Genomic instability in induced stem cells

ABSTRACT The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a

small number of genes in differentiated somatic cells with the _c-Myc_ and _Klf4_ proto-oncogenes typically included in this mix. We have previously shown that expression of oncogenes leads

to DNA replication stress and genomic instability, explaining the high frequency of _p53_ mutations in human cancers. Consequently, we wondered whether stem cell reprogramming also leads to

genomic instability. To test this hypothesis, we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse

mammary cells into mammary stem cells by expressing _c-Myc_. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem cells by

expressing either three genes, _Oct4_, _Sox2_ and _Klf4_, or four genes, OSK plus _c-Myc_. Comparative genomic hybridization analysis of stem cells derived by these protocols revealed the

presence of genomic deletions and amplifications, whose signature was suggestive of oncogene-induced DNA replication stress. The genomic aberrations were to a significant degree dependent on

_c-Myc_ expression and their presence could explain why _p53_ inactivation facilitates stem cell reprogramming. SIMILAR CONTENT BEING VIEWED BY OTHERS ACQUIRED GENETIC CHANGES IN HUMAN

PLURIPOTENT STEM CELLS: ORIGINS AND CONSEQUENCES Article 23 September 2020 TRACING GENOMIC INSTABILITY IN INDUCED MESENCHYMAL STROMAL CELL MANUFACTURE: AN INTEGRATION-FREE TRANSFECTION

APPROACH Article Open access 14 April 2025 RESTRICTING EPIGENETIC ACTIVITY PROMOTES THE REPROGRAMMING OF TRANSFORMED CELLS TO PLURIPOTENCY IN A LINE-SPECIFIC MANNER Article Open access 14

July 2023 MAIN Recent studies have demonstrated that it is possible to reprogram somatic cells into pluripotent stem cells by expressing a specific combination of transcription factors.1, 2,

3, 4, 5 The typical mix of transcription factors used for this purpose includes the oncogenes _c-Myc_ and _Klf4_. Interestingly, oncogenes have the potential to induce genomic instability,6

which raises the possibility that induced pluripotent stem (iPS) cells have aberrant genomes. Given the excitement in the stem cell field by the ability to transform adult differentiated

cells into pluripotent stem cells, it is not surprising that research has focused mainly on methods that enhance reprogramming efficiency, with less attention been paid to the genomic status

of the generated iPS cells.7, 8, 9 However, the presence of genomic aberrations in iPS cells could be one of the reasons why the efficiency with which these cells produce live mice in

tetraploid complementation assays is very low.10 The possibility that aberrant genomes are prevalent in iPS cells became more likely after the demonstration that _p53_ inactivation

facilitates reprogramming.11, 12, 13, 14 One study suggested that the absence of _p53_ enhances reprogramming, because it enhances cell proliferation.15 However, _p53_ is a DNA damage

response gene,16 raising the possibility that reprogramming is accompanied by DNA damage and genomic instability. Consistent with this interpretation, iPS cells generated from mouse embryo

fibroblasts (MEFs) lacking _p53_ function, form malignant tumors when injected in donor mice.17 In this study, we examined directly the genomes of induced stem cells by array-based

comparative genomic hybridization (cGH) analysis. Using three different experimental systems, we report genomic aberrations in induced stem cells. These aberrations were associated with

oncogene-induced DNA replication stress. RESULTS GENOMIC INSTABILITY IN INDUCED MAMMARY STEM CELLS As a first step in exploring whether stem cell reprogramming is accompanied by genomic

instability, we developed a protocol for inducing mammary stem cells. The protocol involves expressing _c-Myc_, one of the four original stem cell reprogramming genes, in mouse mammary cells

and examining whether these cells acquire stem cell properties, such as the ability to form mammospheres _in vitro_ and to repopulate cleared mouse fat pads.18 We first attempted to

reprogram nearly homogeneous populations of mammary progenitor cells. These cells, chosen because they lack stem cell properties, were obtained using a previously described PKH26-based

label-retaining protocol.19 Briefly, primary mammary cells were pulse-labeled with the lipophilic, fluorescent-dye PKH26 and then cultured as mammospheres for two passages. At this time,

PKH26-high cells (about 0.3% of all cells, representing stem cells) and PKH26-negative cells (about 30% of all cells, representing progenitor cells) were isolated by flow sorting.19 The

PKH26-negative cells were then infected with a control lentivirus or a lentivirus-expressing MycER, a c-Myc protein containing a modified estrogen receptor hormone-binding domain at its

C-terminus.20 Five thousand infected cells were cultured under non-adherent conditions to generate mammospheres, which were then passaged on a weekly basis. The control-infected progenitor

cells formed mammospheres with very low efficiency even at the first passage and both the mammosphere number and the cumulative cell number declined to practically zero within a few passages

(Figures 1a–c). In contrast, the MycER-expressing progenitor cells were reprogrammed into mammary stem cells, as ascertained by their ability to form mammospheres and repopulate a cleared

mouse fat pad (Figure 1). Having established that c-Myc can reprogram mammary progenitor cells into stem cells, we repeated these experiments bypassing the PKH26-sorting step. Control

virus-infected mammospheres prepared from wild-type mice could be maintained only for a few passages in tissue culture, suggesting exhaustion of the stem cell population. In contrast,

mammospheres infected with the virus-expressing MycER could be easily expanded with no signs of crisis or stem cell exhaustion and could also repopulate a cleared mouse fat pad (Figure 2).

The reprogramming of mammary progenitor cells into mammary stem cells described above was performed in the absence of 4-hydroxytamoxifen (TAM). When TAM was added to the media, nuclear MycER

protein levels increased (Supplementary Figure 1a) and the MycER-expressing mammosphere cultures were exhausted within one passage (Figure 2a). This was associated with phosphorylation and

stabilization of p53, expression of p53 target genes and apoptosis (Supplementary Figure 1b), suggesting that high levels of MycER protein induced a p53-dependent DNA damage response.16, 21,

22, 23 Consistent with this scenario, MycER-expressing mammospheres prepared from p53−/− mice could be expanded in the presence of TAM (Figure 2a). During stem cell reprogramming, MycER

levels were lower and apparently insufficient to induce an overt DNA damage response (Supplementary Figure 1b), but whether they were low enough to prevent induction of genomic instability

was not clear. To address the issue of genomic instability, mammary cells, reprogrammed into stem cells by infecting them with the lentivirus-expressing MycER, were passaged for 9 weeks in

the absence of tamoxifen. Then, the cells were serially diluted in 48-well plates, to obtain single stem cell clones, which were expanded for 3–6 weeks, again in the absence of tamoxifen,

before preparing genomic DNA (Figure 3a). Control genomic DNA was prepared from non-infected primary mammospheres (passage 5). Eight randomly selected reprogrammed stem cell clones were

subjected to cGH analysis using high-density arrays covering chromosomes 10–13 in their entirety and part of chromosomes 9 and 14, corresponding in total to a quarter of the mouse genome.

Four out of the eight examined clones had focal copy number changes (CNCs) (Figure 3b). The first clone had a deletion of about 100 kbp within the retinoic acid receptor-related orphan

receptor A (_Rora_) gene in chromosome 9. In humans, the _RORA_ gene maps to the common fragile site (CFS) FRA15A.24, 25 The second reprogrammed clone had a deletion of about 250 kbp within

the phosphodiesterase 4D (_Pde4D_) gene in chromosome 13. Like _Rora_, _Pde4D_ is a very large gene. The third clone had a deletion of about 200 kbp within the protein tyrosine phosphatase

receptor type G (_Ptprg_) gene in chromosome 14. In humans, _PTPRG_ maps within the CFS FRA3B,25 although most deletions targeting FRA3B involve the adjacent fragile histidine triad (_FHIT_)

gene. The fourth clone had an amplification of the jumonji AT rich interactive domain 2 (_Jarid2_) and dystrobrevin-binding protein 1 (_Dtnbp1_) genes in chromosome 13. The protein product

of _Jarid2_ associates with the Polycomb repressive complex 2 and regulates the self-renewal of embryonic stem cells,26, 27, 28 suggesting that the amplification of _Jarid2_ may have been

selected in this clone. The observed frequency of genomic aberrations within just a quarter of the mouse genome indicates that reacquisition of stemness features in the reprogramming

protocol is associated with the occurrence of genomic rearrangements. GENOMIC INSTABILITY IN IPS CELLS The analysis of the genomes of induced mammary stem cells, described above, prompted us

to explore the presence of genomic instability in iPS cells prepared by standard protocols.1, 2, 3, 4, 5 We first focused on iPS cells reprogrammed using three factors. Cells prepared from

six independent clones (from two independent experiments, three clones per experiment) expressed pluripotency markers and could be induced to differentiate and form embryoid bodies _in

vitro_ (Supplementary Figure 2). Further, all these clones were diploid, as determined by karyotype analysis (data not shown). cGH was performed for chromosomes 5–13 in their entirety and

for part of chromosomes 4 and 14, corresponding in total to half the mouse genome. Only two clones showed signs of genomic instability. One clone had a small gain within chromosome 8

targeting the odd Oz/ten-m homolog 3 (_Odz3_) gene; a second clone also had a small gain, again within chromosome 8, targeting the cadherin 13 (_Cdh13_) gene (Figure 4). Both _Cdh13_ and

_Odz3_ are large genes. We subsequently examined iPS cells that had been reprogrammed using four factors. Pluripotency was assessed by expression of pluripotency markers (including the

endogenous _Oct4_ locus and alkaline phosphatase), the ability to grow in the presence of ERK and GSK3 kinase inhibitors (the most stringent conditions for the propagation of embryonic stem

cells) and the ability to differentiate into embryoid bodies (Figure 5). The karyotypes of cells from four independent clones were examined; two clones were diploid and two aneuploid (data

not shown). Cells from the diploid clones were subjected to cGH for half the mouse genome, as described above for the OSK iPS cells (Figure 6a). Cells from the first clone had an

amplification in chromosome 7 that involved >20 genes, as well as a deletion in chromosome 8 targeting the fat mass and obesity associated (_Fto_) gene (Figure 6b), a large gene. In

contrast, we could not identify any focal CNCs in cells from the second clone. GENOMIC LESIONS INDUCED BY DNA REPLICATION STRESS Given the conservation of CFS in the mouse and human

genomes,29, 30, 31 it appears that some of the CNCs identified in the induced stem cells map to CFS. Specifically, the deletions in the _Rora_ and _Ptprg_ genes map to the human CFS FRA15A

and FRA3B, respectively. Several of the remaining identified CNCs targeted large genes, again suggesting DNA replication stress as the culprit.30, 31 To explore the spectrum of CNCs

generated by DNA replication stress, we treated cells for 4 weeks with low doses of aphidicolin, a prototypical agent for inducing DNA replication stress, and then monitored CNCs by cGH

(Figure 7a). For this experiment, we employed a mouse–human hybrid cell line, GM11713A, which contains a single copy of human chromosome 3, thus, facilitating the detection of deletions.

This cell line was previously used to map aphidicolin-induced deletions within the _FHIT_ gene.32 Six aphidicolin-treated clones were isolated for cGH analysis spanning the entire chromosome

3. Two clones were essentially identical, except for one small deletion, suggesting that they arose from a common ancestor cell. The concordance of the findings for these two clones

provided a validation of the quality of the cGH analysis, but reduced the number of independent clones from six to five. In addition to deletions targeting the _FHIT_ gene in FRA3B in four

out of the five clones, two clones had deletions within the _PTPRG_ gene, also mapping to the FRA3B CFS (Figure 7b). Deletions were further found in the genes dedicator of cytokinesis 3

(_DOCK3_), cell adhesion molecule 2 (_CADM2_), limbic system-associated membrane protein (_LSAMP_) and _N_-acetylated alpha-linked acidic dipeptidase-like 2 (_NAALADL2_), none of which map

within established CFS (Supplementary Figure 3). Finally, a gain was detected within the ELKS–RAB6-interacting/CAST family member 2 (_ERC2_) gene (Figure 7b). An interesting feature of this

analysis is that all the identified CNCs mapped to large genes. In fact, the CNCs targeted 7 of the 17 largest genes of chromosome 3, whereas no CNCs were detected in any of the remaining

annotated genes of chromosome 3 (Figure 7c). Further, while DNA replication stress is usually associated with deletions, one of the observed CNCs was a gain. Similar findings have been

obtained by another group using a similar experimental approach.32, 33 Thus, based on the spectrum of CNCs induced by aphidicolin, we propose that of the eight CNCs observed in the induced

stem cells (mammary stem cells and iPS cells), six may be linked to DNA replication stress. DISCUSSION Previous analyses of mouse and human iPS cells has demonstrated the presence of

aneuploid karyotypes in some clones.3, 34 However, it was assumed that the clones that had diploid karyotypes did not have genomic aberrations.3, 35 Our analysis and data accumulating in the

literature suggests that this assumption was incorrect.36, 37, 38 Although it is difficult to be conclusive, the most likely mechanism underlying the observed genomic instability in induced

stem cells is oncogene-induced DNA replication stress.6, 39, 40, 41, 42 The presence of CNCs targeting very large genes and the lower frequency of genomic aberrations in stem cells induced

without _c-Myc_ support this tentative conclusion. It is important to note that the mechanisms by which c-Myc and other oncogenes induce DNA replication stress and genomic instability are

not well established. Yet, we know that genomic instability is not directly linked to the number of cell divisions or to proliferation rate.39 In some settings, oncogenes induce genomic

instability within one cell cycle.43 Importantly, the induced stem cells we examined were all early passage and were all derived from very early passage mammary cells or MEFs. A key question

is whether a few genomic aberrations compromise the function and utility of reprogrammed stem cells. To answer this question, it will be important to determine whether the observed genomic

aberrations are due to transient genomic instability during the reprogramming process or whether the genomic instability persists even after reprogramming. Our study does not address this

point. We note that the induced mammary stem cells described here, despite harboring CNCs, were capable of repopulating cleared mouse fat pads and none of the mammary glands derived from

these cells have become cancerous so far (in 20 reconstituted mice; albeit within the short time frame of 7 months after transplantation; data not shown). Also, it has been established that

OSK and OSKC-iPS cells can form viable fertile mice; yet, the efficiency is variable, possibly reflecting the presence of genomic aberrations in the iPS cells.2, 3, 5, 10 Finally, mice

derived from iPS cells often develop tumors and many of them die _in utero_, suggesting developmental abnormalities.2, 10 As considerable effort is being placed to develop more efficient

protocols for inducing stem cells, care must be taken that increased efficiency is not achieved at the expense of genomic stability. For example, _c-Myc_ enhances the efficiency of

reprogramming,1, 4, 5 but in our study it promoted genomic instability. Inactivation of _p53_, which also increases the efficiency of reprogramming,11, 12, 13, 14 probably does so by

allowing genomically unstable cells to escape apoptosis; thus explaining why iPS cells derived from _p53_-deficient fibroblasts form malignant tumors in mice.17 Finally, some of the

chemicals used for reprogramming, such as 5-azacytidine, are known DNA damage-inducing agents.44 On a brighter note, if oncogene-induced DNA replication stress is a significant contributor

to the genomic instability observed in induced stem cells, then modifications to the reprogramming protocols that mitigate oncogenic stress should improve the quality of the generated cells.

Nevertheless, at this time, our results and the results of others,36, 37, 38 suggest that great caution should be exercised when planning human therapies using induced stem cells. MATERIALS

AND METHODS INDUCED MAMMARY STEM CELLS To prepare mammosphere cultures, mammary tissues from 5-month-old virgin female FVB mice (Harlan) or _p53_−/− mice (in the C57/BL6J background,

back-crossed from a 129v background) were collected and dissociated mechanically. Disaggregation of the tissues was completed by enzymatic digestion in DMEM supplemented with 100 U/ml

Hyaluronidase (Sigma, St. Louis, MO, USA) and 200 U/ml Collagenase (Sigma) for approximately 3 h at 37 °C. The digested material was then centrifuged and filtered through 100, 70, 40 and 20 

_μ_m meshes. Red blood cells were lysed by incubating the cell suspension in 0.2% NaCl. The resulting cells were plated on ultralow adhesion plates (Falcon, BD Biosciences, San Jose, CA,

USA) at 100 000 cells/ml in MEBM medium (BioWhittaker, Walkersville, MD, USA) supplemented with 5 _μ_g/ml insulin, 0.5_μ_g/ml hydrocortisone, 2% B27 (Invitrogen, Carlsbad, CA, USA), 20 ng/ml

EGF and bFGF (BD Biosciences, San Jose, CA, USA) and 4 _μ_g/ml heparin (Sigma). Primary mammospheres were allowed to form for 6 days. At passaging, the number of mammospheres was counted,

the mammospheres were then dissociated in single cells and the number of cells counted. A total of 5000 cells were then plated in 24-well plates. At each passage, the number of mammospheres

reflects the number of stem cells that had been plated, because a stem cell can form a mammosphere, whereas, by definition, progenitor cells cannot form mammospheres. For distinguishing

native stem cells from progenitor cells, primary mammary cells were incubated with the PKH26 dye (Sigma) for 5 min, as previously described.19 The reaction was blocked in 1% BSA and the

cells were plated and passaged twice to obtain secondary mammospheres. Single cells obtained from disaggragated secondary mammospheres were sorted by flow cytometry on the basis of PKH26

fluorescence (FACS Vantage SE flow cytometer, Becton and Dickinson, Franklin Lakes, NJ, USA). The PKH26-high population was isolated as the most epifluorescent of the total population (about

0.3% of the cells). PKH26-negative cells were gated at 10 times less fluorescence units with respect to the PKH26-high population (about 30% of the cells). Since stem cells divide once or

just a few times during each passage, they retain high levels of the dye, whereas the progenitor cells that divide many times, become PKH-negative. For non-infected mammospheres obtained

from wild-type mice, the frequency of stem cells in the PKH26-high population is about 1 : 3, whereas the corresponding frequency in the PKH26-negative population is <1 : 80 000.19

Lentiviral infections were performed using the lentiviral vector pWPI (Addgene, Cambridge, MA, USA) carrying the GFP reporter gene and either no other insert (Empty Vector) or MycER as an

insert. Disaggregated primary mammospheres or PKH26-negative cells were plated in Phoenix-generated viral supernatants and infections were carried out in three cycles of 6 h each. After 6

days in culture, secondary mammospheres were collected, disaggregated and FACS sorted for the expression of GFP. For serial passage experiments, 5000 cells from disaggregated mammospheres

were plated in 24-well plates and, after 6 days, counted, and re-plated at the same density. Treatment with 500 nM TAM (Sigma) was performed continuously for 7 days during mammosphere

formation or once on replating of disaggregated mammospheres on day 1 of culture. For transplantation experiments, mammosphere cell suspensions were pelleted into Eppendorf benchtop

microfuge tubes, counted and resuspended in PBS. In all, 30 _μ_l of cell suspensions were transplanted in the cleared fat pad of 3-week-old virgin female FVB mice. The presence of positive

outgrowths was evaluated 2 months later by whole mount analysis. Immunoblot analysis of protein extracts from mammosphere cultures was performed using anti-vinculin (Sigma), anti-p53 (clone

AI25, gift from K Helin), anti-phospho-serine15-p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p21 (clone F5, Santa Cruz Biotechnology) and anti-cleaved caspase3 (clone D175, Cell

Signaling Technology, Danvers, MA, USA) primary antibodies, HRP-conjugated secondary antibodies (Sigma) and the SuperSignal West Pico Substrate detection kit (Pierce, Rockford, IL, USA).

For immunofluorescence, single cell suspensions were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 3% BSA. Cells were stained with anti-Myc antibody

(provided by S Hann, Vanderbilt University School of Medicine, Nashville, TN, USA). IPS CELLS INDUCED BY OSK MEFs containing an _Oct4-GFP_ transgene45 were infected with a lentiviral

vector-expressing human _OCT4_, _SOX2_ and _KLF4_ (OSK) from an SFFV promoter as a single transcript with self-cleaving 2A sequences separating each gene. Colonies of reprogrammed cells were

picked and expanded after 20–25 days. iPS cells were cultured on feeder layers in embryonic stem cell (ESC) medium (DMEM, 15% knock-out serum replacement supplement (Invitrogen),

L-glutamine, penicillin–streptomycin, nonessential amino acids, _β_-mercaptoethanol, and 1000 U/ml LIF). Expression of endogenous pluripotency genes, such as _Nanog_, _Oct4_ and _Sox2_, was

measured in iPS cells and matched MEFs in triplicate by quantitative PCR. Expression levels were normalized to the levels present in embryonic stem (ES) cells. For embryoid body

differentiation, the iPS cells were washed in IMDM medium supplemented with FBS, L-Glu and MonoThioGlycerol to remove LIF. Cells were then resuspended in methylcellulose (40%) containing

IMDM medium and plated. GFP expression from the _Oct4-GFP_ reporter was monitored for 10 days. IPS CELLS INDUCED BY OSKC MEFs containing an _Oct4-GFP_ knock-in reporter allele46 were

infected with a lentiviral vector-expressing mouse _Oct4_, _Sox2_, _Klf4_ and _c-Myc_ (OSKC) from a doxycycline-regulated TetO-mini CMV promoter as a single transcript with self-cleaving 2A

sequences separating each gene.47 After lentiviral infection, the cells were cultured in ESC medium and treated with doxycycline (1 _μ_g/ml) for 23 days. iPS cell colonies were isolated 12

days after doxycycline withdrawal. Genomic DNA was extracted from passage 6 iPS cells. To monitor expression of pluripotency markers, iPS cells were washed in PBS, stained with anti-SSEA-1

phycoerythrin-conjugated antibody (eBioscience, San Diego, CA, USA) and examined for SSEA1 and Oct4-GFP expression by flow cytometry (FACS Calibur flow cytometer, Becton and Dickinson). In

addition, expression of the endogenous genes _Oct4_, _Zfp296_, _Eras_ and _Fgf4_ was monitored in the iPS cells, matched MEFs and ES cells by PCR, as previously described.48 Embryoid body

differentiation was induced, as described above for the iPS-OSK cells. 21 days later, embryoid bodies were collected, fixed and stained with hematoxylin and eosin. APHIDICOLIN-INDUCED DNA

REPLICATION STRESS The GM11713A cell line, a mouse–human hybrid cell line containing a single wild-type copy of human chromosome 3 (Coriell Cell Repository, Camden, NJ, USA), was subjected

to DNA replication stress over a period of 30 days, by adding every 4 days to the tissue culture media 0.4 _μ_ M aphidicolin. At the end of the 30-day period, the cells were trypsinized and

hundred cells were plated on a 10 cm diameter plate. Two weeks later, single colonies were isolated and expanded for 3 weeks, at which time genomic DNA was prepared. CGH ANALYSIS Control DNA

was labeled with Cy5, whereas DNA from the induced stem cells or aphidicolin-treated cells was labeled with Cy3. Equal amounts of Cy5- and Cy3-labeled DNA were hybridized against

high-density tiling microarrays. For analysis of mouse genomic DNA the tiling arrays contained probes covering chromosomes 5–8 and parts of chromosomes 4 and 9 (Roche Nimblegen, Madison, WI,

USA, array MM8 WG CGH2; Build 36) and/or chromosomes 10–13 and parts of chromosomes 9 and 14 (Roche Nimblegen array MM8 WG CGH3; Build 36). The median probe density on these arrays was 1388

 bp. For analysis of human genomic DNA, the tiling arrays contained probes covering chromosome 3 (Roche Nimblegen array HG18 CHR3 FT B3734001-00-01; Build 36). The median probe density on

this array was 475 bp. ABBREVIATIONS * iPS: induced pluripotent stem * OSK: _Oct4_, _Sox2_, _Klf4_ * OSKC: _Oct4_, _Sox2_, _Klf4_, _c-Myc_ * TAM: 4-hydroxytamoxifen * MEF: mouse embryo

fibroblast * CFS: common fragile site * cGH: comparative genomic hybridization * CNC: copy number change * _Rora_ : retinoic acid receptor-related orphan receptor A * _Pde4D_ :

phosphodiesterase 4D * _Ptprg_ : protein tyrosine phosphatase receptor type G * _Fhit_ : fragile histidine triad * _Jarid2_ : jumonji AT rich interactive domain 2 * _Dtnbp1_ :

dystrobrevin-binding protein 1 * _Odz3_ : odd Oz/ten-m homolog 3 * _Cdh13_ : cadherin 13 * _Fto_ : fat mass and obesity associated * _Dock3_ : dedicator of cytokinesis3 * _Cadm2_ : cell

adhesion molecule 2 * _Lsamp_ : limbic system-associated membrane protein * _Naaladl2_ : _N_-acetylated alpha-linked acidic dipeptidase-like 2 * _Erc2_ : ELKS–RAB6-interacting/CAST family

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Google Scholar  Download references ACKNOWLEDGEMENTS Financial support for this project was provided by the Swiss National Foundation to TDH and DT, the National Institutes of Health, USA to

TDH, the European Commission Seventh Framework Programme (GENICA) to TDH and PGP, and the Italian Ministry of Health, Project Giovanni Ricercatori to SC and GT. AUTHOR INFORMATION Author

notes * C E Pasi, A Dereli-Öz and S Negrini: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Experimental Oncology at the IFOM-IEO Campus, Istituto

Europeo di Oncologia, Milan, Italy C E Pasi, G Fragola, G Testa & P G Pelicci * Department of Molecular Biology, University of Geneva, Geneva, Switzerland A Dereli-Öz, S Negrini, G Van

Houwe & T D Halazonetis * School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland M Friedli & D Trono * IFOM, FIRC Institute of Molecular Oncology

Foundation, Milan, Italy G Fragola & S Casola * San Raffaele Telethon Institute for Gene Therapy, Vita Salute San Raffaele University, Milan, Italy A Lombardo & L Naldini *

Dipartimento di Medicina, Chirurgia ed Odontoiatria, Universita degli Studi di Milano, Milan, Italy P G Pelicci * Department of Biochemistry, University of Geneva, Geneva, Switzerland T D

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Correspondence to P G Pelicci or T D Halazonetis. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by G Melino Supplementary

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ABOUT THIS ARTICLE CITE THIS ARTICLE Pasi, C., Dereli-Öz, A., Negrini, S. _et al._ Genomic instability in induced stem cells. _Cell Death Differ_ 18, 745–753 (2011).

https://doi.org/10.1038/cdd.2011.9 Download citation * Received: 10 January 2011 * Revised: 13 January 2011 * Accepted: 13 January 2011 * Published: 11 February 2011 * Issue Date: May 2011 *

DOI: https://doi.org/10.1038/cdd.2011.9 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * induced pluripotent stem cells * genomic instability *

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