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Stem Cell Research (2010) 4, 157–164

REVIEW

Environmental epigenetic modificationsand reprogramming-recalcitrant genesKazuhiro Sakurada ⁎

Sony Computer Science Laboratories Inc., Takanawa Muse Bldg, 3-14-13, Higashigotanda, Shinagawa-ku, Tokyo,141-0022 Japan

Received 13 October 2009; received in revised form 15 January 2010; accepted 15 January 2010

Abstract The term “environmental epigenetic modifications” refers to alterations in phenotype triggered by environmentalstimuli via epigenetic mechanisms. Epidemiologic and animal model studies show that a subset of such environmentalepigenetic marks may affect susceptibility to chronic diseases. A growing body of evidence regarding incompleteness ofreprogramming indicates that the potential retention of pathogenic environmental epigenetics in human induced pluripotentstem cells (iPSCs) should be seriously considered. Given this possibility, the optimization of methods for the generation ofhuman induc pluripotent stem cells may require the identification of epigenetically appropriate somatic cell sources. Similarly,techniques for controlling epigenetic modification by environmental factors may also play a critical role in the development ofepigenetically stable sources of pluripotent stem cells.

© 2010 Elsevier B.V. All rights reserved.

Contents

Environmental epigenetic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159Stem cells as generators and transmitters of environmental epigenetic modifications . . . . . . . . . . . . . . . . . . 160The nature of reprogramming-recalcitrant genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161Challenges in the application of human iPSCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161Optimization of human iPSC generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Although basic biomedical research has yielded an enormousamount of information on disease mechanisms, the recentdecline in the development of new drugs and treatmentssuggests that our fundamental understanding of pathogenesisremains insufficient (Butler, 2008). Themajor characteristics

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of the outputs of scientific investigations and their applica-tions in technology include predictability, reproducibility,and controllability, and, importantly, findings are expectedto be generalizable in the sense that when two systems areuniform and constant, the causes of events in one system canserve as the basis for predictions in its counterpart. Thehuman body, however, is exceedingly complex and diverse,which frustrates the direct use of predictions from models in

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medical care. The genetic sequences of any two individualswill show several millions of single nucleotide substitutions,and the human phenotype also changes as a result ofepigenetic mechanisms. It is thus understandable thatpredicting and controlling such a complex, diverse, dynamic,and irreversible organism as the human body represents atremendous scientific challenge. In clinical studies, popula-tion-based statistics are used to make inferences aboutheterogeneous patient groups, and the uniformity of compo-nents of therapeutic entities in the face of this inter-individual diversity remains a major challenge in cell-basedmedicine.

The advent and advances in the study of human inducedpluripotent stem cells (iPSCs) have raised considerable publicinterest, particularly with respect to their potential applica-tions in stem-cell-based therapy and in vitro cellularmodels ofdisease (Takahashi et al., 2007; Yu et al., 2007; Park et al.,2008; Masaki et al., 2008; Lowry et al., 2008; Park et al., 2008;Dimos et al., 2008; Ebert et al., 2009; Soldner et al., 2009;Raya et al., 2009). This enthusiasm is partially rooted in theassumption that human iPSCs are equivalent to humanembryonic stem (ES) cells. This assumption, however, remainsto be substantiated. In mammalian embryonic development,chromosomal DNA undergoes two different physiologicalreprogramming events: a first round following soon afterfertilization, and a second in primordial germ cells (Reik,2007). Under physiological conditions, only germline-derivedchromosomes are subject to these reprogramming processes(Fig. 1). Technologies that enable artificial reprogramming,such as somatic cell nuclear transfer (SCNT), ES cell-somaticcell fusion, and the induction of pluripotency in somatic cells,are thought to mimic the physiological reprogramming that

Figure 1 Physiological and artificial reprogramming. In physiologicfor reprogramming. Somatic cells receive epigenetic modification durchromosomal DNA of somatic cells and these epigenetic modificatioWeismann Barrier. Environmental epigenetic modifications on chrom

takes places after fertilization, as such methods weredeveloped to establish a post-fertilization environment forsomatic chromosomes (Fig. 1) (Gurdon and Melton, 2008).Although SCNT has shown that the genome state can bereprogrammed to an early embryonic (zygotic) pattern, theuse of somatic cell nuclei from adult animals is inefficient, andfewer than 6% of cloned mice embryos develop into births(Rideout et al., 2001; Kishigami and Wakayama, 2009). Theprocess is also error-prone, and cloned mammals of differentspecies exhibit a variety of abnormal phenotypes, includingplacental hyperplasia, large fetus syndrome, immune dis-orders, and shortened life span (Tanaka et al., 2001;Tamashiro et al., 2002; Ogonuki et al., 2002). Tissue-specificgene expression profiles in cloned mice are also extremelydifferent from those generated by natural reproduction(Kohda et al., 2005). Interestingly, most of the abnormalphenotypes in clonedmice disappear after a single generation,suggesting epigenetic alterations as a possible cause of theabnormalities in gene expression profile and phenotype(Tamashiro et al., 2002). Lending credence to this notion isevidence that the epigenetic memory of active and repressedtranscription in nuclear-donor somatic cells can be inheritedthrough SCNT, in both mouse and Xenopus laevis (Ng andGurdon, 2005; Boiani et al., 2002; Bortvin et al., 2003).

Human iPSC colonies expressing Nanog and/or ALP(alkaline phosphatase) following the forced expression ofOct3/4, Sox-2, c-Myc and Klf4 show significant heterogeneitycharacterized by a number of hallmark features (Fig. 2)(Masaki et al., 2008). Such heterogeneity indicates thedifferent degrees of reprogramming in Nanog-and ALP-positive colonies. Partially reprogrammed cells have alsobeen observed in human iPSCs induced by the forced

al conditions, chromosomal DNA of germ cells are the sole sourceing developmental and postnatal life. In physiological conditions,ns are not inherited by the next generation, which is called theosomes in germline cells and somatic cells must be different.

Figure 2 Heterogeneity of ALP-positive colonies induced by Oct3/4, Sox2, c-Myc and Klf4 genes from human dermis-derived cells.(A) Heterogeneity of ALP-positive colonies was identified by analyzing the gene expression profile of eight marker genes. AlthoughNanog gene expression was observed in 161 of 163 ALP-positive colonies, only four colonies expressed eight human ES cell markergenes. The gene expression profile of the remaining 159 colonies was significantly heterogeneous; however, it had a characteristichierarchical feature. (B) The eight genes are aligned in the order of tendency to be expressed in ALP-positive colonies, NanogNTDGF1N Dnmt3bN Zfp42N FoxD3N GDF3N CYP26A1N TERT. Eight genes were not randomly expressed by their tendency but showed ahierarchical feature that expresses lower-tendency genes in colonies where higher-tendency genes are expressed. (C) Relationbetween the transcriptional factor binding sites and the tendency of marker gene expression. Data was obtained from reference(Masaki et al., 2008).

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expression of a different set of genes (Yu et al., 2007).Recently, Chin et al. identified and described severalimportant features of a number of differentially expressedgenes in human ESCs and iPSCs (Chin et al., 2009). I will referto these as “reprogramming-recalcitrant” genes, as theyresist the induction of a transcriptional state identical tothat seen in embryonic stem cells. The underlying causes canbe categorized broadly as 1) insufficient induction of humanES cell-specific genes; 2) insufficient suppression of somaticcell-specific genes; and 3) induction of human iPSC-specificgenes. Of these, 1 and 2 correspond roughly to the causes ofrecalcitrance to reprogramming by SCNT (Ng and Gurdon,2005). It is important to recognize that genes that are notexpressed in pluripotent cells cannot be detected by geneexpression profiling in self-renewing human iPSCs, meaningthat there may be more reprogramming-recalcitrant genes inhuman iPSCs than have been reported to date. Themeasurement of genome-wide epigenetic marks is onemeans of addressing this limitation. It was recently reported

that, in human iPSCs, the DNA methylation landscapes ofchromosomes 12 and 20 are different from those of the samechromosomes in human ES cells (Deng et al., 2009). Thisraises the question of whether the incomplete resetting ofthe nuclear state is a hallmark of artificial or post-fertilization reprogramming or, in other words, whether itis possible to achieve complete reprogramming by mimickingpost-fertilization reprogramming. The mechanistic basis andfunctional roles of reprogramming-recalcitrant genes in theresetting of somatic nuclei is also of great interest for furtherstudy.

Environmental epigenetic effects

Epigenetics refers to heritable modifications in gene functionwithout alteration of DNA sequences (Goldberg et al., 2007).Chemical modifications to DNA and chromatin proteins arethe underlying drivers of epigenetic modifications. Although

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human DNA methylation occurs exclusively at cytosineresidues in CpG dinucleotides in differentiated cells, non-CpG methylation, which is primarily observed at CpAnucleotides, has recently been detected in stem cells (Listeret al., 2009). Chromatin itself is composed of DNA and histoneproteins, which are subject to more than 100 different typesof modification, including methylation, acetylation, phos-phorylation, and ubiquitination (Goldberg et al., 2007). Theterm “epigenetics”was coined byWaddington to describe themechanisms necessary for the unfolding of genetic informa-tion in ontogenic development (Waddington, 1942). Riggs(Riggs, 1975) and Holiday and Pugh (Holliday and Pugh, 1975)proposed that the programmed methylation and demethyla-tion of DNA might be the molecular mechanism behindWaddington's hypothesis, and it has since been shown that X-chromosome-inactivation, genome imprinting, and lineage-specific gene silencing all rely on DNA methylation.

Epigenetics do not function solely in the establishmentand maintenance of tissue-and cell-type-specific geneexpression. In addition to tissue-specific DNA methylationregions (T-DMRs), alternative DNA methylation regions (A-DMRs) have been identified by analyses of the DNAmethylation profiles of human chromosomes 6, 20, and 22(Eckhardt et al., 2006). In this study, DNA methylation of T-DMRs was found to occur preferentially in evolutionarilyconserved non-protein coding regions (ECRs) several kbpdistant from core promoters, while A-DMRs, which exhibitcell mosaicism, were observed within the promoter. DNAmethylation profiles of A-DMRs vary between differenttissues in the same donor, as well as in the same tissues ororgans in different individuals, indicating that such regionsare acquired throughout life and depend on the specificenvironmental stimuli at the cellular level.

Monozygotic (MZ) twins are highly similar in appearance,especially at birth, yet they are frequently divergent forseveral important phenotypes, including the onset ofcommon diseases (Wong et al., 2005). The concordanceof susceptibility to age-dependent diseases in MZ twins canbe quite low: 15% for breast cancer, 25%-30% for multiplesclerosis, 25%-45% for diabetes, 50% for schizophrenia, and40-70% for Alzheimer's disease (Wong et al., 2005; Petronis,2006). Phenotypic divergence increases with age, andgreater differences are seen between MZ twins raisedand living in separate environments. Several differentmechanisms contribute to these changes, one of which isepigenetic differences. To address this issue, Fraga et al.analyzed global and locus-specific DNA methylation andhistone acetylation of a large cohort of MZ twins andshowed that, although such twins are epigeneticallyindistinguishable in the early years of life, older MZ twinsexhibit remarkable differences in the overall content anddistribution of 5-methylcytosine and histone acetylation(Fraga et al., 2005).

Findings from human epidemiological and animal studiesindicate that environmental stimuli can induce permanentchanges in phenotype, including metabolism and suscepti-bility to chronic disease, at critical periods during pre-andpostnatal development, (Jirtle and Skinner, 2007). It hasalso been suggested that epigenetic mechanisms are likelyinvolved in the developmental origins of health and disease(Waterland and Michels, 2007). Although this remains thesubject of active discussion, environmental epigenetics

may reflect an adaptive response to a given prenatalenvironment that serves to optimize the postnatal pheno-type to meeting the challenges of that environment(Gluckman and Hanson, 2004; Huxley et al., 2007). It hasbeen suggested, for example, mismatches between pre-andpostnatal environments may be linked to increases in therisk of developing chronic diseases, including type IIdiabetes, hypertension, and coronary heart disease (Gluck-man and Hanson, 2004).

Although the molecular mechanisms by which environ-mental stimuli alter gene expression to achieve phenotypicdiversity are largely unknown, changes in the concentrationof fetal hormones, such as glucocorticoids, may serve asmolecular cues. Glucocorticoids have been shown to alterDNA methylation at the promoter of the glucocorticoidreceptor. Maternal behaviors in rat, for example, have beenshown to influence the epigenetic modification of thepromoter of the glucocorticoid receptor in neurons (Weaveret al., 2004). In this study, the glucocorticoid receptor genepromoter was epigenetically repressed in the hippocampusof the offspring of low “licking and arched-back nursing”mothers (Weaver et al., 2004). Another study has shown thatchronic social defeat stress induces methylation of lysine 27(K27) of the histone H3 adjacent to the promoter region ofthe brain-derived neurotrophic factor (BDNF) gene inhippocampal neurons (Tsankova et al., 2006).

Developing organisms are also extremely sensitive tochemical perturbation. Environmental compounds, drugadministration, and physiological stresses in embryogenesisand the early postnatal period have been demonstrated toinfluence the pathogenesis and course of adult onsetdiseases (Hellwig et al., 2000). For example, endocrinedisruptors, such as vinclozolin and diethylstilbestrol, inducereproductive and endocrine defects (Hellwig et al., 2000;Klip et al., 2002). The frequency and reproducibility of suchabnormal phenotypes strongly suggest that epigeneticmodifications, not genetic mutations, are causative, andindeed, aberrant DNA methylation of several genes has beenfound in tissues and cells exposed to diethylstilbestrol(Bromer et al., 2009). Cultured cells exposed to benzopyreneadditionally exhibited genome-wide alterations in histone H3lys9 acetylation (Sadikovic et al., 2008).

Clearly, exposure to different environmental factors,including nutritional, endocrine, and chemical perturbationthroughout life, influences the epigenetic landscape ofeach individual. These epigenetic modifications can bedistinguished with developmental epigenetics and is calledenvironmental epigenetics.

Stem cells as generators and transmitters ofenvironmental epigenetic modifications

Environmental epigenetic modifications introduced intoterminally differentiated cells of post-mitotic tissues ororgans, such as neurons or cardiomyocytes, can playindispensable functional roles (Weaver et al., 2004;Tsankova et al., 2006; Zhang et al., 2002), but are nottransmitted. In contrast, epigenetic modifications insomatic stem cells in mitotically active tissues can bepassed on to progeny cells, meaning that somatic stem cellepigenetic modifications that occur at early developmental

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stages may be transmitted large, tissue-specific cellularpopulations or lineages over the life of the organism.Importantly, proliferating cells, including stem and pro-genitor cells, have a higher tendency to undergo epigeneticmodification (Meissner et al., 2008). During DNA replica-tion, the disruption, transfer, and de novo assembly ofnucleosomes are coordinated to reproduce the epigeneticlandscape (Groth et al., 2007). Because these genome-widerearrangements of chromosome structure occur during DNAreplication, the mitotic S phase may provide a uniqueopportunity for epigenetic modification to occur. As anexample, culture-induced instability of DNA methylation inES and somatic cells has been observed in high-CpGpromoters (Meissner et al., 2008; Allegrucci et al., 2007).The DNA in somatic cells in mitotically active tissues alsotends to be more highly methylated, which has beensuggested as a possible cause of replicative aging (Chu etal., 2007). High-turnover tissues also show higher rates ofcancer development (Rando, 2006; Sakurada et al., 2008).The cell mosaicism of A-DMRs described above supports thenotion that stem cells can serve as generators andtransmitters of environmental epigenetic modifications.

The nature of reprogramming-recalcitrantgenes

Given the evident importance of environmental epigeneticchanges in ontogeny, physiology, and pathogenesis, itseems clear that the extent of reprogramming should beassessed with reference not only to developmentalepigenetics, but environmental epigenetics as well. Alarge body of evidence on the inheritance of environmen-tal epigenetics following physiological reprogramming hasbeen compiled, and indicates that transgenerationalepigenetic effects define phenotypes present in successivegenerations that are not genetically determined (Yongsonand Whitelaw, 2008). Specifically, environmental epige-netic modifications in germline cells may play a major rolein transgenerational phenotypes. A number of examples ofadaptive and non-adaptive transgenerational effects havebeen found in mammals, including humans (Yongson andWhitelaw, 2008). Not all transgenerational epigeneticphenotypes can be explained by the inheritance ofepigenetic marks that avoid zygotic and germline repro-gramming. It has been shown, however, that a patientsuffering hereditary nonpolyposis colorectal cancer(HNPCC) had abnormal DNA methylation of the DNAmismatch repair genes, MLH1, in all three germ layersinherited from parental germ cells that escaped zygoticreprogramming (Hitchins et al., 2007). The incompletereprogramming observed in SCNT and iPSCs could similarlycorrespond to zygotic reprogramming.

As suggested by the heterogeneity of A-DMRs (Eckhardt etal., 2006), environmental epigenetics may mirror commondifferences between germline and somatic cells as deter-mined by differences in niche microenvironments. If so, thisraises the possibility that the artificial reprogramming ofsomatic cells may generate disease phenotypes, which areusually not caused by the reprogramming recalcitrant genesinherited through physiological reprogramming of the germ-line cell nucleus.

Challenges in the application of human iPSCs

Much has been written about potential applications forhuman iPSC technology in the study of disease etiology andpatient-specific cell replacement therapy. The existence ofreprogramming-recalcitrant genes, however, may signifyimportant constraints on such applications. We have seenhow epigenetic modifications can influence disease pheno-types by affecting gene expression levels, irrespective ofDNA sequence. The causes of the phenotype differencesobserved in somatic cells differentiated from patient-specific iPSCs may be complex, as these may be the resultof variations in DNA sequence or the activity of iPSC-specificreprogramming-recalcitrant genes. Environmental epigenet-ic marks inherited from somatic cell sources also exhibit cellmosaicism. This suggests that the only way to reconstitutethe disease-specific phenotypic features resulting frominherited environmental epigenetic modifications in pa-tient-specific human iPSCs may be to induce pluripotencyin somatic cells from the affected tissue.

Neural stem cells are currently being considered aspotential therapies for neurological disorders. It wasrecently reported that a boy suffering ataxia telangiectasiawho had received multiple injections of fetal neural cellsinto the brain developed a multifocal brain tumor derivedfrom the donor cells (Amariglio et al., 2009). Importantly,the fetal neural cells were cultured before transplantation(Amariglio et al., 2009). Culture-induced DNA methylationinstability has been observed in the epigenome of human EScells as well as in that of somatic cells (Meissner et al., 2008;Allegrucci et al., 2007). It is conceivable, therefore, in thisunfortunate case that the cancer-prone epigenetic pheno-type was acquired during culture. The possibility that denovo epigenetic modification during the iPSC-generatingprocess may also induce epigenetic phenotypic changescannot be ruled out.

The role of reprogramming-recalcitrant genes in humaniPSCs needs to be considered seriously. Epigenetics is alatent change in gene function, which cannot be detectedcomprehensively by gene expression profile. At present,there is no technology able to accurately measure genome-wide epigenetic modifications at the single-cell level, andthus it remains impossible to prospectively identifypotentially small populations of cancer-prone cells in acell therapy product. The use of sample-based statisticscan help to address this epigenetic heterogeneity, andshould be applied in the evaluation of cells intended foruse in the study of disease etiology or cell-based therapy.Ideally, a larger number of samples than is typically used inclinical studies should be used to improve accuracy andstatistical power.

Optimization of human iPSC generation

The quality of human iPSCs should be evaluated in respect totheir retention of environmental as well as developmentalepigenetic marks. The definition of human iPSCs remainsambiguous, even in terms of true pluripotency. For example,most publications to date have examined the expression onlyof Nanog or ALP as a surrogate in determining increases inthe efficiency of iPSC generation (Hong et al., 2009; Yoshida

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et al., 2009). However, the presence of partially repro-grammed developmental genes in Nanog-and/or ALP-posi-tive human colonies indicates that the increase in theefficiency of pluripotent cell generation through the use ofnewly developed reprogramming methods cannot be con-firmed without evaluating the expression of multiplepluripotency genes, including Nanog, TDGF1, Dnmt3b,Zfp42, FoxD3, GDF3, Cyp26A1 and TERT, in all coloniesgenerated in the primary induction culture (Masaki et al.,2008) (Fig. 3A). Absent such confirmatory evidence,increases in the number of Nanog-and/or ALP-positivecolonies may only indicate an increase in the number ofpartially reprogrammed cells. To my knowledge, no evalu-ation of environmental epigenetic modifications, such asthose caused by disease states in somatic cells of origin orcell culture, have been conducted on human iPSCs. It shouldbe emphasized that functional analyses using the presentcriteria for pluripotency are insufficient to determine thequality of human iPSCs in terms of their environmentalepigenetics. The differentiative potential of iPSCs does notnecessarily reflect the full set of disease-associated epige-netic modifications that may be inherited from their somaticcells of origin.

Without clear evidence of bona fide complete reprogram-ming, in which all unnecessary epigenetic marks in somaticcells is erased, it is essential to optimize the process of

Figure 3 Evaluation and optimization of degree of epigenetic reprocolonies may reflect the generation of partially reprogrammed cecontribute to the degree of developmental epigenetic reprogramminculture-induced epigenetics will contribute to the degree of environ

human iPSC generation by identifying epigenetically idealsomatic cell sources (Fig. 3B). Although the generation ofpluripotent stem cells from lymphocytes, monocytes/macro-phages, and albumin-or insulin-producing cells has beenproposed, based on the assumption that all somatic cells,including terminally differentiated cells, have the ability tobe reprogrammed to pluripotency, this may be a leap too far(Yamanaka, 2009). Studies of DNA methylation profiles insperm and lymphocytes revealed relatively smaller differ-ences, consistent with the similarities between their geneexpression profiles (Eckhardt et al., 2006; Zeng et al., 2004).Monocytes and macrophages express multiple lineage genesby differentiation or by cell fusion (Zhao et al., 2003;Camargo et al., 2003). These characteristics are notobserved in other terminally-differentiated cells. Addition-ally, the expression of tissue-specific genes, such as thoseencoding albumin or insulin, cannot serve as the basis for thereprogramming of terminally-differentiated cells, since ithas been shown that these genes can also be expressed insomatic stem cells during wound healing and cell culture(Rountree et al., 2007). It has been shown that Bmi-1modification is essential in self-renewing hematopoietic andneural stem cells in an Ink4a-dependent manner (Park et al.,2003; Molofsky et al., 2003). Thus, the increase in theefficiency of colony formation by inhibition of the Ink4/arfpathway means that somatic stem cells are favorable sources

gramming. Increase in the number of Nanog and/or ALP-positivells. The degree of differentiation in somatic cell sources willg. On the other hand, the epigenetic background of a donor, andmental epigenetic reprogramming.

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for iPSC reprogramming (Li et al., 2009). And indeed, thereprogramming of somatic stem cells has been shown to bemore efficient than that of skin-derived cells (Silva et al.,2008; Eminli et al., 2009).

Although human iPSCs have been established fromelderly donors, their quality was not analyzed with respectto environmental epigenetics (Dimos et al., 2008). Globalhypomethylation and local hypermethylation are commonlyobserved in cancer and senescent cells (Suzuki et al.,2006), and age-dependent hypermethylation is observed atA-DMRs, indicating that environmental epigenetics mayincrease with cell senescence. These observations indicatethat young donors may be preferable to elderly donors assources for somatic cells used in the generation of humaniPSCs. Mitotically inactive tissue is also known to undergoless DNA methylation at A-DMRs (Chu et al., 2007). Givenwith these observations regarding DNA methylation and theinstabilities associated with proliferation, it stands toreason that minimally passaged somatic stem cells derivedfrom low-turnover tissues may represent optimal startingcell sources for use in the generation of human iPSCs.

Conclusions

The available evidence indicates that reprogramming isincomplete in human iPSCs, and that reprogramming-recalcitrant genes may influence both human iPSC functionand the phenotypes of their differentiated progeny. It isthus necessary to determine the epigenetic state of“wildtype” pluripotent stem cells in regard to both theirdevelopmental and environmental epigenetics. Under-standing the mechanisms of environmental epigeneticsand transgenerational epigenetic inheritance will offernew methods for manipulating epigenetic marks. The useof chemical compounds that modify the function ofepigenetic enzymes, such as histone deacetylase (HDAC),histone methylase (HMT), and DNA methyltransferase(Dnmt), may help to improve reprogramming efficiencies,and in fact, it has been shown that HDAC and Dnmt1inhibitors do just that (Huangfu et al., 2008). However,these compounds affect genome stability by targetingvarious cellular genomic surveillance mechanisms (Eot-Houllier et al., 2009). Evidence that epigenetics processesare regulated by RNA signaling is also beginning toaccumulate (Mattick et al., 2009). Compared to epigeneticenzymes and chromatin-modifying complexes (Polycomb-group and Trithorax-group), RNA-directed regulation ofchromosome modification may offer the advantage of ahigh degree of sequence-and locus-specificity. Non-proteincoding RNAs (ncRNAs) play a major role in this process. Abetter understanding of the mechanisms of ncRNA-mediat-ed epigenetic modification may provide us with new toolsfor controlling epigenetic modifications associated withdisease susceptibility and pathogenesis.

Acknowledgments

The discussions described in this paper are the outcome ofresearch activities at Sony Computer Science Laboratories.The author is grateful to Dr. Douglas Sipp for his helpfulcritique of this manuscript.

References

Allegrucci, C., et al., 2007. Restriction landmark genome scanningidentifies culture-induced DNA methylation instability in thehuman embryonic stem cell epigenome. Hum. Mol. Genet. 16,1253–1268.

Amariglio, N., et al., 2009. Donor-derived brain tumor followingneural stem cell transplantation in an ataxia telangiectasiapatient. PLoS Med. e1000029, 6.

Boiani, M., et al., 2002. Oct4 distribution and level in mouse clones:consequences for pluripotency. Genes Dev. 16, 1209–1219.

Bortvin, A., et al., 2003. Incomplete reactivation of Oct4-relatedgenes in mouse embryos cloned from somatic nuclei. Develop-ment 130, 1673–1680.

Bromer, J.G., Wu, J., Zhou, Y., Taylor, H.S., 2009. Hypermethyla-tion of homeobox A10 by in utero diethylstilbestrol exposure: anepigenetic mechanism for altered developmental programming.Endocrinology 150, 3376–3382.

Butler, D., 2008. Crossing the valley of death. Nature 453, 840–842.Camargo, F.D., Green, R., Capetanaki, Y., Jackson, K.A., Goodell,

M.A., 2003. Single hematopoietic stem cells generate skeletalmuscle through myeloid intermediates. Nat. Med. 9, 1520–1527.

Chin, M.H., et al., 2009. Induced pluripotent stem cells andembryonic stem cells are distinguished by gene expressionsignatures. Cell Stem Cell 5, 111–123.

Chu, M.W., et al., 2007. Lack of increases in methylation at threeCpG rich genomic loci in non-mitotic adult tissues during aging.BMC Med. Genet. 8, 50.

Deng, J., et al., 2009. Targeted bisulfate sequencing reveals changesin DNA methylation associated with nuclear reprogramming. Nat.Biotechnol. 27, 353–360.

Dimos, J.T., et al., 2008. Induced pluripotent stem cells generatedfrom patients with ALS can be differentiated into motor neurons.Science 321, 1218–1221.

Ebert, A.D., et al., 2009. Induced pluripotent stem cells from aspinal muscular atrophy patient. Nature 457, 277–280.

Eckhardt, F., et al., 2006. DNA methylation profiling of humanchromosomes 6, 20 and 22. Nat. Genet. 12, 1378–1385.

Eminli, S., et al., 2009. Differentiation stage determines potential ofhematopoietic cells for reprogramming into induced pluripotentstem cells. Nat. Genet. 41, 968–976.

Eot-Houllier, G., et al., 2009. Histone deacetylase inhibitors andgenomic instability. Cancer Lett. 274, 169–176.

Fraga, M.F., et al., 2005. Epigenetic differences arise during thelifetime of monozygotic twins. Proc. Natl. Acad. Sci. U.S.A. 102,10604–10609.

Gluckman, P.D., Hanson, M.A., 2004. Living with the past: evolution,development, and patterns of disease. Science 305, 1733–1736.

Goldberg, A.D., Allis, C.D., Bernstein, E., 2007. Epigenetics: Alandscape takes shape. Cell 128, 635–638.

Groth, A., Rocha, W., Verreault, A., Almouzni, G., 2007. Chromatinchallenges during DNA replication and repair. Cell 128, 721–733.

Gurdon, J.B., Melton, D.A., 2008. Nuclear reprogramming in cells.Science 322, 1811–1815.

Hellwig, J., et al., 2000. Pre-andpostonatal oral toxicity of vinclozolin inWistar and Long-Evans rats. Regul. Toxicol. Pharmacol. 32, 42–50.

Hitchins, M.P., et al., 2007. Inheritance of a cancer-associated MLH1germ-line epimutation. N. Engl. J. Med. 356, 697–705.

Holliday, R., Pugh, J.E., 1975. DNA modification mechanisms andgene activity during development. Science 187, 226–232.

Hong, H., et al., 2009. Suppression of induced pluripotent stem cellgeneration by the p53–p21 pathway. Nature 460, 1132–1135.

Huangfu, D., et al., 2008. Induction of pluripotent stem cells bydefined factors is greatly improved by small-molecule com-pounds. Nat. Biotechnol. 26, 795–797.

Huxley, R., et al., 2007. Is birth weight a risk factor for ischemicheart disease in later life? Am. J. Clin. Nutr. 85, 1244–1250.

164 K. Sakurada

Jirtle, R.L., Skinner, M.L., 2007. Environmental epigenomics anddisease susceptibility. Nat. Rev., Genet. 8, 253–262.

Kishigami, S., Wakayama, T., 2009. Somatic cell nuclear transfer inthe mouse. Methods Mol. Biol. 518, 207–218.

Klip, H., Verloop, J., van Gool, J.D., Koster, M.E., Burger, C.W.,van Leeuwen, F.E., 2002. Hypospadias in sons of womenexposed to diethylstilbestrol in utero: a cohort study. Lancet359, 1102–1107.

Kohda, T., et al., 2005. Variation in gene expression and aberrantlyregulated chromosome regions in cloned mice. Biol. Reprod. 73,1302–1311.

Li, H., et al., 2009. The Ink4/Arf locus is a barrier for iPS cellreprogramming. Nature 460, 1149–1153.

Lister, R., et al., 2009. Human DNA methylomes at base resolutionshow widespread epigenomic differences. Nature 462, 315–322.

Lowry, W.E., et al., 2008. Generation of human induced pluripotentstem cells from dermal fibroblasts. Proc. Natl. Acad. Sci. U.S.A.105, 2883–2888.

Masaki, H., et al., 2008. Heterogeneity of pluripotent marker geneexpression in colonies generated in human iPS cell inductionculture. Stem Cell Res. 1, 105–115.

Mattick, J.S., et al., 2009. RNA regulation of epigenetic processes.BioEssays 31, 51–59.

Meissner, A., et al., 2008. Genome-scale DNA methylation maps ofpluripotent and differentiated cells. Nature 454, 766–770.

Molofsky, A.V., et al., 2003. Bmi-1 dependence distinguishes neuralstem cell self-renewal from progenitor proliferation. Nature 425,962–967.

Ng, R.K., Gurdon, J.B., 2005. Epigenetic memory of active genetranscription is inherited through somatic cell nuclear transfer.Proc. Natl. Acad. Sci. U.S.A. 102, 1957–1962.

Ogonuki, N., et al., 2002. Early death of mice cloned from somaticcells. Nat. Genet. 30, 253–254.

Park, I.H., et al., 2008. Disease-specific induced pluripotent stemcells. Cell 134, 877–886.

Park, I.H., et al., 2008. Reprogramming of human somatic cells topluripotency with defined factors. Nature 451, 141–146.

Park, I.K., et al., 2003. Bmi-1 is required for maintenance of adultself-renewing haematopoietic stem cells. Nature 423, 302–305.

Petronis, A., 2006. Epigenetics and twins: three variations on thetheme. Trends Genet. 22, 347–350.

Rando, T.A., 2006. Stem cells, ageing and the quest for immortality.Nature 441, 1080–1086.

Raya, A., et al., 2009. Disease-corrected haematopoietic progeni-tors from Fanconi anaemia induced pluripotent stem cells.Nature 460, 53–59.

Reik, W., 2007. Stability and flexibility of epigenetic gene regulationin mammalian development. Nature 447, 425–432.

Rideout, W.M., Eggan, K., Jaenisch, R., 2001. Nuclear cloning andepigenetic reprogramming of the genome. Science 293,1093–1098.

Riggs, A.D., 1975. X inactivation, differentiation, and DNAmethylation.Cytogenet. Cell Genet. 14, 9–25.

Rountree, C.B., et al., 2007. A CD133-expressing murine liver ovalcell population with bilineage potential. Stem Cells 25,2419–2429.

Sadikovic, B., Andrews, J., Carter, D., Robinson, J., Rodenhiser, D.I.,2008. Genome-wide H3K9 histone acetylation profiles are alteredin benzopyrene-treated MCF7 breast cancer cells. J. Biol. Chem.283, 4051–4060.

Sakurada, K., McDonald, F.M., Shimada, F., 2008. RegenerativeMedicine and stem cell based drug discovery. Angew. Chem. Int.Ed. 47, 5718–5738.

Silva, J., et al., 2008. Promotion of reprogramming to ground statepluripotency by signal inhibition. PLoS Biol. 6, e253.

Soldner, F., et al., 2009. Parkinson's disease patient-derived inducedpluripotent stem cells free of viral reprogramming factors. Cell136, 964–977.

Suzuki, K., et al., 2006. Global DNA demethylation in gastrointes-tinal cancer is age dependent and precedes genomic damage.Cancer Cell 9, 199–207.

Takahashi, K., et al., 2007. Induction of pluripotent stem cells fromadult human fibroblasts by defined factors. Cell 131, 861–872.

Tamashiro, K.L., et al., 2002. Cloned mice have an obese phenotypenot transmitted to their offspring. Nat. Med. 8, 262–267.

Tanaka, S., et al., 2001. Placentomegaly in cloned mouse concepticaused by expansion of the spongiotrophoblast layer. Biol.Reprod. 65, 1813–1821.

Tsankova, N.M., et al., 2006. Sustained hippocampal chromatinregulation in a mouse model of depression and antidepressantaction. Nat. Neruosci. 9, 519–525.

Waddington, C.H., 1942. The epigenotype. Endeavour 1, 18–20.Waterland, R.A., Michels, K.B., 2007. Epigenetic epidemiology of the

developmental origins hypothesis. Annu. Rev. Nutr. 27, 363–388.Weaver, I.C., et al., 2004. Epigenetic programming by maternal

behavior. Nat. Neurosci. 7, 847–854.Wong, A.H.C., Gottesman, I.I., Petronis, A., 2005. Phenotypic

differences in genetically identical organisms: the epigeneticperspective. Hum. Mol. Genet. 14, R11–18.

Yamanaka, S., 2009. Elite and stochastic models for inducedpluripotent stem cell generation. Nature 460, 49–52.

Yongson, N.A., Whitelaw, E., 2008. Transgenerational epigeneticeffects. Annu. Rev. Genomics Hum. Genet. 9, 233–257.

Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., Yamanaka, S.,2009. Hypoxia enhances the generation of induced pluripotentstem cells. Cell Stem Cell 5, 237–241.

Yu, J., et al., 2007. Induced pluripotent stem cell lines derived fromhuman somatic cells. Science 318, 1917–1920.

Zeng, W., et al., 2004. Transcript profile of CD4+and CD8+T cellsfrom the bone marrow of acquired aplastic anemia patients. Exp.Hematol. 32, 806–814.

Zhang, C.L., et al., 2002. Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy. Cell 110, 479–488.

Zhao, Y., Glesne, D., Huberman, E.A., 2003. human peripheral bloodmonocyte-derived subset acts as pluripotent stem cells. Proc.Natl. Acad. Sci. U.S.A. 100, 2426–2431.