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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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Intracellular chloride in TH+ neurons over time.Cells were loaded with the chloride indicator dihydro-MEQ and illuminated at 340 nm, (A) shows a typical field of view. Basal [Cl−] was recorded (and in some cases responses to GABA, 30 µM, not shown in this trace). The addition of the ionophores nigericin (10 µM), tributyltin (10 µM), and valinomycin (5 µM) enabled a standard curve to be constucted, (C), where F0/F is the ratio of fluorescence in Cl− free buffer containing the ionophores over fluorescence intensities at various [Cl−]. Basal and post-GABA [Cl−]i were interpolated from the standard curve. Basal [Cl−]i decreased from day 13 to day 23 of differentiation ((D); one way ANOVA with post-hoc Dunnett's test, P<0.05, n = 4–6). This panel also shows that, at days 13 and 15 GABA (30 µM) reduced [Cl−]i, but by day 21 elevated [Cl−]i (Student's paired t-test, P<0.05, n = 4–6). The fraction of cells to have elevated [Cl−]i in response to GABA increased significantly over differentiation ((E); one way ANOVA with post-hoc Dunnett's test, P<0.05, n = 4–6).
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pone-0031999-g004: Intracellular chloride in TH+ neurons over time.Cells were loaded with the chloride indicator dihydro-MEQ and illuminated at 340 nm, (A) shows a typical field of view. Basal [Cl−] was recorded (and in some cases responses to GABA, 30 µM, not shown in this trace). The addition of the ionophores nigericin (10 µM), tributyltin (10 µM), and valinomycin (5 µM) enabled a standard curve to be constucted, (C), where F0/F is the ratio of fluorescence in Cl− free buffer containing the ionophores over fluorescence intensities at various [Cl−]. Basal and post-GABA [Cl−]i were interpolated from the standard curve. Basal [Cl−]i decreased from day 13 to day 23 of differentiation ((D); one way ANOVA with post-hoc Dunnett's test, P<0.05, n = 4–6). This panel also shows that, at days 13 and 15 GABA (30 µM) reduced [Cl−]i, but by day 21 elevated [Cl−]i (Student's paired t-test, P<0.05, n = 4–6). The fraction of cells to have elevated [Cl−]i in response to GABA increased significantly over differentiation ((E); one way ANOVA with post-hoc Dunnett's test, P<0.05, n = 4–6).

Mentions: Figure 4A shows a typical MEQ fluorescence field of view. The experimental conditions enabling us to calculate [Cl−]i are shown in figure 4B (MEQ fluorescence intensity is inversely proportional to [Cl−]i,). This inverse relationship led to the creation of standard curves (one-phase association, R2 value of 0.84) known as Stern-Volmer plots (figure 4C) from which [Cl−]i was interpolated. Between days 13 and 21 [Cl−]i decreased from 56.0 (55.1; 56.9) to 6.9 (6.8; 7.1) mM (Figure 4D; one way ANOVA with post-hoc Dunnett's test, P<0.001, n = 3–4). Only by day 21 did GABA (30 µM) elicit a significant elevation of [Cl−]i (Figure 4D; Student's paired t-test, P<0.05 and 0.001). Before this time point, GABA either did not affect resting [Cl−]i or reduced it (P<0.05 and 0.001). The percentage of the TH+ population in which GABA elevated [Cl−]i increased from 0% at day 13, to 91±5% at day 23 (Figure 4E; P<0.001, n = 3–4).


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

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

Intracellular chloride in TH+ neurons over time.Cells were loaded with the chloride indicator dihydro-MEQ and illuminated at 340 nm, (A) shows a typical field of view. Basal [Cl−] was recorded (and in some cases responses to GABA, 30 µM, not shown in this trace). The addition of the ionophores nigericin (10 µM), tributyltin (10 µM), and valinomycin (5 µM) enabled a standard curve to be constucted, (C), where F0/F is the ratio of fluorescence in Cl− free buffer containing the ionophores over fluorescence intensities at various [Cl−]. Basal and post-GABA [Cl−]i were interpolated from the standard curve. Basal [Cl−]i decreased from day 13 to day 23 of differentiation ((D); one way ANOVA with post-hoc Dunnett's test, P<0.05, n = 4–6). This panel also shows that, at days 13 and 15 GABA (30 µM) reduced [Cl−]i, but by day 21 elevated [Cl−]i (Student's paired t-test, P<0.05, n = 4–6). The fraction of cells to have elevated [Cl−]i in response to GABA increased significantly over differentiation ((E); one way ANOVA with post-hoc Dunnett's test, P<0.05, n = 4–6).
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pone-0031999-g004: Intracellular chloride in TH+ neurons over time.Cells were loaded with the chloride indicator dihydro-MEQ and illuminated at 340 nm, (A) shows a typical field of view. Basal [Cl−] was recorded (and in some cases responses to GABA, 30 µM, not shown in this trace). The addition of the ionophores nigericin (10 µM), tributyltin (10 µM), and valinomycin (5 µM) enabled a standard curve to be constucted, (C), where F0/F is the ratio of fluorescence in Cl− free buffer containing the ionophores over fluorescence intensities at various [Cl−]. Basal and post-GABA [Cl−]i were interpolated from the standard curve. Basal [Cl−]i decreased from day 13 to day 23 of differentiation ((D); one way ANOVA with post-hoc Dunnett's test, P<0.05, n = 4–6). This panel also shows that, at days 13 and 15 GABA (30 µM) reduced [Cl−]i, but by day 21 elevated [Cl−]i (Student's paired t-test, P<0.05, n = 4–6). The fraction of cells to have elevated [Cl−]i in response to GABA increased significantly over differentiation ((E); one way ANOVA with post-hoc Dunnett's test, P<0.05, n = 4–6).
Mentions: Figure 4A shows a typical MEQ fluorescence field of view. The experimental conditions enabling us to calculate [Cl−]i are shown in figure 4B (MEQ fluorescence intensity is inversely proportional to [Cl−]i,). This inverse relationship led to the creation of standard curves (one-phase association, R2 value of 0.84) known as Stern-Volmer plots (figure 4C) from which [Cl−]i was interpolated. Between days 13 and 21 [Cl−]i decreased from 56.0 (55.1; 56.9) to 6.9 (6.8; 7.1) mM (Figure 4D; one way ANOVA with post-hoc Dunnett's test, P<0.001, n = 3–4). Only by day 21 did GABA (30 µM) elicit a significant elevation of [Cl−]i (Figure 4D; Student's paired t-test, P<0.05 and 0.001). Before this time point, GABA either did not affect resting [Cl−]i or reduced it (P<0.05 and 0.001). The percentage of the TH+ population in which GABA elevated [Cl−]i increased from 0% at day 13, to 91±5% at day 23 (Figure 4E; P<0.001, n = 3–4).

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