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
