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

Optic neuropathies cause irreversible vision loss as retinal ganglion cells (RGCs) die. Transplantation of pluripotent stem cell (PSC)-derived RGCs offers one potential therapeutic avenue to restore vision in patients suffering from optic neuropathy if the donor neurons survive long-term in the recipient eye and develop synaptic connections within the retinal inner plexiform layer (IPL) and subcortical visual centers (1). Thus far, attempts at intravitreal RGC transplantation have been hampered by sequestration on the epiretinal surface without engraftment into the retinal parenchyma. In mouse retinal explant cultures, enzymatic digestion of the retinal internal limiting membrane (ILM) promotes migration of transplanted RGCs into the recipient retina (2). Herein, we examined donor RGC survival and engraftment in living, immunosuppressed mice, rats, and rhesus macaques and in post-mortem human retinal explant cultures. Using 3 separate human PSC lines and 3 independent methods of ILM disruption, we demonstrate that the ILM is a barrier to the retinal engraftment of intravitreally delivered human PSC-derived RGCs. ILM disruption is associated with greater donor RGC survival over 2-8 weeks and enables migration of donor neuronal somas into the endogenous RGC layer where cells elaborate dendrites into the IPL and extend axons that follow the course of the endogenous retinal nerve fiber layer into the optic nerve head. Critically, ILM disruption enables donor RGCs to synaptically integrate into IPL circuits, conferring light responsivity. These findings have important implications for enabling neuronal replacement therapies to restore vision in patients with optic neuropathy. SIGNIFICANCE STATEMENTRetinal ganglion cell (RGC) replacement and optic nerve regeneration through transplantation of stem cell-derived RGCs holds potential for restoring vision lost to optic neuropathies. Here we demonstrate that intravitreally transplanted human RGCs laminate the epiretinal surface without projecting neurites into the retinal parenchyma. However, enzymatic, developmental and surgical disruption of the internal limiting membrane not only improves graft survival, but also enables structural and functional engraftment, with dendrites that stratify the inner plexiform layer, axons that grow into the optic nerve head, and acquired responsivity to light. These observations identify a translatable approach to enable transplantation-based RGC replacement for the treatment of optic neuropathy.