Enzymatic Disruption of the Internal Limiting Membrane Enhances Transplanted Retinal Ganglion Cell Survival

Purpose: In mammals, retinal ganglion cells (RGCs) lack the ability to regenerate after injury, and RGC transplantation is a potential approach for cell replacement therapy. However, the internal limiting membrane (ILM) in the retina serves as a significant barrier to donor cell integration, highlighting the need for strategies to disrupt it. We hypothesize that plasmin can enzymatically disrupt the ILM, thereby enhancing the survival of transplanted donor RGCs. Methods: In vivo, 8- to 10-week-old mice were intravitreally injected with phosphate-buffered saline (PBS) or plasmin (2.3 or 4.6 µg). Immunohistology was performed to verify ILM disruption and assess microglial reactivity and RGC survival. Hematoxylin and eosin (H&E) staining was used to evaluate immune cell infiltration in the vitreous, electrophysiology to assess retinal function, and optical coherence tomography to evaluate retinal structure. Donor RGCs were obtained by differentiating human embryonic stem cells into retinal organoids (ROs). The RO-derived RGCs were transplanted into plasmin-treated eyes 2 weeks after plasmin treatment. Transplanted RGCs were identified using tdTomato fluorescence. Results: Our study identified the optimal dose of plasmin (2.3 µg) that effectively disrupts the ILM without triggering microglia reactivity or immune infiltration in the vitreous. In addition, neither the RGC number nor RGC function was affected after plasmin treatment, whereas increased survival of transplanted cells was observed in the plasmin-treated retinas. Conclusions: Our findings provide a translational insight on plasmin-mediated ILM disruption, demonstrating that an optimal dose can effectively degrade the mouse ILM without compromising RGC integrity, offering a promising strategy to enhance transplanted cells' survival.