Characterization and allogeneic transplantation of a novel transgenic cone-rich donor mouse line.

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.

Damage-Induced Senescent Immune Cells Regulate Regeneration of the Zebrafish Retina.

Zebrafish spontaneously regenerate their retinas in response to damage through the action of Müller glia (MG). Even though MG are conserved in higher vertebrates, the capacity to regenerate retinal damage is lost. Recent work has focused on the regulation of inflammation during tissue regeneration, with temporal roles for macrophages and microglia. Senescent cells that have withdrawn from the cell cycle have mostly been implicated in aging but are still metabolically active, releasing a variety of signaling molecules as part of the senescence-associated secretory phenotype. Here, we discover that in response to retinal damage, a subset of cells expressing markers of microglia/macrophages also express markers of senescence. These cells display a temporal pattern of appearance and clearance during retina regeneration. Premature removal of senescent cells by senolytic treatment led to a decrease in proliferation and incomplete repair of the ganglion cell layer after N-methyl-D-aspartate damage. Our results demonstrate a role for modulation of senescent cell responses to balance inflammation, regeneration, plasticity, and repair as opposed to fibrosis and scarring.

Damage-induced senescent immune cells regulate regeneration of the zebrafish retina.

Zebrafish spontaneously regenerate their retina in response to damage through the action of Müller glia. Even though Müller glia (MG) are conserved in higher vertebrates, the capacity to regenerate retinal damage is lost. Recent work has focused on the regulation of inflammation during tissue regeneration with precise temporal roles for macrophages and microglia. Senescent cells that have withdrawn from the cell cycle have mostly been implicated in aging, but are still metabolically active, releasing proinflammatory signaling molecules as part of the Senescence Associated Secretory Phenotype (SASP). Here, we discover that in response to retinal damage, a subset of cells expressing markers of microglia/macrophages also express markers of senescence. These cells display a temporal pattern of appearance and clearance during retina regeneration. Premature removal of senescent cells by senolytic treatment led to a decrease in proliferation and incomplete repair of the ganglion cell layer after NMDA damage. Our results demonstrate a role for modulation of senescent cell responses to balance inflammation, regeneration, plasticity, and repair as opposed to fibrosis and scarring.

Single-cell transcriptome analysis of xenotransplanted human retinal organoids defines two migratory cell populations of nonretinal origin.

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.

Localized Structural and Functional Deficits in a Nonhuman Primate Model of Outer Retinal Atrophy.

Purpose: Cell-based therapy development for geographic atrophy (GA) in age-related macular degeneration (AMD) is hampered by the paucity of models of localized photoreceptor and retinal pigment epithelium (RPE) degeneration. We aimed to characterize the structural and functional deficits in a laser-induced nonhuman primate model, including an analysis of the choroid. Methods: Macular laser photocoagulation was applied in four macaques. Fundus photography, optical coherence tomography (OCT), dye angiography, and OCT-angiography were conducted over 4.5 months, with histological correlation. Longitudinal changes in spatially resolved macular dysfunction were measured using multifocal electroretinography (MFERG). Results: Lesion features, depending on laser settings, included photoreceptor layer degeneration, inner retinal sparing, skip lesions, RPE elevation, and neovascularization. The intralesional choroid was degenerated. The normalized mean MFERG amplitude within lesions was consistently lower than control regions (0.94 ± 0.35 vs. 1.10 ± 0.27, P = 0.032 at month 1, 0.67 ± 0.22 vs. 0.83 ± 0.15, P = 0.0002 at month 2, and 0.97 ± 0.31 vs. 1.20 ± 0.21, P < 0.0001 at month 3.5). The intertest variation of mean MFERG amplitudes in rings 1 to 5 ranged from 13.0% to 26.0% in normal eyes. Conclusions: Laser application in this model caused localized outer retinal, RPE, and choriocapillaris loss. Localized dysfunction was apparent by MFERG in the first month after lesion induction. Correlative structure-function testing may be useful for research on the functional effects of stem cell-based therapy for GA. MFERG amplitude data should be interpreted in the context of relatively high intertest variability of the rings that correspond to the central macula. Sustained choroidal insufficiency may limit long-term subretinal graft viability in this model.

miRNAs and Müller Glia Reprogramming During Retina Regeneration.

The use of model systems that are capable of robust, spontaneous retina regeneration has allowed for the identification of genetic pathways and components that are required for retina regeneration. Complemented by mouse models in which retina regeneration can be induced after forced expression of key factors, altered chromatin accessibility, or inhibition of kinase/signaling cascades, a clearer picture of the key regulatory events that control retina regeneration is emerging. In all cases, Müller glia (MG) serve as an adult retinal stem cell that must be reprogrammed to allow for regeneration, with the end goal being to understand why regenerative pathways are blocked in mammals, but spontaneous in other vertebrates such as zebrafish. miRNAs have emerged as key gene regulatory molecules that control both development and regeneration in vertebrates. Here, we focus on a small subset of miRNAs that control MG reprogramming during retina regeneration and have the potential to serve as therapeutic targets for treatment of visual disorders and damage.

Quantifiable In Vivo Imaging Biomarkers of Retinal Regeneration by Photoreceptor Cell Transplantation.

Purpose: Short-term improvements in retinal anatomy are known to occur in preclinical models of photoreceptor transplantation. However, correlative changes over the long term are poorly understood. We aimed to develop a quantifiable imaging biomarker grading scheme, using noninvasive multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, to enable serial evaluation of photoreceptor transplantation over the long term. Methods: Photoreceptor cell suspensions or sheets from rhodopsin-green fluorescent protein mice were transplanted subretinally, into either NOD.CB17-Prkdcscid/J or C3H/HeJ-Pde6brd1 mice. Multimodal cSLO imaging was performed serially for up to three months after transplantation. Imaging biomarkers were scored, and a grade was defined for each eye by integrating the scores. Image grades were correlated with immunohistochemistry (IHC) data. Results: Multimodal imaging enabled the extraction of quantitative imaging biomarkers including graft size, GFP intensity, graft length, on-target graft placement, intra-graft lamination, hemorrhage, retinal atrophy, and periretinal proliferation. Migration of transplanted material was observed. Changes in biomarker scores and grades were detected in 14/16 and 7/16 eyes, respectively. A high correlation was found between image grades and IHC parameters. Conclusions: Serial evaluation of multiple imaging biomarkers, when integrated into a per-eye grading scheme, enabled comprehensive tracking of longitudinal changes in photoreceptor cell grafts over time. The application of systematic multimodal in vivo imaging could be useful in increasing the efficiency of preclinical retinal cell transplantation studies in rodents and other animal models. Translational Relevance: By allowing longitudinal evaluation of the same animal over time, and providing quantifiable biomarkers, non-invasive multimodal imaging improves the efficiency of retinal transplantation studies in animal models. Such assays will facilitate the development of cell therapy for retinal diseases.

Isolation and Characterization of Extracellular Vesicles to Activate Retina Regeneration.

Mammals do not possess the ability to spontaneously repair or regenerate damaged retinal tissue. In contrast to teleost fish which are capable of retina regeneration through the action of Müller glia, mammals undergo a process of reactive gliosis and scarring that inhibits replacement of lost neurons. Thus, it is important to discover novel methods for stimulating mammalian Müller glia to dedifferentiate and produce progenitor cells that can replace lost retinal neurons. Inducing an endogenous regenerative pathway mediated by Müller glia would provide an attractive alternative to stem cell injections or gene therapy approaches. Extracellular vesicles (EVs) are now recognized to serve as a novel form of cell-cell communication through the transfer of cargo from donor to recipient cells or by the activation of signaling cascades in recipient cells. EVs have been shown to promote proliferation and regeneration raising the possibility that delivery of EVs could be a viable treatment for visual disorders. Here, we provide protocols to isolate EVs for use in retina regeneration experiments.