Human Reproduction Update

Human Reproduction Update Advance Access originally published online on August 26, 2006
Human Reproduction Update 2007 13(2):103-120; doi:10.1093/humupd/dml041

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 13/2/103    most recent dml041v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (28) Request Permissions Google Scholar Articles by Allegrucci, C. Articles by Young, L.E. Search for Related Content PubMed PubMed Citation Articles by Allegrucci, C. Articles by Young, L.E. Social Bookmarking          What's this?

© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Differences between human embryonic stem cell lines

C. Allegrucci¹ and L.E. Young^1,2

¹ School of Human Development, University of Nottingham, Queens Medical Centre, Nottingham, UK

² To whom correspondence should be addressed at: School of Human Development, University of Nottingham, Queens Medical Centre, East Block, D Floor, Nottingham NG7 2UH, UK. E-mail: lorraine.young{at}nottingham.ac.uk

	Abstract

TOP Abstract Introduction The characterization of hESC... Gene expression signature of... Sources of inter-line variation Epigenetic stability Implications for therapy References

 
The promise of human embryonic stem cell (hESC) lines for treating injuries and degenerative diseases, for understanding early human development, for disease modelling and for drug discovery, has brought much excitement to scientific communities as well as to the public. Although all of the lines derived worldwide share the expression of characteristic pluripotency markers, many differences are emerging between lines that may be more associated with the wide range of culture conditions in current use than the inherent genetic variation of the embryos from which embryonic stem cells were derived. Thus, the validity of many comparisons between lines published thus far is difficult to interpret. This article reviews the evidence for differences between lines, focusing on studies of pluripotency marker molecules, transcriptional profiling, genetic stability and epigenetic stability, for which there is most evidence. Recognition and assessment of environmentally induced differences will be important to facilitate the development of culture systems that maximize stability in culture and provide lines with maximal potential for safety and success in the range of possible applications.

Key words: epigenetics / gene expression / human embryonic stem cells / inter-line variability / karyotype

	Introduction

TOP Abstract Introduction The characterization of hESC... Gene expression signature of... Sources of inter-line variation Epigenetic stability Implications for therapy References

 
To date, human embryonic stem cell (hESC) lines have been obtained from fresh and frozen embryos of varying quality at morula and blastocyst stages of development (Hoffman and Carpenter, 2005a). Embryonic stem cells possess the unique ability of self-renewal that confers indefinite maintenance of the undifferentiated state in vitro while retaining the capacity to generate derivatives of the three embryonic germ layers that precede the formation of all of the tissues of the developing fetus (Semb, 2005; Trounson, 2006). As hESCs can differentiate potentially into all the cell types that make up a human body, they promise exciting potential as a source of cells for regenerative medicine, as well as valuable tools for drug discovery and for understanding human development and disease.

Since the establishment of the first hESC lines (Thomson et al., 1998), some progress has been made in elucidating the biology of these cells and in optimizing conditions for their maintenance in culture. To use hESCs in cell-based therapies, it is necessary not only to develop methods for the production of adequate numbers of differentiated cells for transplantation but also to have enough cell lines to cover the vast spectrum of transplant antigens to avoid rejection (Taylor et al., 2005). These requirements have been the driving force for the derivation of numerous cell lines worldwide. It is also a major advantage for drug discovery applications and toxicology screening that each line represents a unique human genetic complement. The interaction of compounds under test with the variable alleles that individuals inherit often results in adverse drug reactions in a subset of patients in clinical trials (Ingelman-Sundberg and Rodriguez-Antona, 2005). These effects are not identified in preclinical animal tests or in cell-based screens that utilize human, transformed cell lines that were derived from only a few individuals. Thus, the provision of primary human cell types from a wide range of hESC lines that represent genetic diversity represents a unique opportunity.

It was proposed initially that a minimum of 40–50 homozygous hESC lines would be necessary to cover 50% of the HLA isotypes in the American population and thus to minimize the immune rejection of hESC-derived transplants (Faden et al., 2003). More recently, the number of hESC lines needed in a stem cell bank for HLA matching of the UK population has been calculated as 150 (Taylor et al., 2005), but this number may underestimate the lines necessary to match a very ethnically diverse population (Rao and Auerbach, 2006; Rao and Civin, 2006). Two hundred hESC lines were estimated to have been derived worldwide in 2004 (Brimble et al., 2004), and this number is increasing rapidly as more and more embryo donors and fertility clinics become interested in collaborating with scientists. The increasing profile of hESC on national political agendas has also led to more permissive legislation for allowing hESC derivation in some countries. Notably, however, several countries including Italy, Ireland, Norway and Germany still have a complete ban (Rao and Civin, 2006). Of the derived hESC lines, only a limited number are readily available for distribution. These include 22 cell lines of the total 78 listed in the US NIH registry for commercial sale (http://stemcells.nih.gov) and the 17 cell lines derived in Harvard University with funding from the Juvenile Diabetes Research Foundation (Cowan et al., 2004). The remaining lines reside largely within founding laboratories and have limited distribution between close collaborators, presumably due to the considerable resources required for cell expansion and characterization, commercial funding issues and difficulties over producing workable material transfer agreements. Although several national stem cell banks are being established, none are actively distributing hESC lines at the time of writing (March 2006).

Despite a widespread interest in defining the properties of hESCs, the beginnings of comprehensive characterization are available for only a subset of hESC lines (Thomson et al., 1998; Mitalipova et al., 2003; Amit et al., 2004; Brimble et al., 2004; Carpenter et al., 2004; Ginis et al., 2004; Heins et al., 2004; Rosler et al., 2004; Zeng et al., 2004; Xu et al., 2005). Notably, most of all publications pertaining to hESC focus on the first five lines derived in the University of Wisconsin, i.e. H1, H7, H9, H13 and H14 (Denning et al., 2006; Owen-Smith and McCormick, 2006). Although hESC lines are considered to be very similar in terms of self-renewal, expression of pluripotency markers and the ability to differentiate, it is becoming more and more evident that differences between lines also exist (Carpenter et al., 2003; Hoffman and Carpenter, 2005a). Differences in growth rate and genetic and epigenetic stability in long-term culture (Cowan et al., 2004; Hoffman and Carpenter, 2005b; Lee et al., 2005a) have been reported.

It is now been recognized by the scientific community that there is a need for much more comprehensive, parallel characterization of hESC lines than is currently being undertaken to assess their true potential. The safety and efficacy of lines for regenerative medicine applications is of prime concern. To this end, several initiatives are beginning to characterize multiple existing cell lines using standardized assay conditions to allow accurate comparison of the data generated. Such initiatives have been instigated by the International Stem Cell Forum (www.stemcellforum.org; Andrews et al., 2005), the NIH Stem Cell Unit (http://stemcells.nih.gov/research/nihresearch/scunit/) and the American Type Culture Collection (http://stemcells.atcc.org; Rao and Civin, 2005). However, the wide range of feeder cells, culture media, additives and passage methods used to derive lines still confounds the interpretation of inter-line differences and conclusions as to whether these are due to inherent genetic variation or environmental ‘programming’ of the cells (Allegrucci et al., 2005). No standard conditions that are optimal or generically applicable across lines have been established either for their culture or for their differentiation (Hoffman and Carpenter, 2005a). Characteristics described for cell lines cultured in certain conditions (and at a specific passage number) may only be applicable to those specific cells assayed within a specific laboratory. Although some success has been reported in standardizing conditions between independently-derived lines for maintenance and cardiomyocyte differentiation, media used to culture one line were often not transferable to others (Denning et al., 2006). Protocols published for some lines have also proved difficult to transfer to the lines cultured routinely within our laboratory (Figure 1).

View larger version (84K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Morphology of BG01 and HUES-7 human embryonic stem cell (hESC) lines cultured under the proprietary standard protocols (A, B) and published feeder- and conditioned medium-free conditions (C–H). BG01 (A) and HUES-7 (B) cultured under proprietary standard conditions on feeders (Mitalipova et al., 2003; Cowan et al., 2004). BG01 (C) and HUES-7 (D) cultured according to Sato et al. (2004). Cultures of both cell lines could not be maintained due to extensive cell death. BG01 (E) and HUES-7 (F) cultured according to Vallier et al. (2005). Cultures of both cell lines could not be maintained for more than 10 days under these conditions. BG01 (G) and HUES-7 (H) cultured according to Klimanskaya et al. (2005). BG01 cultures could be maintained for at least 10 passages, whereas the same culture conditions were not suitable for HUES-7 maintenance beyond the first passage. Bar, 100 µm. All brightfield images were taken using Hoffman objective lenses. Magnification 160x.

 

	The characterization of hESC lines: pluripotency assessment

TOP Abstract Introduction The characterization of hESC... Gene expression signature of... Sources of inter-line variation Epigenetic stability Implications for therapy References

 
Since the derivation of the first hESC lines, extensive effort has been devoted to compare hESCs with their mouse counterparts (Sato et al., 2003; Ginis et al., 2004; Wei et al., 2005). It is now well recognized that hESCs differ from murine embryonic stem cells (mESCs) in many biological aspects and that no direct extrapolation between the two systems is possible (Rao, 2004; Wobus and Boheler, 2005; Zhan et al., 2005). For this reason, extensive characterization of hESCs has been mandatory to understand the cellular requirements for long-term maintenance of hESC lines in vitro (Boyer et al., 2005; James et al., 2005; Vallier et al., 2005). Many studies have described comparisons between hESC lines derived in the same laboratory and cultured under the same conditions, with no study comparing more than four independently-derived lines (Thomson et al., 1998; Allegrucci et al., 2005). Fewer studies have investigated the characteristics of independently-derived and cultured lines (Abeyta et al., 2004; Amit et al., 2004; Bhattacharya et al., 2005), mainly because of the laborious and costly procedures of maintaining multiple cell lines with different culture requirements but also because of the complexity of interpreting data from cells cultured in different conditions. Despite variable inter-line culture conditions (Hoffman and Carpenter, 2005a), hESCs are considered to possess similar morphology, growth characteristics and expression of a range of ‘pluripotency markers’. Nevertheless, different population doubling rates have been reported for different hESC lines, ranging from 28 to 48 h (Amit et al., 2000; Reubinoff et al., 2000; Xu et al., 2001; Amit et al., 2003; Cowan et al., 2004; Zeng et al., 2004; Kim et al., 2005; Sjogren-Jansson et al., 2005). Expression of SSEA (SSEA-3, SSEA-4) and TRA antigens (TRA-1-60, TRA-1-81), OCT4 and NANOG, is common between the hESC lines and is considered a general criterion for hESC characterization and evaluation of pluripotency (Hoffman and Carpenter, 2005a). However, not all hESC lines are generally tested for the same panels of markers, and differences in relative abundance of pluripotency markers have been described, raising the questions of whether hESC cultures are heterogeneous in nature or whether more than one ‘state of pluripotency’ may exist. For example, significant difference in SSEA-4 expression has been observed between the H1, H7, H9 and H14 lines, such that two populations of SSEA-4-high- and SSEA-4-low-expressing cells can be identified. These populations correlate with the differential expression of additional hESC markers, including TRA-1-60, TR1-81, CD9 and CD133 (Carpenter et al., 2004). Other examples of variable marker expression include the differential expression of OCT4, NODAL and CTNNB1 (ß-Catenin) between Miz-hES1 and SNU-hES3 cell lines (Rho et al., 2006) and of OCT4, NANOG and GDF3 between HS181, HS235, HS237, FES21, FES22, FES29 and FES30 (Skottman et al., 2005a). The variable expression of pluripotency markers could result from the presence of early differentiated progenitors in culture because their down-regulation is gradual upon differentiation (Draper et al., 2002; Bhattacharya et al., 2005), raising the question of whether they can definitively characterize pluripotency. The physiological significance of expression of these markers is not clear, and it is likely that the limited panel of markers in current use may be insufficient to define the state of ‘stemness’ because many of them are not unique to embryonic stem cells. OCT4 is also expressed in germ cells (Niwa et al., 2000), and NANOG has been reported in adult differentiated tissues (Hart et al., 2004). Furthermore, the difficulty in designing RT–PCR primers that distinguish OCT4 and NANOG transcripts from related pseudogene transcripts (Pain et al., 2005) has not been accounted for in many hESC studies, confounding data interpretation.

Considering that no hESC line has been derived clonally from a blastocyst, it is reasonable to hypothesize that hESC cultures represent a heterogeneous population of cells with different characteristics and potential. Studies on the positional effects of the inner cell mass cells within the blastocyst on the phenotype/potential are lacking in any mammalian species. If such positional effects exist, it is not known whether these would likely be reversed by in vitro culture. Some cell lines show differences either in their kinetics of expression of differentiation markers (Reubinoff et al., 2000; Abeyta et al., 2004; Lee et al., 2005a) or in their ability to form specific lineages in vitro (Lee et al., 2005a). Differences in 40% of genes up-regulated in BG02 relative to a pool of H1, H7 and H9 hESC lines have also been described during differentiation into embryoid bodies (EBs) (Bhattacharya et al. 2005). Differentiation potential to any specific lineage in vitro is difficult to assay due to low typical efficiencies of forming most cell types and the inability to apply differentiation strategies across independently-derived lines cultured in different ways. The limited identification of molecular inducing factors and complex 3D tissue environment interactions mean that differentiation strategies are still largely in their infancy. The definitive test for hESC pluripotency is considered to be the ability to form derivatives of the three germ layers in vivo via the formation of teratomas in immunocompromised mice. The formation of teratomas has been reported for a subset of derived hESC lines (http://stemcells.nih.gov), sometimes with different outcomes (Heins et al., 2004). The type of differentiated cells obtained in teratomas is limited and variable and may not reflect the real capacity of hESCs to differentiate into all the cells of an organism, were they in the appropriate tissue environments (niche). To date, only keratinocytes, neurons, glia, cardiomyocytes, haematopoietic precursors, endothelial cells, osteogenic cells, hepatocyte-like cells, insulin-producing cells, adipocytes, trophoblast, germ cells, prostate tissue and melanocytes have been derived from hESCs (Allegrucci et al., 2005; Hoffman and Carpenter, 2005a; Hyslop et al., 2005; Fang et al., 2006; Taylor et al., 2006).

	Gene expression signature of hESCs

TOP Abstract Introduction The characterization of hESC... Gene expression signature of... Sources of inter-line variation Epigenetic stability Implications for therapy References

 
The gene expression profile of hESCs has been explored by several techniques, including serial analysis of gene expression (SAGE), expressed sequence tag (EST) enumeration, microarray analysis and massively parallel signature sequencing (MPSS; Table I). Most of these studies have been undertaken to unravel the key genes that characterize the status of ‘stemness’, regulating pluripotency and maintaining the undifferentiated state. For this reason, research has been focused on those genes that are commonly expressed and enriched in several hESC lines, whereas much less attention has been given to the differences in gene expression observed (Sato et al., 2003; Sperger et al., 2003; Bhattacharya et al., 2004; Brandenberger et al., 2004a; Ginis et al., 2004; Rao et al., 2004; Richards et al., 2004; Cai et al., 2006; Wei et al., 2005).

View this table: [in this window] [in a new window]   Table I. Gene expression differences between human embryonic stem cell (hESC) lines

 
To identify common genes between cell lines, a pool of the cell lines H1, H7, H9 was used in MPSS analysis. When the data generated were compared with other published datasets generated by microarrays and SAGE in different hESC cell lines, (Sato et al., 2003; Sperger et al., 2003; Abeyta et al., 2004; Ginis et al., 2004; Richards et al., 2004; Bhattacharya et al., 2005) similar results were observed with a concordance of 70% (Brandenberger et al., 2004a). However, when a similar approach was undertaken for the generation of ESTs from pooled H1-H7-H9 cell lines (Brandenberger et al., 2004b), only a very limited match was observed in the expression of genes previously described in microarrays as characteristic of ‘stemness’ (Ramalho-Santos et al., 2002) or defining a ‘stem cell signature’ (Ivanova et al., 2002). Studies that compared independently-derived and cultured hESCs showed variation in numbers of differentially expressed genes between cell lines, such as 25% between H1 and BG01 (Rao and Stice, 2004), 48% between HSF-1, HSF-6 and H9 (Abeyta et al., 2004), and 20% between HS181, HS235, HS237, FES21, FES22, FES29 and FES30 (Skottman et al., 2005a; Table I). Variability in gene expression was found in housekeeping genes as well as in several important ‘stem cell’ genes, including GP130, STAT3, FOXD3 and RUNX1 (Abeyta et al., 2004). Quantitative differences in gene expression between hESC lines also exist in a variety of genes, but no direct correlation with function has been made yet (Abeyta et al., 2004; Skottman et al., 2005a). At least some of these differences may have important biological consequences, as demonstrated by the different developmental outcome resulting from a <2-fold variation in the expression of Oct4 in mESCs (Niwa et al., 2000).

	Sources of inter-line variation

TOP Abstract Introduction The characterization of hESC... Gene expression signature of... Sources of inter-line variation Epigenetic stability Implications for therapy References

 
Culture-related effects

Several suggestions have been made to explain the observed variation in gene expression profiles between hESC lines. One possibility is that the variation between independent hESC lines may be the result of different culture conditions and, if so, it would be predicted that hESC lines derived and cultured in the same laboratory may be more similar. Indeed, greater similarities have been observed in four cell lines from Finland (FES21, FES22, FES29 and FES30) compared with three from Sweden (HS181, HS235 and HS237; Skottman et al. 2005a), in HSF1/HSF6 compared with H9 (Abeyta et al., 2004) and in BG01/BG01-MEDII compared with H1 (Rao et al., 2004).

Independently-derived and cultured hESCs generally require different feeder cell types and densities, culture substrates, culture media, growth factors/other additives and passage methods (Allegrucci et al., 2005; Goh et al., 2005). That the culture environment can have a profound effect on the molecular signature of hESCs is shown by the differential expression of 1417 (13.5%) genes between HS237 hESCs cultured in medium containing serum or KnockOut^TM Serum Replacement (Skottman et al., 2005b). Several lines have been exposed to multiple passaging methods during their early history in an attempt to improve their expansion before wide-scale distribution to researchers. Different culture conditions are also adopted frequently by investigators for a few passages to provide appropriate conditions for specific experiments. The long-term effects of these strategies need to be carefully investigated. Examples of such manipulations include the use of Matrigel^TM substrate or lower-density feeders to facilitate genetic modification, enzymatic passaging methods for obtaining a suspension of single cells for freezing or transfection and the use of serum substitutes to provide a more defined and reproducible culture system (Reubinoff et al., 2001; Xu et al., 2001; Vallier et al., 2005; Denning et al., 2006; Ludwig et al., 2006a).

Another important issue to consider when comparing different expression profiles between hESC lines is the passage number at which the assay is performed. Although hESCs maintain the pluripotent state long-term in culture (Amit et al., 2000; Amit et al., 2003; Brimble et al., 2004; Rosler et al., 2004; Lee et al., 2005a), some time-related changes have been observed. Differences in gene expression after continuous passaging, which are independent of aneuploidy, have been reported (Rosler et al., 2004). Many studies do not take passage number into account (and often do not even report the passage number assayed), making the interpretation of inter-line comparisons within a study, as well as comparison between different studies, very difficult.

The presence of contaminating feeder cells typical in hESC culture may represent a further confounding factor in analysis of some parameters. There is also the aforementioned possibility that hESC cultures may not represent a homogeneous population of undifferentiated cells. These problems can be minimized by fluorescence-activated cell sorting (FACS) cells before analysis, as shown by the significant difference in gene expression observed between sorted cells highly positive for the pluripotency marker, SSEA-3, compared with the other fraction of cells that retain multilineage differentiation potential in the same culture but are SSEA-3 negative (Enver et al., 2005). Whether FACS with another ‘pluripotency’ marker, such as TRA-1-60 or TRA-1-81, would yield similar microarray profiles remains to be examined.

Genetic variation

Although more similarity in cell lines derived and cultured in the same laboratory has been observed, even with identical derivation conditions, hESC lines differ to some extent (Cowan et al., 2004; Zeng et al., 2004; Skottman et al., 2005a). SAGE analysis of HES-3 and HES-4 revealed a 27% difference in expressed genes, including the pluripotency-associated REX-1 and GAL (Richards et al., 2004). Similarly, microarray analysis of the cell lines, BG01 and BG02, demonstrated only 36% of genes expressed in common, and of these, only 25% were found expressed in other hESC lines, including TE05, GE01, GE09 and the pooled H1-H7-H9 Wisconsin lines (Zeng et al., 2004). Because differences between lines have been detected despite the same culture conditions, it has been suggested that this could be due to the unique genetic identity of the embryos from which the hESCs were derived (Abeyta et al., 2004; Cai et al., 2006; Wei et al., 2005). Although hESC lines exhibit a distinctive genotype (Brimble et al., 2004; Carpenter et al., 2004; Kim et al., 2005; Lee et al., 2005b; Oh et al., 2005), it is unlikely that such a degree of difference could only be due to genetic variation as <2% variation in gene expression has been found in adult human tissues of different individuals (Hsiao et al., 2001). That hESC lines arise from different quality embryos, which were also cultured to the blastocyst stage in a variety of different media and were donated from patients of undefined infertility, may also contribute to varying degrees (Hoffman and Carpenter, 2005a).

Strategies to obviate comparison between cell lines include the adaptation of cells previously cultured under determined conditions to new ones for few passages before analysis (Skottman et al., 2005a). This approach does not discount epigenetically inherited modifications from previous culture history when differences are observed. Because universal protocols for hESC cultures are not available, it is at present difficult to discriminate the relative contribution of genetic, procedural and/or epigenetic variation to the gene expression signature of each hESC line.

The observed differences cannot be related solely to the gender of lines, as demonstrated by the observation that lines of the same sex are not more similar (Abeyta et al., 2004) and that the expression differences between hESC lines do not cluster on any particular chromosome (Brandenberger et al., 2004a).

Experimental variation

Because of the effect of the microenvironment on hESCs, it is possible that variation in results may be encountered when the same experiment is repeated using a different dish of cells or a different batch post thaw.

However, although many of the studies are carried out with simultaneous replicates (i.e. technical repeats), there are no data on experiments repeated over time or from a different vial of thawed cells. This is likely due to the time scale involved in generating samples for analysis and the cost implications (Aldhous, 2006).

Variance in transcriptome profiles between studies may also be due to methodological variations. The validity of the first published bioinformatic comparisons between transcription profiling reports, undertaken in a search for common, ‘stemness genes’, has been questioned on the basis of differences in outcomes in different studies (Ivanova et al., 2002; Ramalho-Santos et al., 2002; Fortunel et al., 2003). Reproducibility of microarray experiments has been an object of discussion, and this could be due to difference in platforms (Kothapalli et al., 2002), differential gene annotation (Mecham et al., 2004) and variability due to biological material or operational procedures (Yauk et al., 2004). Different platforms for gene expression determination contain array of genes that do not represent the whole genome and may vary between different studies. In addition, variation may also be the result of using the same platform but different ‘control’ samples as reference. The use of a ‘common reference pool’ of relevant lines or cultures may be an appropriate method to limit variability across studies (Beqqali et al., 2006). Computational algorithms and statistical tests applied in microarray experiments are a limiting factor for cross comparisons between different studies, and sometimes the use of raw data may result in a more productive approach (Suarez-Farinas et al., 2005). Another source of variability may arise from the degree of difference in gene expression set as ‘threshold’ in different studies. Many of the experiments consider differential expression when the measurement is 2-fold or higher than the reference sample, but some studies use a higher threshold (Table I).

When considering all these genetic, environmental and methodological factors, directly comparing hESC lines becomes challenging. However, it must be recognized that all the gene expression studies performed so far have been very informative and valuable for the identification of those ‘master’ genes that are highly expressed in hESCs and that are likely to determine hESC function. The future development of standardized conditions for hESC culture and analysis together with stringent and uniform statistical data validation will allow better understanding of the unique ESC characteristics common across different cell lines.

Genetic stability

The preservation of genomic integrity in culture may be a major constraint for stem cell function in vivo and in vitro. Long-term maintenance of stem cells in an environment potentially suboptimal compared with the in vivo situation may lead to the accumulation of genetic defects. Even in optimal culture conditions, the fact that, in vivo, the inner cell mass exists in a pluripotent state for only a few days before forming the tissues of the fetus and placenta may render adaptive genetic changes (Draper et al., 2004), an inevitable consequence of long-term culture. This aspect is of particular relevance if stem cell derivatives are going to be used in cell therapies as the relationship between genomic instability and carcinogenesis is well established (Allegrucci et al., 2004; Rajagopalan and Lengauer, 2004; Storchova and Pellman, 2004).

hESC lines acquire abnormalities in specific chromosomes with extended time in culture, and higher incidence has been reported for aneuploidy of chromosomes 12, 17 and X (Table II; Brimble et al. 2004; Cowan et al., 2004; Draper et al., 2004; Inzunza et al., 2004; Hanson and Caisander, 2005; Maitra et al., 2005; Mitalipova et al., 2005). A propensity for the clonal selection of trisomy 12 was reported in HUES (Cowan et al., 2004) and H1 (Lakshmipathy et al., 2004) lines, whereas trisomy 17 was also observed (Brimble et al., 2004; Mitalipova et al., 2005) in BG01 and BG02. Preferential gains in 12 or 17 in H14 have also varied between laboratories (Liu et al., 1997), while Rosler et al. (2004) reported trisomy 20 as the prevalent aneuploidy in H1, H7 and H9. Trisomy of chromosomes 13 and 3 was only observed in SA002 (Heins et al., 2004; Caisander et al., 2006) and in Miz-hES13 (Kim et al., 2005), respectively.

View this table: [in this window] [in a new window]   Table II. Karyotype reported for human embryonic stem cell (hESC) lines

 
Chromosomal abnormalities are not a general trend in every hESC culture (Buzzard et al., 2004; Darnfors et al., 2005; Mitalipova et al., 2005), and it is not clear whether certain cell lines are intrinsically prone to developing abnormalities or whether their instability is a consequence of certain culture methods. It has been proposed that passage of hESCs by mechanical dissection preserves genomic integrity better than enzymatic methods (Brimble et al., 2004; Buzzard et al., 2004; Mitalipova et al., 2005). This suggestion seems plausible as hESCs express high levels of gap junctions and cell adhesion molecules indicating that cell-to-cell contacts are essential for their function (Xu et al., 2001; Wong et al., 2004; Zeng et al., 2004; Caisander et al., 2006; Huettner et al., in press). Mechanical passage of hESC colonies allows preservation of these connections, given that only a portion of the colony is dissected and re-plated for successive culture with this method. On the contrary, enzymatic dissociation causes destruction of cell contacts to varying extents (e.g. trypsin more than collagenase) and imposes a stress pressure on the isolated hESCs in culture. However, sporadic aneuploidies can also occur in mechanically passaged cells (C. Allegrucci, unpublished data; Buzzard et al., 2004; Caisander et al., 2006), and this may be related to the selection of aberrant, faster-growing cells after passage and/or freezing/ thawing—a phenomenon described as ‘adaptation to culture’ (Draper et al., 2004). During passage by manual dissection, larger colonies (with no visual evidence of differentiation) are generally selected for passage subjectively by the operator, even though there is evidence from mESCs that faster growing colonies are more likely to accumulate genetic alterations and do not give any germline contribution to chimeras (Liu et al., 1997). A chromosome decatenation checkpoint deficiency has been recently discovered in mESCs that could predispose to aneuploidy in conditions of selective pressure such as suboptimal, in vitro culture (Damelin et al., 2005; Damelin and Bestor, 2006). The variable expression of the DNMT3B enzyme observed in hESC lines (Sperger et al., 2003; Bhattacharya et al., 2004; Brandenberger et al., 2004a; Rao et al., 2004; Richards et al., 2004; Skottman et al., 2005a) may also contribute to chromosomal instability due to alteration in genome-wide methylation (Chen et al., 1998; Dodge et al., 2005; Zvetkova et al., 2005). In terms of cryopreservation, vitrification appears to enhance post-thaw survival relative to conventional freezing techniques, but whether it is more likely to induce clonal selection of cell-harbouring defects that enhance survival in culture remains to be investigated. The relative importance of the passage method in the occurrence of chromosomal abnormalities in certain cell lines is not clear. hESCs are derived from supernumerary embryos generated by assisted reproductive technologies (ARTs), and they may be mosaic for aneuploidies from the time of derivation (Hardarson et al., 2003). The derivation of triploid Royan H3, Royan H4 and FC018 cell lines may be the result of such chromosomal mosaicism (Heins et al., 2004; Baharvand et al., 2006).

However, perhaps the major limitation to interpreting ploidy data is the lack of experimental replication. Most published karyotypes refer to only a single culture (Table II), often concluding effects of culture treatment on this limited data. In addition, no technical standards have been fixed for hESC karyotype assessment, and different methods are currently used in different laboratories. The conventional analysis is based on banding of metaphase spreads. With this technique, different conclusions may be reached by analysing variable and statistically insufficient numbers of spreads (Table II) when the aneuploidy does not represent the entire cell population—a typical scenario in hESC cultures (Brimble et al., 2004; Rosler et al., 2004; Mitalipova et al., 2005). According to Hook (1977), examination of 30 cells means that mosaicism at the level of 10% can be ruled out with 95% confidence interval (CI). In addition, G banding, although reliable when performed by an appropriately trained operator, does not permit high resolution. Superior methods, such as fluorescent in-situ hybridization (FISH) and comparative genome hybridization (CGH), have recently been implemented in hESC chromosomal analysis (Amit et al., 2000; Pickering et al., 2003; Heins et al., 2004; Inzunza et al., 2004; Mitalipova et al., 2005; Sjogren-Jansson et al., 2005; Caisander et al., 2006; Ludwig et al., 2006b), and only a combination of different techniques can guarantee ultimately good coverage of all possible genetic abnormalities (Pera, 2004; Speicher and Carter, 2005). For instance, the predominant mechanism of mutation in mESCs is loss and reduplication of chromosomes leading to uniparental disomy (UPD) (Cervantes et al., 2002), and this abnormality is generally not detected by standard karyotyping but can be identified by single-nucleotide polymorphism (SNP) arrays (Speicher and Carter, 2005).

The suggestion that genetic instability in hESCs may arise from the selection of cells with a growth advantage in culture (Draper et al., 2004) is supported by the observed high incidence of trisomies of chromosomes 12 and 17. The presence of isochromosome 12p is also a common feature of human teratocarcinoma cells (hECs), the malignant counterpart of hESCs (Skotheim et al., 2002; Clark et al., 2004; Draper et al., 2004), and the amplification of 17q is associated with neuroblastoma (Westermann and Schwab, 2002). Genes that control self-renewal differentiation and apoptosis, including STELLAR, NANOG, GDF3, STAT3, GRB2, BIRC5, NT3 and p75^NGFR, are located on those chromosomes (Burdon et al., 2002; Chiou et al., 2003; Clark et al., 2004; Pyle et al., 2006), and in vitro constraint may provide the pressure to increase gene copy number. In fact, aneuploid hESC cultures grow faster (Cowan et al., 2004; Enver et al., 2005; Plaia et al., 2005; Herszfeld et al., 2006; Xiao et al., 2006) and are highly clonogenic. Aneuploid cultures also express higher levels of the hESC markers, SSEA-3 and SSEA-4 (Enver et al., 2005; Xiao et al., 2006), and pluripotency genes, OCT4 and NANOG. They also up-regulate self-renewal signalling pathways involving Nodal/Activin, Wnt, FGF and Hedgehog and down-regulate differentiation pathways involving BMP and Notch (Enver et al., 2005; Mitalipova et al., 2005; Xiao et al., 2006). The expression of a truncated form of CD30 is also a feature of karyotypically abnormal hESCs and hECs, and it is associated with the inhibition of apoptosis and increased cell survival (Herszfeld et al., 2006).

Despite these imbalances, aneuploid hESCs seem to maintain the ability to differentiate in vitro (Brimble et al., 2004; Draper et al., 2004) and in vivo (Xiao et al., 2006). However, the presence and isolation of undifferentiated cells from a teratoma generated by the injection of the aneuploid BG01V and HUES-3 cell lines may indicate the formation of an undifferentiated teteratocarcinoma-like tumour and not the completely differentiated benign teratoma expected of euploid hESCs (Plaia et al., 2005; Herszfeld et al., 2006).

A recent, extensive, study demonstrated that 5 of 10 hESC lines analysed presented ploidy alterations (including deletions and amplifications) during prolonged time in culture (Maitra et al., 2005). Of note is the observed amplification of the proto-oncogene, MYC, that is likely to be associated with a selective growth advantage for hESCs, because this transcription factor affects cell proliferation via cell cycle progression (Secombe et al., 2004). Overexpression of MYC has also been observed in the spontaneous transformation of murine and human adult mesenchymal stem cells in vitro (Miura et al., 2005; Rubio et al., 2005). Mitochondrial DNA mutations have also been observed in 2/10 cell lines in the Maitra et al. (2005) study, and it is of interest that these are a common feature of ageing and cancer (Singh, 2004; Czarnecka et al., 2006). Because the alteration in the balance between self-renewal/proliferation and spontaneous differentiation is considered a hallmark of cancerous transformation (Hanahan and Weinberg, 2000; Reya et al., 2001; Pardal et al., 2003; Gudjonsson and Magnusson, 2005; Pathak and Multani, 2006), careful evaluation of the phenomena reviewed above pertaining to hESC ‘adaptation to culture’ must be addressed to ensure safety and efficacy in downstream applications.

	Epigenetic stability

TOP Abstract Introduction The characterization of hESC... Gene expression signature of... Sources of inter-line variation Epigenetic stability Implications for therapy References

 
Although the genetic stability of hESCs has received general attention, little is known about their epigenome. The complex range of epigenetic modifications to DNA and associated histones determine whether and when a particular gene is expressed (Jenuwein and Allis, 2001; Jaenisch and Bird, 2003; Fuks, 2005). Epigenetic changes during culture may have profound implications for the use of hESCs in regenerative medicine by affecting differentiation capacity and tumorigenic potential (Allegrucci et al., 2004).

DNA methylation is a heritable epigenetic modification that contributes to gene silencing, and many cell types accumulate methylation defects during in vitro culture (Antequera et al., 1990; Zhu et al., 1999; Hannula et al., 2001; Smiraglia et al., 2001; Paz et al., 2003). DNA methylation defects can affect different components of the genome, including CpG islands in promoter regions, repetitive sequences and imprinted genes (Robertson, 2005). Methylation alterations in imprinted genes can be induced by cellular stress (Pantoja et al., 2005) and specific culture conditions (Allegrucci et al., 2004). Disrupted imprinting was found in cultures of mESCs (Dean et al., 1998; Humpherys et al., 2001) and monkey ESCs (Fujimoto et al., 2005), but only one line of four examined showed evidence of imprinting instability in undifferentiated hESCs, and then only at high passage number (Rugg-Gunn et al., 2005; Sun et al., 2006). Similarly, no significant sign of imprinting disruption has been found in differentiated human embryonic germ cells (hEGCs; Onyango et al., 2002). Monoallelic expression of the imprinted genes H19, KCNQ1, PEG10 and NDNL1 has been observed in SHhES1 and HUES-7 cell lines at both early and late passage (Sun et al., 2006). Similarly, the aneuploid BG01V cell line shows the expected monoallelic methylation of SNRPN, H19 and DLK1/MEG3 (Plaia et al., 2005). Expected imprinting status of KCNQ1, IGF2, SCL22A18, NESP55 and SNRPN has also been shown in early and late passage H9, H7, HUES-3 and HSF6 hESC lines (Rugg-Gunn et al., 2005). Because the disruption of imprinted genes is associated with disease and carcinogenesis (Lucifero et al., 2004; Holm et al., 2005), more extensive studies examining a larger panel of imprinted genes in additional hESC lines would be still prudent to conclude the widespread stability of genomic imprinting in these cells.

DNA methylation alteration at non-imprinted genomic loci can induce genomic instability and is associated with cancer (Gaudet et al., 2003; Herman and Baylin, 2003; Baylin, 2005; Esteller, 2005). Both DNA hypomethylation (activating oncogenes) and hypermethylation (inactivating tumour suppressors) at diverse genomic sequences are found in tumours, often associated with altered expression of the DNA methyltransferase enzymes (Jones and Baylin, 2002). A high incidence hypermethylation of CpG islands in the promoter region of the tumour suppressor genes, RASSF1 and/or PTPN6, has been associated with long-term culture of the BG01, BG02, BG03, HUES-2, HUES-3, H7, H9, SA001 and SA002 cell lines. Methylation of TNFRSF10C was observed but only in HUES-2 and SA002 and not in the other seven lines examined (Maitra et al., 2005). Differential methylation in 5'-flanking regions of pluripotency-associated genes DPPA3 and DPPA5 between ESM01, ESM02 and ESM03 hESC lines upon differentiation is also indicative of variation in the epigenetic status of hESCs (Lagarkova et al., 2006).

The de-novo DNA methyltransferase, DNMT3B, plays a fundamental role in the methylation of CpG islands in mESCs (Hattori et al., 2004). High expression of this enzyme has been demonstrated in several hESC lines, although expression levels between lines are variable (Sperger et al., 2003; Bhattacharya et al., 2004; Brandenberger et al., 2004a; Rao et al., 2004; Richards et al., 2004; Skottman et al., 2005a). Expression of DNMT3B varies over 2-fold between undifferentiated hESC lines (Rao and Stice, 2004; Skottman et al., 2005a), and its expression can be influenced by the presence of serum in the culture medium (Skottman et al., 2005b). DNMT3B is generally down-regulated in hESCs during differentiation (Richards et al., 2004; Bhattacharya et al., 2005; Enver et al., 2005), but this is not observed when hESC lines are cultured in feeder-free conditions compared with cultures on mouse embryonic fibroblast feeders (Noaksson et al., 2005). It is possible that variations in the level of this key enzyme could account for the epigenetic diversity of hESCs as de-novo methylation of target sequences may be regulated by an instructive mechanism depending on the levels on the DNA methyltransferase enzymes (Keshet et al., 2006). However, because the family of DNA methyltransferases is known to co-operate to methylate DNA (Kim et al., 2002), studies of other methyltransferases are also likely to be informative. Some nutrients that regulate the availability of methyl group donors for cellular methylation reactions are also present in high and variable levels in hESC culture media, and in the range of media used for human embryo culture (Steele et al., 2005). Interaction between methyl metabolism and methyltransferases provides another potential route to altering the epigenetic status of cultured hESC over time (Allegrucci et al., 2005).

Epigenetic variation between hESCs may also perturb X chromosome inactivation. In order that female embryos express similar levels of X-linked genes to males, epigenetic regulation of the X chromosome in mammalian females constitutes an important mechanism for gene dosage compensation. Inactivation of one of the two female chromosomes is achieved via expression of the non-coding XIST mRNA, by histone modifications and by DNA methylation (Chang et al., 2006). Discordant data report variable failures of X chromosome inactivation in different hESC lines. Hoffman et al. (2005) observed the expected XIST expression indicative of X-inactivation in both undifferentiated and differentiated cells of H9 and CyT25, but not H7. The cell lines, H7 and H13, also expressed XIST in the undifferentiated state, whereas H9 did not (Sperger et al., 2003). Variations have also been reported within a line; undifferentiated H9 cells in an independent study exhibited only active X chromosomes, although X-inactivation was established after differentiation (Dhara and Benvenisty, 2004). Furthermore, undifferentiated and euploid H7 cells demonstrated XIST expression that was lost in high passage, aneuploid cells from the same line even after their differentiation (Enver et al., 2005). Failure to silence one of the X chromosomes is also observed frequently in female mESC lines (Sado et al., 1996). As a consequence, complete loss of one of the two active X chromosomes is observed together with global hypomethylation of the genome (Zvetkova et al., 2005). For this reason, establishment of stable male ESC lines may be more successful in the mouse. In contrast, the frequency of derivation and maintenance of female and male hESCs is almost equivalent (http://stemcells.nih.gov), and it is not clear why differences in X chromosome inactivation between hESC lines are observed, nor is the functional significance defined.

One possibility is that these variations may depend on the status of X-inactivation at the blastocyst stage at the time of hESC derivation (Enver et al., 2005), because X-inactivation is thought to occur in the human embryo around this time (Reik and Lewis, 2005). However, this would not account for the disruption of X chromosome inactivation observed in the H7 cell line after prolonged culture, and it is likely instead that the occurrence is a consequence of in vitro culture conditions. This hypothesis agrees with the observation that the same hESC line can behave differently in different independent experiments where varying culture conditions are used, like for instance H7 and H9 hESCs cultured on feeders cells or feeder-free conditions (Dhara and Benvenisty, 2004; Enver et al., 2005; Hoffman et al., 2005). However, until the degree of replication is increased in more hESC experiments, the possibility of variations even between culture dishes within an experiment cannot be ruled out.

	Implications for therapy

TOP Abstract Introduction The characterization of hESC... Gene expression signature of... Sources of inter-line variation Epigenetic stability Implications for therapy References

 
Collectively, all of the observations reviewed above regarding differences between hESC lines point to a degree of additive inherited variation between the lines that depends on the environment(s) to which the cells have been exposed since the time of embryo culture and derivation (Figure 2). Only by applying genome-wide analyses of hESC epigenotype, genotype, transcriptome, proteome and metabolome across many cell lines derived and cultured in a range of comparable means can future studies resolve the impact of environmentally induced effects. With the current cost, material transfer agreement and intensive manual labour limitations of current hESC culture, this type of comprehensive analysis is only likely to occur through large-scale, collaborative efforts and with the development of automated culture systems. Without high-throughput, multi-line hESC culture, many of the developments currently being made by individual laboratories on their own lines are unlikely to be generically applicable—a pitiful waste of our scientific and financial resources and a strategy that can only lengthen developmental timelines for transferring hESC-based therapies to the clinic.

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. Possible sources of environmental influence and potential effects on human embryonic stem cells (hESCs) upon long-term culture.

 
In this context, it is important to consider that an optimal protocol for obtaining cells that will be suitable for producing a particular therapeutic cell type or cell lines that represent key alleles for drug screening or immune haplotype may not be ideal for all applications. For basic science applications of these cells, such as studying early human development and modelling disease, lines that resemble as closely as possible the embryo in vivo are the ideal goal (Rossant, 2001; Smith, 2001; Zwaka and Thomson, 2005). Considering that no available line has been made to the good manufacturing practice (GMP) and clinical grade standards required for transplantation (Rodriguez et al., 2006), the effort required to ensure that new lines are derived in conditions optimal for applications, in addition to being GMP-compliant, must surely be a high international priority.

	Acknowledgements

TOP Abstract Introduction The characterization of hESC... Gene expression signature of... Sources of inter-line variation Epigenetic stability Implications for therapy References

 
The authors thank Chris Denning for helpful discussion. This work was funded by BBSRC (grant number BBS/B/06164) and the University of Nottingham.

	References

TOP Abstract Introduction The characterization of hESC... Gene expression signature of... Sources of inter-line variation Epigenetic stability Implications for therapy References

 

Abeyta MJ, Clark AT, Rodriguez RT, Bodnar MS, Pera RA and Firpo MT (2004) Unique gene expression signatures of independently-derived human embryonic stem cell lines. Hum Mol Genet 13,601–608.[Abstract/Free Full Text]

Aldhous P (2006) Stem-cells: Miracle postponed? New Scientist 2542,42–46.

Allegrucci C, Denning C, Priddle H and Young L (2004) Stem-cell consequences of embryo epigenetic defects. Lancet 364,206–208.[CrossRef][Web of Science][Medline]

Allegrucci C, Denning CN, Burridge P, Steele W, Sinclair KD and Young LE (2005) Human embryonic stem cells as a model for nutritional programming: an evaluation. Reprod Toxicol 20,353–367.[CrossRef][Web of Science][Medline]

Amit M and Itskovitz-Eldor J (2002) Derivation and spontaneous differentiation of human embryonic stem cells. J Anat 200,225–232.[CrossRef][Web of Science][Medline]

Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J and Thomson JA (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 227,271–278.[CrossRef][Web of Science][Medline]

Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R and Itskovitz-Eldor J (2003) Human feeder layers for human embryonic stem cells. Biol Reprod 68,2150–2156.[Abstract/Free Full Text]

Amit M, Shariki C, Margulets V and Itskovitz-Eldor J (2004) Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 70,837–845.[Abstract/Free Full Text]

Andrews PW, Benvenisty N, McKay R, Pera MF, Rossant J, Semb H and Stacey GN (2005) The international stem cell Initiative: toward benchmarks for human embryonic stem cell research. Nat Biotechnol 23,795–797.[CrossRef][Web of Science][Medline]

Antequera F, Boyes J and Bird A (1990) High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 62,503–514.[CrossRef][Web of Science][Medline]

Baharvand H, Ashtiani SK, Valojerdi MR, Shahverdi A, Taee A and Sabour D (2004) Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst. Differentiation 72,224–229.[CrossRef][Web of Science][Medline]

Baharvand H, Ashtiani SK, Taee A, Massumi M, Valojerdi MR, Yazdi PE, Moradi SZ and Farrokhi A (2006) Generation of new human embryonic stem cell lines with diploid and triploid karyotypes. Dev Growth Differ 48,117–128.[CrossRef][Web of Science][Medline]

Baylin SB (2005) DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol 2,S4–S11.

Beqqali A, Kloots J, Ward-van Oostwaard D, Mummery C and Passier R (2006) Genome-wide transcriptional profiling of human embryonic stem cells differentiating to cardiomyocytes. Stem Cells 24,1956–1967.[CrossRef][Medline]

Bhattacharya B, Miura T, Brandenberger R, Mejido J, Luo Y, Yang AX, Joshi BH, Ginis I, Thies RS, Amit M et al. (2004) Gene expression in human embryonic stem cell lines: unique molecular signature. Blood 103,2956–2964.[Abstract/Free Full Text]

Bhattacharya B, Cai J, Luo Y, Miura T, Mejido J, Brimble SN, Zeng X, Schulz TC, Rao MS and Puri RK (2005) Comparison of the gene expression profile of undifferentiated human embryonic stem cell lines and differentiating embryoid bodies. BMC Dev Biol 5,22.

Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG et al. (2005) Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122,947–956.[CrossRef][Web of Science][Medline]

Brandenberger R, Khrebtukova I, Thies RS, Miura T, Jingli C, Puri R, Vasicek T, Lebkowski J and Rao M (2004a) MPSS profiling of human embryonic stem cells. BMC Dev Biol 4,10.[CrossRef][Medline]

Brandenberger R, Wei H, Zhang S, Lei S, Murage J, Fisk GJ, Li Y, Xu C, Fang R, Guegler K et al. (2004b) Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat Biotechnol 22,707–716.[CrossRef][Web of Science][Medline]

Brimble SN, Zeng X, Weiler DA, Luo Y, Liu Y, Lyons IG, Freed WJ, Robins AJ, Rao MS and Schulz TC (2004) 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.[CrossRef][Web of Science][Medline]

Burdon T, Smith A and Savatier P (2002) Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol 12,432–438.[CrossRef][Web of Science][Medline]

Buzzard JJ, Gough NM, Crook JM and Colman A (2004) Karyotype of human ES cells during extended culture. Nat Biotechnol 22,381–382; author reply 382.[CrossRef][Web of Science][Medline]

Cai J, Chen J, Liu Y, Miura T, Luo Y, Loring JF, Freed WJ, Rao MS and Zeng X (2005) Assessing self-renewal and differentiation in hESC lines. Stem Cells 24,516–530.

Caisander G, Park H, Frej K, Lindqvist J, Bergh C, Lundin K and Hanson C (2006) Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture. Chromosome Res 14,131–137.[CrossRef][Web of Science][Medline]

Carpenter MK, Rosler E and Rao MS (2003) Characterization and differentiation of human embryonic stem cells. Cloning Stem Cells 5,79–88.[CrossRef][Web of Science][Medline]

Carpenter MK, Rosler ES, Fisk GJ, Brandenberger R, Ares X, Miura T, Lucero M and Rao MS (2004) Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 229,243–258.[CrossRef][Web of Science][Medline]

Cervantes RB, Stringer JR, Shao C, Tischfield JA and Stambrook PJ (2002) Embryonic stem cells and somatic cells differ in mutation frequency and type. Proc Natl Acad Sci USA 99,3586–3590.[Abstract/Free Full Text]

Chang SC, Tucker T, Thorogood NP and Brown CJ (2006) Mechanisms of X-chromosome inactivation. Front Biosci 11,852–866.[Web of Science][Medline]

Chen RZ, Pettersson U, Beard C, Jackson-Grusby L and Jaenisch R (1998) DNA hypomethylation leads to elevated mutation rates. Nature 395,89–93.[CrossRef][Medline]

Chiou SK, Jones MK and Tarnawski AS (2003) Survivin – an anti-apoptosis protein: its biological roles and implications for cancer and beyond. Med Sci Monit 9,PI25–29.[Medline]

Clark AT, Rodriguez RT, Bodnar MS, Abeyta MJ, Cedars MI, Turek PJ, Firpo MT and Reijo Pera RA (2004) 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.[CrossRef][Web of Science][Medline]

Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, Wang S, Morton CC, McMahon AP, Powers D et al. (2004) Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 350,1353–1356.[Free Full Text]

Czarnecka AM, Golik P and Bartnik E (2006) Mitochondrial DNA mutations in human neoplasia. J Appl Genet 47,67–78.[Web of Science][Medline]

Damelin M and Bestor TH (2006) Decatenation checkpoint deficiency destabilizes the stem cell genome. Cell Cycle 5,345–346.[Web of Science][Medline]

Damelin M, Sun YE, Sodja VB and Bestor TH (2005) Decatenation checkpoint deficiency in stem and progenitor cells. Cancer Cell 8,479–484.[CrossRef][Web of Science][Medline]

Darnfors C, Flodin A, Andersson K, Caisander G, Lindqvist J, Hyllner J, Wahlstrom J and Sartipy P (2005) High-resolution analysis of the subtelomeric regions of human embryonic stem cells. Stem Cells 23,483–488.[CrossRef][Web of Science][Medline]

Dean W, Bowden L, Aitchison A, Klose J, Moore T, Meneses JJ, Reik W and Feil R (1998) Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 125,2273–2282.[Abstract]

Denning C, Allegrucci C, Priddle H, Barbadillo-Munoz MD, Anderson D, Self T, Smith NM, Parkin CT and Young LE (2006) Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7. Int J Dev Biol 50,27–37.[CrossRef][Web of Science][Medline]

Dhara SK and Benvenisty N (2004) Gene trap as a tool for genome annotation and analysis of X chromosome inactivation in human embryonic stem cells. Nucleic Acids Res 32,3995–4002.[Abstract/Free Full Text]

Dodge JE, Okano M, Dick F, Tsujimoto N, Chen T, Wang S, Ueda Y, Dyson N and Li E (2005) Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem 280,17986–17991.[Abstract/Free Full Text]

Draper JS, Pigott C, Thomson JA and Andrews PW (2002) Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat 200,249–258.[CrossRef][Web of Science][Medline]

Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson J, Meisner L, Zwaka TP, Thomson JA and Andrews PW (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 22,53–54.[CrossRef][Web of Science][Medline]

Dvash T, Mayshar Y, Darr H, McElhaney M, Barker D, Yanuka O, Kotkow KJ, Rubin LL, Benvenisty N and Eiges R (2004) Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies. Hum Reprod 19,2875–2883.[Abstract/Free Full Text]

Enver T, Soneji S, Joshi C, Brown J, Iborra F, Orntoft T, Thykjaer T, Maltby E, Smith K, Dawud RA et al. (2005) Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells. Hum Mol Genet 14,3129–3140.[Abstract/Free Full Text]

Esteller M (2005) Aberrant DNA methylation as a cancer-inducing mechanism. Annu Rev Pharmacol Toxicol 45,629–656.

Faden RR, Dawson L, Bateman-House AS, Agnew DM, Bok H, Brock DW, Chakravarti A, Gao XJ, Greene M, Hansen JA et al. (2003) Public stem cell banks: considerations of justice in stem cell research and therapy. Hastings Cent Rep 33,13–27.[Web of Science][Medline]

Fang D, Leishear K, Nguyen TK, Finko R, Cai K, Fukunaga M, Li L, Brafford PA, Kulp AN, Xu X et al. (in press) Defining the conditions for the generation of melanocytes from human embryonic stem cells. Stem Cells 24,1668–1677.

Fortunel NO, Otu HH, Ng HH, Chen J, Mu X, Chevassut T, Li X, Joseph M, Bailey C, Hatzfeld JA et al. (2003) Comment on ‘ "Stemness": transcriptional profiling of embryonic and adult stem cells’ and ‘a stem cell molecular signature’. Science 302,393.

Fujimoto A, Mitalipov SM, Kuo HC and Wolf DP (2005) Aberrant genomic imprinting in rhesus monkey ES cells. Stem Cells 24,595–603.

Fuks F (2005) DNA methylation and histone modifications: teaming up to silence genes. Curr Opin Genet Dev 15,490–495.[CrossRef][Web of Science][Medline]

Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW, Leonhardt H and Jaenisch R (2003) Induction of tumors in mice by genomic hypomethylation. Science 300,489–492.[Abstract/Free Full Text]

Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J et al. (2004) Differences between human and mouse embryonic stem cells. Dev Biol 269,360–380.[CrossRef][Web of Science][Medline]

Goh G, Self T, Barbadillo Munoz MD, Hall IP, Young L and Denning C (2005) Molecular and phenotypic analyses of human embryonic stem cell-derived cardiomyocytes: opportunities and challenges for clinical translation. Thromb Haemost 94,728–737.[Web of Science][Medline]

Golan-Mashiach M, Dazard JE, Gerecht-Nir S, Amariglio N, Fisher T, Jacob-Hirsch J, Bielorai B, Osenberg S, Barad O, Getz G et al. (2005) Design principle of gene expression used by human stem cells: implication for pluripotency. Faseb J 19,147–149.[Abstract/Free Full Text]

Gudjonsson T and Magnusson MK (2005) Stem cell biology and the cellular pathways of carcinogenesis. Apmis 113,922–929.[CrossRef][Web of Science][Medline]

Hanahan D and Weinberg RA (2000) The hallmarks of cancer. Cell 100,57–70.[CrossRef][Web of Science][Medline]

Hannula K, Lipsanen-Nyman M, Scherer SW, Holmberg C, Hoglund P and Kere J (2001) Maternal and paternal chromosomes 7 show differential methylation of many genes in lymphoblast DNA. Genomics 73,1–9.[CrossRef][Web of Science][Medline]

Hanson C and Caisander G (2005) Human embryonic stem cells and chromosome stability. Apmis 113,751–755.[CrossRef][Web of Science][Medline]

Hardarson T, Caisander G, Sjogren A, Hanson C, Hamberger L and Lundin K (2003) A morphological and chromosomal study of blastocysts developing from morphologically suboptimal human pre-embryos compared with control blastocysts. Hum Reprod 18,399–407.[Abstract/Free Full Text]

Hart AH, Hartley L, Ibrahim M and Robb L (2004) Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human. Dev Dyn 230,187–198.[CrossRef][Web of Science][Medline]

Hattori N, Abe T, Hattori N, Suzuki M, Matsuyama T, Yoshida S, Li E and Shiota K (2004) Preference of DNA methyltransferases for CpG islands in mouse embryonic stem cells. Genome Res 14,1733–1740.[Abstract/Free Full Text]

Heins N, Englund MC, Sjoblom C, Dahl U, Tonning A, Bergh C, Lindahl A, Hanson C and Semb H (2004) Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells 22,367–376.[CrossRef][Web of Science][Medline]

Heins N, Lindahl A, Karlsson U, Rehnstrom M, Caisander G, Emanuelsson K, Hanson C, Semb H, Bjorquist P, Sartipy P et al. (2006) Clonal derivation and characterization of human embryonic stem cell lines. J Biotechnol 122,2867–2874.

Herman JG and Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349,2042–2054.[Free Full Text]

Herszfeld D, Wolvetang E, Langton-Bunker E, Chung TL, Filipczyk AA, Houssami S, Jamshidi P, Koh K, Laslett AL, Michalska A et al. (2006) CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells. Nat Biotechnol 24,351–357.[CrossRef][Medline]

Hoffman LM and Carpenter MK (2005a) Characterization and culture of human embryonic stem cells. Nat Biotechnol 23,699–708.[CrossRef][Web of Science][Medline]

Hoffman LM and Carpenter MK (2005b) Human embryonic stem cell stability. Stem Cell Rev 1,139–144.

Hoffman LM, Hall L, Batten JL, Young H, Pardasani D, Baetge EE, Lawrence J and Carpenter MK (2005) X-inactivation status varies in human embryonic stem cell lines. Stem Cells 23,1468–1478.[CrossRef][Web of Science][Medline]

Holm TM, Jackson-Grusby L, Brambrink T, Yamada Y, Rideout WM III and Jaenisch R (2005) Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell 8,275–285.[CrossRef][Web of Science][Medline]

Hook EB (1977) Exclusion of chromosomal mosaicism: tables of 90%, 95% and 99% confidence limits and comments on use. Am J Hum Genet 29,94–97.[Web of Science][Medline]

Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M and Ahrlund-Richter L (2003) A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 18,1404–1409.[Abstract/Free Full Text]

Hsiao LL, Dangond F, Yoshida T, Hong R, Jensen RV, Misra J, Dillon W, Lee KF, Clark KE, Haverty P et al. (2001) A compendium of gene expression in normal human tissues. Physiol Genomics 7,97–104.[Abstract/Free Full Text]

Huettner JE, Lu A, Qu Y, Wu Y, Kim M and McDonald Iii JW (2006) Gap junctions and connexon hemichannels in human embryonic stem cells. Stem Cells 24,1654–1667.[CrossRef][Web of Science][Medline]

Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM III, Biniszkiewicz D, Yanagimachi R and Jaenisch R (2001) Epigenetic instability in ES cells and cloned mice. Science 293,95–97.[Abstract/Free Full Text]

Hyslop LA, Armstrong L, Stojkovic M and Lako M (2005) Human embryonic stem cells: biology and clinical implications. Expert Rev Mol Med 7,1–21.[Medline]

Ingelman-Sundberg M and Rodriguez-Antona C (2005) Pharmacogenetics of drug-metabolizing enzymes: implications for a safer and more effective drug therapy. Philos Trans R Soc Lond B Biol Sci 360,1563–1570.[Abstract/Free Full Text]

Inzunza J, Sahlen S, Holmberg K, Stromberg AM, Teerijoki H, Blennow E, Hovatta O and Malmgren H (2004) 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.[Abstract/Free Full Text]

Inzunza J, Gertow K, Stromberg MA, Matilainen E, Blennow E, Skottman H, Wolbank S, Ahrlund-Richter L and Hovatta O (2005) Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells 23,544–549.[CrossRef][Web of Science][Medline]

Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA and Lemischka IR (2002) A stem cell molecular signature. Science 298,601–604.[Abstract/Free Full Text]

Jaenisch R and Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33,245–254.

James D, Levine AJ, Besser D and Hemmati-Brivanlou A (2005) TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132,1273–1282.[Abstract/Free Full Text]

Jenuwein T and Allis CD (2001) Translating the histone code. Science 293,1074–1080.[Abstract/Free Full Text]

Jones PA and Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3,415–428.[Web of Science][Medline]

Keshet I, Schlesinger Y, Farkash S, Rand E, Hecht M, Segal E, Pikarski E, Young RA, Niveleau A, Cedar H et al. (2006) Evidence for an instructive mechanism of de novo methylation in cancer cells. Nat Genet 38,149–153.[CrossRef][Web of Science][Medline]

Kim GD, Ni J, Kelesoglu N, Roberts RJ and Pradhan S (2002) Co-operation and communication between the human maintenance and de novo DNA (cytosine-5) methyltransferases. Embo J 21,4183–4195.[CrossRef][Web of Science][Medline]

Kim SJ, Lee JE, Park JH, Lee JB, Kim JM, Yoon BS, Song JM, Roh SI, Kim CG and Yoon HS (2005) Efficient derivation of new human embryonic stem cell lines. Mol Cells 19,46–53.[Web of Science][Medline]

Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD and Lanza R (2005) Human embryonic stem cells derived without feeder cells. Lancet 365,1636–1641.[CrossRef][Web of Science][Medline]

Kothapalli R, Yoder SJ, Mane S and Loughran TP Jr (2002) Microarray results: how accurate are they? BMC Bioinformatics 3,22.[CrossRef][Medline]

Lagarkova MA, Volchkov PY, Lyakisheva AV, Philonenko ES and Kiselev SL (2006) Diverse epigenetic profile of novel human embryonic stem cell lines. Cell Cycle 5,416–420.[Web of Science][Medline]

Lakshmipathy U, Pelacho B, Sudo K, Linehan JL, Coucouvanis E, Kaufman DS and Verfaillie CM (2004) Efficient transfection of embryonic and adult stem cells. Stem Cells 22,531–543.[CrossRef][Web of Science][Medline]

Lee JB, Kim JM, Kim SJ, Park JH, Hong SH, Roh SI, Kim MK and Yoon HS (2005a) Comparative characteristics of three human embryonic stem cell lines. Mol Cells 19,31–38.[Web of Science][Medline]

Lee JB, Lee JE, Park JH, Kim SJ, Kim MK, Roh SI and Yoon HS (2005b) Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol Reprod 72,42–49.[Abstract/Free Full Text]

Li T, Zhou CQ, Mai QY and Zhuang GL (2005) Establishment of human embryonic stem cell line from gamete donors. Chin Med J (Engl) 118,116–122.

Liu X, Wu H, Loring J, Hormuzdi S, Disteche CM, Bornstein P and Jaenisch R (1997) Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission. Dev Dyn 209,85–91.[CrossRef][Web of Science][Medline]

Lucifero D, Chaillet JR and Trasler JM (2004) Potential significance of genomic imprinting defects for reproduction and assisted reproductive technology. Hum Reprod Update 10,3–18.[Abstract/Free Full Text]

Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS et al. (2006a) Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24,185–187.[CrossRef][Web of Science][Medline]

Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS et al. (2006b) Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24,185–187.[CrossRef][Web of Science][Medline]

Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V, Kassauei K, Sui G, Cutler DJ, Liu Y, Brimble SN et al. (2005) Genomic alterations in cultured human embryonic stem cells. Nat Genet 37,1099–1103.[CrossRef][Web of Science][Medline]

Mandal A, Tipnis S, Pal R, Ravindran G, Bose B, Patki A, Rao MS and Khanna A (2006) Characterization and in vitro differentiation potential of a new human embryonic stem cell line, ReliCellhES1. Differentiation 74,81–90.[CrossRef][Web of Science][Medline]

Mecham BH, Klus GT, Strovel J, Augustus M, Byrne D, Bozso P, Wetmore DZ, Mariani TJ, Kohane IS and Szallasi Z (2004) Sequence-matched probes produce increased cross-platform consistency and more reproducible biological results in microarray-based gene expression measurements. Nucleic Acids Res 32, e74.[Abstract/Free Full Text]

Mitalipova M, Calhoun J, Shin S, Wininger D, Schulz T, Noggle S, Venable A, Lyons I, Robins A and Stice S (2003) Human embryonic stem cell lines derived from discarded embryos. Stem Cells 21,521–526.[CrossRef][Web of Science][Medline]

Mitalipova MM, Rao RR, Hoyer DM, Johnson JA, Meisner LF, Jones KL, Dalton S and Stice SL (2005) Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol 23,19–20.[CrossRef][Web of Science][Medline]

Miura M, Miura Y, Padilla-Nash HM, Molinolo AA, Fu B, Patel V, Seo BM, Sonoyama W, Zheng JJ, Baker CC et al. (2005) Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 24,1095–1103.

Niwa H, Miyazaki J and Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24,372–376.[CrossRef][Web of Science][Medline]

Noaksson K, Zoric N, Zeng X, Rao MS, Hyllner J, Semb H, Kubista M and Sartipy P (2005) Monitoring differentiation of human embryonic stem cells using real-time PCR. Stem Cells 23,1460–1467.[CrossRef][Web of Science][Medline]

Oh SK, Kim HS, Ahn HJ, Seol HW, Kim YY, Park YB, Yoon CJ, Kim DW, Kim SH and Moon SY (2005) Derivation and characterization of new human embryonic stem cell lines: SNUhES1, SNUhES2, and SNUhES3. Stem Cells 23,211–219.[CrossRef][Web of Science][Medline]

Onyango P, Jiang S, Uejima H, Shamblott MJ, Gearhart JD, Cui H and Feinberg AP (2002) Monoallelic expression and methylation of imprinted genes in human and mouse embryonic germ cell lineages. Proc Natl Acad Sci USA 99,10599–10604.[Abstract/Free Full Text]

Owen-Smith J and McCormick J (2006) An international gap in human ES cell research. Nat Biotechnol 24,391–392.[CrossRef][Web of Science][Medline]

Pain D, Chirn GW, Strassel C and Kemp DM (2005) Multiple retropseudogenes from pluripotent cell-specific gene expression indicates a potential signature for novel gene identification. J Biol Chem 280,6265–6268.[Abstract/Free Full Text]

Pantoja C, de Los Rios L, Matheu A, Antequera F and Serrano M (2005) Inactivation of imprinted genes induced by cellular stress and tumorigenesis. Cancer Res 65,26–33.[Abstract/Free Full Text]

Pardal R, Clarke MF and Morrison SJ (2003) Applying the principles of stem-cell biology to cancer. Nat Rev Cancer 3,895–902.[CrossRef][Web of Science][Medline]

Park SP, Lee YJ, Lee KS, Ah Shin H, Cho HY, Chung KS, Kim EY and Lim JH (2004) Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers. Hum Reprod 19,676–684.[Abstract/Free Full Text]

Pathak S and Multani AS (2006) Aneuploidy, stem cells and cancer. EXS 96,49–64.

Paz MF, Fraga MF, Avila S, Guo M, Pollan M, Herman JG and Esteller M (2003) A systematic profile of DNA methylation in human cancer cell lines. Cancer Res 63,1114–1121.[Abstract/Free Full Text]

Pera MF (2004) Unnatural selection of cultured human ES cells? Nat Biotechnol 22,42–43.[CrossRef][Web of Science][Medline]

Pickering SJ, Braude PR, Patel M, Burns CJ, Trussler J, Bolton V and Minger S (2003) Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. Reprod Biomed Online 7,353–364.[Medline]

Plaia TW, Josephson R, Liu Y, Zeng X, Ording C, Toumadje A, Brimble SN, Sherrer ES, Uhl EW, Freed WJ et al. (2005) Characterization of a new NIH registered variant human embryonic stem cell line BG01V: a tool for human embryonic stem cell research. Stem Cells 24,531–546.

Pyle AD, Lock LF and Donovan PJ (2006) Neurotrophins mediate human embryonic stem cell survival. Nat Biotechnol 24,344–350.[CrossRef][Web of Science][Medline]

Rajagopalan H and Lengauer C (2004) Aneuploidy and cancer. Nature 432,338–341.[CrossRef][Medline]

Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC and Melton DA (2002) ‘Stemness’: transcriptional profiling of embryonic and adult stem cells. Science 298,597–600.[Abstract/Free Full Text]

Rao M (2004) Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells. Dev Biol 275,269–286.[CrossRef][Web of Science][Medline]

Rao RR and Stice SL (2004) Gene expression profiling of embryonic stem cells leads to greater understanding of pluripotency and early developmental events. Biol Reprod 71,1772–1778.[Abstract/Free Full Text]

Rao MS and Civin CI (2005) Translational research: toward better characterization of human embryonic stem cell lines. Stem Cells 23,1453.[Web of Science][Medline]

Rao MS and Auerbach JM (2006) Estimating human embryonic stem-cell numbers. Lancet 367,650.[Web of Science][Medline]

Rao MS and Civin CI (2006) How many human embryonic stem cell lines are sufficient? A US Perspective. Stem Cells 24,800–803.[CrossRef][Web of Science][Medline]

Rao RR, Calhoun JD, Qin X, Rekaya R, Clark JK and Stice SL (2004) Comparative transcriptional profiling of two human embryonic stem cell lines. Biotechnol Bioeng 88,273–286.[CrossRef][Web of Science][Medline]

Reik W and Lewis A (2005) Co-evolution of X-chromosome inactivation and imprinting in mammals. Nat Rev Genet 6,403–410.[CrossRef][Web of Science][Medline]

Reubinoff BE, Pera MF, Fong CY, Trounson A and Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18,399–404.[CrossRef][Web of Science][Medline]

Reubinoff BE, Pera MF, Vajta G and Trounson AO (2001) Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 16,2187–2194.[Abstract/Free Full Text]

Reya T, Morrison SJ, Clarke MF and Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414,105–111.[CrossRef][Medline]

Rho JY, Yu K, Han JS, Chae JI, Koo DB, Yoon HS, Moon SY, Lee KK and Han YM (2006) Transcriptional profiling of the developmentally important signalling pathways in human embryonic stem cells. Hum Reprod 21,405–412.[Abstract/Free Full Text]

Richards M, Tan SP, Tan JH, Chan WK and Bongso A (2004) The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 22,51–64.[CrossRef][Web of Science][Medline]

Robertson KD (2005) DNA methylation and human disease. Nat Rev Genet 6,597–610.[CrossRef][Web of Science][Medline]

Rodriguez CI, Galan A, Valbuena D and Simon C (2006) Derivation of clinical-grade human embryonic stem cells. Reprod Biomed Online 12,112–118.[Web of Science][Medline]

Rosler ES, Fisk GJ, Ares X, Irving J, Miura T, Rao MS and Carpenter MK (2004) Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 229,259–274.[CrossRef][Web of Science][Medline]

Rossant J (2001) Stem cells from the Mammalian blastocyst. Stem Cells 19,477–482.[CrossRef][Web of Science][Medline]

Rubio D, Garcia-Castro J, Martin MC, de la Fuente R, Cigudosa JC, Lloyd AC and Bernad A (2005) Spontaneous human adult stem cell transformation. Cancer Res 65,3035–3039.[Abstract/Free Full Text]

Rugg-Gunn PJ, Ferguson-Smith AC and Pedersen RA (2005) Epigenetic status of human embryonic stem cells. Nat Genet 37,585–587.[CrossRef][Web of Science][Medline]

Sado T, Tada T and Takagi N (1996) Mosaic methylation of Xist gene before chromosome inactivation in undifferentiated female mouse embryonic stem and embryonic germ cells. Dev Dyn 205,421–434.[CrossRef][Web of Science][Medline]

Sato N, Sanjuan IM, Heke M, Uchida M, Naef F and Brivanlou AH (2003) Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol 260,404–413.[CrossRef][Web of Science][Medline]

Sato N, Meijer L, Skaltsounis L, Greengard P and Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10,55–63.[CrossRef][Web of Science][Medline]

Secombe J, Pierce SB and Eisenman RN (2004) Myc: a weapon of mass destruction. Cell 117,153–156.[CrossRef][Web of Science][Medline]

Semb H (2005) Human embryonic stem cells: origin, properties and applications. Apmis 113,743–750.[CrossRef][Web of Science][Medline]

Simon C, Escobedo C, Valbuena D, Genbacev O, Galan A, Krtolica A, Asensi A, Sanchez E, Esplugues J, Fisher S et al. (2005) First derivation in Spain of human embryonic stem cell lines: use of long-term cryopreserved embryos and animal-free conditions. Fertil Steril 83,246–249.[CrossRef][Web of Science][Medline]

Singh KK (2004) Mitochondrial dysfunction is a common phenotype in aging and cancer. Ann N Y Acad Sci 1019,260–264.[CrossRef][Web of Science][Medline]

Sjogren-Jansson E, Zetterstrom M, Moya K, Lindqvist J, Strehl R and Eriksson PS (2005) Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system. Dev Dyn 233,1304–1314.[CrossRef][Web of Science][Medline]

Skotheim RI, Monni O, Mousses S, Fossa SD, Kallioniemi OP, Lothe RA and Kallioniemi A (2002) New insights into testicular germ cell tumorigenesis from gene expression profiling. Cancer Res 62,2359–2364.[Abstract/Free Full Text]

Skottman H, Mikkola M, Lundin K, Olsson C, Stromberg AM, Tuuri T, Otonkoski T, Hovatta O and Lahesmaa R (2005a) Gene expression signatures of seven individual human embryonic stem cell lines. Stem Cells 23,1343–1356.[CrossRef][Web of Science][Medline]

Skottman H, Stromberg AM, Matilainen E, Inzunza J, Hovatta O and Lahesmaa R (2005b) Unique gene expression signature by human embryonic stem cells cultured under serum free conditions correlates with their enhanced and prolonged growth in an undifferentiated stage. Stem Cells 24,151–167.

Smiraglia DJ, Rush LJ, Fruhwald MC, Dai Z, Held WA, Costello JF, Lang JC, Eng C, Li B, Wright FA et al. (2001) Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies. Hum Mol Genet 10,1413–1419.[Abstract/Free Full Text]

Smith AG (2001) Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 17,435–462.[CrossRef][Web of Science][Medline]

Speicher MR and Carter NP (2005) The new cytogenetics: blurring the boundaries with molecular biology. Nat Rev Genet 6,782–792.[CrossRef][Web of Science][Medline]

Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, Jones SB, Brooks JD, Andrews PW, Brown PO and Thomson JA (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci USA 100,13350–13355.[Abstract/Free Full Text]

Steele W, Allegrucci C, Singh R, Lucas E, Priddle H, Denning C, Sinclair K and Young L (2005) Human embryonic stem cell methyl cycle enzyme expression: modelling epigenetic programming in assisted reproduction? Reprod Biomed Online 10,755–766.[Web of Science][Medline]

Stojkovic M, Lako M, Stojkovic P, Stewart R, Przyborski S, Armstrong L, Evans J, Herbert M, Hyslop L, Ahmad S et al. (2004) Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture. Stem Cells 22,790–797.[CrossRef][Web of Science][Medline]

Storchova Z and Pellman D (2004) From polyploidy to aneuploidy, genome instability and cancer. Nat Rev Mol Cell Biol 5,45–54.[CrossRef][Web of Science][Medline]

Suarez-Farinas M, Noggle S, Heke M, Hemmati-Brivanlou A and Magnasco MO (2005) Comparing independent microarray studies: the case of human embryonic stem cells. BMC Genomics 6,99.[CrossRef][Medline]

Sun BW, Yang AC, Feng Y, Sun YJ, Zhu Y, Zhang Y, Jiang H, Li CL, Gao FR, Zhang ZH et al. (2006) Temporal and parental-specific expression of imprinted genes in a newly derived Chinese human embryonic stem cell line and embryoid bodies. Hum Mol Genet 15,65–75.[Abstract/Free Full Text]

Suss-Toby E, Gerecht-Nir S, Amit M, Manor D and Itskovitz-Eldor J (2004) Derivation of a diploid human embryonic stem cell line from a mononuclear zygote. Hum Reprod 19,670–675.[Abstract/Free Full Text]

Taylor CJ, Bolton EM, Pocock S, Sharples LD, Pedersen RA and Bradley JA (2005) Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching. Lancet 366,2019–2025.[CrossRef][Web of Science][Medline]

Taylor RA, Cowin PA, Cunha GR, Pera M, Trounson AO, Pedersen J and Risbridger GP (2006) Formation of human prostate tissue from embryonic stem cells. Nat Methods 3,179–181.[CrossRef][Web of Science][Medline]

Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS and Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282,1145–1147.[Abstract/Free Full Text]

Trounson A (2006) The production and directed differentiation of human embryonic stem cells. Endocr Rev 27,208–219.[Abstract/Free Full Text]

Vallier L, Alexander M and Pedersen RA (2005) Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci 118,4495–4509.[Abstract/Free Full Text]

Wei CL, Miura T, Robson P, Lim SK, Xu XQ, Lee MY, Gupta S, Stanton L, Luo Y, Schmitt J et al. (2005) Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 23,166–185.[CrossRef][Web of Science][Medline]

Westermann F and Schwab M (2002) Genetic parameters of neuroblastomas. Cancer Lett 184,127–147.[CrossRef][Web of Science][Medline]

Wobus AM and Boheler KR (2005) Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev 85,635–678.[Abstract/Free Full Text]

Wong RC, Pebay A, Nguyen LT, Koh KL and Pera MF (2004) Presence of functional gap junctions in human embryonic stem cells. Stem Cells 22,883–889.[CrossRef][Web of Science][Medline]

Xiao L, Yuan X and Sharkis SJ (2006) Activin A maintains self-renewal and regulates FGF, Wnt and BMP pathways in human embryonic stem cells. Stem Cells 24,1476–1486.[CrossRef][Web of Science][Medline]

Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD and Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19,971–974.[CrossRef][Web of Science][Medline]

Xu RH, Peck RM, Li DS, Feng X, Ludwig T and Thomson JA (2005) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Meth 2,185–190.

Yauk CL, Berndt ML, Williams A and Douglas GR (2004) Comprehensive comparison of six microarray technologies. Nucleic Acids Res 32, e124.[Abstract/Free Full Text]

Zeng X, Miura T, Luo Y, Bhattacharya B, Condie B, Chen J, Ginis I, Lyons I, Mejido J, Puri RK et al. (2004) Properties of pluripotent human embryonic stem cells BG01 and BG02. Stem Cells 22,292–312.[CrossRef][Web of Science][Medline]

Zhan M, Miura T, Xu X and Rao MS (2005) Conservation and variation of gene regulation in embryonic stem cells assessed by comparative genomics. Cell Biochem Biophys 43,379–405.[CrossRef][Web of Science][Medline]

Zhu X, Deng C, Kuick R, Yung R, Lamb B, Neel JV, Richardson B and Hanash S (1999) Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome. Proc Natl Acad Sci USA 96,8058–8063.[Abstract/Free Full Text]

Zvetkova I, Apedaile A, Ramsahoye B, Mermoud JE, Crompton LA, John R, Feil R and Brockdorff N (2005) Global hypomethylation of the genome in XX embryonic stem cells. Nat Genet 37,1274–1279.[CrossRef][Web of Science][Medline]

Zwaka TP and Thomson JA (2005) A germ cell origin of embryonic stem cells? Development 132,227–233.[Abstract/Free Full Text]

Received on April 18, 2006; revised July 12, 2006; accepted on July 29, 2006

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				I. Mateizel, C. Spits, A. Verloes, A. Mertzanidou, I. Liebaers, and K. Sermon Characterization of CD30 expression in human embryonic stem cell lines cultured in serum-free media and passaged mechanically Hum. Reprod., October 1, 2009; 24(10): 2477 - 2489. [Abstract] [Full Text] [PDF]

				A. Raya, I. Rodriguez-Piza, B. Aran, A. Consiglio, P.N. Barri, A. Veiga, and J.C. Izpisua Belmonte Generation of Cardiomyocytes from New Human Embryonic Stem Cell Lines Derived from Poor-quality Blastocysts Cold Spring Harb Symp Quant Biol, November 26, 2008; (2008) sqb.2008.73.038v2. [Abstract] [PDF]

				K. K. Intemann and Inmaculada de Melo-Martin Regulating scientific research: should scientists be left alone? FASEB J, March 1, 2008; 22(3): 654 - 658. [Abstract] [Full Text] [PDF]

				K.-P. Kim, A. Thurston, C. Mummery, D. Ward-van Oostwaard, H. Priddle, C. Allegrucci, C. Denning, and L. Young Gene-specific vulnerability to imprinting variability in human embryonic stem cell lines Genome Res., December 1, 2007; 17(12): 1731 - 1742. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 13/2/103    most recent dml041v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (28) Request Permissions Google Scholar Articles by Allegrucci, C. Articles by Young, L.E. Search for Related Content PubMed PubMed Citation Articles by Allegrucci, C. Articles by Young, L.E. Social Bookmarking          What's this?

Online ISSN 1460-2369 - Print ISSN 1355-4786

Copyright © 2009 European Society of Human Reproduction and Embryology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics