Functional development of embryonic stem cell-derived midbrain dopaminergic neurons

Because of their unique properties, embryonic stem cells (ESCs) promise to deliver an indefinite number of any type of somatic cell. Unfortunately, current differentiation protocols only result in heterogeneous mixtures of cells, often with a low percentage of the desired cell type. Consequently, much stem cell research is currently devoted to understanding the nature of, or improving the yield from differentiation, which has often meant that detailed functional studies of the cells obtained from ESCs have been ignored. In this study I generated dopaminergic neurons, the cell type lost from the midbrain of patients with Parkinson’s disease, and investigated some of the functional characteristics these neurons possess throughout differentiation

Functional development of embryonic stem cell-derived midbrain dopaminergic neurons

Because of their unique properties, embryonic stem cells (ESCs) promise to deliver an indefinite number of any type of somatic cell. Unfortunately, current differentiation protocols only result in heterogeneous mixtures of cells, often with a low percentage of the desired cell type. Consequently, much stem cell research is currently devoted to understanding the nature of, or improving the yield from differentiation, which has often meant that detailed functional studies of the cells obtained from ESCs have been ignored. In this study I generated dopaminergic neurons, the cell type lost from the midbrain of patients with Parkinson’s disease, and investigated some of the functional characteristics these neurons possess throughout differentiation

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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

Morphological and transcriptomic analyses of stem cell-derived cortical neurons reveal mechanisms underlying synaptic dysfunction in schizophrenia

Abstract Background Postmortem studies in schizophrenia consistently show reduced dendritic spines in the cerebral cortex but the mechanistic underpinnings of these deficits remain unknown. Recent genome-wide association studies and exome sequencing investigations implicate synaptic genes and processes in the disease biology of schizophrenia. Methods We generated human cortical pyramidal neurons by differentiating iPSCs of seven schizophrenia patients and seven healthy subjects, quantified dendritic spines and synapses in different cortical neuron subtypes, and carried out transcriptomic studies to identify differentially regulated genes and aberrant cellular processes in schizophrenia. Results Cortical neurons expressing layer III marker CUX1, but not those expressing layer V marker CTIP2, showed significant reduction in dendritic spine density in schizophrenia, mirroring findings in postmortem studies. Transcriptomic experiments in iPSC-derived cortical neurons showed that differentially expressed genes in schizophrenia were enriched for genes implicated in schizophrenia in genome-wide association and exome sequencing studies. Moreover, most of the differentially expressed genes implicated in schizophrenia genetic studies had lower expression levels in schizophrenia cortical neurons. Network analysis of differentially expressed genes led to identification of NRXN3 as a hub gene, and follow-up experiments showed specific reduction of the NRXN3 204 isoform in schizophrenia neurons. Furthermore, overexpression of the NRXN3 204 isoform in schizophrenia neurons rescued the spine and synapse deficits in the cortical neurons while knockdown of NRXN3 204 in healthy neurons phenocopied spine and synapse deficits seen in schizophrenia cortical neurons. The antipsychotic clozapine increased expression of the NRXN3 204 isoform in schizophrenia cortical neurons and rescued the spine and synapse density deficits. Conclusions Taken together, our findings in iPSC-derived cortical neurons recapitulate cell type-specific findings in postmortem studies in schizophrenia and have led to the identification of a specific isoform of NRXN3 that modulates synaptic deficits in schizophrenia neurons

Human pluripotent stem cell derived midbrain PITX3eGFP/w neurons: a versatile tool for pharmacological screening and neurodegenerative modeling

AbstractPITX3 expression is confined to adult midbrain dopaminergic neurons. In this study we describe the generation and basic functional characteristics of midbrain dopaminergic neurons derived from a human pluripotent stem cell line expressing eGFP under the control of the PITX3 promoter. Flow cytometry shows that eGFP is evident in 15% of the neuron population at day 12 of differentiation and this level is maintained until at least day 80. From day 20-80 of differentiation intracellular chloride decreases and throughout this period around ~20% of PITX3eGFP/w neurons exhibit spontaneous Ca2+ transients (from 3.3+/-0.3 to 5.0+/-0.1 min-1, respectively). These neurons also respond to any of ATP, glutamate, acetylcholine or noradrenaline with elevations of intracellular calcium. As neuronal cultures mature more dopamine is released and single PITX3eGFP/w neurons begin to respond to more than one neurotransmitter. MPP+ and tumor necrosis factor(TNF), but not prostaglandin E2, caused death of the ~50% of PITX3eGFP/w neurons (day 80). Tracking eGFP using time lapse confocal microscopy over 24 hours demonstrated significant TNF-mediated neurite retraction over time. These PITX3eGFP/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

Characterisation of [Ca²⁺]_i and [Cl⁻]_i in <i>Pitx3</i>-eGFP⁺ cells.

<p>eGFP was highly co-localised with TH at all days; (<b>A</b>) shows a day 23 culture of TH⁺GFP⁺ cells. (<b>B</b>) Basal [Ca²⁺]_i was 103 (91,115) nM at day 23. KCl (30 mM) elevated [Ca²⁺]_i to 806 (716,919) nM (P<0.001, Student's unpaired t-test, n = 4). (<b>C</b>) Basal [Cl⁻]_i fell from 38 (36, 40) mM at day 13 to 2.7 (2.4,3.0) mM at day 23. GABA (30 µM) caused a significant reduction in [Cl⁻]_i at day 13 (Student's paired t-test, P<0.05, n = 4) and a significant increase in [Cl⁻]_i at day 23 (Student's paired t-test, P<0.05, n = 4).</p

Calcium imaging methods and immunocytochemical characterization of neurons.

<p>Cultures were loaded with the calcium sensitive fluorophore, fluo-4 AM (10 µM; blue overlay on 10× bright field image in (<b>A</b>)). Drugs were added to culture and fluorescent recordings taken. Post experiment immunocytochemistry allowed for TH⁺ cells to be identified (red overlay in (<b>A</b>)). Regions were drawn within cells enabling fluorescence intensity to be measured after replaying the initial experiment (regions 2–8 are TH⁺ cells and region 1 is for the subtraction of background fluorescence). TH⁺ cells in mounds were generally larger, and excluded from analysis (see (<b>A</b>), top left corner). A typical fluorescent response after application of ATP (300 µM) at day 13, and at day 23, is shown in (<b>B</b>). (<b>C</b>) shows TH⁺ immunoreactivity and (<b>D</b>) shows β3-tubulin⁺ immunoreactivity for the same field of view at day 21. Note the different morphologies of the TH⁺ cells (left most arrows), as well as TH/β3-tubulin⁺ varicosities (right arrow). Scale bars; (<b>A</b>) 100 µm, (<b>C and D</b>) 50 µm.</p

Spontaneous calcium oscillations in TH⁺ neurons during differentiation.

<p>(<b>A</b>) typical traces showing the regular spontaneous oscillations of [Ca²⁺]_i evident at day 23, but not at day 13 of differentiation. (<b>B</b>) shows the mean ± SEM of spontaneous oscillations observed per minute during differentiation (one way ANOVA with post-hoc Dunnett's test, P<0.05, n = 4).</p