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In vitro maturation of dopaminergic neurons derived from mouse embryonic stem cells: implications for transplantation.

Watmuff B, Pouton CW, Haynes JM - PLoS ONE (2012)

Bottom Line: At days 13 and 15 TH(+) neurons responded to GABA (30 µM) with reductions in intracellular Cl(-) ([Cl(-)](i)); from day 21 the majority of neurons responded to GABA (30 µM) with elevations of [Cl(-)](i).As [Cl(-)](i) reduced, the ability of GABA (30 µM) to elevate intracellular Ca(2+) ([Ca(2+)](i)) did also.By day 23 cultures were resistant to the effects of both GABA and L-glutamate.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Group, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

ABSTRACT
The obvious motor symptoms of Parkinson's disease result from a loss of dopaminergic neurons from the substantia nigra. Embryonic stem cell-derived neural progenitor or precursor cells, adult neurons and fetal midbrain tissue have all been used to replace dying dopaminergic neurons. Transplanted cell survival is compromised by factors relating to the new environment, for example; hypoxia, mechanical trauma and excitatory amino acid toxicity. In this study we investigate, using live-cell fluorescence Ca(2+) and Cl(-) imaging, the functional properties of catecholaminergic neurons as they mature. We also investigate whether GABA has the capacity to act as a neurotoxin early in the development of these neurons. From day 13 to day 21 of differentiation [Cl(-)](i) progressively dropped in tyrosine hydroxylase positive (TH(+)) neurons from 56.0 (95% confidence interval, 55.1, 56.9) mM to 6.9 (6.8, 7.1) mM. At days 13 and 15 TH(+) neurons responded to GABA (30 µM) with reductions in intracellular Cl(-) ([Cl(-)](i)); from day 21 the majority of neurons responded to GABA (30 µM) with elevations of [Cl(-)](i). As [Cl(-)](i) reduced, the ability of GABA (30 µM) to elevate intracellular Ca(2+) ([Ca(2+)](i)) did also. At day 13 of differentiation a three hour exposure to GABA (30 µM) or L-glutamate (30 µM) increased the number of midbrain dopaminergic (TH(+) and Pitx3(+)) neurons labeled with the membrane-impermeable nuclear dye TOPRO-3. By day 23 cultures were resistant to the effects of both GABA and L-glutamate. We believe that neuronal susceptibility to amino acid excitotoxicity is dependent upon neuronal maturity, and this should be considered when isolating cells for transplantation studies.

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Exposure to amino acid neurotransmitters causes cell death in early GFP+ cultures.Cultures containing Pitx3-eGFP+ cells were incubated with the membrane impermeable dye TOPRO-3 and exposed to GABA (30 µM), Glut (30 µM), or a combination of the two. (A) shows the effects of GABA exposure on cells at day 15 and day 23. The panels show, from top to bottom, eGFP and TOPRO-3 at time = 0, after 3 hours of constant incubation, and overlays of regions showing increased TOPRO-3 reactivity. After 3 hours of amino acid incubation there were a significantly higher number of red overlays in non-vehicle control treated cells from day 15 (One way ANOVA with post-hoc Dunnett's test, P<0.05, n = 5) (B) than were present in cultures from day 23 (C).
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pone-0031999-g008: Exposure to amino acid neurotransmitters causes cell death in early GFP+ cultures.Cultures containing Pitx3-eGFP+ cells were incubated with the membrane impermeable dye TOPRO-3 and exposed to GABA (30 µM), Glut (30 µM), or a combination of the two. (A) shows the effects of GABA exposure on cells at day 15 and day 23. The panels show, from top to bottom, eGFP and TOPRO-3 at time = 0, after 3 hours of constant incubation, and overlays of regions showing increased TOPRO-3 reactivity. After 3 hours of amino acid incubation there were a significantly higher number of red overlays in non-vehicle control treated cells from day 15 (One way ANOVA with post-hoc Dunnett's test, P<0.05, n = 5) (B) than were present in cultures from day 23 (C).

Mentions: Finally, we investigated the possibility that the excitation of neurons early in differentiation would cause cell death. At day 15, a 3 hour incubation of cells with GABA (30 µM), Glut (30 µM), or GABA and Glut in combination (both 30 µM) increased (P<0.05; one-way ANOVA with post-hoc Dunnett's test, n = 5) the number of TOPRO-3+ eGFP+ cells (Figure 8A shows typical fields of view at days 15 and 23). Figure 8B shows mean (± SEM) number of TOPRO-3+ eGFP+ cells, expressed as a percentage of the original number of eGFP+ cells. Figure 8C shows that by day 23 incubation with GABA or Glut did not increase TOPRO-3+ labeling (n = 5).


In vitro maturation of dopaminergic neurons derived from mouse embryonic stem cells: implications for transplantation.

Watmuff B, Pouton CW, Haynes JM - PLoS ONE (2012)

Exposure to amino acid neurotransmitters causes cell death in early GFP+ cultures.Cultures containing Pitx3-eGFP+ cells were incubated with the membrane impermeable dye TOPRO-3 and exposed to GABA (30 µM), Glut (30 µM), or a combination of the two. (A) shows the effects of GABA exposure on cells at day 15 and day 23. The panels show, from top to bottom, eGFP and TOPRO-3 at time = 0, after 3 hours of constant incubation, and overlays of regions showing increased TOPRO-3 reactivity. After 3 hours of amino acid incubation there were a significantly higher number of red overlays in non-vehicle control treated cells from day 15 (One way ANOVA with post-hoc Dunnett's test, P<0.05, n = 5) (B) than were present in cultures from day 23 (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285205&req=5

pone-0031999-g008: Exposure to amino acid neurotransmitters causes cell death in early GFP+ cultures.Cultures containing Pitx3-eGFP+ cells were incubated with the membrane impermeable dye TOPRO-3 and exposed to GABA (30 µM), Glut (30 µM), or a combination of the two. (A) shows the effects of GABA exposure on cells at day 15 and day 23. The panels show, from top to bottom, eGFP and TOPRO-3 at time = 0, after 3 hours of constant incubation, and overlays of regions showing increased TOPRO-3 reactivity. After 3 hours of amino acid incubation there were a significantly higher number of red overlays in non-vehicle control treated cells from day 15 (One way ANOVA with post-hoc Dunnett's test, P<0.05, n = 5) (B) than were present in cultures from day 23 (C).
Mentions: Finally, we investigated the possibility that the excitation of neurons early in differentiation would cause cell death. At day 15, a 3 hour incubation of cells with GABA (30 µM), Glut (30 µM), or GABA and Glut in combination (both 30 µM) increased (P<0.05; one-way ANOVA with post-hoc Dunnett's test, n = 5) the number of TOPRO-3+ eGFP+ cells (Figure 8A shows typical fields of view at days 15 and 23). Figure 8B shows mean (± SEM) number of TOPRO-3+ eGFP+ cells, expressed as a percentage of the original number of eGFP+ cells. Figure 8C shows that by day 23 incubation with GABA or Glut did not increase TOPRO-3+ labeling (n = 5).

Bottom Line: At days 13 and 15 TH(+) neurons responded to GABA (30 µM) with reductions in intracellular Cl(-) ([Cl(-)](i)); from day 21 the majority of neurons responded to GABA (30 µM) with elevations of [Cl(-)](i).As [Cl(-)](i) reduced, the ability of GABA (30 µM) to elevate intracellular Ca(2+) ([Ca(2+)](i)) did also.By day 23 cultures were resistant to the effects of both GABA and L-glutamate.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Group, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

ABSTRACT
The obvious motor symptoms of Parkinson's disease result from a loss of dopaminergic neurons from the substantia nigra. Embryonic stem cell-derived neural progenitor or precursor cells, adult neurons and fetal midbrain tissue have all been used to replace dying dopaminergic neurons. Transplanted cell survival is compromised by factors relating to the new environment, for example; hypoxia, mechanical trauma and excitatory amino acid toxicity. In this study we investigate, using live-cell fluorescence Ca(2+) and Cl(-) imaging, the functional properties of catecholaminergic neurons as they mature. We also investigate whether GABA has the capacity to act as a neurotoxin early in the development of these neurons. From day 13 to day 21 of differentiation [Cl(-)](i) progressively dropped in tyrosine hydroxylase positive (TH(+)) neurons from 56.0 (95% confidence interval, 55.1, 56.9) mM to 6.9 (6.8, 7.1) mM. At days 13 and 15 TH(+) neurons responded to GABA (30 µM) with reductions in intracellular Cl(-) ([Cl(-)](i)); from day 21 the majority of neurons responded to GABA (30 µM) with elevations of [Cl(-)](i). As [Cl(-)](i) reduced, the ability of GABA (30 µM) to elevate intracellular Ca(2+) ([Ca(2+)](i)) did also. At day 13 of differentiation a three hour exposure to GABA (30 µM) or L-glutamate (30 µM) increased the number of midbrain dopaminergic (TH(+) and Pitx3(+)) neurons labeled with the membrane-impermeable nuclear dye TOPRO-3. By day 23 cultures were resistant to the effects of both GABA and L-glutamate. We believe that neuronal susceptibility to amino acid excitotoxicity is dependent upon neuronal maturity, and this should be considered when isolating cells for transplantation studies.

Show MeSH
Related in: MedlinePlus