Engineering the retinal microenvironment to improve donor and host retinal neuron survival in vivo

Introduction: The degeneration or loss of retinal ganglion cells (RGCs) in glaucoma is a leading cause of irreversible blindness worldwide, with no available therapies to restore vision once these cells are lost. RGCs, the neurons in the retina that transmit visual information from the eye to the brain, can regenerate in some lower vertebrates but lack this capacity in the mammalian retina.
Cell transplantation presents a promising approach to replace RGCs lost in advanced retinal disease. However, poor post-transplantation cell survival rates have hindered the effectiveness and development of this strategy. A significant barrier to successful RGC replacement is the glaucomatous retinal microenvironment. Typically characterized by elevated intraocular pressure (IOP), the glaucomatous retina can experience inflammation, immune cell infiltration, and host cell reactivity. Even after IOP normalization, a toxic microenvironment can persist, posing challenges to the survival of transplanted donor cells. Additionally, donor cells face an environment deficient in oxygenation and metabolic support until they integrate into the host retina, exacerbating their stress and further decreasing survival rates.
To address these challenges and create a more favorable microenvironment, we have identified and delivered pro-survival factors into the retina to enhance donor RGC survival following transplantation.

Materials and

Methods: We performed an in silico analysis of the single-cell transcriptome of developing human retinas to identify molecular features present during development that diminish in adulthood. We focused on changes in gene expression of various neurotrophic factors and RGC receptors throughout development. We also examined their roles in reducing apoptosis pathway gene expression and included ligand-receptor interactions using the CellChat package to differentiate between autocrine and paracrine pathways. Since optic neuropathies lead to RGC death, there is no proneuronal autocrine singling to support donor RGCs post-transplantation.
For transplantation experiments, RGCs were differentiated from Thy1-GFP mouse iPSCs and Brn3b-tdTomato hESCs in 3D retinal organoid cultures. To support donor RGCs and create a proneuronal microenvironment, we formulated mouse or human donor RGCs with selected pro-survival factors. To avoid the need for repeated intraocular injections, which could induce additional inflammation and reduce donor RGC viability, we used slow-release neurotrophic factors (growth factor-loaded polyhedrin-based particles (PODs)). Donor RGCs (10,000 – 20,000 RGCs/retina) were transplanted into an RGC-deficient mouse retina (either inherited loss or neurotoxic model) with and without PODs. Three to fourteen days post-transplantation, whole-mounted retinas were stained to assess host and donor cell integration and survival (8 – 11 mice/group). Eyes were enucleated three days after human donor RGC transplantation to limit the immune reaction associated with xenotransplantation and fourteen days after mouse donor RGC transplantation to allow for neurite outgrowth. Electroretinography (ERG) was performed six weeks after transplantation to assess host retinal function and measure functional recovery following transplantation.

Results, Conclusions, and Discussions: Our in silico analysis identified Brain-Derived Neurotrophic Factor (BDNF) and Glial Cell-Derived Neurotrophic Factor (GDNF) as critical for RGC development but absent in adulthood (Fig. 1A). This suggests the need for their supplementation to support regeneration. Additionally, the negative correlation between BDNF/GDNF receptor expression and apoptosis pathway gene expression during development implies that upregulating these factors can enhance RGC survival (Fig. 1B). Our findings also reveal that RGCs are the primary cell type secreting BDNF and GDNF, underscoring the importance of replacing these factors after RGC death in glaucoma to create a supportive microenvironment for transplantation (Fig. 1C).
Transplantation experiments showed that co-delivery of BDNF/GDNF-loaded PODs with donor RGCs significantly improved outcomes. For mouse donor RGC transplants, the success rate increased from 37% to 73%, with a 2.7-fold increase in donor RGC coverage and extensive neurite outgrowth towards the optic nerve head (Fig. 1D). This is a crucial finding as it indicates the potential for transplanted cells to establish connections with their postsynaptic targets in the brain, a necessary step for functional recovery after injury. Furthermore, PODs treatment, with or without RGC transplantation, partially preserved RGC function (32 µV in the PODS group vs. 17 µV in the control group), indicating a neuroprotective effect on host RGCs (Fig. 1E).
For human donor RGC transplants, PODs co-treatment led to a 15-fold increase in the number of detected human donor RGCs and 50% of transplants exhibited neurite extension towards the optic nerve head, with an average length of 1008.44 ± 264.12 µm (Fig. 1F-G). These findings demonstrate the potential of PODs to enhance the survival and integration of transplanted human RGCs, which could facilitate functional recovery in retinal degeneration patients.
In conclusion, modulating the retinal microenvironment with slow-release growth factor co-treatment is an effective strategy for improving RGC transplantation outcomes. This approach enhances donor RGC morphology, axon outgrowth, and survival. Further research is needed to optimize donor RGC integration, but incorporating neuroprotective factors like BDNF and GDNF shows promise for restoring vision in patients with retinal degeneration.

Acknowledgements (Optional): NIH/NEI–F32EY033211 (Soucy), NIH/NEI–L70EY034355 (Soucy), NIH/NEI–T32EY007145 (MBED fellowship, Soucy), NIH/NEI–5U24EY029893-03 (Baranov), NIH/NEI–P30EY003790 (Core Facility Grant), Bright Focus Foundation (Baranov), Gilbert Family Foundation (Baranov)