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Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

Epsztejn-Litman S, Cohen-Hadad Y, Aharoni S, Altarescu G, Renbaum P, Levy-Lahad E, Schonberger O, Eldar-Geva T, Zeligson S, Eiges R - PLoS ONE (2015)

Bottom Line: By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome.Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation.As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, Shaare Zedek Medical Center affiliated with the Hebrew University School of Medicine, Jerusalem, Israel.

ABSTRACT
We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

No MeSH data available.


Related in: MedlinePlus

Methylation levels at H19, SNRPN and MEST imprinted loci.Bisulfite single colony sequencing was performed on 3 different imprinted regions (H19, SNRPN and MEST) to determine methylation levels in normal (WT) and SZ-SMA5 HESC lines. Each line represents a single DNA molecule. Full circles correspond to methylated CpGs while empty circles represent unmethylated CpGs.
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pone.0138893.g003: Methylation levels at H19, SNRPN and MEST imprinted loci.Bisulfite single colony sequencing was performed on 3 different imprinted regions (H19, SNRPN and MEST) to determine methylation levels in normal (WT) and SZ-SMA5 HESC lines. Each line represents a single DNA molecule. Full circles correspond to methylated CpGs while empty circles represent unmethylated CpGs.

Mentions: To explain the absence of paternal SMN1-flanking alleles in an embryo with a 46(XX) karyotype and homozygosity for the SMN1 deletion, we speculated that either SZ-SMA5 had maternal uniparental isodisomy (UPD) for chromosome 5 (SMN1 is located in 5q13.2) or alternatively, it may be a product of parthenogenesis. To distinguish between these two genomic states, we determined methylation levels of the cells at three different previously characterized imprinted loci. DNA methylation status was determined for the maternal imprinted loci SNRPN, and MEST (expected to be differentially methylated when transmitted by the mother) and for the paternal imprinted locus H19 (expected to be differentially methylated when transmitted by the father). DNA bisulfite colony sequencing showed that, in contrast to normal HESCs (which reveal approximately 50% methylation in all of the aforementioned loci), the SNRPN and MEST were totally methylated (100%); while the H19 was completely unmethylated (0%) (Fig 3). These methylation patterns are characteristic to cells that contain maternal but not paternal genomes and are in line with the differential expression of maternal, but not paternal, inherited genes such as H19 and SNRPN, respectively (S1 Fig). Therefore, based on the methylation status of three different imprinted genomic regions and the expression of two oppositely imprinted genes, we hypothesized that SZ-SMA5 was established from a maternal uniparental embryo.


Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

Epsztejn-Litman S, Cohen-Hadad Y, Aharoni S, Altarescu G, Renbaum P, Levy-Lahad E, Schonberger O, Eldar-Geva T, Zeligson S, Eiges R - PLoS ONE (2015)

Methylation levels at H19, SNRPN and MEST imprinted loci.Bisulfite single colony sequencing was performed on 3 different imprinted regions (H19, SNRPN and MEST) to determine methylation levels in normal (WT) and SZ-SMA5 HESC lines. Each line represents a single DNA molecule. Full circles correspond to methylated CpGs while empty circles represent unmethylated CpGs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608834&req=5

pone.0138893.g003: Methylation levels at H19, SNRPN and MEST imprinted loci.Bisulfite single colony sequencing was performed on 3 different imprinted regions (H19, SNRPN and MEST) to determine methylation levels in normal (WT) and SZ-SMA5 HESC lines. Each line represents a single DNA molecule. Full circles correspond to methylated CpGs while empty circles represent unmethylated CpGs.
Mentions: To explain the absence of paternal SMN1-flanking alleles in an embryo with a 46(XX) karyotype and homozygosity for the SMN1 deletion, we speculated that either SZ-SMA5 had maternal uniparental isodisomy (UPD) for chromosome 5 (SMN1 is located in 5q13.2) or alternatively, it may be a product of parthenogenesis. To distinguish between these two genomic states, we determined methylation levels of the cells at three different previously characterized imprinted loci. DNA methylation status was determined for the maternal imprinted loci SNRPN, and MEST (expected to be differentially methylated when transmitted by the mother) and for the paternal imprinted locus H19 (expected to be differentially methylated when transmitted by the father). DNA bisulfite colony sequencing showed that, in contrast to normal HESCs (which reveal approximately 50% methylation in all of the aforementioned loci), the SNRPN and MEST were totally methylated (100%); while the H19 was completely unmethylated (0%) (Fig 3). These methylation patterns are characteristic to cells that contain maternal but not paternal genomes and are in line with the differential expression of maternal, but not paternal, inherited genes such as H19 and SNRPN, respectively (S1 Fig). Therefore, based on the methylation status of three different imprinted genomic regions and the expression of two oppositely imprinted genes, we hypothesized that SZ-SMA5 was established from a maternal uniparental embryo.

Bottom Line: By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome.Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation.As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, Shaare Zedek Medical Center affiliated with the Hebrew University School of Medicine, Jerusalem, Israel.

ABSTRACT
We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

No MeSH data available.


Related in: MedlinePlus