Generation of induced pluripotent stem cells for patient-specific modelling of glaucoma in vitro

© 2016 Dr. Katherine Patrice GillGlaucoma is a group of optic neurodegenerative diseases of the retinal ganglion cells (RGCs) leading to reduced vision loss (Quigley, 1999). Despite glaucoma being prevalent in the developed world (Quigley and Broman, 2006), the aetiology of glaucoma remains unknown. Several known risk factors exist including: age, increased intraocular pressure, genetics and oxidative stress (Almasieh et al., 2012, Agarwal et al., 2009). As glaucoma is insidious, and coupled with the anatomical location of the RGCs, pre-mortem studies of its pathophysiology in vivo remains difficult. Induced pluripotent stem cell (iPSC) technology has revolutionized the medical research field enabling a generation of in vitro cellular models. Human induced pluripotent stem cells (hiPSCs) can therefore be used for interrogation of disease pathophysiology, such as for glaucoma. Addressing the aims of this project, this dissertation has two main parts: optimization of an RGC differentiation protocol and disease modelling. Part A describes the testing and optimization of differentiation protocols for generating RGCs from a commercially available human embryonic stem cell (hESC) line, showing that these methods can be applied to hiPSCs. During this process, an adherent retinal differentiation protocol was optimized to include an RGC enrichment step by magnetic activated cell sorting (MACS) utilizing an retina-specific RGC surface marker, Thy1.1. Following enrichment, hESC-derived RGCs were comprehensively characterized, including by: gene and protein expression; electrophysiology; and mitochondrial transport assays, a feature that is underwhelming performed in other RGC protocols. Further, RNA sequencing (RNAseq) analysis revealed that the hESC-derived RGCs exhibit a similar gene expression profile to publicly available human ganglion cell layer (GCL) data confirming the enrichment of the hESC-differentiated population of retinal cells. Part B describes the generation of iPSC lines reprogrammed from the fibroblasts of individuals harbouring genetic risk variants associated with primary open angle glaucoma (POAG), the most common form of glaucoma. To elucidate a potential molecular mechanism for POAG, RNAseq was performed upon iPSC-derived RGCs harbouring different genetic variants. This data indicated that significant misexpression of genes involved in extracellular matrix organization, collagen synthesis and integrin signalling, may be the cause for differences between POAG affected and unaffected individual iPSC lines. These findings support the mechanical theory for glaucoma (Morgan, 2000). To investigate a disease-associated phenotype, apoptotic death of the iPSC-derived RGCs in response to an oxidative stress insult was analysed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The results indicated a statistically insignificant (p=0.0503) trend towards increased susceptibility of the POAG affected iPSC-derived RGCs. Instead, these findings may indicate a subtle effect of oxidative stress that may be suitable for the slow, progressive development of POAG in vivo. As POAG is an age-related disease, increased levels of oxidative stress occurring during the normal ageing process may expose individuals with underlying genetic predispositions. Therefore, individuals predisposed towards POAG may develop RGCs with a reduced threshold for ROS and other by-products of oxidative stress. In summary, the data presented within this PhD describes an in vitro disease model for POAG with the use of patient-specific iPSCs that has advanced our understanding of potential molecular mechanisms for its development in vivo