Jones, MF; (2013) Development of Methods for Retinal Ganglion Cell Replacement in Glaucoma. Doctoral thesis (PhD), UCL (University College London).

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Abstract

A sub population of human Müller glia contained within the neural retina is known to exhibit stem cell characteristics. Previous studies in the host laboratory have investigated their ability to differentiate into different retinal populations in vitro, providing a potential source of cells for development of therapies to treat retinal degenerative diseases. This study investigated molecular factors involved in differentiation of retina ganglion cells (RGCs) derived from human Müller stem cells. It also aimed to explore the feasibility of transplanting RGC derived from Müller stem cells into large mammalian eyes, using collagen based cellular scaffolds. These objectives ultimately aimed to study the potential application of Müller stem cells for treatment of late stage glaucoma. As determined by microarray analysis of total RNA specimens, in vitro culture of Müller stem cells undergoing Notch inhibition in the presence or absence of bFGF, led to alteration in microRNA (miRNA) profiles. These are short RNA molecules synthesised within cells that play a role in various cellular processes. MicroRNAs associated with Notch signalling, cell cycling and differentiation, were enriched in Müller cell populations undergoing Notch inhibition, conditions previously shown to induce RGC development. Elevated expression of these specific miRNAs suggests the emergence of novel targets under the regulation of the Notch pathway in Müller stem cells. These constitute factors that could be potentially used to develop therapies which facilitate endogenous neural differentiation of the latent Müller stem cell population in the human neural retina. Collagen based cellular scaffolds were developed to deliver RGCs derived from Müller stem cells onto the inner retina of the large mammalian eye. Collagen is a ubiquitous protein found within numerous tissues types, acting as a framework for cellular support and adhesion. Delivery of grafted cells onto retinal explants in vitro, and transplantation of scaffolds into the rabbit eye in vivo, was examined by confocal microscopy and showed that plastic compressed collagen scaffolds served as a potential suitable substrate for transfer of cells to the host retina. Integration was observed in some cases and rarely observed into host tissue, facilitated by degradation of the extracellular matrix by chondroitinase ABC and by the use of the anti-inflammatory agent triamcinolone in vivo. In conclusion, the present study showed that standardised protocols used to differentiate Müller stem cells into RGC promoted alterations in Müller stem cell miRNAs associated with RGC development and maturation. In addition, compressed collagen scaffolds were shown to aid delivery of RGCs onto the inner retinal surface. These observations pave the way for further investigations to promote endogenous retina regeneration and refinement of transplantation strategies to apply these cells to the development of human therapies.

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