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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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Electrophysiologically active PG neurons.Representative electrophysiological analysis of hpESC-derived neurons (LLC9P) after in vitro differentiation for 28 days. (A). Representative current traces in whole cell configuration responding to step depolarization. Insert shows sodium currents. Stimulation via stepwise increase of membrane potential (−80 mV to +55 mV, step size 15 mV) in VC-mode. (B) Representative traces of membrane potential responding to step depolarization by current injection in CC mode; depolarization - black line, hyperpolarization - grey line. (C) Current (I)/voltage (V) curves of voltage clamp (VC)-stimulation. Stimulation potential [mV] is plotted against the highest and the lowest measured current (current is normalized to cell size [pA/pF]). (D) I/V curves of VC-stimulation before and after treatment with tetrodotoxin (TTX, sodium channel blocker) or tetraethylamonium (TEA, potassium channel blocker). Stimulation potential [mV] is plotted against the highest and the lowest measured current (current was normalized to cell size [pA/pF]). n = 3.
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pone-0042800-g004: Electrophysiologically active PG neurons.Representative electrophysiological analysis of hpESC-derived neurons (LLC9P) after in vitro differentiation for 28 days. (A). Representative current traces in whole cell configuration responding to step depolarization. Insert shows sodium currents. Stimulation via stepwise increase of membrane potential (−80 mV to +55 mV, step size 15 mV) in VC-mode. (B) Representative traces of membrane potential responding to step depolarization by current injection in CC mode; depolarization - black line, hyperpolarization - grey line. (C) Current (I)/voltage (V) curves of voltage clamp (VC)-stimulation. Stimulation potential [mV] is plotted against the highest and the lowest measured current (current is normalized to cell size [pA/pF]). (D) I/V curves of VC-stimulation before and after treatment with tetrodotoxin (TTX, sodium channel blocker) or tetraethylamonium (TEA, potassium channel blocker). Stimulation potential [mV] is plotted against the highest and the lowest measured current (current was normalized to cell size [pA/pF]). n = 3.

Mentions: We further investigated whether PG neurons can functionally mature in vitro. As shown in Table S1, electrophysiological properties of PG neurons at 28 days of differentiation were comparable to those reported in literature for human in vitro induced neuronal cells [32]. PG neurons showed typical neuronal Na+/K+ currents in voltage clamp mode (vc stimulation pattern: −80 mV to +55 mV, step size 15 mV, stimulation time 20 ms) (Fig. 4A). Depolarizing step current injections over a 500 ms time period elicited multiple action potentials with a maximum frequency of 30 Hz (Fig. 4B). When maximum in- and outward currents were plotted against the corresponding stimulation voltage, PG neurons depicted a typical neuron-like current pattern (Fig. 4C) that responded to selective pharmacological blockers of sodium (tetrodotoxin) and potassium (tetraethylammonium) channels (Fig. 4D).


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)

Electrophysiologically active PG neurons.Representative electrophysiological analysis of hpESC-derived neurons (LLC9P) after in vitro differentiation for 28 days. (A). Representative current traces in whole cell configuration responding to step depolarization. Insert shows sodium currents. Stimulation via stepwise increase of membrane potential (−80 mV to +55 mV, step size 15 mV) in VC-mode. (B) Representative traces of membrane potential responding to step depolarization by current injection in CC mode; depolarization - black line, hyperpolarization - grey line. (C) Current (I)/voltage (V) curves of voltage clamp (VC)-stimulation. Stimulation potential [mV] is plotted against the highest and the lowest measured current (current is normalized to cell size [pA/pF]). (D) I/V curves of VC-stimulation before and after treatment with tetrodotoxin (TTX, sodium channel blocker) or tetraethylamonium (TEA, potassium channel blocker). Stimulation potential [mV] is plotted against the highest and the lowest measured current (current was normalized to cell size [pA/pF]). n = 3.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412801&req=5

pone-0042800-g004: Electrophysiologically active PG neurons.Representative electrophysiological analysis of hpESC-derived neurons (LLC9P) after in vitro differentiation for 28 days. (A). Representative current traces in whole cell configuration responding to step depolarization. Insert shows sodium currents. Stimulation via stepwise increase of membrane potential (−80 mV to +55 mV, step size 15 mV) in VC-mode. (B) Representative traces of membrane potential responding to step depolarization by current injection in CC mode; depolarization - black line, hyperpolarization - grey line. (C) Current (I)/voltage (V) curves of voltage clamp (VC)-stimulation. Stimulation potential [mV] is plotted against the highest and the lowest measured current (current is normalized to cell size [pA/pF]). (D) I/V curves of VC-stimulation before and after treatment with tetrodotoxin (TTX, sodium channel blocker) or tetraethylamonium (TEA, potassium channel blocker). Stimulation potential [mV] is plotted against the highest and the lowest measured current (current was normalized to cell size [pA/pF]). n = 3.
Mentions: We further investigated whether PG neurons can functionally mature in vitro. As shown in Table S1, electrophysiological properties of PG neurons at 28 days of differentiation were comparable to those reported in literature for human in vitro induced neuronal cells [32]. PG neurons showed typical neuronal Na+/K+ currents in voltage clamp mode (vc stimulation pattern: −80 mV to +55 mV, step size 15 mV, stimulation time 20 ms) (Fig. 4A). Depolarizing step current injections over a 500 ms time period elicited multiple action potentials with a maximum frequency of 30 Hz (Fig. 4B). When maximum in- and outward currents were plotted against the corresponding stimulation voltage, PG neurons depicted a typical neuron-like current pattern (Fig. 4C) that responded to selective pharmacological blockers of sodium (tetrodotoxin) and potassium (tetraethylammonium) channels (Fig. 4D).

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