Embryonic stem (ES) cells constitute a very important tool for regenerative medicine today. Human ES cells, in particular, are almost all derived from embryos obtained by
2. Epigenetic regulation by DNA methylation
In vertebrate genomic DNA, the 5' cytosine residues in CpG sequences are often methylated . DNA methylation plays an essential role in the normal development of mammalian embryos by regulating gene expression through genomic imprinting and X chromosome inactivation, and confers genomic stability [4-7]. In this chapter, we focus primarily on genomic imprinting, which is the preferential silencing of one of the parental alleles of a gene by epigenetic DNA methylation since epigenetic modifications to some imprinted genes cause diseases such as Beckwith-Wiedemann syndrome and Prader-Willie syndrome. For example, the expression level of the
3. Epigenetic instability in preimplantation embryos
In general, ES cells, especially human ES cells, are generated from blastocyst stage embryos that are produced by
4. Epigenetic instability in ES cells during prolonged culture
ES cells are established from the inner cell mass (ICM) of blastocyst stage embryos [18,19]. Once ES cell lines are established, they can be maintained for long periods of time and used for several applications. However, ES cells lose their pluripotency during prolonged
5. Epigenetic differences between male and female ES cells
Large differences in epigenetic drift have been observed between male (XY) and female (XX) mouse ES cells. Global demethylation, including imprinted genes and satellite repeats, occurred more frequently in female ES cell lines compared to male ES cell lines [21, 25]. This global demethylation reflects the number and state of X chromosomes in ES cells. In general, both X chromosomes are active in female ES cells, whereas male ES cells have only one active X chromosome. The X chromosome state in female ES cells is thought to lead to downregulation of DNA methyltransferases (Dnmt3a and Dnmt3b) and, ultimately, to global hypomethylation . Thus, DNA methylation of imprinted genes and repetitive sequences are gained or lost at high rates even in clonal populations of ES cells, and these alterations may have deleterious effects on phenotypes of ES cell-derived animals or tissues.
6. Epigenetic differences between
vivoand vitroES cells
6.1. Methylation state of
vivoand vitroES cells
In human ES cells, several studies have recently provided evidence for the efficient induction of endoderm, mesoderm, and ectoderm, and many of their downstream derivatives , and these reports offer broad possibilities for regenerative medicine. However, all human ES cell lines are established from
Although the genomic imprinting is maintained during preimplantation development, normal imprinting can occasionally be disrupted in preimplantation embryos during IVC, resulting in biallelic expression of the
In additional experiments, both Vivo and Vitro ES cells were passaged several more times, and the methylation state of imprinted genes and satellite repeats was investigated at later passages (P5) (Fig. 3). Results from COBRA analysis at P5 showed no significant differences between Vivo and Vitro ES cells. Even Vivo ES cells exhibited highly demethylated alleles. In contrast, some Vitro ES cells had an almost normally methylated allele. This result indicates that the methylation state of ES cells at later passages depends more on the character of the individual cell lines than on the origin of the ES cells.
6.2. Gene expression of
vivoand vitroES cells
We assessed gene expression patterns in ES cells at early and late passages by quantitative real-time RT-PCR. The expression of
7. Epigenetic instability in SCNT and uniparental ES cells
7.1. SCNT ES cells
Maintenance of the normal epigenetic state in SCNT-ES cells is crucial for their use in therapeutic applications. We established two SCNT-ES cell lines from embryos that were produced by introducing mouse embryonic fibroblast (MEF) donor cells into enucleated oocytes. Only two ES cell lines were generated by SCNT, which give a small sample size to examine, but the DNA methylation state of imprinted genes seems to be more severely altered compared to normal ES cell lines at early passages (Fig. 2 and Fig. 4). The abnormal DNA methylation in SCNT-ES cells undergoes further changes during prolonged culture (P10 and P30). For example, the imprinting methylation of the
7.2. Uniparental (parthenogenetic) ES cells
We and other groups have suggested that parthenogenetic ES (PgES) cells may be a pluripotent stem cell that could serve as a source of tissue for transplantation [34-36]. PgES cells do not require the destruction of viable biparental embryos as do normal ES cells. In addition, PgES cells do not need viruses or expression plasmids for the establishment of iPS cells. These are very powerful advantages for therapeutic applications. However, the biased epigenetic status and poor pluripotency of parthenogenetic cells are major issues to be overcome. PgES cells are established from parthenogenetic embryos that are produced by the artificial activation of the oocyte. Therefore, PgES cells that possess only maternal genomes could exhibit biallelic or silenced expression of imprinted genes, which causes poor pluripotency. Indeed, parthenogenetic embryos show poor growth and restricted tissue contribution in chimeras [37,38]. However, established PgES cells exhibit an improved contribution to chimeras, compared to chimeras derived from parthenogenetic embryos . Recent reports have shown that loss of imprinting occurred in PgES cells and derivative somatic cells in chimeras and led to changes in the gene expression of imprinted genes and improved pluripotency [2,40]. For example,
8. Effect of altered DNA methylation on pluripotency and disease
In humans, a growing number of reports suggest that children born following ART have an increased risk of developing epigenetic diseases such as Beckwith-Wiedemann syndrome [41,42] and Angelman Syndrome , which are caused by epigenetic modifications of imprinted genes. In sheep, epigenetic changes in the
How do these abnormalities in ES cells affect chimeric mice or ES cell-derived tissues? Several studies have indicated that the accumulation of epigenetic alterations during prolonged culture causes a loss of pluripotency in ES cells [21,49]. In chimeras, prolonged culture of ES cells gives rise to abnormalities and frequently results in postnatal death of chimeras possessing a high ES cell contribution . One reason for these problems could be that a loss of imprinting enhances tumorigenesis. In fact, mice derived from ES cells that had a global loss of DNA methylation display widespread cancer formation .
9. Candidate genes that cause altered DNA methylation
9.1. DNA methyltransferases
The most important factors for the maintenance of DNA methylation are the DNA methyltransferases. Three CpG DNA methyltransferases, Dnmt1, Dnmt3a and Dnmt3b, coordinately regulate CpG methylation in the genome [12-14]. Deletion of Dnmt1, Dnmt3a or Dnmt3b induces hypomethylation of genomic DNA [14,51], and forced expression of Dnmts causes genomic hypermethylation [52-54]. One of the Dnmt family members, Dnmt3L, is not expressed in differentiated somatic cells but is expressed in ES cells. Although Dnmt3L lacks the functional domains required for catalytic activity, overexpression or downregulation of Dnmt3L results in changes in DNA methylation in ES cells . Thus, the upregulation or downregulation of Dnmts could cause epigenetic instability in ES cells. Indeed, hypomethylation in XX ES cells is associated with reduced levels of Dnmt3a and Dnmt3b, which is the result of both X chromosomes being in the active state . Among Dnmts, a number of alternative splicing variants that lack the regulatory and/or catalytic regions have been reported. In particular, Dnmt3b has nearly 40 different isoforms generated by alternative splicing and/or alternative promoter usage. We recently reported that murine Dnmt3b lacking exon 6 (exon 5 in human) is highly expressed in
9.2. Other methylation factors
Other new methylation factors are Stella (PGC7) and Zfp57. Stella (PGC7), a primordial germ cell and ES cell marker, protects against DNA demethylation in early embryogenesis . Zfp57, a putative KRAB zinc finger protein, is also required for the post-fertilization maintenance of maternal and paternal methylation at multiple imprinted domains . Reductions of the levels of these factors could induce hypomethylation of DNA in ES cells.
9.3. Active demethylation factors
Active DNA demethylation via the base excision repair pathway has recently been proposed in mammals. In zebrafish, the coupling of a deaminase (activation-induced cytidine deaminase, AID), a glycosylase (methyl-CpG binding domain protein 4, MBD4), and Gadd45 is involved in DNA demethylation . In mammals, AID is indeed required for reprogramming of the somatic cell genome by demethylation of pluripotency genes in ES-somatic cell fusion . Gadd45 also promotes epigenetic gene activation by repair-mediated demethylation in mammals [28,29]. A
9.4. Chromatin structure specific to ES cells
In ES cells, bivalent domains of chromatin, that regulate several key developmental genes, contain both repressive (histone H3 lysine 27 methylation) and activating (histone H3 lysine 4 methylation) histone modifications that are usually mutually exclusive . Bivalent domains silence developmental genes in ES cells while preserving their potential to become activated upon initiation of specific differentiation programs. DNA methylation was thought to determine the chromatin structure; however, recent reports suggest that chromatin can affect DNA methylation and demethylation [66-67]. Therefore, bivalent chromatin modifications specific to ES cells could be associated with DNA methylation instability.
ES cells exhibit instabilities in DNA methylation that are correlated with the origin of the blastocysts from which they were derived (
5-hmC, 5-hydroxymethylcytosine; 5-mC, 5-methylcytosine; AID, activation-induced cytidine deaminase; COBRA, Combined bisulfite restriction analysis; DMR, differentially methylated region; Dnmt, DNA methyltransferase; ES, embryonic stem; Gadd45, Growth arrest and DNA damage-inducible protein 45; ICM, inner cell mass; IVC,
AcknowledgmentsWe thank Mr. Eikichi Yanagisawa, Dr. Sumiyo Morita, Ms. Mika Kimura, Mr. Daiki Tamura, Mr. Ryohei Kobayashi and Dr. Yasumitsu Nagao for technical assistance and helpful comments on the manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labour and Welfare of Japan; the National Institute of Biomedical Innovation; and the Takeda Science Foundation.
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