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 Ca2+ 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 Ca2+ ([Ca2+]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.
Highlights
Embryonic stem cells (ESCs) can be propagated in culture and can differentiate into any cell type of the adult form [1]
In this study we examine, using live-cell calcium and chloride imaging, the function of ESC-derived tyrosine hydroxylase (TH)+ and TH+/Pitx3+ neurons as they develop in monolayer culture
Monolayer dopaminergic differentiation was initiated by seeding 3.56103 cells cm22 in maintenance media and incubating cultures at 37uC in a humidified 5% CO2-in-air atmosphere for at least 24 h before the maintenance medium was replaced with N2B27 medium, a 1:1 mixture of DMEM/F-12 supplemented with N2 additives, 50 mg mL21 bovine albumin fraction V (Invitrogen, Australia), 25 mg mL21 insulin (Sigma-Aldrich, USA) and Neurobasal media supplemented with B-27 serum-free additive (Invitrogen, Australia), and cultures maintained for 5 days
Summary
Embryonic stem cells (ESCs) can be propagated in culture and can differentiate into any cell type of the adult form [1]. Transplantation studies aim to correct the functional deficit that becomes evident as the resident neurons die. To date, these studies have used cells at all levels of neural differentiation, from neuronal stem cells to post-mitotic Pitx3-expressing neurons [8,9,10,11,12,13,14,15]. One problem that continues to plague transplantation therapies is the low survival rate of transplanted neurons [16,17,18,19]. This is not surprising since transplanted neurons will be subject to a wide variety of insults, from hypoxia to mechanical trauma, free radical production, growth factor deprivation and amino acid excitotoxicity
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