Internal limiting membrane disruption facilitates engraftment of transplanted human stem cell derived retinal ganglion cells

Abstract

Retinal ganglion cell (RGC) death causes irreversible vision loss in patients with glaucoma and other forms of optic neuropathy because the mammalian retina and optic nerve lack endogenous regenerative capacity. RGC transplantation and optic nerve regeneration represent a potential translational approach to vision restoration in glaucoma secondary to RGC loss. Functional RGC replacement requires that 1) donor RGCs integrate into the recipient retina and receive synaptic input from afferent bipolar and amacrine cells and that 2) donor RGCs extend their axons and establish synaptic connections to appropriate neurons in the brain. Here, in an effort to address retinal integration, we demonstrate that the internal limiting membrane (ILM) acts as a physical barrier to the integration of transplanted human stem cell-derived RGCs (hRGCs) into the recipient retina following intravitreal transplantation in vivo. To circumvent the ILM barrier, we intravitreally injected the nonspecific protease pronase-E in immunosuppressed adult C57BL/6J mice prior to transplantation of hRGCs. Separately, we also transplanted hRGCs into adult Lama1nmf223mice, which harbor a point mutation in their Lama-α1 gene that causes developmental ILM dysgenesis. We assessed donor hRGC survival and engraftment using 3D reconstructions of confocal z-stacks in retinal flatmounts. Migration of surviving donor RGC somas into the recipient RGC layer significantly increased after proteolytic or developmental ILM disruption. Moreover, lamination of dendritic arbors into the recipient inner plexiform layer was observed exclusively following ILM disruption. To assess the clinical translatability of this finding, we transplanted hRGCs onto postmortem organotypic human retinal explant cultures and observed significantly increased engraftment following proteolytic ILM digestion. These findings enhance our understanding of the barriers faced by transplanted hRGCs in the adult murine and human retina and provide an avenue for clinically translatable regenerative medicine approaches to vision restoration in optic neuropathy.