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[email protected] Human embryonic stem cells and genomic instability Owing to their original properties, pluripotent human embryonic stem cells (hESCs) and their progenies are highly valuable not only for regenerative medicine, but also as tools to study development and pathologies or as cellular substrates to screen and test new drugs. However, ensuring their genomic integrity is one important prerequisite for both research and therapeutic applications. Until recently, several studies about the genomic stability of cultured hESCs had described chromosomal or else large genomic alterations detectable with conventional karyotypic methods. In the past year, several laboratories have reported many small genomic alterations, in the megabase-sized range, using more sensitive karyotyping methods, showing that hESCs are prone to acquire focal genomic abnormalities in culture. As these alterations were found to be nonrandom, these findings strongly advocate for high-resolution monitoring of human pluripotent stem cell lines, especially when intended to be used for clinical applications KEYWORDS: culture adaptation n genomic alteration n human pluripotent stem cells n karyotype n regenerative medicine
Human pluripotent stem cells Human embryonic stem cells (hESCs) are derived from the inner cell mass of a human blastocyst (stage 5.5–7.5 days postfertilization). hESCs are pluripotent and self-renewable. These two essential properties make hESCs capable of virtually endless division while maintaining their capacity to differentiate, under specific conditions, into all cell types of the organism. Undifferentiated hESCs are characterized by the expression of three key transcription factors implicated in stemness regulation: POU5F1 (formerly known as OCT‑4), SOX2 and NANOG [1] . In addition, hESCs express a panel of surface markers, including tumor rejection antigens (TRA‑1–60 and TRA‑1–81), stage-specific embryonic antigens (SSEA‑3 and SSEA‑4 but not SSEA‑1, in contrast to mouse ESCs) and alkaline phosphatase [1] . Telomerase is highly activated after fertilization and its expression is maintained in hESCs. The first ESC lines were isolated from mouse embryos in the early 1980s [2,3] , but the first report on the derivation of hESCs was only published nearly 20 years later in 1998 [4] . To date, ESCs from at least three additional species have been isolated: monkey [5] , rat [6,7] and dog [8] . Pre-implantation genetic diagnosis procedure is a screening procedure that allows embryos produced by in vitro fertilization to be tested for the presence of a specific genetic defect prior to uterine implantation. Since 2005, several hESC lines carrying specific gene mutations related to genetic diseases have been derived from
pre-implantation genetic diagnosed embryos [9–12] . These cell lines represent an extremely powerful model to unravel the mechanisms of pathogenesis and uncover new therapeutic targets. More recently, a new type of human pluripotent stem cells, induced pluripotent stem cells (iPSCs), has become accessible to the scientific community. Human iPSCs exhibit the same phenotypical characteristics as the hESC. iPSCs were first produced in 2006 from mouse cells [13] and in 2007 from human cells [14,15] . Human iPSCs are derived from somatic cells such as adult fibroblasts [14] or, more recently, adult blood cells [16] that have been reprogrammed by forcing the transgenic expression of defined transcription factors. In the first study [13] , carried out on mouse fibroblasts, a cocktail of four transcription factors (Oct4, Sox2, c‑Myc and Klf4) was successfully used to fully reprogram the somatic cells into pluripotent stem cells. The same four factors were later successfully used to fully reprogram human somatic cells. Moreover, a similar approach used for mouse and human cells allows the generation of iPSCs from adult skin fibroblasts of rhesus monkey [17] , from rat WBF344 cells [18] , or from rat primary ear fibroblast or bone marrow [19] . iPSC lines can be derived from somatic cells isolated from patients affected with a particular disease, thereby providing an in vitro model of the pathology [20,21] . Human ESCs and iPSCs offer the opportunity to recapitulate human cell ontogenesis in vitro, and to investigate the efficacy of drug
10.2217/RME.09.63 © 2009 Future Medicine Ltd
Regen. Med. (2009) 4(6), 899–909
Nathalie Lefort†, Anselme L Perrier, Yacine Laâbi, Christine Varela & Marc Peschanski Author for correspondence: INSERM/UEVE UMR-861, I-STEM, AFM, Institute for Stem cell Therapy and Exploration of Monogenic diseases, 5 rue Henri Desbruères, 91030 Evry cedex, France Tel.: +33 169 908 588; Fax: +33 169 908 521;
[email protected] †
ISSN 1746-0751
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therapies. Moreover they represent a promising source for substitutive and regenerative allogenic (hESCs) or autologous (iPSCs) cell therapies. However, their unlimited self-renewal property bears the risk of tumor formation after engrafment, which could potentially be exacerbated by the presence of genomic abnormalities. Quality control of human pluripotent stem cells is, therefore, essential for the reproducibility of research experiments and for the safety of clinical applications based on these cells. As human pluripotent stem cells can and are often propagated for extended periods of time, these necessary controls include monitoring and controlling the integrity of the genome of these cells.
Methods to unravel chromosome abnormalities in cultured hESC There are various methods to assess karyotypic integrity of hESCs, which differ both in their sensitivity and resolution [22] . Conventional Giemsa-stained karyotypes (Figure 1A) method (also called G‑banding), where cells are blocked in metaphase with colchicine, is the reference in laboratories working on hESCs and is strongly advised as a regular control for genomic integrity [23] . G‑banding allows the identification of abnormal chromosome number (aneuploidy or polyploidy) and structural chromosome changes, such as translocations and gains or losses of large parts of chromosomes. G‑banding yields 300–400 lightly and darkly stained bands in a normal human genome, resulting in an average resolution around 5–10 Mb depending on the location of the region of interest in the genome. Conditions for chromosome preparation need to be slightly adapted to each cell line. The quality of the chromosome preparation and thus of the final interpretation depend greatly on the skill and experience of the cytogeneticist in charge of the analyses. Spectral karyotype and multicolor fluorescent in situ hybridization (mFISH), two chromosome painting methods, are a modification to traditional karyotyping that have been introduced by Schrock et al. [24] . Spectral karyotype and mFISH have a resolution of approximately 1–2 Mb. Using a series of whole-chromosome painting probes labeled with different fluorochromes or fluorochrome combinations, different pairs of chromosomes have unique color profiles (Figure 1B) [25] . These methods are useful since they allow a simpler and automatable pairing of the chromosomes. These conventional karyotypes may not be sufficient to evaluate genome integrity as many of the intrachromosomal 900
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rearrangements and submicroscopic alterations in DNA can be missed. FISH is used to detect structural chromosome abnormalities such as submicroscopic deletions or amplifications that are beyond the resolution of conventional karyotyping and mFISH. However, a prior knowledge of the genomic region of interest is required (Figure 1C) . As compared with chromosome banding and painting technologies, molecular karyotyping such as array-based comparative genomic hybridization (array‑CGH) [26] or single nucleotide polymorphism (SNP) array [27] has a much higher resolution, ranging from less than 1 Mb to less than 100 kb depending on the number of probes spotted on the arrays (Figure 1D) . Deep sequencing, a more extensive genomic analysis technique, could in theory be applied to identify acquired rearrangements at the nucleotide level [28] . However, the cost and logistics required for the routine use of this technique still prevent this type of application. Array‑CGH and SNP array technologies do not require that cells be in metaphase, abnormal cells with a lower mitotic rate can be revealed [29] . Because test and control samples contain the same proportion of haploid sets per hybridized DNA, ploidy abnormalities (e.g., triploidy) are undetectable with array‑CGH technology. Compared with the array-CGH, SNP array technology also has the added advantage of allowing the identification of copy-neutral loss of heterozygosity (LOH). Copy-neutral LOH events occur when one chromosome or a chromosomal region has been duplicated and its homolog deleted. Copy-neutral LOH is not detectable when using either conventional cytogenetics or array-CGH. It has not been described in cultured hESCs, perhaps because adequate means to detect these types of changes has simply not been applied to many hESCs yet. However, this technique has already helped in determining the genetic origin of an ESC line. The hESC line SCNT‑hES‑1, previously claimed to have been derived by somatic cell nuclear transfer was proven to be a human parthenogenetic ESC line by SNP ana lysis [30] . However, one main downside of these molecular techniques – besides their relative cost as compared with cytogenetic techniques – is their poor sensitivity. The analysis represents a mean of many cells diluted in a potentially hetero geneous cell population. Chromosomal mosaicism can be detected by molecular karyotype technologies only if the abnormality is present at a frequency of at least 20% [31] . A much higher sensitivity can be achieved by using metaphase cytogenetic technologies since it is a cell-by-cell analysis. Routine G‑band or FISH analyses screen future science group
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Figure 1 Methods for diagnosis of stem cell genomic and chromosomal abnormalities. (A) Representative pictures of G-banding of a female cell line and (B) multicolor fluorescent in situ hybridization karyotypes of a male cell line; in which there is a gain of entire chromosome 20. (C) Representative picture of interphase nucleus showing a cell containing an amplification at 20q11.21 revealed by fluorescent in situ hybridization. (D) Representative array‑CGH and SNP profiles indicating an amplification at 20q11.21. CGH: Comparative genomic hybridization; SNP: Single nucleotide polymorphism.
as many metaphases as required for genetic abnormalities, thereby increasing the sensitivity of the detection. Furthermore, molecular karyotyping fails to detect balanced translocations and inversions. In conclusion, conventional and molecular cytogenetic technologies are complementary approaches that provide meaningful information when combined. future science group
Genetic instability & ‘Darwinian’ selection of hESCs during in vitro culture The mechanisms involved in maintaining chromosome integrity concern various pathways implicated in cell division and fidelity of DNA replication [32] . In vivo, the rate of spontaneous mutations in normal human cells is approximately www.futuremedicine.com
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10 ‑7 to 10‑8 per nucleotide per cell division. This rate has been approximated with indirect estimates, obtained by comparison of orthologous human and chimpanzee pseudogenes [33] and with direct estimates of human per nucleotide mutation rates at loci causing Mendelian diseases [34] . In vitro studies performed with mouse [35] or human [36] cell lines established that the rate of spontaneous mutation was close to 10‑6 to 10‑8 per nucleotide per generation. In humans there are approximately 3 × 109 nucleotides per haploid genome, which means that we may count between 30 and 3000 mutations per cell at each cell cycle. As with any other cell lines growing in culture, hESC lines exhibit genomic alterations when maintained in vitro. Some of these changes are thought to provide a proliferative or survival advantage to the affected cells, which will eventually outgrow the original cell line. Since 2004, several studies have reported that culture conditions used to amplify undifferentiated pluripotent hESCs can have a significant impact on chromosomal stability [37] . Gains of chromosome arms or of entire chromosomes were the most frequent changes observed, most likely because of the karyotyping methods that were used. Before 2008, most major studies aiming at the identification of genetic changes in hESCs were conducted on cells in metaphase using conventional cytogenetic techniques of banding [38–53] or FISH [39,46–50] , but rarely using molecular karyotyping [54,55] . A study published by Baker et al. in 2007 listed the abnormal karyotypes reported by different groups [51] . The most frequent alterations described were gains of entire chromosomes (12, 17 and X) or gains of chromosome arms (12p and 17q). The same chromosomal additions are very common in embryonal carcinoma cells from human terato carcinomas [56–58] . Other abnormalities such as trisomies for chromosomes 8 [43,51] and 20 [45,51] were observed at a much lower frequency. Studies using molecular karyotyping published over the past year [59–61] have changed the list of nonrandom abnormal karyotypes profile and revealed the existence of a new region subject to recurrent defects. Recurring abnormalities occur not infrequently, thus they are most likely nonrandom, in a number of different cell lines at 20q11.21 [54,59–62] . Very few data on genomic integrity of human iPSCs are currently available, which makes it difficult to identify regions of recurrent instability. A t(17;20)(13;p11.2) chromosome translocation involving chromosomes 17 and 20 has been detected in human 902
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iPSCs derived from human keratinocytes [63] . Few additional genomic alterations have also been identified in human iPSCs derived from fibroblasts [64] . The existence of genetic alterations, even when recurrent, does not mean that the cells are genetically unstable. The word ‘instability’ rather suggests a context of general instability. A first mutation occurs and causes further mutations. In the case of hESCs, the word instability may be incorrect since it is not really demonstrated that there is a context of general instability. However, there is currently a consensus in published articles dealing with genomic alterations in cultured hESCs to use this term. In summary, specific chromosomal abnormalities occur nonrandomly and quite commonly in hESC lines. These specific alterations are not necessarily hotspots of genetic instabilities but are rather the consequence of the selective pressure imposed by the in vitro culture condition of hESCs. This pressure might simply have favored growth of cells harboring these abnormalities even though these specific types of mutations arose at no greater rate than the others. Recurrent karyotypic abnormalities in hESCs The karyotypic abnormalities affecting hESCs recurrently affect the same regions of the genome. The prevailing hypothesis is that the genetic changes lead to modifications in the gene-expression profile of the mutated cells, which will confer a selective advantage for selfrenewal of undifferentiated hESCs. This selective advantage can be an increased survival or an increased rate of proliferation. Gain of chromosomes 12 & 17
Gain of an entire chromosome 12 or its short arm is one of the most frequent abnormalities observed in culture [41,43,44,46,47,49,51,53,65] . Genes that are implicated in cell growth or survival are present in this chromosome, such as STELLAR, GDF3 and the pluripotency gene NANOG, all of which are located on 12p13 [60] . Cells trisomic for chromosome 12 still maintain the expression of the undifferentiated markers SSEA‑3, SSEA‑4, TRA‑1–60, TRA‑1–81, OCT-4 and NANOG, and can give rise to the three embryonic germ layers [66] . Additional copies of an entire chromosome 17 or its long arm are also frequently found in hESCs [41,43,46,51,52,54] . Consistently, studies performed on mouse ESCs have shown that a number of them were trisomic for chromosome 11, which future science group
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is syntenic to human chromosome 17 [67,68] . Taken together, these observations suggest that one or more genes located on chromosome 17 may provide a selective advantage to hESCs in culture. The BIRC5 gene, located on 17q25, is such a candidate. BIRC5 codes for the survivin protein, which inhibits apoptosis and regulates cell division [69] . Recently, it has been shown that survivin contributes to teratoma formation by hESCs [69] . The miRNA hsa‑mir‑21, located in 17q23, is another candidate since aberrant expression of miRNA has been linked to some cancers, suggesting their role as a novel class of oncogenes or tumor suppressors [70,71] . Furthermore, Bcl2, a mitochondrial protein involved in cell survival, is one of the putative targets of hsa‑mir‑21. hsa‑mir‑21 is frequently overexpressed in breast cancer and patients with elevated hsa‑mir‑21 expression were found to have a significantly worse prognosis [72] . However, each of chromosome 12 and chromosome 17 contain approximately 1400 genes (NCBI Mapviewer). It is therefore not surprising to find that some of them are implicated in cell death, survival, proliferation or stemness. Similarly, all other chromosomes also harbor genes coding for cell-cycle regulators and oncogenes. Chromosome X abnormalities
Xist is a X‑linked gene, which produces a noncoding RNA. This RNA is the master regulator of X inactivation. Interestingly, it has been established that in mouse ESCs, Nanog, Oct‑4 and Sox2 cooperate to repress Xist [73] . How aneuploidies have an impact on the coupling of Xist regulation and pluripotency remain to be elucidated. Most of the genomic alterations involving X chromosome are trisomies [40,43,46,47] . These are mostly associated with trisomy 17 with or without an additional trisomy 12 [43,46] . It could be hypothesized that X trisomy itself does not confer any selective advantage. There could be a context of genomic instability giving rise to both X and 17 trisomies. The growth advantage may be conferred by the association of both chromosome X and chromosome 17 gain. Recently, however, loss of chromosome X has also been described [Lefort N et al., Unpublished Data] [60] . In Spits and coworkers’ study, the loss of chromosome X was associated with previous karyotypic changes (dup(5)(q21.3qter), del(18)(q12.1qter), see later in the ‘Other abnormalities’ section). In our laboratory, loss of chromosome X has been observed in two different hESC lines as the only genetic change present in the cells. Several future science group
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studies have shown an increase in X chromosome loss in peripheral blood lymphocytes and skin fibroblasts in the human population over time in both males and females [74,75] . Thus, loss of X chromosome seems to be a relatively frequent event in vivo. Gain of whole or part of chromosome 20
Entire gain of a chromosome 20 [45,51] or duplication of the 20q11.21 region ranging from 2.5 to 4.6 Mb [54,59–62] , encompassing between 25 and around 80 genes, respectively (NCBI Mapviewer), have been reported. It is not clear thus far that the same target gene(s) are responsible for the selective advantage conferred to the cells that have acquired these two abnormalities. Indeed, duplication events of a megabasesized region generate a breakpoint in the DNA sequence that could have specific consequences absent in the case of a complete trisomy. DNA breaking point could modify an evolutionarily conserved regulatory region of noncoding DNA, located far from its target gene [76] , the latter not necessarily being located within the duplicated region and therefore hard to identify. To date, five different groups have reported the existence of genomic instability at 20q11.21 in at least eleven distinct hESC lines [54,59–62] . Because studies reporting genetic instabilities in hESCs are often based on routine quality controls and not from a purpose-driven systematic survey, the actual frequency of each genetic alteration is difficult to estimate and its current measurement is biased as it is performed on metaanalyses of published reports. Nonetheless, the 20q11.21 duplication seems to occur at least as often as trisomies 12, 17 and X in hESCs. hESC lines with the 20q11.21 amplification appeared normal as they express the archetypal hESC markers [Lefort N et al., Unpublished Data] [61] . A recent study, unfortunately describing only one occurrence of 20q11.21 duplication, has shown that cells displayed some features of neoplastic progression, including a growth factor independence, an increase in the frequency of tumor-initiating cells and an aberrant lineage specification [61] . The 20q11.21 region corresponds to the mouse 2region H, which has been considered as ‘hypermutable’, perhaps owing to the high instability of microsatellite D2MIT140 [77,78] . The 20q11.21 region is also amplified in a variety of cancers such as breast carcinomas [79,80] , lung cancer [81] , melanoma [82] , hepatocellular carcinoma [83] and bladder cancer [84] . Moreover, it has been shown that the acquisition of 20q11.2 occurs at an early stage in cervical www.futuremedicine.com
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cancer [85] . Several genes located in the duplicated region could be responsible for the selective advantage conferred to the cells. One or more of these genes could modulate cell proliferation, survival or death, thus providing either growth or survival advantages or both to the mutated cells. Potential candidate genes include BCL2L1, implicated in cell survival and death [86] , ASXL1, which is overexpressed in cell lines derived from carcinomas [87] , the stemness gene DNMT3B [88] , which is included in the largest duplications (4.6 Mb), POFUT1, which is highly expressed in gliomas [89] , and the miRNA hsa-mir-1825 located in the 7th exon of POFUT1.
show a stable karyotype for up to 38 passages. Nevertheless, the existence of submicroscopic abnormalities cannot be excluded. Medium conditions The cell culture medium commonly used contains knockout serum replacement more often than fetal calf serum. The knockout serum replacement is a poorly disclosed serum substitute composed of a mix of protein, mix of trace elements, bovine insulin, porcine transferrin, sodium selenite and single amino acids. hESCs maintained in culture in serum-containing medium displayed a noteworthy stable karyotype using G‑banding analyses [65] .
Other abnormalities
Abnormalities on chromosome 18 have been observed in five different hESC lines. Hemizygous deletion and loss of entire chromosome 18 were found in H1 [54] and VUB01 [60] cell lines, respectively. Spits and coauthors noted the occurrence of a derivative chromosome 18, which contains part of chromosome 18, including the centromere, and part of chromosomes 5q or 7q. Indeed, VUB04_ CF exhibited a deletion at 18q21.2qter associated with a duplication at 5q14.2qter. VUB13_FXS presented a deletion at 18q12.1qter and a duplication at 5q21.3qter, and VUB26 revealed a deletion at 18q23qter and a duplication at 7q33qter. A recent paper has also reported a loss of the 5q34a– 5q34b region associated with a mosaic gain of chromosome 12 [61] .
Effect of in vitro environmental conditions on chromosomal stability It has been reported that the hESC lines H1 and H14 grown at the University of Wisconsin (USA) were subject to trisomy 12 whereas at the University of Sheffield (UK) gain of chromosome 17 predominated [61] . The diversity of culture conditions applied in the hESC laboratories may create different selection and constraint events opening alternative routes to adaptation. Feeders Human ESC lines are either grown on a feeder cell layer or on defined extracellular matrices. Feeder cells commonly used are derived from mouse fibroblasts (STO and MEF) or human fibroblasts (foreskin). Whatever the conditions, genetic alterations have been reported [41,49,54,59] . hESCs have also been derived and maintained on mesenchymal stem cell feeders [90] or in a feeder-free culture system using Matrigel™ and mesenchymal stem cell-conditioned medium [91] . Using conventional G‑banding analysis hESCs 904
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Technique used for cell passaging Methods used to dissociate the cells during passaging have often been incriminated in the appearance of karyotypic aberrations. Passaging of cell lines with enzymatic treatment (collagenase, dispase and trypsine) is believed to generate more abnormalities than when cells are passaged by mechanical cutting [42,46] . Recently, this widely accepted theory has been questioned by two studies published in 2008 by laboratories expanding hESCs using only mechanical methods, as nine distinct hESC lines showed genomic changes at 20q11.21 [59,60] . This can be explained by the fact that most hESC laboratories use conventional G‑banding karyotyping and, consequently, submicroscopic changes had been overlooked. Therefore, it seems that all passaging methods may be associated with genomic alterations but with different patterns. hESC lines expanded with enzymatic methods are prone to generate aneuploidy whereas cells expanded by mechanical cutting exhibit mostly more focal genomic changes. Unfortunately, the size of the genomic defect does not always correspond to its functional significance. In the case of mechanical cutting, a bias could be introduced by the technician who carefully selects the colonies. Consequently, colonies with the best morphological criteria are selected at the expense of the ‘less beautiful’ ones. Cells harboring selective advantage mutations are particularly easy to grow [Lefort N, Unpublished Data] , which can lead to their artificial selection (Figure 2A) . Enzymatic cell dissociation does not introduce a bias due to the subjectivity of the technician since all colonies are passaged but rather naturally selects cells that proliferate faster or survive better (Figure 2B) . Therefore, mechanical cutting may artificially select for chromosomal changes that give rise to cells with future science group
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Cell with chromosomal abnormalities Cell with ‘the best’ morphological criteria Cell with ‘the less beautiful’ morphological criteria
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Figure 2. Karyotype abnormalities in cultured human embryonic stem cells resulting from (A) artificial or (B) natural selection. (A) The operator chooses to cut the colonies with the ‘best’ morphological criteria. In this way, cells that have acquired genomic changes that give a stemness or survival advantage are selected. After several passages the proportion of mutated colonies dramatically increases. (B) The operator uses an enzymatic method to passage the cells. In this way each colony has the same probability to be passaged whatever its morphology. After several passages cells that have acquired genomic changes giving rise to a higher or faster proliferation capacity will quickly predominate over the original cell line. Figure was produced using Servier Medical Art.
a stemness phenotype, whereas enzymatic passaging naturally selects those that make cells proliferate faster or survive better. Freeze–thawing It is a complex issue to determine whether genomic alterations observed after cryopreservation and thawing processes are due to an increased mutation rate or rather to a selection of cells already carrying a mutation that confers a selective advantage. hESCs are poorly clonogenic and therefore must be cryopreserved as cell clusters. Methods for hESC cryopreservation, either by slow cooling or vitrification, rely on the use of penetrating cryoprotectants such as dimethylsulfoxide (DMSO) and ethylene glycol. However, DMSO, for example, is a known differentiation-inducing future science group
substance that affects cell fate and can in some instances modify the epigenetic profile of cells [92] . DMSO likely acts on one or more DNA methyl transferases as well as on enzymes that modify histones. Freezing/thawing techniques could also lead to alterations in DNA replication and/or chromatin structure by increasing the production of free radicals [93] . Moreover, thawing is responsible for the formation of ice and gas bubbles, which can induce abnormal segregation of chromosomes by disruption of spindle [93] , contrary to trehalose, which preserves cellular integrity [94,95] .
Future perspective Owing to their unique properties, pluripotent stem cells are relevant candidates for cell therapy. However, karyotypic changes often occur www.futuremedicine.com
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and may have an effect on cell differentiation and behavior. Cells with an amplification of the 20q11.21 region display some features of neoplastic progression [61] . Working with cells at low passage is not, however, a guarantee of their quality. Indeed, genomic alteration may appear even at low passages, although it tends to accrue over time in culture. In terms of safety, iPSCs have not been examined as extensively, but there is no reason to think that they will suffer fewer chromosomal abnormalities than hESCs. To ensure the quality of the cell lines, we advocate periodic monitoring of pluripotent cells using both molecular and conventional karyotype technologies, particularly as novel culture methods are introduced. It is also recommended that several batches of the lines that have been previously checked are freezed in order to secure them. Some other genomic and epigenomic changes may affect genomic stability. miRNAs are small RNA molecules that act as regulators of gene expression in a variety of cellular processes. They bind to their target mRNAs and induce their degradation or inhibit their translation. The cluster miR302–367 has been identified as a potential stemness regulator in hESCs [96–98] . Any perturbation in the regulation of miRNAs implicated in the maintenance of stem cell self-renewal and pluripotency can contribute to genomic
instability. Aberrant promoter methylation patterns are observed in many cancers [99] . These phenomena are also a natural consequence of aging in some tissues [100] . Studies have shown that in vitro culture of hESCs affects their methylation profiles over time [54,101,102] . Whether miRNA deregulation and aberrant promoter methylation are causes or consequences of genomic instability remain to be elucidated. Karyotype analyses are desirable for both in vitro and in vivo experiments. The use of hESC-derived progenies as a source for cell therapy cannot be safely envisioned without specific measures of genomic integrity. Acknowledgement The authors thank M Melkus for assistance with the grammatical editing of the manuscript.
Financial & competing interests disclosure Studies by the authors have been supported by additional grants from MediCen (IngeCell program), FP6 of the EC (STEM-HD program) and ANR (hESCREEN program). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or mate‑ rials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Executive summary Pluripotent stem cells Human embryonic stem cells (hESCs) have the ability to self-renew while retaining the potential to generate cell types from the three germ layers. Consequently, they have a strong potential for substitutive and regenerative cell therapies. How to analyze abnormalities in cultured hESCs Conventional cytogenetics allows the identification of abnormal chromosome number and structural changes of large parts of chromosomes. Molecular technologies such as array-based comparative genomic hybridization and single nucleotide polymorphism array have a higher resolution but are less sensitive. Altogether, these methods allow complementary approaches and provide meaningful information when combined. Genetic instability & ‘Darwinian’ selection of hESCs during in vitro culture As any other cell lines growing in culture, hESC lines exhibit genomic alterations when maintained in vitro. Some of these changes provide a proliferative or survival advantage to the affected cells that will eventually replace the original cell line. hESC predisposition to recurrent karyotypic abnormalities Chromosome abnormalities occur nonrandomly and frequently in cultured hESCs. Gains of chromosome 12, 17 and X, duplications of 20q11.21 region or losses of part of chromosome 18 have been reported. These genomic regions may contain genes that are implicated in cell growth or survival. Effect of in vitro environmental conditions on chromosomal stability hESCs are subjected to a wide range of selective pressure due to the environmental conditions associated with in vitro culture: - Culture medium: there may be essential components present in fetal calf serum that are lacking in the composition knockout serum replacement, making the cells more vulnerable to a wide range of factors. - Technique used for passaging the cells: enzymatic, chemical and mechanical methods induce mutations but with different profiles. hESC lines expanded with enzymatic methods are prone to generate aneuploidy whereas cells expanded by mechanical cutting exhibit mostly focal genomic changes. - Freeze–thawing: cryoprotectant such as dimethylsulfoxide affects the epigenetic profile of the cells. Future perspective hESCs are relevant candidates for cell therapy. However, without specific measures being taken to ensure genomic integrity, their use as a cellular source for cell therapy may be compromised for safety reasons.
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Bibliography
14
Takahashi K, Tanabe K, Ohnuki M et al.: Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
15
Yu J, Vodyanik MA, Smuga-Otto K et al.: Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).
16
Loh YH, Agarwal S, Park IH et al.: Generation of induced pluripotent stem cells from human blood. Blood 113, 5476–5479 (2009).
Papers of special note have been highlighted as: n of interest nn of considerable interest 1
Adewumi O, Aflatoonian B, Ahrlund-Richter L et al.: Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat. Biotechnol. 25, 803–816 (2007).
2
Evans MJ, Kaufman MH: Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).
3
Martin GR: Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634–7638 (1981).
4
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al.: Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
5
Thomson JA, Kalishman J, Golos TG et al.: Isolation of a primate embryonic stem cell line. Proc. Natl Acad. Sci. USA 92, 7844–7848 (1995).
17
18
19
Liu H, Zhu F, Yong J et al.: Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3, 587–590 (2008). Li W, Wei W, Zhu S et al.: Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4, 16–19 (2009). Liao J, Cui C, Chen S et al.: Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4, 11–15 (2009). Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221 (2008).
21
7
Buehr M, Meek S, Blair K et al.: Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287–1298 (2008).
22 Catalina P, Cobo F, Cortes JL et al.:
8
Hayes B, Fagerlie SR, Ramakrishnan A et al.: Derivation, characterization, and in vitro differentiation of canine embryonic stem cells. Stem Cells 26, 465–473 (2008).
9
Verlinsky Y, Strelchenko N, Kukharenko V et al.: Human embryonic stem cell lines with genetic disorders. Reprod. Biomed. Online 10, 105–110 (2005).
10
11
Stephenson EL, Mason C, Braude PR: Preimplantation genetic diagnosis as a source of human embryonic stem cells for disease research and drug discovery. BJOG 116, 158–165 (2009). Catalina P, Bueno C, Montes R et al.: Genetic stability of human embryonic stem cells: a first-step toward the development of potential hESC-based systems for modeling childhood leukemia. Leuk. Res. 33, 980–990 (2009).
12 Tropel P, Tournois J, Côme J et al.: High
efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis. In Vitro Cell. Dev. Biol. Anim. (2009) (In Press). 13 Takahashi K, Yamanaka S: Induction of
pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
future science group
genome-wide massively parallel paired-end sequencing. Nat. Genet. 40, 722–729 (2008). 29 Cheung SW, Shaw CA, Scott DA et al.:
Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics. Am. J. Med. Genet. A 143A, 1679–1686 (2007). 30 Kim K, Ng K, Rugg-Gunn PJ et al.:
Recombination signatures distinguish embryonic stem cells derived by parthenogenesis and somatic cell nuclear transfer. Cell Stem Cell 1, 346–352 (2007). 31
Park IH, Arora N, Huo H et al.: Disease-specific induced pluripotent stem cells. Cell 134, 877–886 (2008). Conventional and molecular cytogenetic diagnostic methods in stem cell research: a concise review. Cell Biol. Int. 31, 861–869 (2007).
23 Loring JF, Rao MS: Establishing standards
for the characterization of human embryonic stem cell lines. Stem Cells 24, 145–150 (2006). 24 Schrock E, du Manoir S, Veldman T et al.:
Multicolor spectral karyotyping of human chromosomes. Science 273, 494–497 (1996). 25 Bueno C, Catalina P, Melen GJ et al.:
Etoposide induces MLL rearrangements and other chromosomal abnormalities in human embryonic stem cells. Carcinogenesis 30(9), 1628–1637 (2009). 26 Sanlaville D, Lapierre JM, Turleau C et al.:
Molecular karyotyping in human constitutional cytogenetics. Eur. J. Med. Genet. 48, 214–231 (2005). 27 Peiffer DA, Le JM, Steemers FJ et al.:
High-resolution genomic profiling of chromosomal aberrations using Infinium whole-genome genotyping. Genome Res. 16, 1136–1148 (2006). 28 Campbell PJ, Stephens PJ, Pleasance ED
et al.: Identification of somatically acquired rearrangements in cancer using
www.futuremedicine.com
Vermeesch JR, Melotte C, Froyen G et al.: Molecular karyotyping: array CGH quality criteria for constitutional genetic diagnosis. J. Histochem. Cytochem. 53, 413–422 (2005).
32 Jefford CE, Irminger-Finger I: Mechanisms of
chromosome instability in cancers. Crit. Rev. Oncol. Hematol. 59, 1–14 (2006). 33 Nachman MW, Crowell SL: Estimate of the
mutation rate per nucleotide in humans. Genetics 156, 297–304 (2000). 34 Kondrashov AS: Direct estimates of human
20 Dimos JT, Rodolfa KT, Niakan KK et al.:
Li P, Tong C, Mehrian-Shai R et al.: Germline competent embryonic stem cells derived from rat blastocysts. Cell 135, 1299–1310 (2008).
6
Review
per nucleotide mutation rates at 20 loci causing Mendelian diseases. Hum. Mutat. 21, 12–27 (2003). 35
Boesen JJ, Niericker MJ, Dieteren N, Simons JW: How variable is a spontaneous mutation rate in cultured mammalian cells? Mutat. Res. 307, 121–129 (1994).
36 Kuick RD, Neel JV, Strahler JR et al.:
Similarity of spontaneous germinal and in vitro somatic cell mutation rates in humans: implications for carcinogenesis and for the role of exogenous factors in “spontaneous” germinal mutagenesis. Proc. Natl Acad. Sci. USA 89, 7036–7040 (1992). 37 Pera MF: Unnatural selection of cultured
human ES cells? Nat. Biotechnol. 22, 42–43 (2004). 38 Amit M, Carpenter MK, Inokuma MS et al.:
Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278 (2000). 39 Amit M, Margulets V, Segev H et al.:
Human feeder layers for human embryonic stem cells. Biol. Reprod. 68, 2150–2156 (2003). 40 Inzunza J, Sahlen S, Holmberg K et al.:
Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol. Hum. Reprod. 10, 461–466 (2004). 41 Draper JS, Smith K, Gokhale P et al.:
Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22, 53–54 (2004).
907
Review n
Lefort, Perrier, Laâbi, Varela & Peschanski
First study to report the occurrence of chromosome 12 and 17 gain in cultured human embryonic stem cells (hESCs).
54 Maitra A, Arking DE, Shivapurkar N et al.:
42 Buzzard JJ, Gough NM, Crook JM,
Colman A: Karyotype of human ES cells during extended culture. Nat. Biotechnol. 22, 381–382; author reply 382 (2004).
nn
43 Brimble SN, Zeng X, Weiler DA et al.:
Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. Stem Cells Dev. 13, 585–597 (2004).
55
n
44 Cowan CA, Klimanskaya I, McMahon J
et al.: Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350, 1353–1356 (2004). 45
Rosler ES, Fisk GJ, Ares X et al.: Long-term culture of human embryonic stem cells in feeder-free conditions. Dev. Dyn. 229, 259–274 (2004).
49 Imreh MP, Gertow K, Cedervall J et al.:
et al.: Cytogenetic and molecular analysis of human male germ cell tumors: chromosome 12 abnormalities and gene amplification. Genes Chromosomes Cancer 1, 289–300 (1990).
Trisomy 12 in hESC leads to no selective in vivo growth advantage in teratomas, but induces an increased abundance of renal development. J. Cell. Biochem. 100, 1518–1525 (2007). 51
nn
52
Baker DE, Harrison NJ, Maltby E et al.: Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat. Biotechnol. 25, 207–215 (2007).
New insights into testicular germ cell tumorigenesis from gene expression profiling. Cancer Res. 62, 2359–2364 (2002). 59 Lefort N, Feyeux M, Bas C et al.: Human
n
One of the first two studies revealing the recurrent abnormality at 20q11.21. chromosomal abnormalities in human embryonic stem cells. Nat. Biotechnol. 26, 1361–1363 (2008).
n
Pandolfi PP: The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res. 6, 321–328 (1997). 69 Blum B, Bar-Nur O, Golan-Lev T,
Benvenisty N: The anti-apoptotic gene survivin contributes to teratoma formation by human embryonic stem cells. Nat. Biotechnol. 27, 281–287 (2009). 70 Caldas C, Brenton JD: Sizing up miRNAs as
cancer genes. Nat. Med. 11, 712–714 (2005). 71 Calin GA, Sevignani C, Dumitru CD et al.:
Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl Acad. Sci. USA 101, 2999–3004 (2004). 72 Yan LX, Huang XF, Shao Q et al.: MicroRNA
miR‑21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA 14, 2348–2360 (2008).
61
nn
73 Navarro P, Chambers I, Karwacki-Neisius V
et al.: Molecular coupling of Xist regulation and pluripotency. Science 321, 1693–1695 (2008).
One of the first two studies revealing the recurrent abnormality at 20q11.21. Werbowetski-Ogilvie TE, Bosse M, Stewart M et al.: Characterization of human embryonic stem cells with features of neoplastic progression. Nat. Biotechnol. 27, 91–97 (2009). Demonstrates that hESC lines with submicroscopic genetic abnormalities can display altered growth and differentiation properties.
Osafune K, Caron L, Borowiak M et al.: Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26, 313–315 (2008).
63 Aasen T, Raya A, Barrero MJ et al.: Efficient
908
68 Longo L, Bygrave A, Grosveld FG,
60 Spits C, Mateizel I, Geens M et al.: Recurrent
62 Wu H, Kim KJ, Mehta K et al.: Copy number
ESCs predisposition to karyotypic instability: is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties? Mol. Cancer 7, 76 (2008).
Ito T: Current status of chromosomal abnormalities in mouse embryonic stem cell lines used in Japan. Comp. Med. 56, 31–34 (2006).
embryonic stem cells reveal recurrent genomic instability at 20q11.21. Nat. Biotechnol. 26, 1364–1366 (2008).
Thorough general overview of karyotypic aberrations observed in hESCs in culture.
53 Catalina P, Montes R, Ligero G et al.: Human
67 Sugawara A, Goto K, Sotomaru Y, Sofuni T,
58 Skotheim RI, Monni O, Mousses S et al.:
In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells. J. Cell. Biochem. 99, 508–516 (2006). 50 Gertow K, Cedervall J, Unger C et al.:
Describes a molecular characterization of multiple hESC lines and proposes a set of tests allowing comparison across cell lines and laboratories.
Human STELLAR, NANOG, and GDF3 genes are expressed in pluripotent cells and map to chromosome 12p13, a hotspot for teratocarcinoma. Stem Cells 22, 169–179 (2004).
57 Samaniego F, Rodriguez E, Houldsworth J
48 Caisander G, Park H, Frej K et al.:
Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture. Chromosome Res. 14, 131–137 (2006).
Josephson R, Sykes G, Liu Y et al.: A molecular scheme for improved characterization of human embryonic stem cell lines. BMC Biol. 4, 28 (2006).
variant analysis of human embryonic stem cells. Stem Cells 26, 1484–1489 (2008). and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat. Biotechnol. 26, 1276–1284 (2008). 64 Chin MH, Mason MJ, Xie W et al.: Induced
pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell 5, 111–123 (2009). Regen. Med. (2009) 4(6)
Herszfeld D, Wolvetang E, Langton-Bunker E et al.: CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells. Nat. Biotechnol. 24, 351–357 (2006).
66 Clark AT, Rodriguez RT, Bodnar MS et al.:
chromosome change, i(12p), in testicular tumours? Lancet 2, 1349 (1982).
47 Ludwig TE, Levenstein ME, Jones JM et al.:
Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006).
Describes genomic alterations in cultured hESCs including aberrations in copy number, mitochondrial DNA sequence and gene promoter methylation.
56 Atkin NB, Baker MC: Specific
46 Mitalipova MM, Rao RR, Hoyer DM et al.:
Preserving the genetic integrity of human embryonic stem cells. Nat. Biotechnol. 23, 19–20 (2005).
65
Genomic alterations in cultured human embryonic stem cells. Nat. Genet. 37, 1099–1103 (2005).
74
Guttenbach M, Koschorz B, Bernthaler U, Grimm T, Schmid M: Sex chromosome loss and aging: in situ hybridization studies on human interphase nuclei. Am. J. Hum. Genet. 57, 1143–1150 (1995).
75 Russell LM, Strike P, Browne CE, Jacobs PA:
X chromosome loss and ageing. Cytogenet. Genome Res. 116, 181–185 (2007). 76 Benko S, Fantes JA, Amiel J et al.: Highly
conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence. Nat. Genet. 41, 359–364 (2009). 77 Rithidech K, Bond VP, Cronkite EP,
Thompson MH, Bullis JE: Hypermutability of mouse chromosome 2 during the development of x-ray-induced murine myeloid leukemia. Proc. Natl Acad. Sci. USA 92, 1152–1156 (1995).
future science group
Human embryonic stem cells & genomic instability
78 Rithidech KN, Dunn JJ, Gordon CR,
Cronkite EP, Bond VP: Evidence for an uncommon microsatellite instability on mouse chromosomes 2 and 4 and its possible role in radiation leukemogenesis. Blood Cells Mol. Dis. 23, 99–109 (1997). 79 Tanner MM, Tirkkonen M, Kallioniemi A
et al.: Independent amplification and frequent co-amplification of three nonsyntenic regions on the long arm of chromosome 20 in human breast cancer. Cancer Res. 56, 3441–3445 (1996).
by an integrative genomic approach in cervical cancer: potential role in progression. Genes Chromosomes Cancer 47, 755–765 (2008). n
86 Kim R: Unknotting the roles of Bcl‑2 and
Bcl‑xL in cell death. Biochem. Biophys. Res. Commun. 333, 336–343 (2005). 87 Fisher CL, Berger J, Randazzo F, Brock HW:
A human homolog of Additional sex combs, ADDITIONAL SEX COMBS‑LIKE 1, maps to chromosome 20q11. Gene 306, 115–126 (2003).
80 Guan XY, Xu J, Anzick SL, Zhang H,
Trent JM, Meltzer PS: Hybrid selection of transcribed sequences from microdissected DNA: isolation of genes within amplified region at 20q11‑q13.2 in breast cancer. Cancer Res. 56, 3446–3450 (1996). 81
Tonon G, Wong KK, Maulik G et al.: High-resolution genomic profiles of human lung cancer. Proc. Natl Acad. Sci. USA 102, 9625–9630 (2005).
88 Richards M, Tan SP, Tan JH, Chan WK,
Bongso A: The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 22, 51–64 (2004). 89 Kroes RA, Dawson G, Moskal JR:
Focused microarray analysis of glyco-gene expression in human glioblastomas. J. Neurochem. 103(Suppl. 1), 14–24 (2007).
82 Koynova DK, Jordanova ES, Milev AD et al.:
Gene-specific fluorescence in situ hybridization analysis on tissue microarray to refine the region of chromosome 20q amplification in melanoma. Melanoma Res. 17, 37–41 (2007). 83 Midorikawa Y, Yamamoto S, Ishikawa S et al.:
Molecular karyotyping of human hepatocellular carcinoma using singlenucleotide polymorphism arrays. Oncogene 25, 5581–5590 (2006). 84 Hurst CD, Fiegler H, Carr P, Williams S,
Carter NP, Knowles MA: High-resolution analysis of genomic copy number alterations in bladder cancer by microarray-based comparative genomic hybridization. Oncogene 23, 2250–2263 (2004). 85 Scotto L, Narayan G, Nandula SV et al.:
Identification of copy number gain and overexpressed genes on chromosome arm 20q
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Demonstrates that the 20q acquisition occurs at an early stage in cervical cancer.
90 Cortes JL, Sanchez L, Ligero G et al.:
Mesenchymal stem cells facilitate the derivation of human embryonic stem cells from cryopreserved poor-quality embryos. Hum. Reprod. 24, 1844–1851 (2009). 91
Montes R, Ligero G, Sanchez L et al.: Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF‑b and IGF‑II. Cell Res. 19, 698–709 (2009).
92 Iwatani M, Ikegami K, Kremenska Y et al.:
Dimethyl sulfoxide has an impact on epigenetic profile in mouse embryoid body. Stem Cells 24, 2549–2556 (2006). 93 Diaferia GR, Dessi SS, Deblasio P, Biunno I:
Is stem cell chromosomes stability affected by cryopreservation conditions? Cytotechnology 58, 11–16 (2008).
www.futuremedicine.com
Review
94 Eroglu A, Russo MJ, Bieganski R et al.:
Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nat. Biotechnol. 18, 163–167 (2000). 95 Guo N, Puhlev I, Brown DR, Mansbridge J,
Levine F: Trehalose expression confers desiccation tolerance on human cells. Nat. Biotechnol. 18, 168–171 (2000). 96 Rosa A, Spagnoli FM, Brivanlou AH:
The miR‑430/427/302 family controls mesendodermal fate specification via species-specific target selection. Dev. Cell 16, 517–527 (2009). 97 Barroso-del Jesus A, Lucena-Aguilar G,
Menendez P: The miR‑302–367 cluster as a potential stemness regulator in ESCs. Cell Cycle 8, 394–398 (2009). 98 Barroso-del Jesus A, Romero-Lopez C,
Lucena-Aguilar G et al.: Embryonic stem cell-specific miR302–367 cluster: human gene structure and functional characterization of its core promoter. Mol. Cell. Biol. 28, 6609–6619 (2008). 99 Baylin SB, Herman JG: DNA
hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet. 16, 168–174 (2000). 100 Toyota M, Issa JP: CpG island methylator
phenotypes in aging and cancer. Semin Cancer Biol 9, 349–357 (1999). 101 Bibikova M, Chudin E, Wu B et al.:
Human embryonic stem cells have a unique epigenetic signature. Genome Res. 16, 1075–1083 (2006). 102 Bibikova M, Laurent LC, Ren B, Loring JF,
Fan JB: Unraveling epigenetic regulation in embryonic stem cells. Cell Stem Cell 2, 123–134 (2008).
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