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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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Differentiation of hpNSCs towards dopaminergic neurons and motoneurons.(A) Schematic representation of in vitro neural differentiation of hpESCs (LLC9P) towards dopaminergic neurons and motoneurons (adapted from [26]). (B) RT-PCR analysis for the expression of midbrain transcripts (Nurr1, En1 and Pax2). Immunostaining with antibodies specific for En1, Pitx3 and TH. Cells were co-stained with DAPI. Percentages of DAPI and En1, Pitx3 or TH positive cells are indicated. (C) RT-PCR analysis for the expression HoxA1 and HoxA2. Shown are immunostainings with antibodies specific for: Isl1, Nkx2.2 and MAP2/HB9. Cells were co-stained with DAPI. Shown are percentages of DAPI and Isl1, Nkx2.2 and HB9- and DAPI-positive cells. Scale bars: 50 µm; n = 3.
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pone-0042800-g003: Differentiation of hpNSCs towards dopaminergic neurons and motoneurons.(A) Schematic representation of in vitro neural differentiation of hpESCs (LLC9P) towards dopaminergic neurons and motoneurons (adapted from [26]). (B) RT-PCR analysis for the expression of midbrain transcripts (Nurr1, En1 and Pax2). Immunostaining with antibodies specific for En1, Pitx3 and TH. Cells were co-stained with DAPI. Percentages of DAPI and En1, Pitx3 or TH positive cells are indicated. (C) RT-PCR analysis for the expression HoxA1 and HoxA2. Shown are immunostainings with antibodies specific for: Isl1, Nkx2.2 and MAP2/HB9. Cells were co-stained with DAPI. Shown are percentages of DAPI and Isl1, Nkx2.2 and HB9- and DAPI-positive cells. Scale bars: 50 µm; n = 3.

Mentions: Next we analyzed whether hpNSCs remain responsive to instructive regionalization cues known to induce dopaminergic [27] or motoneuron differentiations [28]. To induce formation of TH+ (tyrosine hydroxylase)-neurons, hpNSCs were first cultured in media supplemented with sonic hedgehog (SHH) and FGF8b, followed by culture in media containing BDNF and GDNF (Fig. 3A, top). After 28 days of differentiation, cells expressed transcripts for the midbrain-specific Nurr1 (nuclear receptor related 1 protein), En1 (engrailed homeobox 1) and Pax2 (paired box gene 2) (Fig. 3B). Immunocytochemical staining verified upregulation of the midbrain markers En1 and Pitx3 (paired-like homeodomain 3), which are transcription factors required for differentiation and survival of midbrain dopaminergic neurons, and for TH (dopamine biogenesis) (Fig. 3B). We observed 79.8±3.2% En1, 10.8±0.6% Pitx3 and 13.3±1.6% TH positive cells (Fig. 3B).


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)

Differentiation of hpNSCs towards dopaminergic neurons and motoneurons.(A) Schematic representation of in vitro neural differentiation of hpESCs (LLC9P) towards dopaminergic neurons and motoneurons (adapted from [26]). (B) RT-PCR analysis for the expression of midbrain transcripts (Nurr1, En1 and Pax2). Immunostaining with antibodies specific for En1, Pitx3 and TH. Cells were co-stained with DAPI. Percentages of DAPI and En1, Pitx3 or TH positive cells are indicated. (C) RT-PCR analysis for the expression HoxA1 and HoxA2. Shown are immunostainings with antibodies specific for: Isl1, Nkx2.2 and MAP2/HB9. Cells were co-stained with DAPI. Shown are percentages of DAPI and Isl1, Nkx2.2 and HB9- and DAPI-positive cells. Scale bars: 50 µm; n = 3.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412801&req=5

pone-0042800-g003: Differentiation of hpNSCs towards dopaminergic neurons and motoneurons.(A) Schematic representation of in vitro neural differentiation of hpESCs (LLC9P) towards dopaminergic neurons and motoneurons (adapted from [26]). (B) RT-PCR analysis for the expression of midbrain transcripts (Nurr1, En1 and Pax2). Immunostaining with antibodies specific for En1, Pitx3 and TH. Cells were co-stained with DAPI. Percentages of DAPI and En1, Pitx3 or TH positive cells are indicated. (C) RT-PCR analysis for the expression HoxA1 and HoxA2. Shown are immunostainings with antibodies specific for: Isl1, Nkx2.2 and MAP2/HB9. Cells were co-stained with DAPI. Shown are percentages of DAPI and Isl1, Nkx2.2 and HB9- and DAPI-positive cells. Scale bars: 50 µm; n = 3.
Mentions: Next we analyzed whether hpNSCs remain responsive to instructive regionalization cues known to induce dopaminergic [27] or motoneuron differentiations [28]. To induce formation of TH+ (tyrosine hydroxylase)-neurons, hpNSCs were first cultured in media supplemented with sonic hedgehog (SHH) and FGF8b, followed by culture in media containing BDNF and GDNF (Fig. 3A, top). After 28 days of differentiation, cells expressed transcripts for the midbrain-specific Nurr1 (nuclear receptor related 1 protein), En1 (engrailed homeobox 1) and Pax2 (paired box gene 2) (Fig. 3B). Immunocytochemical staining verified upregulation of the midbrain markers En1 and Pitx3 (paired-like homeodomain 3), which are transcription factors required for differentiation and survival of midbrain dopaminergic neurons, and for TH (dopamine biogenesis) (Fig. 3B). We observed 79.8±3.2% En1, 10.8±0.6% Pitx3 and 13.3±1.6% TH positive cells (Fig. 3B).

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