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Generation of stem cell derived photoreceptors for the treatment and modelling of retinal degeneration

Final Report Summary - STEMRD (Generation of stem cell derived photoreceptors for the treatment and modelling of retinal degeneration)

The objective of this project was to determine if pluripotent stem (PS) cell-derived photoreceptors could be utilised as a therapeutic source of cells to repair the degenerate retina and to model photoreceptor disorders in vitro. During our investigations, we made observations that led us to question the underlying cellular mechanisms behind functional rescue following donor-derived photoreceptor transplantation. While donor photoreceptor cell migration and integration occurs, it accounts for far fewer of the reporter-labeled cells observed than previously thought. In addition we have demonstrated that donor-derived and PS cell-derived photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells (Pearson et al., 2016; Waldron et al., 2018). The cellular mechanisms by which this occurs have yet to be determined, but it appears that a wide array of either RNAs and/or proteins might be exchanged in sufficient quantities to render the recipient cells functional. Although we find that material transfer accounts for a significant proportion of rescued cells, following cone photoreceptor transplantation into non-degenerative hosts, substantial numbers of cones integrated into degenerate retinas that involve disruption of the outer limiting membrane. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome (Waldron et al., 2018).

Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. To provide a source of cone photoreceptor precursors for retinal cell therapy we initially investigated the generation of cones from mouse embryonic stem (ES) cells using a 3D retinal differentiation method. We found that the specification of cone photoreceptor cell fate followed the same pathways as established in vivo and adapting our differentiation method accordingly, we were able to produce and purify sufficient quantities of stage-specific cone photoreceptor precursors to investigate their transplantation to the adult degenerate retina. ES cell-derived cone photoreceptors matured in vivo to express photopigments that they lacked in vitro and survived for at least 3 weeks in the subretinal space (Kruczek et al., 2017). Mouse ES cell-derived photoreceptors can be generated in less than 30 days, however for human photoreceptors the differentiation process takes at least 100 days, in line with natural development. Despite this lengthy culture period we established a robust method to generate both cone and rod human photoreceptors from PS cell sources. Following transplantation, we have observed the survival of these cells in close apposition with host retinal neurons in mouse models of retinal degeneration for up to 6 weeks (Gonzalez-Cordero et al., 2017). Further work to investigate the functional restoration of light responses attributable to mouse and human PS cell-derived transplanted cells is currently in progress. These findings will determine the ability of PS cell-derived photoreceptor precursors to repair the degenerate adult retina.

Finally, the majority of inherited retinal disorders affect photoreceptor structure and function, therefore to model photoreceptor disease effectively differentiated human photoreceptors need to demonstrate mature morphology, which includes the formation of inner and outer segments connected by a cilium. We examined human PS cell-derived photoreceptor development for over 200 days and have found that using our 2D/3D retinal differentiation method, photoreceptors exhibit mature structural morphology by 190 days in culture in all PS cell lines tested (Gonzalez-Cordero et al., 2017). In parallel, we have established a robust platform for reprogramming patient blood samples and have established multiple retinal disease iPS cell lines with which to study inherited retinal degeneration. We have further improved our long term culture methods to enable PS cell-derived photoreceptors to efficiently form outer segments with more organised membranous disc structures. This has allowed us to model retinal disease phenotypes more reliably, confirm the disease phenotypes observed using viral vectors and/or gene editing and test possible therapeutic treatments for specific inherited retinal disorders in vitro.