Loss of Genomic Imprinting in Mouse Parthenogenetic Embryonic Stem Cells

Horii, Takuro; Kimura, Masashi; Morita, Sumiyo; Nagao, Yoshimi; Hatada, Izuho

doi:10.1634/stemcells.2006-0635

Cited by 43 publications

(45 citation statements)

References 41 publications

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“…Although with decreased numbers, these Nanog þ /Sox2 þ epiblast cells still exist in the parthenote ICM and can be used to derive patient-specific ES cells for stem cell therapies. On the other hand, there are other drawbacks of parthenogenetic embryos, which had been previously reported, including aberrant expression of imprinted and development-related genes that disrupt full development of organisms (Humpherys et al, 2001;Zvetkova et al, 2005;Bonk et al, 2007;Jiang et al, 2007;Mitalipov et al, 2007;Horii et al, 2008). Our study showed the abnormal gene expression in the preimplantation parthenogenetic embryo, which Fig.…”

Section: Discussionsupporting

confidence: 54%

Ectopic expression of Fgf3 leads to aberrant lineage segregation in the mouse parthenote preimplantation embryos

Chen

2012

Developmental Dynamics

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Background: Parthenogenetic mammalian embryos were reported to die in utero no later than the 25-somite stage due to abnormal development of both embryonic and extraembryonic lineages. Interestingly, it has been shown that parthenogenetic ICM cells tend to differentiate more into primitive endoderm cells and less into epiblast and ES cells. Hence we are interested in studying the molecular mechanisms underlying lineage defects of parthenotes. Results: We found that parthenote inner cell masses (ICMs) contained decreased numbers of Sox2 1 /Nanog 1 epiblast cells but increased numbers of Gata4 1 primitive endoderm cells, indicating an unusual lineage segregation. We demonstrate for the first time that the increased Gata4 level in parthenotes may be explained by the strong up-regulation of Fgf3 and Fgfr2 phosphorylation. Inhibition of Fgfr2 activation by SU5402 in parthenotes restored normal Nanog and Gata4 levels without affecting Fgf3, indicating that Fgf3 is upstream of Fgfr2 activation. In parthenote trophectoderm, we detected normal Cdx2 but ectopic Gata4 expression and reduced Elf5 and Tbr2(Eomes) levels. Conclusions: Taken together, our work provides for the first time the insight into the molecular mechanisms of the developmental defects of parthenogenetic embryos in both the trophectoderm and ICM.

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Section: Discussionsupporting

confidence: 54%

Ectopic expression of Fgf3 leads to aberrant lineage segregation in the mouse parthenote preimplantation embryos

Chen

2012

Developmental Dynamics

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“…In contrast, the in vivo-developed blastocysts were collected from uteri 96 h after hCG. The methods used to produce parthenogenetic embryos [35] and SCNT embryos have been described previously [36]. Some of the IVC and in vivo-developed blastocysts were transferred to the uterine horns of pseudopregnant recipient females (CD1) 2.5 days postcoitus (dpc), and these embryos were harvested after 7 days (9.5 dpc).…”

Section: Embryo Collection Culture and Transfermentioning

confidence: 99%

“…The methods used for generation of ES cell lines have been previously described [35,38]. Briefly, blastocysts were transferred into gelatinized tissue culture wells (2-3 blastocysts per well of a 4-well multiplate, Nunc, Rochester, NY, USA) and cultured for 7 days in ES medium and DMEM (Gibco, Gland Island, NY, USA) containing 17.5% Knockout SR (Gibco) following standard procedures [39,40].…”

Section: Generation Of Es Cell Linesmentioning

confidence: 99%

The Dnmt3b Splice Variant is Specifically Expressed in In Vitro-manipulated Blastocysts and Their Derivative ES Cells

Horii

Suetake

Yanagisawa

et al. 2011

Journal of Reproduction and Development

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Abstract. Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos. Key words: DNA methylation, Dnmt3b, In vitro culture (J. Reprod. Dev. 57: 579-585, 2011) he 5th position cytosine residues in CpG sequences are often methylated in vertebrate genomic DNA [1]. DNA methylation plays an essential role in the normal development of mammalian embryos by regulating gene expression through genomic imprinting, X chromosome inactivation and genomic stability [2][3][4][5][6]. In vertebrates, two types of DNA methyltransferase activity have been reported, the de novo and maintenance types. In mice, de novo-type DNA methylation activity creates gene-specific methylation patterns during the implantation stage of embryogenesis, while maintenance-type activity ensures clonal transmission of lineage-specific methylation patterns during replication. Dnmt1 is responsible for the latter activity. On the other hand, two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for creation of methylation patterns during the early stages of embryogenesis [7,8] and have been shown to possess de novo-type DNA methylation activity in vitro [9][10][11][12]. Recent studies have shown that Dnmts function in cooperation with each other to facilitate DNA methylation in both humans and mice [13][14][15].In mice, Dnmt3b is the major de novo DNA methyltransferase in E (embryonic stage) 4.5-7.5 embryos, and its expression is downregulated after midgestation [8,16]. Disruption of Dnmt3b results in embryonic lethality at E13.5 and hypomethylation of centromeric minor satellite repeats [8]. In humans, DNMT3B mutations have been shown to cause ICF (immunodeficiency, chromosomal instabi...

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“…The use of ESP cells is limited by lack of the expression of paternally imprinted genes that are associated with fetal and placental growth due to the absence of the paternal genome. Therefore, parthenotes cannot develop to term (10,11). Kono et al (12) have demonstrated that parthenogenetic mice with a 13-kb deletion in the maternal imprinting gene H19, which is located on the same chromosome as the Igf2 gene, could develop to term.…”

Section: Introductionmentioning

confidence: 99%

Parthenogenetic embryonic stem cells with H19 siRNA-mediated knockdown as a potential resource for cell therapy

Kwak

Hong²,

Yu³

et al. 2011

Int J Mol Med

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Abstract. Embryonic stem (ES) cells are used in cell therapy and tissue engineering due to their ability to produce different cells types. However, studies of ES cells that are derived from fertilized embryos have raised concerns about the limitations imposed by ethical and political considerations. Therefore, many studies of stem cells use the stem cells that are derived from unfertilized oocytes and adult tissue. Although parthenogenetic embryonic stem (ESP) cells also avoid ethical and political dilemmas and can be used in cell-based therapy, the ESP cells exhibit growth retardation problems. Therefore, to investigate the potential for muscle growth from genetically modified ESP cells, we established four ES cell types, including normal embryonic stem (ESN) cells, ESP cells, ESP cells that overexpress the insulin-like growth factor 2 (Igf2) gene (ESI) and ESP cells with down-regulated H19 gene expression (ESH). Using these cells, we examined the expression profiles of genes that were related to imprinting and muscle using microarrays. The gene expression patterns of ESI and ESH cells were similar and were more closely related to the ESN pattern than that of the ESP cells. Differentiated ESH cells exhibited increased expression of bone morphologic protein 4 (BMP4), which is a mesoderm marker, compared with the differentiated ESI cells. We showed that Igf2 expression was induced by H19 silencing in the ESP cells via hypermethylation of the H19 imprinting control region 1 (ICR1). Moreover, the proportion of ESH-derived chimera was slightly higher than those produced from the ESP cells. In addition, we detected increased cell proliferation in the MEF cells following H19 knock-down. These results indicate that the ESH cells may be a source of cell-based therapy for conditions such as muscular atrophy.

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Loss of Genomic Imprinting in Mouse Parthenogenetic Embryonic Stem Cells

Cited by 43 publications

References 41 publications

Ectopic expression of Fgf3 leads to aberrant lineage segregation in the mouse parthenote preimplantation embryos

Ectopic expression of Fgf3 leads to aberrant lineage segregation in the mouse parthenote preimplantation embryos

The Dnmt3b Splice Variant is Specifically Expressed in In Vitro-manipulated Blastocysts and Their Derivative ES Cells

Parthenogenetic embryonic stem cells with H19 siRNA-mediated knockdown as a potential resource for cell therapy

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