Electrophysiological characterization of human stem cell-derived neurones and glia in models of neurodevelopmental and neurodegenerative diseases
James, Owain Thomas
Human pluripotent stem cell (hPSC)-derived neuronal and glial material presents a relatively new opportunity to model human neurophysiology in both health, and disease. Validation of regionally-defined hPSC-derived neurones and glia cultures thus represents the founding blocks of technology that aims to complement existing models. Principally, the relevance of in vitro hPSC-derived material is determined by how representative it is of native material, yet at present the physiology of these cells remains underexplored. Here, electrophysiology and pharmacology are used to functionally assess hPSC-derived excitatory cortical neurones (hECNs), motorneurones (MNs) and oligodendrocyte-lineage cells in the context of regional-specific properties and maturation. These properties are then examined in material derived from hPSCs generated from patients with neurological disorders. This thesis examines of the properties of GABAARs and strychnine-sensitive glycine receptors (GlyRs) in hECNs by assessing their subunit composition, and compares these with studies which have made comparable investigations of rodent tissue where maturation is associated with a shift in GABAA and GlyR compositions. Using pharmacology and RNAseq analysis, GABAAR and GlyRs in hECNs were found to possess receptor populations typical of those reported in the immature cortex. hECNs generated from patients harbouring a mutation to the Disrupted-in-schizophrenia-gene 1 (DISC1), a candidate schizophrenia gene, were then examined. Imbalances in the excitation/inhibition balance are suspected in schizophrenia and, in this regard, the intrinsic excitability properties alongside expression and composition of major neurotransmitter receptors and intracellular chloride concentration were assessed. No obvious differences in excitability or functional expression of AMPARs, GABAARs or NMDARs were observed between case and control derived neurones. Receptor composition and intracellular chloride concentrations were found to be predominantly immature-like, however, AMPAR composition and intracellular chloride concentration were found to be like that of adult cortical neurones. These data are discussed in the context of modelling DISC1-associated pathologies. Thirdly, MNs from hPSCs generated from ALS patients harbouring mutations on the C9ORF72 gene were examined. The hypothesis that increased glutamate-mediated excitoxicity could, in part, be explained by increased expression of Ca2+- permeable AMPARs was examined. The estimated mean single-channel conductance of AMPARs was found to be high in MNs derived from ALS patients, reminiscent of Ca2+-permeable AMPARs and was reversed by gene-editing of the C9ORF72 mutation. Finally, oligodendrocytes generated from ALS patients harbouring TARDBP mutations were examined. Distinctive electrophysiological shifts in oligodendrocytes-lineage cell development are reported. A similar AMPAR phenotype of elevated Ca2+-permeable AMPAR expression was observed in oligodendrocytes derived from two patient hPSC lines and was rescued in an isogenic, gene-edited line, raising the intriguing possibility of convergence in pathophysiologies in the nature of the overlap between cell-type, AMPAR pathology and excitotoxicity in ALS disease progression mechanisms.