Psychiatry - Theses
Permanent URI for this collection
Search Results
1 - 1 of 1
-
ItemFunctional in vitro modelling of the nervous system using human pluripotent stem cells; a platform to study brain disorders( 2016)Human pluripotent stem cells (hPSC) constitute a valuable resource for establishing in vitro models of nervous system development, function and dysfunction. This is particularly needed for complex developmental brain disorders whereby animal models and access to postmortem human brain tissue may be limited. A major prerequisite step to developing suitable in vitro models of the human nervous system is to establish hPSC induction protocols to derive regionally specific neuronal populations. The first major aim of this thesis was to characterize the phenotype and maturation stage of neural progenitor cell types derived from the following induction protocols: the dual SMAD inhibition protocol (DS), sonic hedgehog pathway agonist protocol (SAG), and SMAD/GSK3b inhibition protocol (CHIR). Our studies show that the dual SMAD inhibition protocol results predominantly in cortical progenitors representative of deep and intermediate cortical layer neurons, while combined early treatment of sonic hedgehog agonist during neural induction give rise to progenitors of ventral cortical identity. Using an induction protocol involving inhibition of the SMAD and GSK3b pathways, followed by later exposure to BMP2/4, hPSC are directed towards neural crest lineages, which upon further differentiation give rise to peripheral sensory neurons. Another essential component for in vitro modelling of the human nervous system using hPSC is to demonstrate neuronal activity and connectivity as measures of functionality. Whilst intercellular activity and characteristics of hPSC-derived neurons are well documented, reports on their potential to form functional network is scarce. This feature is critical for using hPSC to model psychiatric disorders such as Autism spectrum disorder (ASD) in which abnormal connectivity is one of the major characteristics of the disease. Using microelectrode arrays (MEA), the second major aim of this thesis was to assess the functional maturation rate and neuronal network activities of hPSC-derived cortical and peripheral sensory neuronal populations. Our data demonstrate that functional maturation of hPSC-derived cortical neurons occurs at a slower rate relative to hPSC-derived sensory neurons with no evidence of network activities detected over 8 weeks of differentiation. In contrast, hPSC-derived sensory neurons show faster maturation and form functional networks in vitro by week 6-7 post differentiation. Further functional characterization on MEA reveals the capability of sensory neuronal subtypes to respond to appropriate stimuli including heat, capsaicin and hypoosmotic induced stretch. The final major objective of the thesis was to employ our in vitro model of corticogenesis to interrogate neurodevelopmental disorders, specifically microcephaly and ASD. Accordingly, one aim was to investigate the function of WD Repeat Domain 62 (WDR62) in neurogenesis, a major candidate gene of autosomal recessive primary microcephaly. Studies of WDR62 utilizing human based in vitro models are lacking. Our studies show that WDR62 expression coincides with SOX2 and PAX6 expression during hPSC neural induction, the prime period of neurogenesis. Furthermore, knockdown of WDR62 expression in hPSC impacted on neurogenic and gliogenic differentiation, as shown by a reduction in TBR2 (EOMES) and S100b+ progenitor populations, respectively. The final aim was to characterize idiopathic ASD induced pluripotent stem cell (iPSC)-derived cortical neurons, focusing on the expression of genes associated with corticogenesis, glia specification, synaptic function, cell proliferation and cell death. Our data suggests higher expression of TBR1 and lower expression of MAP2AB, GFAP and GRM5 in ASD neurons compared to controls. These results implicate processes related to deep cortical layer neuronal differentiation, dendritic formation, glial differentiation and synaptic function in ASD core pathophysiology, paving the way for future studies utilizing these ASD iPSC lines. In summary, these studies provide key information for utilizing hPSC to model neural circuitry systems and demonstrate hPSC robustness as a complementary in vitro model system to investigate developmental brain disorders.