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Characterisation of neurons derived from a cortical human neural stem cell line CTX0E16.

Anderson GW, Deans PJ, Taylor RD, Raval P, Chen D, Lowder H, Murkerji S, Andreae LC, Williams BP, Srivastava DP - Stem Cell Res Ther (2015)

Bottom Line: Differentiation of CTX0E16 hNPCs predominately resulted in the generation of neurons expressing markers of cortical and glutamatergic (excitatory) fate, and with a typical polarized neuronal morphology.Taken together, these findings demonstrate that the CTX0E16 hNPC line is a robust source of cortical neurons, which display functional properties consistent with a glutamatergic phenotype.Thus CTX0E16 neurons can be used to study cortical cell function, and furthermore, as these neurons express a range of disease-associated genes, they represent an ideal platform with which to investigate neurodevelopmental mechanisms in native human cells in health and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic and Clinical Neuroscience, Institute of Psychiatry Psychology & Neuroscience, King's College London, London, SE5 8AF, UK. greg.anderson@kcl.ac.uk.

ABSTRACT

Introduction: Conditionally immortalised human neural progenitor cells (hNPCs) represent a robust source of native neural cells to investigate physiological mechanisms in both health and disease. However, in order to recognise the utility of such cells, it is critical to determine whether they retain characteristics of their tissue of origin and generate appropriate neural cell types upon differentiation. To this end, we have characterised the conditionally immortalised, cortically-derived, human NPC line, CTX0E16, investigating the molecular and cellular phenotype of differentiated neurons to determine whether they possess characteristics of cortical glutamatergic neurons.

Methods: Differentiated CTX0E16 cells were characterised by assessing expression of several neural fates markers, and examination of developing neuronal morphology. Expression of neurotransmitter receptors, signalling proteins and related proteins were assessed by q- and RT-PCR and complemented by Ca(2+) imaging, electrophysiology and assessment of ERK signalling in response to neurotransmitter ligand application. Finally, differentiated neurons were assessed for their ability to form putative synapses and to respond to activity-dependent stimulation.

Results: Differentiation of CTX0E16 hNPCs predominately resulted in the generation of neurons expressing markers of cortical and glutamatergic (excitatory) fate, and with a typical polarized neuronal morphology. Gene expression analysis confirmed an upregulation in the expression of cortical, glutamatergic and signalling proteins following differentiation. CTX0E16 neurons demonstrated Ca(2+) and ERK1/2 responses following exogenous neurotransmitter application, and after 6 weeks displayed spontaneous Ca(2+) transients and electrophysiological properties consistent with that of immature neurons. Differentiated CTX0E16 neurons also expressed a range of pre- and post-synaptic proteins that co-localized along distal dendrites, and moreover, displayed structural plasticity in response to modulation of neuronal activity.

Conclusions: Taken together, these findings demonstrate that the CTX0E16 hNPC line is a robust source of cortical neurons, which display functional properties consistent with a glutamatergic phenotype. Thus CTX0E16 neurons can be used to study cortical cell function, and furthermore, as these neurons express a range of disease-associated genes, they represent an ideal platform with which to investigate neurodevelopmental mechanisms in native human cells in health and disease.

No MeSH data available.


Development of functional properties in CTX0E16 neurons. a Representative image of Fluo-4 AM-loaded CTX0E16 neurons used for single cell Ca2+ imaging. b Representative time series of 18 neurons displaying spontaneous Ca2+ transients. Spontaneous activity was classified as a somatic calcium event greater than 5 % ΔF/F0: 38.0 ± 6.89 % cells displayed spontaneous activity over an 80-second period of imaging (n = 155 cells from 14 coverslips). c, d Representative traces of intracellular Ca2+ in responses to 50 mM KCl (c) or 1 μM tetrodotoxin (TTX) (d). e Resting membrane potential (Vm) recorded in current clamp progressively becomes more negative as CTX0E16 neurons become more mature (day of differentiation (DD) 29−DD 61); error bars represent SD. f Representative action potential recorded in voltage clamp in the cell attached configuration, recorded from DD 50 CTX0E16 neuron. g Representative voltage clamp recording at a holding potential of −70 mV in DD 36 CTX0E16 neurons. The downward deflections indicate the presence of AMPA receptor-mediated spontaneous excitatory postsynaptic currents (EPSCs). h Example of a spontaneous N-methyl-D-aspartate (NMDA) receptor-mediated EPSC recorded in voltage clamp at +40 mV from a DD 33 CTX0E16 neuron; n = 3–6 cells from at least three independent coverslips
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Fig5: Development of functional properties in CTX0E16 neurons. a Representative image of Fluo-4 AM-loaded CTX0E16 neurons used for single cell Ca2+ imaging. b Representative time series of 18 neurons displaying spontaneous Ca2+ transients. Spontaneous activity was classified as a somatic calcium event greater than 5 % ΔF/F0: 38.0 ± 6.89 % cells displayed spontaneous activity over an 80-second period of imaging (n = 155 cells from 14 coverslips). c, d Representative traces of intracellular Ca2+ in responses to 50 mM KCl (c) or 1 μM tetrodotoxin (TTX) (d). e Resting membrane potential (Vm) recorded in current clamp progressively becomes more negative as CTX0E16 neurons become more mature (day of differentiation (DD) 29−DD 61); error bars represent SD. f Representative action potential recorded in voltage clamp in the cell attached configuration, recorded from DD 50 CTX0E16 neuron. g Representative voltage clamp recording at a holding potential of −70 mV in DD 36 CTX0E16 neurons. The downward deflections indicate the presence of AMPA receptor-mediated spontaneous excitatory postsynaptic currents (EPSCs). h Example of a spontaneous N-methyl-D-aspartate (NMDA) receptor-mediated EPSC recorded in voltage clamp at +40 mV from a DD 33 CTX0E16 neuron; n = 3–6 cells from at least three independent coverslips

Mentions: A hallmark of the generation of neuronal cell types is the emergence of spontaneous Ca2+ oscillations due to neuronal activity [43, 44]. No spontaneous Ca2+ oscillations were observed in DD 28 CTX0E16 cultures. We therefore differentiated CTX0E16 cells for 6 weeks (DD 42) and performed single cell Ca2+ imaging using Fluo-4 (Fig. 5a). This revealed that 38.0 ± 6.89 % of CTX0E16 neurons displayed spontaneous activity, defined as a sharp transient Ca2+ flux in a neuronal cell soma (Fig. 5b). In addition, application of KCl, a depolarising agent, resulted in a large Ca2+ flux in 79.1 ± 4.2 % of DD 42 CTX0E16 neurons (Fig. 5c), indicating the presence of voltage-gated Ca2+ channels, consistent with the observed increase in CACNA1C expression levels upon differentiation (Fig. 4a). Furthermore, addition of 1 μm tetrodotoxin (TTX) blocked spontaneous Ca2+ transients (Fig. 5d).Fig. 5


Characterisation of neurons derived from a cortical human neural stem cell line CTX0E16.

Anderson GW, Deans PJ, Taylor RD, Raval P, Chen D, Lowder H, Murkerji S, Andreae LC, Williams BP, Srivastava DP - Stem Cell Res Ther (2015)

Development of functional properties in CTX0E16 neurons. a Representative image of Fluo-4 AM-loaded CTX0E16 neurons used for single cell Ca2+ imaging. b Representative time series of 18 neurons displaying spontaneous Ca2+ transients. Spontaneous activity was classified as a somatic calcium event greater than 5 % ΔF/F0: 38.0 ± 6.89 % cells displayed spontaneous activity over an 80-second period of imaging (n = 155 cells from 14 coverslips). c, d Representative traces of intracellular Ca2+ in responses to 50 mM KCl (c) or 1 μM tetrodotoxin (TTX) (d). e Resting membrane potential (Vm) recorded in current clamp progressively becomes more negative as CTX0E16 neurons become more mature (day of differentiation (DD) 29−DD 61); error bars represent SD. f Representative action potential recorded in voltage clamp in the cell attached configuration, recorded from DD 50 CTX0E16 neuron. g Representative voltage clamp recording at a holding potential of −70 mV in DD 36 CTX0E16 neurons. The downward deflections indicate the presence of AMPA receptor-mediated spontaneous excitatory postsynaptic currents (EPSCs). h Example of a spontaneous N-methyl-D-aspartate (NMDA) receptor-mediated EPSC recorded in voltage clamp at +40 mV from a DD 33 CTX0E16 neuron; n = 3–6 cells from at least three independent coverslips
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Fig5: Development of functional properties in CTX0E16 neurons. a Representative image of Fluo-4 AM-loaded CTX0E16 neurons used for single cell Ca2+ imaging. b Representative time series of 18 neurons displaying spontaneous Ca2+ transients. Spontaneous activity was classified as a somatic calcium event greater than 5 % ΔF/F0: 38.0 ± 6.89 % cells displayed spontaneous activity over an 80-second period of imaging (n = 155 cells from 14 coverslips). c, d Representative traces of intracellular Ca2+ in responses to 50 mM KCl (c) or 1 μM tetrodotoxin (TTX) (d). e Resting membrane potential (Vm) recorded in current clamp progressively becomes more negative as CTX0E16 neurons become more mature (day of differentiation (DD) 29−DD 61); error bars represent SD. f Representative action potential recorded in voltage clamp in the cell attached configuration, recorded from DD 50 CTX0E16 neuron. g Representative voltage clamp recording at a holding potential of −70 mV in DD 36 CTX0E16 neurons. The downward deflections indicate the presence of AMPA receptor-mediated spontaneous excitatory postsynaptic currents (EPSCs). h Example of a spontaneous N-methyl-D-aspartate (NMDA) receptor-mediated EPSC recorded in voltage clamp at +40 mV from a DD 33 CTX0E16 neuron; n = 3–6 cells from at least three independent coverslips
Mentions: A hallmark of the generation of neuronal cell types is the emergence of spontaneous Ca2+ oscillations due to neuronal activity [43, 44]. No spontaneous Ca2+ oscillations were observed in DD 28 CTX0E16 cultures. We therefore differentiated CTX0E16 cells for 6 weeks (DD 42) and performed single cell Ca2+ imaging using Fluo-4 (Fig. 5a). This revealed that 38.0 ± 6.89 % of CTX0E16 neurons displayed spontaneous activity, defined as a sharp transient Ca2+ flux in a neuronal cell soma (Fig. 5b). In addition, application of KCl, a depolarising agent, resulted in a large Ca2+ flux in 79.1 ± 4.2 % of DD 42 CTX0E16 neurons (Fig. 5c), indicating the presence of voltage-gated Ca2+ channels, consistent with the observed increase in CACNA1C expression levels upon differentiation (Fig. 4a). Furthermore, addition of 1 μm tetrodotoxin (TTX) blocked spontaneous Ca2+ transients (Fig. 5d).Fig. 5

Bottom Line: Differentiation of CTX0E16 hNPCs predominately resulted in the generation of neurons expressing markers of cortical and glutamatergic (excitatory) fate, and with a typical polarized neuronal morphology.Taken together, these findings demonstrate that the CTX0E16 hNPC line is a robust source of cortical neurons, which display functional properties consistent with a glutamatergic phenotype.Thus CTX0E16 neurons can be used to study cortical cell function, and furthermore, as these neurons express a range of disease-associated genes, they represent an ideal platform with which to investigate neurodevelopmental mechanisms in native human cells in health and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic and Clinical Neuroscience, Institute of Psychiatry Psychology & Neuroscience, King's College London, London, SE5 8AF, UK. greg.anderson@kcl.ac.uk.

ABSTRACT

Introduction: Conditionally immortalised human neural progenitor cells (hNPCs) represent a robust source of native neural cells to investigate physiological mechanisms in both health and disease. However, in order to recognise the utility of such cells, it is critical to determine whether they retain characteristics of their tissue of origin and generate appropriate neural cell types upon differentiation. To this end, we have characterised the conditionally immortalised, cortically-derived, human NPC line, CTX0E16, investigating the molecular and cellular phenotype of differentiated neurons to determine whether they possess characteristics of cortical glutamatergic neurons.

Methods: Differentiated CTX0E16 cells were characterised by assessing expression of several neural fates markers, and examination of developing neuronal morphology. Expression of neurotransmitter receptors, signalling proteins and related proteins were assessed by q- and RT-PCR and complemented by Ca(2+) imaging, electrophysiology and assessment of ERK signalling in response to neurotransmitter ligand application. Finally, differentiated neurons were assessed for their ability to form putative synapses and to respond to activity-dependent stimulation.

Results: Differentiation of CTX0E16 hNPCs predominately resulted in the generation of neurons expressing markers of cortical and glutamatergic (excitatory) fate, and with a typical polarized neuronal morphology. Gene expression analysis confirmed an upregulation in the expression of cortical, glutamatergic and signalling proteins following differentiation. CTX0E16 neurons demonstrated Ca(2+) and ERK1/2 responses following exogenous neurotransmitter application, and after 6 weeks displayed spontaneous Ca(2+) transients and electrophysiological properties consistent with that of immature neurons. Differentiated CTX0E16 neurons also expressed a range of pre- and post-synaptic proteins that co-localized along distal dendrites, and moreover, displayed structural plasticity in response to modulation of neuronal activity.

Conclusions: Taken together, these findings demonstrate that the CTX0E16 hNPC line is a robust source of cortical neurons, which display functional properties consistent with a glutamatergic phenotype. Thus CTX0E16 neurons can be used to study cortical cell function, and furthermore, as these neurons express a range of disease-associated genes, they represent an ideal platform with which to investigate neurodevelopmental mechanisms in native human cells in health and disease.

No MeSH data available.