Anatomy and Neuroscience - Theses
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ItemUsing induced pluripotent stem cell-derived retinal pigment epithelium cells to characterise phenotypic differences associated with reticular pseudodrusen in age-related macular degeneration( 2024-02)Age-Related Macular Degeneration (AMD) is one of the leading causes of severe vision loss in individuals aged over fifty in Western populations. A hallmark of AMD pathogenesis is the accumulation of lipid and protein deposits, termed drusen, in the macula. Past studies have established that retinal pigment epithelium (RPE) dysfunction alone initiates drusen-like deposit formation, with disease lines exhibiting greater volume and number with respect to deposit formation in vitro. Despite this, automated quantification methods for these deposits were lacking in the literature. While conventional drusen on the basal side of the RPE is a hallmark of AMD pathogenesis, the recognition of reticular pseudodrusen (RPD) on the apical side indicates a distinct AMD phenotype. This thesis focuses on the modelling of RPD using human induced pluripotent stem cells (hiPSCs) as part of this extensive investigation into molecular distinctions between conventional AMD and RPD. Initial identity confirmation of hiPSC-derived RPE cells involved rigorous characterisation and functional assays to establish baseline phenotypic presentations. Subsequent investigations delved into pathological AMD signatures, initially confirming the appearance of hallmark drusen deposits in culture. Recognising the time-intensive and manual nature of existing quantification methods, significant efforts were made to develop and publish two semi-automated quantification tools, fostering standardised approaches in the scientific community. These tools facilitated stressor experiments with N-Retinylidene-N-Retinylethanolamine (A2E), revealing its potential exacerbation of AMD phenotypes in vitro. A large-scale proteomics study aimed to identify differentially expressed proteins between AMD and RPD samples uncovered distinctions in extracellular matrix proteins, the spliceosome complex, and cellular homeostasis, including mitochondrial health. Validation of mitochondrial hits involved assays comparing baseline oxidative states. Utilising the developed pipelines for quantifying drusen-like deposits at baseline and post-stressor treatment, and investigating baseline oxidative stress in RPE cultures derived from patient-specific iPSCs revealed differences between AMD cohorts with and without RPD. This study underscores the utility of patient-specific iPSCs for in vitro modelling of AMD pathogenesis, elucidating variations in phenotypic presentations within stratified AMD disease cohorts. The data suggests discernible differences in disease profiles concerning mitochondrial dysfunction and drusen accumulation between AMD samples with or without RPD, emphasising the potential of iPSCs in unravelling the complexities of AMD.