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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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hpNSC differentiation into neuronal and glial cell types.(A) Schematic representation of neural in vitro differentiation of hpESCs (LLC9P) towards neural subtypes (adapted from [26]). (B) RT-PCR analysis of hpESCs, hpNSC-derived neural cell cultures (hp neural cells) and of an hFB sample for the expression of neural transcripts: Tuj1, GFAP, S100B and Olig2. (C) Immunostainings of hpNSC-derived neural cell cultures with antibodies specific for: Tuj1, NeuN, MAP2, GFAP, O4, Synapsin1/Map2/Tau (insert shows higher magnification), Tuj1/GABA. Cells were counterstained with DAPI. (D) Percentages of neural subtypes after differentiation are given. The percentages were determined by counting neuronal or glial marker- and DAPI-positive cells. ImageJ software was used for counting. Scale bars: 50 µm; n≥3.
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pone-0042800-g002: hpNSC differentiation into neuronal and glial cell types.(A) Schematic representation of neural in vitro differentiation of hpESCs (LLC9P) towards neural subtypes (adapted from [26]). (B) RT-PCR analysis of hpESCs, hpNSC-derived neural cell cultures (hp neural cells) and of an hFB sample for the expression of neural transcripts: Tuj1, GFAP, S100B and Olig2. (C) Immunostainings of hpNSC-derived neural cell cultures with antibodies specific for: Tuj1, NeuN, MAP2, GFAP, O4, Synapsin1/Map2/Tau (insert shows higher magnification), Tuj1/GABA. Cells were counterstained with DAPI. (D) Percentages of neural subtypes after differentiation are given. The percentages were determined by counting neuronal or glial marker- and DAPI-positive cells. ImageJ software was used for counting. Scale bars: 50 µm; n≥3.

Mentions: To study the neural differentiation potential of hpNSCs (LLC9P), cells were subjected to growth factor withdrawal to induce terminal differentiation (Fig. 2A). In contrast to undifferentiated hpESCs and similar to a human fetal brain isolate, hpNSC-derived cells (differentiated for 28 days) expressed neuronal (Tuj1 - class III beta-tubulin), astrocyte (GFAP - glial fibrillary acidic protein; S100B - S100 calcium binding protein B) and oligodendrocyte (Olig2 -oligodendrocyte transcription factor 2) lineage-specific transcripts (Fig. 2B) and cell type-specific protein markers Tuj1, NeuN, MAP2 (microtubule-associated protein 2, neurons), GFAP (astrocytes) and O4 (oligodendrocytes) (Fig. 2C). Expression of the presynaptic vesicle protein Synapsin1, the dendritic marker MAP2 and the axonal marker Tau was also detectable at this stage of differentiation (Fig. 2C). 95±1.3% of Tuj1/DAPI positive cells co-expressed the neurotransmitter GABA (γ-aminobutyric acid) (Fig. 2C). Overall, we observed that hpNSCs favor neuronal differentiation (61±1.6% of cells), whereas glial cells were less frequently detectable (17±0.3% of cells). Oligodendrocytes were detected only after 6 weeks of differentiation (2±0.3% of cells) (Fig. 2D). Similar results of neuronal and astroglial differentiation were observed for the hpESC line LLC6P (. Fig. S3 A, B), with the exception that O4-positive cells were not detected (Fig. S3 C).


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)

hpNSC differentiation into neuronal and glial cell types.(A) Schematic representation of neural in vitro differentiation of hpESCs (LLC9P) towards neural subtypes (adapted from [26]). (B) RT-PCR analysis of hpESCs, hpNSC-derived neural cell cultures (hp neural cells) and of an hFB sample for the expression of neural transcripts: Tuj1, GFAP, S100B and Olig2. (C) Immunostainings of hpNSC-derived neural cell cultures with antibodies specific for: Tuj1, NeuN, MAP2, GFAP, O4, Synapsin1/Map2/Tau (insert shows higher magnification), Tuj1/GABA. Cells were counterstained with DAPI. (D) Percentages of neural subtypes after differentiation are given. The percentages were determined by counting neuronal or glial marker- and DAPI-positive cells. ImageJ software was used for counting. Scale bars: 50 µm; n≥3.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412801&req=5

pone-0042800-g002: hpNSC differentiation into neuronal and glial cell types.(A) Schematic representation of neural in vitro differentiation of hpESCs (LLC9P) towards neural subtypes (adapted from [26]). (B) RT-PCR analysis of hpESCs, hpNSC-derived neural cell cultures (hp neural cells) and of an hFB sample for the expression of neural transcripts: Tuj1, GFAP, S100B and Olig2. (C) Immunostainings of hpNSC-derived neural cell cultures with antibodies specific for: Tuj1, NeuN, MAP2, GFAP, O4, Synapsin1/Map2/Tau (insert shows higher magnification), Tuj1/GABA. Cells were counterstained with DAPI. (D) Percentages of neural subtypes after differentiation are given. The percentages were determined by counting neuronal or glial marker- and DAPI-positive cells. ImageJ software was used for counting. Scale bars: 50 µm; n≥3.
Mentions: To study the neural differentiation potential of hpNSCs (LLC9P), cells were subjected to growth factor withdrawal to induce terminal differentiation (Fig. 2A). In contrast to undifferentiated hpESCs and similar to a human fetal brain isolate, hpNSC-derived cells (differentiated for 28 days) expressed neuronal (Tuj1 - class III beta-tubulin), astrocyte (GFAP - glial fibrillary acidic protein; S100B - S100 calcium binding protein B) and oligodendrocyte (Olig2 -oligodendrocyte transcription factor 2) lineage-specific transcripts (Fig. 2B) and cell type-specific protein markers Tuj1, NeuN, MAP2 (microtubule-associated protein 2, neurons), GFAP (astrocytes) and O4 (oligodendrocytes) (Fig. 2C). Expression of the presynaptic vesicle protein Synapsin1, the dendritic marker MAP2 and the axonal marker Tau was also detectable at this stage of differentiation (Fig. 2C). 95±1.3% of Tuj1/DAPI positive cells co-expressed the neurotransmitter GABA (γ-aminobutyric acid) (Fig. 2C). Overall, we observed that hpNSCs favor neuronal differentiation (61±1.6% of cells), whereas glial cells were less frequently detectable (17±0.3% of cells). Oligodendrocytes were detected only after 6 weeks of differentiation (2±0.3% of cells) (Fig. 2D). Similar results of neuronal and astroglial differentiation were observed for the hpESC line LLC6P (. Fig. S3 A, B), with the exception that O4-positive cells were not detected (Fig. S3 C).

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