Baron, M.; (2012) Towards cone photoreceptor transplantation for retinal repair. Doctoral thesis , UCL (University College London).

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Abstract

Photoreceptors, the light sensing neurons of the retina, are pivotal to human vision. Degenerative diseases of the retina leading to photoreceptor death are a prime cause of untreatable blindness. In particular, loss of cone photoreceptors, which convey high visual acuity and colour perception, most severely a�ffects central vision. This thesis investigates the possibility of replacement of lost cone photoreceptors by transplantation. The cone-rod homoeobox gene (Crx) is expressed in developing cone and rod photoreceptors cells. This thesis examines the potential of Crxexpressing cells to generate new cone and rod photoreceptors after transplantation into adult retinae. The expression of a novel photoreceptorspeci�c CrxGFP transgenic reporter mouse was characterised. In order to confi�rm the nuclear retinoid x receptor gamma (Rxrγ) as an early cone marker, BrdU pulse chase time-course experiments were performed utilising the reporter line as a marker for post-mitotic photoreceptors. To assess the efficacy of CrxGFP photoreceptor precursors for therapy and determine a suitable ontogenetic stage for cone transplantation, precursor cells were isolated from embryonic and early postnatal retinae by FACS and transplanted into adult retinae. Cells integrated and developed into mature cone and rod photoreceptors, distinguished by morphology and Rxrγ staining. Cone photoreceptors were only detected when embryonic donor cells were used, while rods integrated at all time-points, although integration efficiency was highest with post-natal donors. Integrated photoreceptors stained for a variety of functional rod and cone proteins, as well as several synaptic markers. In order to expose precursor cells to di�fferent recipient environments, embryonic precursors were also transplanted into two models of retinal degeneration. While the outer limiting membrane defects of the Crb1rd8/rd8 mouse had no signi�ficant impact on photoreceptor integration, the lack of cones in the Gucy2e-/- model led to signi�ficantly increased cone integration. Attempts were also made to derive an equivalent transplantable photoreceptor precursor cell species from renewable stem cell sources. Ciliary epithelium-derived cells, which can be isolated from the adult eye and were previously attributed with retinal stem cell qualities, were expanded in culture, but no evidence for photoreceptor diff�erentiation was found. Embryonic stem cells were di�fferentiated towards a retinal lineage and an RxrγRFP transgene reporter was introduced in order to label ES cell derived cone photoreceptors. These studies show for the �first time that new cone cells can integrate into the adult retina and contribute to the development of stem cell therapy for retinal degenerative disease.

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