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Human pluripotent stem cell derived midbrain PITX3(eGFP/w) neurons: a versatile tool for pharmacological screening and neurodegenerative modeling.

Watmuff B, Hartley BJ, Hunt CP, Fabb SA, Pouton CW, Haynes JM - Front Cell Neurosci (2015)

Bottom Line: From days 20 to 80 of differentiation intracellular chloride decreased and throughout this period around ∼20% of PITX3(eGFP/w) neurons exhibited spontaneous Ca(2+) transients (from 3.3 ± 0.3 to 5.0 ± 0.1 min(-1), respectively).As neuronal cultures matured more dopamine was released and single PITX3(eGFP/w) neurons began to respond to more than one neurotransmitter.Tracking eGFP using time lapse confocal microscopy over 24 h demonstrated significant TNF-mediated neurite retraction over time.

View Article: PubMed Central - PubMed

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

ABSTRACT
PITX3 expression is confined to adult midbrain dopaminergic (mDA) neurons. In this study we describe the generation and basic functional characteristics of mDA neurons derived from a human pluripotent stem cell (hPSC) line expressing eGFP under the control of the PITX3 promoter. Flow cytometry showed that eGFP was evident in 15% of the neuron population at day 12 of differentiation and this level was maintained until at least day 80. From days 20 to 80 of differentiation intracellular chloride decreased and throughout this period around ∼20% of PITX3(eGFP/w) neurons exhibited spontaneous Ca(2+) transients (from 3.3 ± 0.3 to 5.0 ± 0.1 min(-1), respectively). These neurons also responded to any of ATP, glutamate, acetylcholine, or noradrenaline with elevations of intracellular calcium. As neuronal cultures matured more dopamine was released and single PITX3(eGFP/w) neurons began to respond to more than one neurotransmitter. MPP(+) and tumor necrosis factor (TNF), but not prostaglandin E2, caused death of the ∼50% of PITX3(eGFP/w) neurons (day 80). Tracking eGFP using time lapse confocal microscopy over 24 h demonstrated significant TNF-mediated neurite retraction over time. This work now shows that these PITX3(eGFP/w) neurons are amenable to flow cytometry, release dopamine and respond to multiple neurotransmitters with elevations of intracellular calcium, we believe that they represent a versatile system for neuropharmacological and neurotoxicological studies.

No MeSH data available.


Related in: MedlinePlus

Functional characterization of PITX3eGFP/w neurons reveals development of maturity during terminal differentiation.(A) Ca2+ and Cl- imaging was employed to probe the function of PITX3eGFP/w neurons. These panels show, from left to right, eGFP in a field of view prior to imaging, the Ca2+ -sensitive probe Fluo-4 loaded into cells of the same field, and TH and eGFP after post-imaging immunocytochemistry. (B) Shows a representative trace from a Ca2+ imaging experiment. Spontaneous oscillations of [Ca2+]i, typical of in vitro mDA neurons are highlighted by arrows. The y-axis represents change in fluorescence: baseline fluorescence (ΔF/F). (C) The frequency of these oscillations did not change significantly by day 80 (one-way ANOVA with post hoc Dunnett’s test, indicates p > 0.05, n = 3 wells, 60–80 cells). (D) Intracellular calcium ion concentration ([Ca2+]i) in PITX3eGFP/w neurons was calculated at rest and after stimulation with KCl (30 mM). (E) Basal and KCl-induced elevations of [Ca2+]i increased during differentiation (one-way ANOVA with post hoc Dunnett’s test, ∗∗∗ indicates p < 0.001, n = 3 experiments, 40–80 cells). (F) Similarly, GABA (30 mM) decreased [Cl-]i at day 20 and 40 of differentiation, by day 80 GABA induced a significant increase in [Cl]i (Student’s paired t-test, ∗∗∗ indicates p < 0.001, n = 4, 60–80 cells). (G) By day 80 most PITX3eGFP/w neurons respond to GABA with an elevation of [Cl-]i (blue; R2 = 0.99), rather than the decrease seen at earlier days of differentiation (red; R2 = 0.99). (H) Shows the change in dopamine concentration in the supernatant of eGFP+ cultures following vehicle addition (i.e., basal level), GABA (30 mM), l-glutamate (30 mM) or KCl (30 mM) at different days of culture. Dopamine release in response to GABA (30 μM), but not l-glutamate or KCl significantly decreased from days 20 to 80 of differentiation (one-way ANOVA with post hoc Dunnett’s test compared to day 20, ∗∗, ∗∗∗ indicates p < 0.01 and 0.001, respectively, n = 3 wells).
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Figure 5: Functional characterization of PITX3eGFP/w neurons reveals development of maturity during terminal differentiation.(A) Ca2+ and Cl- imaging was employed to probe the function of PITX3eGFP/w neurons. These panels show, from left to right, eGFP in a field of view prior to imaging, the Ca2+ -sensitive probe Fluo-4 loaded into cells of the same field, and TH and eGFP after post-imaging immunocytochemistry. (B) Shows a representative trace from a Ca2+ imaging experiment. Spontaneous oscillations of [Ca2+]i, typical of in vitro mDA neurons are highlighted by arrows. The y-axis represents change in fluorescence: baseline fluorescence (ΔF/F). (C) The frequency of these oscillations did not change significantly by day 80 (one-way ANOVA with post hoc Dunnett’s test, indicates p > 0.05, n = 3 wells, 60–80 cells). (D) Intracellular calcium ion concentration ([Ca2+]i) in PITX3eGFP/w neurons was calculated at rest and after stimulation with KCl (30 mM). (E) Basal and KCl-induced elevations of [Ca2+]i increased during differentiation (one-way ANOVA with post hoc Dunnett’s test, ∗∗∗ indicates p < 0.001, n = 3 experiments, 40–80 cells). (F) Similarly, GABA (30 mM) decreased [Cl-]i at day 20 and 40 of differentiation, by day 80 GABA induced a significant increase in [Cl]i (Student’s paired t-test, ∗∗∗ indicates p < 0.001, n = 4, 60–80 cells). (G) By day 80 most PITX3eGFP/w neurons respond to GABA with an elevation of [Cl-]i (blue; R2 = 0.99), rather than the decrease seen at earlier days of differentiation (red; R2 = 0.99). (H) Shows the change in dopamine concentration in the supernatant of eGFP+ cultures following vehicle addition (i.e., basal level), GABA (30 mM), l-glutamate (30 mM) or KCl (30 mM) at different days of culture. Dopamine release in response to GABA (30 μM), but not l-glutamate or KCl significantly decreased from days 20 to 80 of differentiation (one-way ANOVA with post hoc Dunnett’s test compared to day 20, ∗∗, ∗∗∗ indicates p < 0.01 and 0.001, respectively, n = 3 wells).

Mentions: To determine whether PITX3eGFP/w neurons were functional we performed live-cell fluorescent imaging to measure intracellular calcium ([Ca2+]i) and chloride ion ([Cl-]i) activity, as well as DA release during differentiation (Figure 5A). From day 20 around 20% of PITX3eGFP/w neurons (which were subsequently shown to be immunoreactive to TH) showed regular spontaneous elevations of [Ca2+]i (Figure 5B) at a frequency of 3.3 ± 0.3 min-1 (Figure 5C). By day 80 the frequency of these events was 5.0 ± 0.1 min-1 (Figure 5C; change from days 20 to 80 was not significant, one-way ANOVA with post hoc Dunnett’s test, p> 0.05, n = 3). [Ca2+]i in PITX3eGFP/w neurons was calculated at rest and after stimulation with KCl (30 mM; Figure 5D). Basal [Ca2+]i increased during differentiation from 45 ± 13 nM at day 20 to 169 ± 25 nM at day 80 (Figure 5E; one-way ANOVA, p < 0.001, n = 4, 60–80 cells). KCl (30 mM) increased maximal [Ca2+]i from 357 ± 80 nM at day 20 to 931 ± 119 nM at day 80 (Figure 5E; one-way ANOVA, p < 0.001, n = 4, 60–80 cells).


Human pluripotent stem cell derived midbrain PITX3(eGFP/w) neurons: a versatile tool for pharmacological screening and neurodegenerative modeling.

Watmuff B, Hartley BJ, Hunt CP, Fabb SA, Pouton CW, Haynes JM - Front Cell Neurosci (2015)

Functional characterization of PITX3eGFP/w neurons reveals development of maturity during terminal differentiation.(A) Ca2+ and Cl- imaging was employed to probe the function of PITX3eGFP/w neurons. These panels show, from left to right, eGFP in a field of view prior to imaging, the Ca2+ -sensitive probe Fluo-4 loaded into cells of the same field, and TH and eGFP after post-imaging immunocytochemistry. (B) Shows a representative trace from a Ca2+ imaging experiment. Spontaneous oscillations of [Ca2+]i, typical of in vitro mDA neurons are highlighted by arrows. The y-axis represents change in fluorescence: baseline fluorescence (ΔF/F). (C) The frequency of these oscillations did not change significantly by day 80 (one-way ANOVA with post hoc Dunnett’s test, indicates p > 0.05, n = 3 wells, 60–80 cells). (D) Intracellular calcium ion concentration ([Ca2+]i) in PITX3eGFP/w neurons was calculated at rest and after stimulation with KCl (30 mM). (E) Basal and KCl-induced elevations of [Ca2+]i increased during differentiation (one-way ANOVA with post hoc Dunnett’s test, ∗∗∗ indicates p < 0.001, n = 3 experiments, 40–80 cells). (F) Similarly, GABA (30 mM) decreased [Cl-]i at day 20 and 40 of differentiation, by day 80 GABA induced a significant increase in [Cl]i (Student’s paired t-test, ∗∗∗ indicates p < 0.001, n = 4, 60–80 cells). (G) By day 80 most PITX3eGFP/w neurons respond to GABA with an elevation of [Cl-]i (blue; R2 = 0.99), rather than the decrease seen at earlier days of differentiation (red; R2 = 0.99). (H) Shows the change in dopamine concentration in the supernatant of eGFP+ cultures following vehicle addition (i.e., basal level), GABA (30 mM), l-glutamate (30 mM) or KCl (30 mM) at different days of culture. Dopamine release in response to GABA (30 μM), but not l-glutamate or KCl significantly decreased from days 20 to 80 of differentiation (one-way ANOVA with post hoc Dunnett’s test compared to day 20, ∗∗, ∗∗∗ indicates p < 0.01 and 0.001, respectively, n = 3 wells).
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Related In: Results  -  Collection

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Show All Figures
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Figure 5: Functional characterization of PITX3eGFP/w neurons reveals development of maturity during terminal differentiation.(A) Ca2+ and Cl- imaging was employed to probe the function of PITX3eGFP/w neurons. These panels show, from left to right, eGFP in a field of view prior to imaging, the Ca2+ -sensitive probe Fluo-4 loaded into cells of the same field, and TH and eGFP after post-imaging immunocytochemistry. (B) Shows a representative trace from a Ca2+ imaging experiment. Spontaneous oscillations of [Ca2+]i, typical of in vitro mDA neurons are highlighted by arrows. The y-axis represents change in fluorescence: baseline fluorescence (ΔF/F). (C) The frequency of these oscillations did not change significantly by day 80 (one-way ANOVA with post hoc Dunnett’s test, indicates p > 0.05, n = 3 wells, 60–80 cells). (D) Intracellular calcium ion concentration ([Ca2+]i) in PITX3eGFP/w neurons was calculated at rest and after stimulation with KCl (30 mM). (E) Basal and KCl-induced elevations of [Ca2+]i increased during differentiation (one-way ANOVA with post hoc Dunnett’s test, ∗∗∗ indicates p < 0.001, n = 3 experiments, 40–80 cells). (F) Similarly, GABA (30 mM) decreased [Cl-]i at day 20 and 40 of differentiation, by day 80 GABA induced a significant increase in [Cl]i (Student’s paired t-test, ∗∗∗ indicates p < 0.001, n = 4, 60–80 cells). (G) By day 80 most PITX3eGFP/w neurons respond to GABA with an elevation of [Cl-]i (blue; R2 = 0.99), rather than the decrease seen at earlier days of differentiation (red; R2 = 0.99). (H) Shows the change in dopamine concentration in the supernatant of eGFP+ cultures following vehicle addition (i.e., basal level), GABA (30 mM), l-glutamate (30 mM) or KCl (30 mM) at different days of culture. Dopamine release in response to GABA (30 μM), but not l-glutamate or KCl significantly decreased from days 20 to 80 of differentiation (one-way ANOVA with post hoc Dunnett’s test compared to day 20, ∗∗, ∗∗∗ indicates p < 0.01 and 0.001, respectively, n = 3 wells).
Mentions: To determine whether PITX3eGFP/w neurons were functional we performed live-cell fluorescent imaging to measure intracellular calcium ([Ca2+]i) and chloride ion ([Cl-]i) activity, as well as DA release during differentiation (Figure 5A). From day 20 around 20% of PITX3eGFP/w neurons (which were subsequently shown to be immunoreactive to TH) showed regular spontaneous elevations of [Ca2+]i (Figure 5B) at a frequency of 3.3 ± 0.3 min-1 (Figure 5C). By day 80 the frequency of these events was 5.0 ± 0.1 min-1 (Figure 5C; change from days 20 to 80 was not significant, one-way ANOVA with post hoc Dunnett’s test, p> 0.05, n = 3). [Ca2+]i in PITX3eGFP/w neurons was calculated at rest and after stimulation with KCl (30 mM; Figure 5D). Basal [Ca2+]i increased during differentiation from 45 ± 13 nM at day 20 to 169 ± 25 nM at day 80 (Figure 5E; one-way ANOVA, p < 0.001, n = 4, 60–80 cells). KCl (30 mM) increased maximal [Ca2+]i from 357 ± 80 nM at day 20 to 931 ± 119 nM at day 80 (Figure 5E; one-way ANOVA, p < 0.001, n = 4, 60–80 cells).

Bottom Line: From days 20 to 80 of differentiation intracellular chloride decreased and throughout this period around ∼20% of PITX3(eGFP/w) neurons exhibited spontaneous Ca(2+) transients (from 3.3 ± 0.3 to 5.0 ± 0.1 min(-1), respectively).As neuronal cultures matured more dopamine was released and single PITX3(eGFP/w) neurons began to respond to more than one neurotransmitter.Tracking eGFP using time lapse confocal microscopy over 24 h demonstrated significant TNF-mediated neurite retraction over time.

View Article: PubMed Central - PubMed

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

ABSTRACT
PITX3 expression is confined to adult midbrain dopaminergic (mDA) neurons. In this study we describe the generation and basic functional characteristics of mDA neurons derived from a human pluripotent stem cell (hPSC) line expressing eGFP under the control of the PITX3 promoter. Flow cytometry showed that eGFP was evident in 15% of the neuron population at day 12 of differentiation and this level was maintained until at least day 80. From days 20 to 80 of differentiation intracellular chloride decreased and throughout this period around ∼20% of PITX3(eGFP/w) neurons exhibited spontaneous Ca(2+) transients (from 3.3 ± 0.3 to 5.0 ± 0.1 min(-1), respectively). These neurons also responded to any of ATP, glutamate, acetylcholine, or noradrenaline with elevations of intracellular calcium. As neuronal cultures matured more dopamine was released and single PITX3(eGFP/w) neurons began to respond to more than one neurotransmitter. MPP(+) and tumor necrosis factor (TNF), but not prostaglandin E2, caused death of the ∼50% of PITX3(eGFP/w) neurons (day 80). Tracking eGFP using time lapse confocal microscopy over 24 h demonstrated significant TNF-mediated neurite retraction over time. This work now shows that these PITX3(eGFP/w) neurons are amenable to flow cytometry, release dopamine and respond to multiple neurotransmitters with elevations of intracellular calcium, we believe that they represent a versatile system for neuropharmacological and neurotoxicological studies.

No MeSH data available.


Related in: MedlinePlus