Abstract
Purpose :
Retinal ganglion cell (RGC) transplantation holds potential for restoring lost vision in glaucoma and other optic neuropathies, but attempts thus far have been limited by low integration into the recipient retina. In organotypic retinal explants, we recently showed that disruption of the internal limiting membrane (ILM) allows donor RGCs to enter the retina. Here, we assess the effects of proteolytic ILM digestion and traumatic optic nerve injury on retinal localization of RGC somas and neurites following intravitreal transplantation in living mice.
Methods :
Human embryonic stem cells expressing tdTomato at the BRN3B locus were differentiated into RGCs using an established protocol. Pronase-E was injected intravitreally into mice at various concentrations and ILM integrity was assessed 2 weeks later using laminin immunofluorescence. Optic nerve crush (ONC) was performed using cross-action forceps for 5 seconds. RGCs (50,000 cells) were transplanted intravitreally 2 weeks after pronase injection or optic nerve crush (N≥13 per group). Localization of donor RGC somas and neurites was quantified 2 weeks after transplantation using high-resolution 3D reconstructions of confocal microscopy z-stacks.
Results :
Intravitreal pronase had a narrow effective concentration window: <0.06U/mL showed little effect on the ILM, >0.10U/mL induced frequent intraocular hemorrhage, but 0.08U/mL induced sizeable ILM defects without bleeding. Whereas >90% of transplanted eyes harbored surviving donor RGCs, <1% of transplanted cells remained at 2 weeks in control retina. By contrast, ONC and pronase treatment each doubled donor RGC survival (p<0.05). The percentage of surviving donor RGC somas migrating into the host RGC layer (1.4±0.7% in control eyes) increased by >4-fold with ONC (5.8±1.5%, p<0.01) and >12-fold with pronase (18.1±7.2%, p<0.001). Appropriate donor RGC neurite localization to the inner plexiform layer occurred only after pronase treatment.
Conclusions :
Both proteolytic ILM digestion and endogenous RGC injury through ONC increased survival and structural migration of transplanted human donor RGCs into the retinal parenchyma. Targeted interventions to promote functional integration of RGCs into the recipient retina, including ILM circumvention, may be required for RGC replacement to restore visual function in human patients with optic neuropathy.
This is a 2021 ARVO Annual Meeting abstract.