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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

Characterization of SZ-SMA5.(A) Expression of undifferentiated cell specific markers by immunostaining for OCT4 (red nuclear staining, merged onto Hoechst (blue)), for the cell surface marker Tra 1–60 (red, merged onto Hoechst (blue)); and (B) for alkaline phosphatase activity (AP). (C) Expression of undifferentiated cell specific markers: OCT4, REX1, NANOG and SOX2 in SZ-SMA5 and a wild-type (WT) HESC control by RT-PCR. GAPDH expression served as a loading control. (D) Teratoma sections stained by H&E from SZ-SMA5 demonstrating multi-cellular structures derived from the three different embryonic germ layers. (E) A representative normal 46(XX) karyotype in SZ-SMA5 as identified by Giemsa staining of 20 different spreads of metaphase chromosomes.
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pone.0138893.g001: Characterization of SZ-SMA5.(A) Expression of undifferentiated cell specific markers by immunostaining for OCT4 (red nuclear staining, merged onto Hoechst (blue)), for the cell surface marker Tra 1–60 (red, merged onto Hoechst (blue)); and (B) for alkaline phosphatase activity (AP). (C) Expression of undifferentiated cell specific markers: OCT4, REX1, NANOG and SOX2 in SZ-SMA5 and a wild-type (WT) HESC control by RT-PCR. GAPDH expression served as a loading control. (D) Teratoma sections stained by H&E from SZ-SMA5 demonstrating multi-cellular structures derived from the three different embryonic germ layers. (E) A representative normal 46(XX) karyotype in SZ-SMA5 as identified by Giemsa staining of 20 different spreads of metaphase chromosomes.

Mentions: The SMA pHESC line that we established was derived from an embryo, obtained by PGD, to a couple that carries a common deletion of the SMN1 gene. Following haplotype analysis using 8 tightly linked informative markers, the embryo was typed as potentially affected since it carried the mutant chromosome of the mother, but failed to reveal the alleles of the father. As it had a 50% risk of being affected, it was classified as disqualified for embryo transfer and donated for HESC lines derivation by the parents (IRB 87/07). The newly established cell line (termed SZ-SMA5) displays all key features of pluripotent cells. It has an ES-like cell morphology, is capable of unrestricted growth in culture, and it expresses the typical markers of undifferentiated cells (Fig 1A–1C). In addition, SZ-SMA5 has the potential to differentiate into a wide range of cell types since it can be used to induce teratomas in immune-compromised mice (Fig 1D). In addition, Giemsa staining of 20 different high quality metaphase spreads demonstrated, without exception, a normal diploid 46(XX) karyotype (Fig 1E).


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)

Characterization of SZ-SMA5.(A) Expression of undifferentiated cell specific markers by immunostaining for OCT4 (red nuclear staining, merged onto Hoechst (blue)), for the cell surface marker Tra 1–60 (red, merged onto Hoechst (blue)); and (B) for alkaline phosphatase activity (AP). (C) Expression of undifferentiated cell specific markers: OCT4, REX1, NANOG and SOX2 in SZ-SMA5 and a wild-type (WT) HESC control by RT-PCR. GAPDH expression served as a loading control. (D) Teratoma sections stained by H&E from SZ-SMA5 demonstrating multi-cellular structures derived from the three different embryonic germ layers. (E) A representative normal 46(XX) karyotype in SZ-SMA5 as identified by Giemsa staining of 20 different spreads of metaphase chromosomes.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138893.g001: Characterization of SZ-SMA5.(A) Expression of undifferentiated cell specific markers by immunostaining for OCT4 (red nuclear staining, merged onto Hoechst (blue)), for the cell surface marker Tra 1–60 (red, merged onto Hoechst (blue)); and (B) for alkaline phosphatase activity (AP). (C) Expression of undifferentiated cell specific markers: OCT4, REX1, NANOG and SOX2 in SZ-SMA5 and a wild-type (WT) HESC control by RT-PCR. GAPDH expression served as a loading control. (D) Teratoma sections stained by H&E from SZ-SMA5 demonstrating multi-cellular structures derived from the three different embryonic germ layers. (E) A representative normal 46(XX) karyotype in SZ-SMA5 as identified by Giemsa staining of 20 different spreads of metaphase chromosomes.
Mentions: The SMA pHESC line that we established was derived from an embryo, obtained by PGD, to a couple that carries a common deletion of the SMN1 gene. Following haplotype analysis using 8 tightly linked informative markers, the embryo was typed as potentially affected since it carried the mutant chromosome of the mother, but failed to reveal the alleles of the father. As it had a 50% risk of being affected, it was classified as disqualified for embryo transfer and donated for HESC lines derivation by the parents (IRB 87/07). The newly established cell line (termed SZ-SMA5) displays all key features of pluripotent cells. It has an ES-like cell morphology, is capable of unrestricted growth in culture, and it expresses the typical markers of undifferentiated cells (Fig 1A–1C). In addition, SZ-SMA5 has the potential to differentiate into a wide range of cell types since it can be used to induce teratomas in immune-compromised mice (Fig 1D). In addition, Giemsa staining of 20 different high quality metaphase spreads demonstrated, without exception, a normal diploid 46(XX) karyotype (Fig 1E).

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