Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James A.; Ferguson, Betsy; Gokhale, Sumita; Penedo, M. C. T.; Wolf, Don P.; Mitalipov, Shoukhrat

doi:10.1634/stemcells.2007-0869

Cited by 61 publications

(73 citation statements)

References 37 publications

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“…Methods for isolation and culture of monkey ESCs from in vitro fertilization, SCNT, and parthenogenetic embryos used in this study were reported previously (Mitalipov et al 2006;Byrne et al 2007;Dighe et al 2008). Briefly, ESCs were grown on feeder layers (mouse embryonic fibroblasts, mEFs) in DMEM/F12 medium with glucose and without sodium pyruvate, supplemented with 1% nonessential amino acids, 2 mM L-glutamine, 0.1 mM b-mercaptoethanol, and 15% FBS at 37°C, 3% CO 2, 5% O 2 , and 92% N 2 .…”

Section: Stem Cells Culturementioning

confidence: 99%

DNA methylation programming and reprogramming in primate embryonic stem cells

et al. 2009

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DNA methylation is an important epigenetic mechanism, affecting normal development and playing a key role in reprogramming epigenomes during stem cell derivation. Here we report on DNA methylation patterns in native monkey embryonic stem cells (ESCs), fibroblasts, and ESCs generated through somatic cell nuclear transfer (SCNT), identifying and comparing epigenome programming and reprogramming. We characterize hundreds of regions that are hyper-or hypomethylated in fibroblasts compared to native ESCs and show that these are conserved in human cells and tissues. Remarkably, the vast majority of these regions are reprogrammed in SCNT ESCs, leading to almost perfect correlation between the epigenomic profiles of the native and reprogrammed lines. At least 58% of these changes are correlated in cis to transcription changes, Polycomb Repressive Complex-2 occupancy, or binding by the CTCF insulator. We also show that while epigenomic reprogramming is extensive and globally accurate, the efficiency of adding and stripping DNA methylation during reprogramming is regionally variable. In several cases, this variability results in regions that remain methylated in a fibroblast-like pattern even after reprogramming.[Supplemental material is available online at http://www.genome.org. DNA methylation profiles from this study have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE17981.]DNA methylation is considered a key factor in the formation of cellular memory and identity, but owing to experimental and conceptual limitations, we still do not truly understand how the cell writes and erases DNA methylation marks in the course of normal cellular differentiation, and how these marks revert to their original embryonic stem cell (ESC)-like form following somatic cell nuclear transfer or induced pluripotent stem cell (iPS) reprogramming (Reik 2007). Progress in the field was hampered for years by lack of quality methods for high-throughput DNA methylation profiling, but recently several effective assays for profiling DNA methylation in large fractions of the mammalian genome were developed and applied successfully (Weber et al. 2005;Keshet et al. 2006;Rollins et al. 2006;Cokus et al. 2008;Irizarry et al. 2008;Meissner et al. 2008). Another major source of confusion and difficulty in understanding the role of mammalian DNA methylation is the nonuniform CpG content of the genome, which led most of the experimental attention toward regions with high CpG content (CpG islands). Recent evidence suggests that classical CpG islands of high CpG content are almost never methylated under normal conditions, yet much dynamic DNA methylation (manifested as differentially methylated regions, DMRs) can be found in regions with intermediate CpG content, some of which are classically defined as CpG islands and some of which are not (Irizarry et al. 2009;Straussman et al. 2009). Adding to these difficulties, multiple studies have shown that DNA methylation is stably acquired in culture, forming s...

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Section: Stem Cells Culturementioning

confidence: 99%

DNA methylation programming and reprogramming in primate embryonic stem cells

et al. 2009

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“…Since the expression of GFP and MeCP2 transgenes was driven by the same promoter, the expression of GFP is a good indicator for the expression of MeCP2. Short tandem repeat (STR) genotyping and single-nucleotide polymorphism (SNP) analysis were performed to examine the parental origin of the F1 monkeys [11]. The analysis of 23 STR loci of the 7 XG monkeys confirmed their paternal origin (Supplementary information, Table S1B).…”

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confidence: 92%

Generation of macaques with sperm derived from juvenile monkey testicular xenografts

et al. 2015

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“…Recently, a considerable number of studies has reported similar abnormal karyotypes, with y chromosome deletion observed in SCNT monkey ES cell lines (8), and recurrent genomic instability has been reported in hES cell lines over long-term culture (9). Genomic imprinting is a genetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner.…”

Section: Discussionmentioning

confidence: 86%

Epigenetic signatures of somatic cell nuclear transfer-derived embryonic stem cells

Jung

Son²,

Jeung³

et al. 2011

Int J Mol Med

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Abstract. Some human embryonic stem cell lines have shown genomic instabilities over long-term culture. To study the controversial origin of the SCNT-hES-1 line, which was derived from autologous somatic cell nuclear transfer (SCNT), we compared the expression and methylation patterns of imprinted genes in the SCNT-hES-1 cells with the donor's somatic cells by semi-quantitative RT-PCR, real-time PCR and bisulfite sequencing. Examined imprinted genes were H19, GNAS, SLC22A18, UBE3A and ZNF264 for maternally expressed genes, and IGF2, SNRPN, PEG3, PEG10, MEST, MAGEL2 and ARHI for paternally expressed genes, respectively. We found that the expression of imprinted genes in the SCNT-hES-1 cell line is comparable to that in the donor's somatic cells, and that its methylation patterns are similar to those of other SCNT-products. Therefore, the present study indicates that the SCNT-hES-1 line was derived from SCNT. IntroductionThe somatic cell nuclear transfer (SCNT) technique has been widely acknowledged for its potential utility for regenerative medicine (1-5). Hwang et al (4) claimed that they produced the first human embryonic stem cell line, SCNT-hES-1, via autologous SCNT, by transferring the donor's own somatic cell into the donor's own enucleated oocyte. Because autologous SCNT provides the molecular and genetic identity for a reconstructed SCNT embryo, it may decrease deficient epigenetic reprogramming of the donor cell nucleus in an enucleated oocyte. Also, to overcome inefficiencies of human SCNT, autologous SCNT was performed in the production of SCNT-hES-1 (4).However, for this reason, there have been disputes over its origin.The Seoul National University Investigation Committee (SNUIC) has insisted that parthenogenetic SCNT-hES-1 were generated by the re-influx of the first polar body using only an analysis of short tandem repeat (STR) DNA. However, Kim et al (6) claimed that there was no scientific evidence for SNUIC's assumption stated above, the re-influx of the first polar body, and that SCNT-hES-1 was created by parthenogenesis derived from inhibition of second polar body's extrusion as supported by analysis of single nucleotide polymorphisms (SNP) and methylation patterns in the cell line. Their SNP results indicated that the line appeared to be homozygous in the centromeric region of nearly every chromosome (6). Therefore, the parthenogenic origin of SCNT-hES-1 is unclear. They also insisted that the SCNT-hES-1 line originated from parthenogenesis based on an analysis of DNA methylation patterns, despite the absence of comparison with the donor's somatic cells or of the IVF-ES line as a control (6). Indeed, it is difficult to determine the origin of SCNT products (cloned embryos or ES cells) by methylation patterns alone, because there are aberrant methylation patterns in various genomic regions after SCNT (7). It has also been questioned why they used the SCNT-hES-1 line with an abnormal karyotype (45, XO) for their experiments (6), despite the existence of the line with normal karyotype (46, XX).T...

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Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

Cited by 61 publications

References 37 publications

DNA methylation programming and reprogramming in primate embryonic stem cells

DNA methylation programming and reprogramming in primate embryonic stem cells

Generation of macaques with sperm derived from juvenile monkey testicular xenografts

Epigenetic signatures of somatic cell nuclear transfer-derived embryonic stem cells

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