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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

Whole genome view of Cytoscan SNP Array data.Genome wide SNP array results obtained from (A) reference DNA from a male with a normal karyotype (46; XY); and (B) SZ-SMA5 HESC line DNA. The X axis represents chromosomes 1–22, X and Y. (I) The Y axis represents the copy number, determined by the log2 ratio (grey dots) on the left side of the graph, and it's smoothed ratio (red line) on the right. The expected copy number is 2 for autosomal chromosomes (log2 of 0 and smooth signal of 2). The log2 ratio and the smooth signal are determined from both the nonpolymorphic copy number probes and the polymorphic SNP probes. (II) The Y-axis corresponds to homozygote calls (AA or BB) and heterozygote calls (AB). Allele peaks of 1, 0, and -1 indicate a copy number of two, while allele peaks of 0.5 and -0.5 indicate a copy number of one. Allele peaks are calculated from SNP probes. The distinction between XY (reference DNA) and XX (SZ-SMA5) cells is clearly illustrated by the difference in X chromosome copy number. In addition, the overall 0.49% inherent heterozygote call error rate in SZ-SMA5 is below even the expected array genotyping error of ~1% (as determined by dividing the number of heterozygous calls by the total number of SNP probes on the array). Therefore, these data indicate that SZ-SMA5 features a completely homozygous diploid genome. (C) Fraction of SNP heterozygote calls in WT male reference and SZ-SMA5 DNA. Chromosomes are indicated in the X axis and the Y axis indicates the fraction of heterozygous SNP calls per total SNP calls on each chromosome.
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pone.0138893.g004: Whole genome view of Cytoscan SNP Array data.Genome wide SNP array results obtained from (A) reference DNA from a male with a normal karyotype (46; XY); and (B) SZ-SMA5 HESC line DNA. The X axis represents chromosomes 1–22, X and Y. (I) The Y axis represents the copy number, determined by the log2 ratio (grey dots) on the left side of the graph, and it's smoothed ratio (red line) on the right. The expected copy number is 2 for autosomal chromosomes (log2 of 0 and smooth signal of 2). The log2 ratio and the smooth signal are determined from both the nonpolymorphic copy number probes and the polymorphic SNP probes. (II) The Y-axis corresponds to homozygote calls (AA or BB) and heterozygote calls (AB). Allele peaks of 1, 0, and -1 indicate a copy number of two, while allele peaks of 0.5 and -0.5 indicate a copy number of one. Allele peaks are calculated from SNP probes. The distinction between XY (reference DNA) and XX (SZ-SMA5) cells is clearly illustrated by the difference in X chromosome copy number. In addition, the overall 0.49% inherent heterozygote call error rate in SZ-SMA5 is below even the expected array genotyping error of ~1% (as determined by dividing the number of heterozygous calls by the total number of SNP probes on the array). Therefore, these data indicate that SZ-SMA5 features a completely homozygous diploid genome. (C) Fraction of SNP heterozygote calls in WT male reference and SZ-SMA5 DNA. Chromosomes are indicated in the X axis and the Y axis indicates the fraction of heterozygous SNP calls per total SNP calls on each chromosome.

Mentions: To provide an additional assay that distinguishes between maternal UPD and parthenogenesis, we determined the parental identity and degree of heterozygosity of the cell line by genome-wide SNP microarray analysis (Fig 4). Indeed, this comprehensive assessment confirmed homozygosity across the entire genome of SZ-SMA5 (Fig 4A and 4B, S2 Fig and S1 File). Strikingly, this complete absence of heterozygosity (<1%) is evident in each chromosome of SZ-SMA5 as compared with reference DNA (which averages 23%-30% heterozygous SNPs per chromosome; Fig 4C). Therefore, these data unambiguously demonstrate that SZ-SMA5 has originated from a reduplication of a haploid set of chromosomes. This experiment, together with bisulfite sequencing, imprinted gene expression, and haplotype data provides firm evidence that the HESC line in question was in fact derived from a maternal uniparental embryo. Since we could not find any indication for recombination events by SNP array, we conclude that parthenogenesis was achieved through the activation of a mature unfertilized mutant oocyte. Thus, our SMA pHESC line represents a proof-of-principle outcome of mutant oocyte conversion into diploid homozygous mutation-bearing HESCs without need for gene manipulation.


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)

Whole genome view of Cytoscan SNP Array data.Genome wide SNP array results obtained from (A) reference DNA from a male with a normal karyotype (46; XY); and (B) SZ-SMA5 HESC line DNA. The X axis represents chromosomes 1–22, X and Y. (I) The Y axis represents the copy number, determined by the log2 ratio (grey dots) on the left side of the graph, and it's smoothed ratio (red line) on the right. The expected copy number is 2 for autosomal chromosomes (log2 of 0 and smooth signal of 2). The log2 ratio and the smooth signal are determined from both the nonpolymorphic copy number probes and the polymorphic SNP probes. (II) The Y-axis corresponds to homozygote calls (AA or BB) and heterozygote calls (AB). Allele peaks of 1, 0, and -1 indicate a copy number of two, while allele peaks of 0.5 and -0.5 indicate a copy number of one. Allele peaks are calculated from SNP probes. The distinction between XY (reference DNA) and XX (SZ-SMA5) cells is clearly illustrated by the difference in X chromosome copy number. In addition, the overall 0.49% inherent heterozygote call error rate in SZ-SMA5 is below even the expected array genotyping error of ~1% (as determined by dividing the number of heterozygous calls by the total number of SNP probes on the array). Therefore, these data indicate that SZ-SMA5 features a completely homozygous diploid genome. (C) Fraction of SNP heterozygote calls in WT male reference and SZ-SMA5 DNA. Chromosomes are indicated in the X axis and the Y axis indicates the fraction of heterozygous SNP calls per total SNP calls on each chromosome.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138893.g004: Whole genome view of Cytoscan SNP Array data.Genome wide SNP array results obtained from (A) reference DNA from a male with a normal karyotype (46; XY); and (B) SZ-SMA5 HESC line DNA. The X axis represents chromosomes 1–22, X and Y. (I) The Y axis represents the copy number, determined by the log2 ratio (grey dots) on the left side of the graph, and it's smoothed ratio (red line) on the right. The expected copy number is 2 for autosomal chromosomes (log2 of 0 and smooth signal of 2). The log2 ratio and the smooth signal are determined from both the nonpolymorphic copy number probes and the polymorphic SNP probes. (II) The Y-axis corresponds to homozygote calls (AA or BB) and heterozygote calls (AB). Allele peaks of 1, 0, and -1 indicate a copy number of two, while allele peaks of 0.5 and -0.5 indicate a copy number of one. Allele peaks are calculated from SNP probes. The distinction between XY (reference DNA) and XX (SZ-SMA5) cells is clearly illustrated by the difference in X chromosome copy number. In addition, the overall 0.49% inherent heterozygote call error rate in SZ-SMA5 is below even the expected array genotyping error of ~1% (as determined by dividing the number of heterozygous calls by the total number of SNP probes on the array). Therefore, these data indicate that SZ-SMA5 features a completely homozygous diploid genome. (C) Fraction of SNP heterozygote calls in WT male reference and SZ-SMA5 DNA. Chromosomes are indicated in the X axis and the Y axis indicates the fraction of heterozygous SNP calls per total SNP calls on each chromosome.
Mentions: To provide an additional assay that distinguishes between maternal UPD and parthenogenesis, we determined the parental identity and degree of heterozygosity of the cell line by genome-wide SNP microarray analysis (Fig 4). Indeed, this comprehensive assessment confirmed homozygosity across the entire genome of SZ-SMA5 (Fig 4A and 4B, S2 Fig and S1 File). Strikingly, this complete absence of heterozygosity (<1%) is evident in each chromosome of SZ-SMA5 as compared with reference DNA (which averages 23%-30% heterozygous SNPs per chromosome; Fig 4C). Therefore, these data unambiguously demonstrate that SZ-SMA5 has originated from a reduplication of a haploid set of chromosomes. This experiment, together with bisulfite sequencing, imprinted gene expression, and haplotype data provides firm evidence that the HESC line in question was in fact derived from a maternal uniparental embryo. Since we could not find any indication for recombination events by SNP array, we conclude that parthenogenesis was achieved through the activation of a mature unfertilized mutant oocyte. Thus, our SMA pHESC line represents a proof-of-principle outcome of mutant oocyte conversion into diploid homozygous mutation-bearing HESCs without need for gene manipulation.

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