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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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hpESCs generate NSCs.(A) Time-lapse, phase-contrast images illustrating the individual stages of neural in vitro differentiation of hpESC line LLC9P towards hpNSCs. Starting from hpESCs grown on human foreskin fibroblasts to hpESC-derived floating embryoid bodies, attached embryoid bodies (insert indicates rosette-like structures), floating neurospheres and NSCs. Scale bars, left panel: 0.5 mm; other panels: 0.25 mm. (B) RT-PCR analysis for the expression of pluripotency (Oct4 and Nanog) and neural stem cell markers (Sox1, Nestin, Pax6 and MS1) in undifferentiated hESC and hpESC cultures, and in hNSC and hpNSC cultures. Also shown is a RT-PCR analysis for the expression of the neural crest cell markers Snai2 and FoxD3 and the mesodermal marker Acta1 in undifferentiated hESCs, hNSCs, hpESCs, and hpNSCs at passages 5, 10 and 15. Controls shown are analyses of human parthenogenetic neural crest stem cells (hpNCSCs), human fetal brain (hFB) and human adipose tissue-derived mesenchymal stromal cells (hMSCs). (C) Representative confocal images of hpNSC cultures immunostained with antibodies specific for Nestin, Sox1, Sox2, and Vimentin are shown. Scale bars: 50 µm; n = 3.
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pone-0042800-g001: hpESCs generate NSCs.(A) Time-lapse, phase-contrast images illustrating the individual stages of neural in vitro differentiation of hpESC line LLC9P towards hpNSCs. Starting from hpESCs grown on human foreskin fibroblasts to hpESC-derived floating embryoid bodies, attached embryoid bodies (insert indicates rosette-like structures), floating neurospheres and NSCs. Scale bars, left panel: 0.5 mm; other panels: 0.25 mm. (B) RT-PCR analysis for the expression of pluripotency (Oct4 and Nanog) and neural stem cell markers (Sox1, Nestin, Pax6 and MS1) in undifferentiated hESC and hpESC cultures, and in hNSC and hpNSC cultures. Also shown is a RT-PCR analysis for the expression of the neural crest cell markers Snai2 and FoxD3 and the mesodermal marker Acta1 in undifferentiated hESCs, hNSCs, hpESCs, and hpNSCs at passages 5, 10 and 15. Controls shown are analyses of human parthenogenetic neural crest stem cells (hpNCSCs), human fetal brain (hFB) and human adipose tissue-derived mesenchymal stromal cells (hMSCs). (C) Representative confocal images of hpNSC cultures immunostained with antibodies specific for Nestin, Sox1, Sox2, and Vimentin are shown. Scale bars: 50 µm; n = 3.

Mentions: Uniparental hpESCs (ESC lines LLC9P and LLC6P [10]) were cultured using a multi-step in vitro differentiation protocol that can produce NSCs from pluripotent stem cells [26]. Initial differentiation of hpESCs produced floating embryoid bodies (EB) that formed neural rosettes after attachment (Fig. 1A, Fig. S1 A). Isolated neural rosettes could be expanded as floating neurospheres that formed monolayers with NSC-like homogeneous morphology after plating onto polyornithine/laminin-coated plates (Fig. 1A right panel). The NSC identity of monolayer cells was confirmed by gene expression analysis revealing upregulation of NSC markers Sox1, Nestin, Pax6, and Musashi1 (MS1) (Fig. 1B,. Fig. S1 B), silencing of pluripotency marker genes (Oct4 or Nanog), and absence of activation of markers of non-neural lineage commitment, including neural crest (Snai2, FoxD3) and mesoderm (Acta1). Expression of the neural stem cell markers Nestin, Sox1, Sox2 and Vimentin in hpNSC cultures was ubiquitous and not limited to subsets of cells (Fig. 1C and in. Fig. S1 C). Upon differentiation, two 10 cm2 plate dishes of LLC9P hpESCs yielded a mean of 29 (±3.5) million hpNSCs whereas LLC6P hpESCs generated 11.8 (±1.7) million cells. As a recent report described aberrant expression levels of molecules related to spindle formation and chromosome segregation in hpESCs [20], we verified expression of these markers in undifferentiated LLC6P and LLC9P hpESCs, and detected variations in gene expression not only between PG and N cells but also between individual hpESCs (Fig. S2 A). Additionally, reduced levels of extracellular matrix (ECM) transcripts had been detected in PG compared to N (biparental) ESCs [22]. We observed variation in ECM transcript levels of ECM molecules between individual PG and N cell lines, with lower expression in LLC6P hpESCs compared to LLC9P cells and to hESCs (Fig. S2 B). In conclusion, hpESCs can differentiate into hpNSCs that express neural stem cell markers in the absence of pluripotency or neural crest cell marker expression.


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)

hpESCs generate NSCs.(A) Time-lapse, phase-contrast images illustrating the individual stages of neural in vitro differentiation of hpESC line LLC9P towards hpNSCs. Starting from hpESCs grown on human foreskin fibroblasts to hpESC-derived floating embryoid bodies, attached embryoid bodies (insert indicates rosette-like structures), floating neurospheres and NSCs. Scale bars, left panel: 0.5 mm; other panels: 0.25 mm. (B) RT-PCR analysis for the expression of pluripotency (Oct4 and Nanog) and neural stem cell markers (Sox1, Nestin, Pax6 and MS1) in undifferentiated hESC and hpESC cultures, and in hNSC and hpNSC cultures. Also shown is a RT-PCR analysis for the expression of the neural crest cell markers Snai2 and FoxD3 and the mesodermal marker Acta1 in undifferentiated hESCs, hNSCs, hpESCs, and hpNSCs at passages 5, 10 and 15. Controls shown are analyses of human parthenogenetic neural crest stem cells (hpNCSCs), human fetal brain (hFB) and human adipose tissue-derived mesenchymal stromal cells (hMSCs). (C) Representative confocal images of hpNSC cultures immunostained with antibodies specific for Nestin, Sox1, Sox2, and Vimentin are shown. Scale bars: 50 µm; n = 3.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412801&req=5

pone-0042800-g001: hpESCs generate NSCs.(A) Time-lapse, phase-contrast images illustrating the individual stages of neural in vitro differentiation of hpESC line LLC9P towards hpNSCs. Starting from hpESCs grown on human foreskin fibroblasts to hpESC-derived floating embryoid bodies, attached embryoid bodies (insert indicates rosette-like structures), floating neurospheres and NSCs. Scale bars, left panel: 0.5 mm; other panels: 0.25 mm. (B) RT-PCR analysis for the expression of pluripotency (Oct4 and Nanog) and neural stem cell markers (Sox1, Nestin, Pax6 and MS1) in undifferentiated hESC and hpESC cultures, and in hNSC and hpNSC cultures. Also shown is a RT-PCR analysis for the expression of the neural crest cell markers Snai2 and FoxD3 and the mesodermal marker Acta1 in undifferentiated hESCs, hNSCs, hpESCs, and hpNSCs at passages 5, 10 and 15. Controls shown are analyses of human parthenogenetic neural crest stem cells (hpNCSCs), human fetal brain (hFB) and human adipose tissue-derived mesenchymal stromal cells (hMSCs). (C) Representative confocal images of hpNSC cultures immunostained with antibodies specific for Nestin, Sox1, Sox2, and Vimentin are shown. Scale bars: 50 µm; n = 3.
Mentions: Uniparental hpESCs (ESC lines LLC9P and LLC6P [10]) were cultured using a multi-step in vitro differentiation protocol that can produce NSCs from pluripotent stem cells [26]. Initial differentiation of hpESCs produced floating embryoid bodies (EB) that formed neural rosettes after attachment (Fig. 1A, Fig. S1 A). Isolated neural rosettes could be expanded as floating neurospheres that formed monolayers with NSC-like homogeneous morphology after plating onto polyornithine/laminin-coated plates (Fig. 1A right panel). The NSC identity of monolayer cells was confirmed by gene expression analysis revealing upregulation of NSC markers Sox1, Nestin, Pax6, and Musashi1 (MS1) (Fig. 1B,. Fig. S1 B), silencing of pluripotency marker genes (Oct4 or Nanog), and absence of activation of markers of non-neural lineage commitment, including neural crest (Snai2, FoxD3) and mesoderm (Acta1). Expression of the neural stem cell markers Nestin, Sox1, Sox2 and Vimentin in hpNSC cultures was ubiquitous and not limited to subsets of cells (Fig. 1C and in. Fig. S1 C). Upon differentiation, two 10 cm2 plate dishes of LLC9P hpESCs yielded a mean of 29 (±3.5) million hpNSCs whereas LLC6P hpESCs generated 11.8 (±1.7) million cells. As a recent report described aberrant expression levels of molecules related to spindle formation and chromosome segregation in hpESCs [20], we verified expression of these markers in undifferentiated LLC6P and LLC9P hpESCs, and detected variations in gene expression not only between PG and N cells but also between individual hpESCs (Fig. S2 A). Additionally, reduced levels of extracellular matrix (ECM) transcripts had been detected in PG compared to N (biparental) ESCs [22]. We observed variation in ECM transcript levels of ECM molecules between individual PG and N cell lines, with lower expression in LLC6P hpESCs compared to LLC9P cells and to hESCs (Fig. S2 B). In conclusion, hpESCs can differentiate into hpNSCs that express neural stem cell markers in the absence of pluripotency or neural crest cell marker expression.

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