(262f) Inhibiting Molecular Cues to Improve Human Stem Cell-Derived Donor Retinal Neuron Transplantation in the Mouse Retina

Glaucoma is a leading cause of irreversible blindness, affecting an estimated 3 million people in the United States. As the population ages, the socio-economic impact of this disease is expected to increase. The mammalian retina lacks the ability to regenerate, and the death of retinal ganglion cells (RGCs), the neurons in the retina that relay all visual information from the eye to the brain, leads to permanent vision loss. While no therapies currently exist to mitigate or reverse this loss, recent studies have demonstrated the feasibility of cell replacement therapy using RGCs isolated from a developing retina or derived from stem cells. However, poor structural and functional integration of transplanted RGCs into the existing circuitry remains a significant challenge for successful RGC replacement. Previously, we have shown that donor RGC survival and integration within the retina can be improved by altering the host microenvironment with neurotropic factors and chemokines. However, despite these improvements, most donor RGCs fail to integrate into the ganglion cell layer (GCL). One possible explanation for this limitation is the presence of homophilic molecular cues limiting donor RGC integration into the existing retinal neurocircuitry. Understanding the molecular mechanisms governing donor RGC integration is critical to improving cell replacement therapies and restoring vision.

Methods:

To identify the molecular cue that limits the integration of donor RGCs, we examined the transcriptome of integrated donor RGCs after transplantation and discovered a significant decrease in the expression of DSCAM, a molecule that is known to regulate neuronal self-avoidance during retinal development. DSCAM's role in preserving the mosaic spacing of RGC subtypes in the retina during development may limit the ability of donor RGCs to migrate toward their natural connecting points within the retina. To disrupt this molecular cue in donor RGCs, we established an inducible DSCAM shRNA human stem cell line using lentiviral particles. RGCs were differentiated in 3D organoid cultures, and DSCAM knockout was induced by administering doxycycline 24 hours before transplantation. Slow-release neurotrophic factors were combined with 2x10⁴ donor RGCs and injected subretinally into mice. To assess the combined effect of controlling the microenvironment and intrinsic cell state, SDF1 (10ng) was injected intravitreally immediately after transplantation. Three days after transplantation, retinas were stained for donor and host RGCs and evaluated by tracking the position of each donor RGC in 3D reconstructions of retinal flat mounts (5 – 9 mice/group).

Results:

In our study, we found that knocking out DSCAM in donor RGCs led to a significant increase in the number of cells that spontaneously migrated out of the subretinal space. Specifically, 92% of DSCAM KO donor RGCs migrated out of the subretinal space compared to only 52% of wild-type RGCs. Additionally, we observed that treatment with SDF1 resulted in 69% of wild-type donor RGCs migrating out of the subretinal space, which is in agreement with our previous results. When DSCAM KO and SDF1 treatment were combined, we observed a synergistic effect, with 86% of donor RGCs migrating out of the subretinal space and 57% of donor RGCs integrating into the GCL. This is compared to only 37% of donor RGCs integrating into the GCL when DSCAM was knocked out alone. These results suggest that the combination of DSCAM KO and SDF1 treatment can significantly enhance the integration of donor RGCs into the host retina, which may have important implications for the development of cell replacement therapies for glaucoma and other retinal diseases.

Conclusion:

Our study indicates that the successful integration of donor RGCs into the host retina is dependent not only on the microenvironment but also on the intrinsic molecular state of the donor RGCs. Although we found that SDF1 gradients across the retina can improve the structural integration of donor RGCs, our results also confirm that SDF1 alone is not enough to overcome the intrinsic cytophobic molecular cues such as DSCAM within the retina. Our results suggest that blocking these molecular cues may hold promise as a therapeutic approach to improve the integration of donor RGCs into the host retina. Further research will focus on validating the role of DSCAM in donor RGC integration and developing blocking antibodies to enhance the integration of transplanted RGCs. Ultimately, these findings have the potential to lead to the development of more translatable therapeutic approaches to neuron transplantation aimed at treating glaucoma and other optic neuropathies.