, and until detailed analyses of other iPS cells are carried out, it would seem premature to rule out a contribution of retroviral insertional mutagenesis to iPS-cell generation. Indeed, to rule out a contribution of vector-insertion events to the generation or biology of iPS cells, one would first have to demonstrate that all integration sites have been identified. A number of PCR-based protocols have been developed for integration site recovery, but a limitation of all widely used methods is a bias introduced by the choice of restriction enzyme for genomic DNA cleavage, which may result in failure to recover a significant proportion of insertions.13Wang GP Garrigue A Ciuffi A Ronen K Leipzig J Berry C et al.DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.Nucleic Acids Res. 2008; 36: e49Crossref PubMed Scopus (92) Google Scholar On the other hand—taking lessons from the gene therapy field—it is not unreasonable to imagine that comprehensive elaboration of retroviral integration sites in iPS cells (notwithstanding the caveat regarding restriction-enzyme bias) may provide new insights into the mechanisms of somatic cell reprogramming.14Kustikova OS Geiger H Li Z Brugman MH Chambers SM Shaw CA et al.Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways.Blood. 2007; 109: 1897-1907Crossref PubMed Scopus (79) Google Scholar Download .doc (.18 MB) Help with doc files Table S1. Retroviral integration sites in iPS-Hep and iPS-Stm cells listed in the Mouse Retrovirus Tagged Cancer Gene Database. This work was supported in part by National Institutes of Health grants R01HL65519, R01HL66305, and R24RR16209, and by an Elaine H. Snyder Cancer Research Award and a King Fahd Endowed Professorship from The George Washington University Medical Center.