Abstract
IntroductionConditionally 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.MethodsDifferentiated 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 Ca2+ 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.ResultsDifferentiation 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 Ca2+ and ERK1/2 responses following exogenous neurotransmitter application, and after 6 weeks displayed spontaneous Ca2+ 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.ConclusionsTaken 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0136-8) contains supplementary material, which is available to authorized users.
Highlights
Immortalised human neural progenitor cells represent a robust source of native neural cells to investigate physiological mechanisms in both health and disease
Human neural stem cells and the neural progenitor cells (NPCs) that they generate have become a major focus of interest as they provide a renewable and accessible model system in which to investigate basic human neurodevelopment mechanisms and complex neurodevelopmental disorders [1,2,3,4]
Previous studies have demonstrated that human neural progenitor cell (hNPC) are self-renewing and are multipotent, being able to differentiate into multiple neural cell types, including different types of neurons, astroctyes and oligodendrocytes [11,12,13,14]
Summary
Immortalised human neural progenitor cells (hNPCs) represent a robust source of native neural cells to investigate physiological mechanisms in both health and disease. 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. Immortalised hNPCs isolated from first trimester human fetal spinal cord, midbrain, hippocampus and cortex have been successfully differentiated into functional neurons and interneurons both in vitro and in vivo [12, 14, 18, 19] These hNPC lines have already been used to investigate the mechanisms of antidepressant drug action [18], to characterise the biological functions of susceptibility genes for schizophrenia and bipolar disorder [20] and are currently in trial for engraftment following ischaemic stroke [21]. In order to fully understand how such mechanisms may impact early neurodevelopment, and in particular corticogenesis, it is critical to determine whether these cell lines can a) robustly differentiate into glutamatergic neurons in vitro; b) display functional responses to activation of neurotransmitter signalling systems; c) form putative synapses; and d) are able to respond to activity-dependent stimulations in the anticipated manner
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