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Human retinal organoid transplantation could potentially be a treatment for degenerative retinal diseases. How the recipient retina regulates the survival, maturation, and proliferation of transplanted organoid cells is unknown. We transplanted human retinal organoid-derived cells into photoreceptor-deficient mice and conducted histology and single-cell RNA sequencing alongside time-matched cultured retinal organoids. Unexpectedly, we observed human cells that migrated into all recipient retinal layers and traveled long distances. Using an unbiased approach, we identified these cells as astrocytes and brain/spinal cord-like neural precursors that were absent or rare in stage-matched cultured organoids. In contrast, retinal progenitor-derived rods and cones remained in the subretinal space, maturing more rapidly than those in the cultured controls. These data suggest that recipient microenvironment promotes the maturation of transplanted photoreceptors while inducing or facilitating the survival of migratory cell populations that are not normally derived from retinal progenitors. These findings have important implications for potential cell-based treatments of retinal diseases., Competing Interests: Conflict of interests M.S.S. is/was a paid advisor to Revision Therapeutics, Johnson & Johnson, Third Rock Ventures, Bayer Healthcare, Novartis Pharmaceuticals, W. L. Gore & Associates, Deerfield, Trinity Partners, Kala Pharmaceuticals, Janssen, and Acucela. M.S.S. has received sponsored research support from Bayer for other research. S.B. receives research support from Genentech, is a co-founder and shareholder in CDI Labs, LLC, and is/was a consultant for Third Rock Ventures and Tenpoint Therapeutics. M.S.S. and R.J.J are co-founders and shareholders in Agnos Therapeutics. These arrangements have been reviewed and approved by the Johns Hopkins University in accordance with its conflict-of-interest policies. M.S.S., S.B., J.Q., R.J.J., Y.V.L., C.P.S., M.V.C.-S., M.F.-B., S.A.-D., and K.V.L. are named as inventors on patents or patent applications assigned to their respective universities., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Cone photoreceptor transplantation is a potential treatment for macular diseases. The optimal conditions for cone transplantation are poorly understood, partly because of the scarcity of cones in donor mice. To facilitate allogeneic cone photoreceptor transplantation studies in mice, we aimed to create and characterize a donor mouse model containing a cone-rich retina with a cone-specific enhanced green fluorescent protein (EGFP) reporter., Methods: We generated OPN1LW-EGFP/NRL -/- mice by crossing NRL -/- and OPN1LW-EGFP mice. We characterized the anatomical phenotype of OPN1LW-EGFP/NRL -/- mice using multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, immunohistology, and transmission electron microscopy. We evaluated retinal function using electroretinography (ERG), including 465 and 525 nm chromatic stimuli. Retinal sheets and cell suspensions from OPN1LW-EGFP/NRL -/- mice were transplanted subretinally into immunodeficient Rd1 mice., Results: OPN1LW-EGFP/NRL -/- retinas were enriched with OPN1LW-EGFP + and S-opsin + cone photoreceptors in a dorsal-ventral distribution gradient. Cone photoreceptors co-expressing OPNL1W-EGFP and S-opsin significantly increased in OPN1LW-EGFP/NRL -/- compared to OPN1LW-EGFP mice. Temporal dynamics of rosette formation in the OPN1LW-EGFP/NRL -/- were similar as the NRL -/- with peak formation at P15. Rosettes formed preferentially in the ventral retina. The outer retina in P35 OPN1LW-EGFP/NRL -/- was thinner than NRL -/- controls. The OPN1LW-EGFP/NRL -/- ERG response amplitudes to 465 nm stimulation were similar to, but to 535 nm stimulation were lower than, NRL -/- controls. Three months after transplantation, the suspension grafts showed greater macroscopic degradation than sheet grafts. Retinal sheet grafts from OPN1LW-EGFP/NRL -/- mice showed greater S-opsin  +  cone survival than suspension grafts from the same strain., Conclusions: OPN1LW-EGFP/NRL -/- retinae were enriched with S-opsin + photoreceptors. Sustained expression of EGFP facilitated the longitudinal tracking of transplanted donor cells. Transplantation of cone-rich retinal grafts harvested prior to peak rosette formation survived and differentiated into cone photoreceptor subtypes. Photoreceptor sheet transplantation may promote greater macroscopic graft integrity and S-opsin + cone survival than cell suspension transplantation, although the mechanism underlying this observation is unclear at present. This novel cone-rich reporter mouse strain may be useful to study the influence of graft structure on cone survival., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)