Novel cell transplantation strategies for treatment of degenerative retinal disease
Retinal degenerations, both hereditary and acquired, are major causes of visual morbidity world-wide. Two of the principal disease categories are age-related macular degeneration (AMD) and retinitis pigmentosa (RP). AMD is the major cause of blind registrations in the over 65 age group in the western world and retinitis pigmentosa has an incidence of 1:3500 but, significantly, affects much younger patients. As yet there are no treatments available for these diseases that are either definitively preventative or curative. Retinal cell transplantation is one treatment strategy that has been proposed for such patients: retinal pigment epithelium (RPE) grafts for AMD and neural retina for retinal dystrophies. The genetic heterogeneity of retinitis pigmentosa and the lack of understanding of the key pathobiological mechanisms in AMD make treating the final common pathway with retinal cell transplantation an attractive option. To date results in humans have not been encouraging, highlighting the need for further studies in animal models in terms of visual function, the basic cellular and molecular mechanisms involved and exploration of other potential cell types for transplantation. This thesis examines the potential of several different donor cell types, both immortalised allogeneic (LD7.4) and xenogeneic (human: hlRPE7 & ARPE19) RPE cell lines for treating retinal degeneration in the dystrophic Royal College of Surgeons (RCS) rat. The dystrophic RCS rat has a primary RPE defect resulting in failure of rod outer segment ingestion and subsequent secondary photoreceptor degeneration. Additionally, syngeneic neonatal Schwann cells were transplanted into the subretinal space of the rhodopsin knockout (Rho -/-) mouse, a knockout model that never elaborates outer segments. Dystrophic RCS rats (21 days old) received subretinal grafts of immortalised allogeneic (LD7.4) RPE cell lines in non-immunosuppressed rats or immortalised xenogeneic (hlRPE-7 or ARPE19) cell lines in immunosuppressed rats. Appropriate sham control injections were delivered to age-matched groups of rats. The animals' visual function was assessed longitudinally using a head-tracking system at 16, 20, and 26 weeks of age. Retinal sensitivity maps were produced for selected animals from each group at 20 weeks (LD 7.4 only) and 26 weeks (hlRPE7 and LD7.4 cell). Further groups of animals received grafts of ARPE19 or LD7.4 cells pre-labelled with bromodeoxyuridine (BrdU) in order to identify donor cells in vivo. Another labelling technique, transfecting LD7.4 cells with Human Foamy Virus (HFV) expressing green fluorescent protein (GFP), was also tested. Syngeneic neonatal Schwann cells were grafted to the Rho -/- mouse at 5 weeks of age, prior to total photoreceptor loss and analysed for functional (electroretinogram, ERG) and histological rescue at 10 weeks. Each cell type demonstrated behavioural, functional and morphological rescue in the RCS rat model. In the human cell-BrdU labelled group (i.e. with immunosuppression), grafted cells were seen integrated between host RPE cells on Bruch's membrane in association with areas of photoreceptor rescue up to five months post-grafting. In contrast, animals that received allografted cells prelabelled with BrdU (without immunopuppression) were not detectable on the host Bruch's membrane immunohistochemically by 4 weeks post-transplantation. Even though no donor cells were detectable, host visual function and photoreceptors were preserved. This result was repeated in the LD7.4/GFP labelled cell group. The Schwann cell grafted Rho -/- mice showed significant photoreceptor preservation over sham-injected animals at 10 weeks of age. These results demonstrate a number of key points regarding cell transplantation strategies for retinal degeneration. In some circumstances it is possible to achieve integration of grafted RPE cells on host Bruch's membrane with preservation of photoreceptors and visual function in the RCS rat. Allografts of immortalised RPE cells do not appear to survive in the subretinal space of the RCS rat beyond 4 weeks. Schwann cell transplantation to two different rodent models (RCS rat and Rho -/- mouse) preserved visual function in the former and morphology in both groups. Cell transplantation for retinal degeneration is an exciting prospect for treating these diseases, RPE transplantation for AMD or primary RPE disease and Schwann cell transplantation for hereditary retinal disease (RP). Before these strategies become clinically useful a number of issues need to be addressed including integration of the grafted material with the host, appropriate functioning of the grafted tissue in the subretinal space (SRS) and long-term survival of the transplants.