Structural incorporation of transplanted human retinal ganglion cell neurites following proteolytic digestion or transgenic disruption of retinal extracellular matrix components

Abstract

Purpose : Retinal ganglion cell (RGC) replacement has the potential to restore lost vision in optic neuropathies including glaucoma. Functional integration of transplanted neurons into the retinal neurocircuitry is a key step necessary for clinical translation. We hypothesize that extracellular matrix components including the inner limiting membrane (ILM) obstruct ingrowth of dendrites from intravitreally transplanted RGCs into the inner plexiform layer (IPL), thereby hindering engraftment.

Methods : Human RGCs were differentiated and purified from a reporter ES cell line that expresses tdTomato at the BRN3B locus, using an established protocol. The ILM surface of adult CD1 mouse retinal explants were pre-treated with pronase E (5μL of 0.6U/mL) for 60 minutes, which did not affect tissue viability in culture. RGCs were co-cultured for 1 week on the ILM surface of retinal explants from control, pronase-treated, and Lama1^nmf223 mutant mice, which exhibit developmental ILM defects. 3D structural morphology of transplanted RGCs in relation to recipient retinal tissue was visualized and quantified using confocal microscopy and Imaris software.

Results : Immunofluorescence revealed that pronase disrupted laminin and collagen IV at the ILM without affecting the vasculature or inhibiting glial reactivity. Transplanted RGC survival was 16.5±10.4% (mean±SD) and not significantly (p>0.05) affected by pronase treatment (24.1±10.7%) or recipient retinal Lama1 mutation (17.1±0.4%). Pronase treatment was associated with increased number (69.0±20.8 vs 1.8±1.3 segments/RGC/mm², p<0.001) and length (1543.5±643.7 vs 43.8±14.8 μm/RGC/mm², p<0.001) of transplanted RGC neurites penetrating the IPL vs controls. Transplanted RGC neurite number (16.3±14.6 segments/RGC/mm²) and length (469.4±188.2 μm/RGC/mm²) were also greater within Lama1^nmf223 mutant IPL vs wild type (p<0.05).

Conclusions : The ILM may represent a barrier to the structural integration of transplanted RGCs into the retina, and anatomic ILM disruption is associated with greater neurite ingrowth into the IPL where synaptogenesis might potentially occur. As the ILM is dispensable following neurodevelopment and can be safely surgically peeled to treat human macular disease, ILM removal may be helpful for clinical translation of RGC transplantation approaches for optic neuropathy.

This is a 2020 ARVO Annual Meeting abstract.