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Modelling Usher Syndrome using patient-derived pluripotent stem cells in 3D organoid and Retinal Pigment Epithelium (RPE) monolayer culture systems to investigate the role of RPE in retinal degeneration



Wong, Rachel Rui Ching;

(2022)

Modelling Usher Syndrome using patient-derived pluripotent stem cells in 3D organoid and Retinal Pigment Epithelium (RPE) monolayer culture systems to investigate the role of RPE in retinal degeneration.

Doctoral thesis (Ph.D), UCL (University College London).


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Rachel Rui Wong Thesis – Corrected 200322.pdf
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Abstract

Usher Syndrome is a debilitating autosomal recessive genetic disease and leading cause of deafblindness. Hearing loss is corrected using cochlear implants but the retinitis pigmentosa is currently untreatable. Usher type I displays the most severe phenotype and MYO7A mutations represent the most common cause. Previous studies suggest the encoded MYO7A is a carrier motor protein that moves along actin filaments. The Myo7a-null mouse model, Shaker-1, faithfully displayed a deafness phenotype but not degeneration of the retina, which has hampered study of the disease mechanism.
This thesis aimed to use Usher patient-derived induced pluripotent stem cells (iPSC) to investigate the effect of MYO7A mutation on human retinal cells; specifically, to examine whether the retinal pigment epithelium (RPE) may be affected in Usher patients contributing to retinal degeneration of the adjacent photoreceptor cells. RPE monolayer and 3D retinal organoids culture systems were established on embryonic stem cells and applied to MYO7A-affected patient-derived iPSC to investigate RPE dysfunction and gene expression profiles. Characterization of control and patient iPSC-RPE via transepithelial-electrical resistance analysis, transmission electron microscopy and immunohistochemistry confirmed formation of tight junctions, normal RPE morphology and expression of RPE markers. Lower uptake of bovine outer segments was recorded in patient iPSCRPE, which may be due to loss of MYO7A negatively affecting the transport of outer segment-containing phagosomes or enzymecontaining melanosomes that breakdown phagosomes. Transcriptome profiles associated with patient iPSC-RPE cells were identified through RNAseq analysis. Upregulation of RPE-specific genes confirmed the iPSC-RPE specification and differentiation. Gene set enrichment analysis indicated dysregulation in genesets associated with extracellular matrix and collagen fibril organisation, and epithelial-mesenchymal transition. The SLC30A8 zinc transporter was also differentially expressed in patient iPSC-RPE. In summary, 2 this study showed that patient iPSC-RPE recapitulated native RPE characteristics but displayed reduced phagocytosis and alterations in gene expression profiles that suggests a pathological RPE phenotype.

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