Waldron, PV; (2017) Developing retinal cell therapy: cones and cone-like cells in transplantation and development. Doctoral thesis , UCL (University College London).

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	Video Supplementary Movie 1 Single stack.avi - Supplemental Material Download (8MB)
	Video Supplementary Movie 2 Time lapse stack.avi - Supplemental Material Download (4MB)
	Video Supplementary Movie 3 RPE cell death.avi - Supplemental Material Download (3MB)
	Video Supplementary Movie 4 P14 retina.avi - Supplemental Material Download (267kB)
	Video Supplementary Movie 5 P6 Chrnb4.eGFP.avi - Supplemental Material Download (481kB)
	Video Supplementary Movie 6 Cells moving both ways.avi - Supplemental Material Download (159kB)
	Video Supplementary Movie 7 Cell being lost basally.avi - Supplemental Material Download (153kB)
	Video Supplementary Movie 8 Ciliobrevin D treated P5 retina.avi - Supplemental Material Download (580kB)

Abstract

Retinal cell replacement therapy aims to restore vision in retinal degenerative diseases by replacing dead photoreceptors. Injecting GFP+ rod photoreceptor precursors into the subretinal space of recipient mice leads to GFP+ cells being seen in the recipient retina. In models of retinal degeneration, replacement of proteins lacking in the recipient, as well as functional improvement, has been seen. This thesis aimed to extend this work to use cone photoreceptors, which are more important for human vision. The Chrnb4.eGFP model was selected as a source of cones, as well as the Nrl-/- and Nr2e3rd7/rd7 models which generate an increased number of cone-like cells. Cells were injected into the subretinal space of several different mouse recipients, and a number of retinal degenerative models representing a range of cone to rod ratios and functionalities. GFP+ photoreceptors, including unambiguous morphology and immunohistochemical markers were seen in recipient retinas after transplantation into all tested recipient types. The highest numbers occurred in Nrl-deficient and Prph2 mutant mice. The majority of GFP+ cells resembled rod photoreceptors in morphology, and did not express cone-specific markers, except in Nrl-deficient recipients. Evidence from these and other experiments showed that these results were most likely due not to cell integration but instead to the transfer of material including GFP from injected cells to existing recipient photoreceptors. To investigate functional outcomes of transplantation, electroretinography and multi-electrode array (MEA) techniques were used. MEA data showed no clear improvement in light response in treated retinas. Time-lapse imaging of explanted early postnatal retinal tissue using multi-photon microscopy was used to investigate the migratory behaviour of developing cone photoreceptor precursors around the ages of transplantation. This revealed a cyclical pattern of migration similar to interkinetic nuclear migration, with slow basal and rapid apical movements. Pharmacological intervention implicated the dynein/kinesin motor proteins in the apical movement seen.

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