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Functional neuronal cells generated by human parthenogenetic stem cells.

Ahmad R, Wolber W, Eckardt S, Koch P, Schmitt J, Semechkin R, Geis C, Heckmann M, Brüstle O, McLaughlin JK, Sirén AL, Müller AM - PLoS ONE (2012)

Bottom Line: Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation.Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes.Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Radiation and Cell Research in the Center for Experimental Molecular Medicine, University of Würzburg, Würzburg, Germany.

ABSTRACT
Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.

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Analysis of the methylation status of differentially methylated regions (DMRs) and expression analysis of imprinted genes.Shown are comparative bisulfite sequencing analysis and analysis of imprinted gene expression in hESCs, hNSCs (I3) and in hpESCs, hpNSCs (LLC9P) respectively. (A) Bisulfite sequencing of KvDMR1 (position: 66531–66801) in N and PG cells. The location of KvDMR1 and the transcriptional start sites of Cdkn1c, Kcnq1ot1 and Kcnq1 are indicated (maternal allele). Black boxes represent methylated CpGs; grey boxes show unmethylated CpGs; white boxes: not analyzed. Percentages of CpG methylation are indicated. (B) Shown are RT-PCR analyses of imprinted genes regulated by Kcnq1ot1 long non-coding RNA in PG and N cells. The relative expression represents the fold change of gene expression in PG compared to N cells, respectively. Fold change was calculated by the 2−ΔΔCt method. The housekeeping gene GAPDH was used as a reference gene. Expression levels of N cells were set to 1. n = 3. (C) Shown are the location of DMR1 (position: 66531–66801), gene regions of Igf2 and H19 (maternal allel) and results of bisulfite sequencing analyses of DMR1. (D) Shown are gene expression analyses of Igf2 and H19 in N and PG cells. n = 3. (E) Expression analysis of imprinted genes of other loci by quantitative RT-PCR. n = 3, * p<0.05, ** p<0.01, *** p<0.001 by Student's t-test.
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pone-0042800-g005: Analysis of the methylation status of differentially methylated regions (DMRs) and expression analysis of imprinted genes.Shown are comparative bisulfite sequencing analysis and analysis of imprinted gene expression in hESCs, hNSCs (I3) and in hpESCs, hpNSCs (LLC9P) respectively. (A) Bisulfite sequencing of KvDMR1 (position: 66531–66801) in N and PG cells. The location of KvDMR1 and the transcriptional start sites of Cdkn1c, Kcnq1ot1 and Kcnq1 are indicated (maternal allele). Black boxes represent methylated CpGs; grey boxes show unmethylated CpGs; white boxes: not analyzed. Percentages of CpG methylation are indicated. (B) Shown are RT-PCR analyses of imprinted genes regulated by Kcnq1ot1 long non-coding RNA in PG and N cells. The relative expression represents the fold change of gene expression in PG compared to N cells, respectively. Fold change was calculated by the 2−ΔΔCt method. The housekeeping gene GAPDH was used as a reference gene. Expression levels of N cells were set to 1. n = 3. (C) Shown are the location of DMR1 (position: 66531–66801), gene regions of Igf2 and H19 (maternal allel) and results of bisulfite sequencing analyses of DMR1. (D) Shown are gene expression analyses of Igf2 and H19 in N and PG cells. n = 3. (E) Expression analysis of imprinted genes of other loci by quantitative RT-PCR. n = 3, * p<0.05, ** p<0.01, *** p<0.001 by Student's t-test.

Mentions: To assess the status of epigenetic marks involved in the control of imprinted gene expression during neural differentiation of hpESCs, we analyzed the methylation status of CpG islands of two differentially methylated regions, the 5′ region of the long non-coding RNA Kcnq1ot1 (KvDMR1) and the H19 DMR1 (Fig. 5). Methylation of KvDMR1 on the maternal allele, acquired during germ cell development, is associated with silencing of Kcnq1ot1, whereas Kcnq1ot1 expression from the unmethylated paternal allele is involved in domain-wide chromatin repression of a cluster of genes including Cdkn1c and Kcnq1[33]. Consistent with PG origin, CpGs of the KvDMR1 were mostly methylated in hpESCs and hpNSCs, while conventional hESCs and hNSCs exhibited 50% methylation, indicating the presence of maternal and paternal alleles (Fig. 5A). Quantitative RT-PCR analysis revealed absence of Kcnq1ot1 RNA in hpESCs and hpNSCs, and higher expression of Kcnq1 but not Cdkn1c in PG compared to N cells (Fig. 5B).


Functional neuronal cells generated by human parthenogenetic stem cells.

Ahmad R, Wolber W, Eckardt S, Koch P, Schmitt J, Semechkin R, Geis C, Heckmann M, Brüstle O, McLaughlin JK, Sirén AL, Müller AM - PLoS ONE (2012)

Analysis of the methylation status of differentially methylated regions (DMRs) and expression analysis of imprinted genes.Shown are comparative bisulfite sequencing analysis and analysis of imprinted gene expression in hESCs, hNSCs (I3) and in hpESCs, hpNSCs (LLC9P) respectively. (A) Bisulfite sequencing of KvDMR1 (position: 66531–66801) in N and PG cells. The location of KvDMR1 and the transcriptional start sites of Cdkn1c, Kcnq1ot1 and Kcnq1 are indicated (maternal allele). Black boxes represent methylated CpGs; grey boxes show unmethylated CpGs; white boxes: not analyzed. Percentages of CpG methylation are indicated. (B) Shown are RT-PCR analyses of imprinted genes regulated by Kcnq1ot1 long non-coding RNA in PG and N cells. The relative expression represents the fold change of gene expression in PG compared to N cells, respectively. Fold change was calculated by the 2−ΔΔCt method. The housekeeping gene GAPDH was used as a reference gene. Expression levels of N cells were set to 1. n = 3. (C) Shown are the location of DMR1 (position: 66531–66801), gene regions of Igf2 and H19 (maternal allel) and results of bisulfite sequencing analyses of DMR1. (D) Shown are gene expression analyses of Igf2 and H19 in N and PG cells. n = 3. (E) Expression analysis of imprinted genes of other loci by quantitative RT-PCR. n = 3, * p<0.05, ** p<0.01, *** p<0.001 by Student's t-test.
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getmorefigures.php?uid=PMC3412801&req=5

pone-0042800-g005: Analysis of the methylation status of differentially methylated regions (DMRs) and expression analysis of imprinted genes.Shown are comparative bisulfite sequencing analysis and analysis of imprinted gene expression in hESCs, hNSCs (I3) and in hpESCs, hpNSCs (LLC9P) respectively. (A) Bisulfite sequencing of KvDMR1 (position: 66531–66801) in N and PG cells. The location of KvDMR1 and the transcriptional start sites of Cdkn1c, Kcnq1ot1 and Kcnq1 are indicated (maternal allele). Black boxes represent methylated CpGs; grey boxes show unmethylated CpGs; white boxes: not analyzed. Percentages of CpG methylation are indicated. (B) Shown are RT-PCR analyses of imprinted genes regulated by Kcnq1ot1 long non-coding RNA in PG and N cells. The relative expression represents the fold change of gene expression in PG compared to N cells, respectively. Fold change was calculated by the 2−ΔΔCt method. The housekeeping gene GAPDH was used as a reference gene. Expression levels of N cells were set to 1. n = 3. (C) Shown are the location of DMR1 (position: 66531–66801), gene regions of Igf2 and H19 (maternal allel) and results of bisulfite sequencing analyses of DMR1. (D) Shown are gene expression analyses of Igf2 and H19 in N and PG cells. n = 3. (E) Expression analysis of imprinted genes of other loci by quantitative RT-PCR. n = 3, * p<0.05, ** p<0.01, *** p<0.001 by Student's t-test.
Mentions: To assess the status of epigenetic marks involved in the control of imprinted gene expression during neural differentiation of hpESCs, we analyzed the methylation status of CpG islands of two differentially methylated regions, the 5′ region of the long non-coding RNA Kcnq1ot1 (KvDMR1) and the H19 DMR1 (Fig. 5). Methylation of KvDMR1 on the maternal allele, acquired during germ cell development, is associated with silencing of Kcnq1ot1, whereas Kcnq1ot1 expression from the unmethylated paternal allele is involved in domain-wide chromatin repression of a cluster of genes including Cdkn1c and Kcnq1[33]. Consistent with PG origin, CpGs of the KvDMR1 were mostly methylated in hpESCs and hpNSCs, while conventional hESCs and hNSCs exhibited 50% methylation, indicating the presence of maternal and paternal alleles (Fig. 5A). Quantitative RT-PCR analysis revealed absence of Kcnq1ot1 RNA in hpESCs and hpNSCs, and higher expression of Kcnq1 but not Cdkn1c in PG compared to N cells (Fig. 5B).

Bottom Line: Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation.Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes.Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Radiation and Cell Research in the Center for Experimental Molecular Medicine, University of Würzburg, Würzburg, Germany.

ABSTRACT
Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.

Show MeSH
Related in: MedlinePlus