In Vitro Maturation of Dopaminergic Neurons Derived from Mouse Embryonic Stem Cells: Implications for Transplantation

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.

Functional Upregulation of Ca2+ -Activated K+ Channels in the Development of Substantia Nigra Dopamine Neurons

“…It is known that early in development the action of psychostimulants such as amphetamine has a paradoxical effect on the firing rate of dopamine neurons [72] and this effect could be interesting evidence of the changes of excitability and its regulation by dopamine in the transformation from inactive to active processing of action selection regulated by dopamine. Recent experiments in mice show that -currents are upregulated in DA neurons in culture [73] and this observation together with our reported findings suggests a state of rapid changes in excitability in SN DA neurons indicating that these neurons and perhaps all DA neurons in the midbrain undergo a process where they transition from a non-functional immature state to a functional mature one in the first month of postnatal life. Given all of these findings, it would be interesting to examine the effects of dopamine on the synaptic plasticity mechanisms at cortico-striatal synapses early in development.…”

Engraftment of Mouse Embryonic Stem Cells Differentiated by Default Leads to Neuroprotection, Behaviour Revival and Astrogliosis in Parkinsonian Rats

“…It has been recently demonstrated that neuronal survivability and viability is directly related to a great extent on neuronal maturity, and relatively maturing neurons will have higher survivability in an environment in the host brain that is rich in excitatory amino acids and other debilitating factors resulting from oxidative stress and immunological responses to tissue trauma [19]. Since 10-days differentiated cultures showed a lower population of progenitors and a higher population of mature neurons as compared to 7-days differentiated cells (Figure S1), the latter was considered for transplantation studies.…”

In vitro Differentiation of Pluripotent Stem Cells towards either Forebrain GABAergic or Midbrain Dopaminergic Neurons

“…The majority of cells in murine cultures will be mature at this point (Watmuff et al 2012); it is likely that human cells at day 20 are still maturing ( …”