Epigenetic Differences between Embryonic Stem Cells Generated from Blastocysts Developed<i>In Vitro</i>and<i>In Vivo</i>

Horii, Takuro; Yanagisawa, Eikichi; Kimura, Masashi; Morita, Sumiyo; Hatada, Izuho

doi:10.1089/cell.2009.0104

Cited by 15 publications

(23 citation statements)

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“…In mouse and human ESCs, epigenetic status can be affected by in vitro culture [44]. Surprisingly, Horii et al reported that epigenetic modification can occur within as few as 5 passages, over which time differences in expression patterns between ESCs established from embryos produced via IVF and in ESCs from in vivo embryos can disappear [45]. In our study, it is possible that in vitro culture of the fESCs altered their epigenetic profile, resulting in expression characteristics that were comparable to control ESCs.…”

Section: Discussionmentioning

confidence: 99%

Generation of Embryonic Stem Cell Lines from Immature Rabbit Ovarian Follicles

Teramura

Sugimoto

Frampton

et al. 2013

Stem Cells and Development

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In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection followed by in vitro culture. Although only weak expression of POU5f1 was observed in the inner cell masses of in-vitro-cultured follicle-derived embryos, repeated careful cloning enabled establishment of 3 stable ESC lines. These ESC lines displayed the morphological characteristics of primed pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1, and Sox2. Further, these ESCs could be differentiated into each of the 3 different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.

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Section: Discussionmentioning

confidence: 99%

Generation of Embryonic Stem Cell Lines from Immature Rabbit Ovarian Follicles

Teramura

Sugimoto

Frampton

et al. 2013

Stem Cells and Development

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“…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%

“…For densitometry, the Quantity One software (Bio-Rad, Hercules, CA, USA) was used according to the manufacturer's instructions, and the expression ratios of -exon 6/+exon 6 (-exon 5/+exon 5) were calculated. Quantitative real-time RT-PCR for Dnmt3b was also carried out as previously reported [38]. …”

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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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“…For example, a suboptimal culture medium can cause aberrant genomic imprinting of the Snrpn and H19 gene, whereas embryos cultured in potassium simplex optimized medium with added amino acids (KSOMAA) show global gene expression, genomic imprinting and embryo development resembling that found in in vivo developed embryos (Doherty et al, 2000;Mann et al, 2004). Furthermore, long term culture of ES cells also affects the methylation status of imprinted genes and their totipotency (Dean et al, 1998;Horii et al, 2010). A second consideration is that XX ES cells including PGES cells are more susceptible to demethylation than XO and XY ES cells (Zvetkova et al, 2005).…”

Section: Why Epigenetic Reprogramming Occurred In Pges Cells?mentioning

confidence: 99%