Generation and characterisation of transplantable hiPSC-derived photoreceptors for treatment of retinal degeneration

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Vision impairment and blindness caused by the degeneration and loss of the non-regenerating light-sensing photoreceptors in the retina affects millions of patients with no curative therapy currently established. Transplantation of healthy photoreceptors represents a promising treatment approach. Within the BMBF-funded consortium ‘ReSight’ a platform for the robust and reproducible generation and isolation of induced pluripotent stem cell (iPSC)-derived human photoreceptors and their transplantation into pre-clinical mouse models of retinal degeneration has been established. Using photoreceptor-specific reporter iPSC lines, retinal organoids containing up to 60% fluorescently-labelled photoreceptors are generated and FACS enriched for transplantation studies. Retinal organoids generated from the Crx-mCherry-iPSC line contain at least three principle cell-types - photoreceptor precursors, rods and cones - with changing proportions at different developmental stages. Given the heterogeneity of this reporter-labeled cell population, expression profiling at the single cell level will be necessary to assess the exact cell composition of retinal organoid-derived transplants of different ages and their differentiation and maturation within different disease environments over time. Indeed, donor human photoreceptors survive up to 9 months in mouse recipients, express photoreceptor-specific markers, contact host bipolar cells and interact with host Müller glia. With prolonged time after transplantation, human photoreceptors further mature, forming ribbon synapses, mitochondria-rich inner segments and membrane-discs containing outer segments oriented towards the retinal pigment epithelium. However, histological analysis shows obvious differences in regard to structure and maturation of donor photoreceptors after transplantation, correlating with (i) different developmental stages of the transplants (donor photoreceptor isolation from younger vs. older retinal organoids), (ii) time within the recipient, (iii) chosen retinal degeneration model (moderate vs. complete degeneration), and (iv) integrating vs. non-interacting graft areas within the same host retina. Analysis by scRNAseq will therefore provide detailed insights into human photoreceptor transplant characteristics, indispensable for defining and optimizing utilizable donor cell populations as an essential prerequisite for successful photoreceptor transplantation and translation towards clinical application.