Role of the Internal Limiting Membrane in Structural Engraftment and Topographic Spacing of Transplanted Human Stem Cell-Derived Retinal Ganglion Cells

Abstract

Summary Retinal ganglion cell (RGC) replacement holds potential for restoring vision lost to optic neuropathy. Transplanted RGCs must undergo neuroretinal integration to receive afferent visual signals for processing and efferent transmission. To date, retinal integration following RGC transplantation has been limited. We sought to overcome key barriers to transplanted human stem cell-derived RGC integration. Following co-culture ex vivo on organotypic mouse retinal explants, human RGCs cluster and extend bundled neurites that remain superficial to the neuroretina, hindering afferent synaptogenesis. To enhance integration, we increased the cellular permeability of the internal limiting membrane (ILM). Extracellular matrix digestion using proteolytic enzymes achieved ILM disruption while minimizing retinal toxicity and preserving glial reactivity. ILM disruption is associated with dispersion rather than clustering of co-cultured RGC bodies and neurites, and increased parenchymal neurite ingrowth. The ILM represents a significant obstacle to transplanted RGC connectivity and its circumvention may be necessary for functional RGC replacement.

Retinal Ganglion Cell Transplantation: Approaches for Overcoming Challenges to Functional Integration

“…A secondary marker that outlines the boundary of the retinal parenchyma should be included when possible, such as immunofluorescent laminin delineation of the ILM (Figure 3). In our experience, we observe negligible spontaneous donor human RGC integration into the host RGCL when the ILM remains intact, whereas somal integration is greatly enhanced by enzymatic disruption of ILM [16], suggesting the inner retinal extracellular matrix is a significant structural barrier to transplanted RGCs integration. A chronic progressive mouse model of glaucoma demonstrated pathologic retinal ECM remodeling and upregulation of several families of glycoproteins, including laminin and fibronectin [152].…”

“…The ability of transplanted RGCs to migrate spontaneously into the recipient retina is inconsistent across published studies, which may be related to differences in graft and host species, or to the methodologies of determining graft localization. Our previous ex vivo work has identified that the ILM is a major barrier to both human RGC somal and neurite engraftment; we have found that donor human RGC migration into the host mouse retinal parenchyma rarely occurs spontaneously [16]. Disrupting ILM integrity can profoundly reverse donor sequestration outside the retinal parenchyma.…”

“…Donor cells arriving at the retina from the vitreous cavity encounter the retinal basement membrane (the ILM), local astrocytic and Müller glial reactivity, and local inflammatory cells that impede spontaneous cell migration into the retina [16,147]. Endogenous molecules from the extracellular matrix contribute to the migratory blockade of transplanted cells.…”

“…We have observed a propensity for transplanted RGCs to cluster when cultured on organotypic retinal explants with intact ILM [16]. Proteolytic ILM digestion eliminates cell clumping and achieves greater donor cell coverage of the retinal surface.…”

“…Intravitreal transplantation might provide more direct access to the inner retina, but introduces new obstacles including dispersion of cells into a much larger three-dimensional space, without sequestration adjacent to retinal tissue, and behind physical barriers not present in the subretinal space. The internal limiting membrane (ILM) at the vitreoretinal junction impedes transplanted RGC migration into the host retina [16] and should be mitigated in a safe manner. Once localized to the host RGCL, donor RGCs must establish proper topographical spacing, while targeting dendrites to the relevant IPL sublamina to form synaptic connections with amacrine and bipolar cells.…”