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Kevin Y Zhang, Caitlyn Tuffy, Joseph L Mertz, Harry A Quigley, Donald J Zack, Thomas Vincent Johnson; Enzymatic disruption of retinal extracellular matrix affects surface topology of transplanted human retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):635.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cell (RGC) transplantation may be a viable approach to restoring vision in optic neuropathies including glaucoma. Past work suggested that retinal extracellular matrix (ECM) obstructs the structural integration of transplanted neurons, and that proteolytic digestion enhances transplanted RGC neurite ingrowth. Here we describe how enzymatic inner limiting membrane (ILM) digestion affects the topologic patterning of transplanted human stem cell-derived RGCs.
Organotypic retinal explants from adult CD1 mice were cocultured with human embryonic stem cell-derived RGCs (expressing tdTomato driven by the BRN3B promoter) for 1 week. ILM was digested by collagenase or pronase treatment (5uL for 60 min) to the vitreous surface before RGC transplantation. Topology of transplanted RGCs was visualized by tiled epifluorescent microscopy. Spatial statistics were characterized by nearest neighbor distance (NND) and density recovery profiles (DRP). DRP defined each RGC as a reference cell and measured annuli of progressively increasing radii around that reference cell; the overall RGC density at each distance around the reference RGC was measured and binned at 10mm intervals. Because dissection procedures physically disrupted ILM at the retinal edge and promoted artifactual clustering of transplanted cells, a 150mm distance from the true edge was defined in ImageJ to exclude these clusters from analyses.
Transplanted RGC survival rate was not significantly affected by pronase treatment (10.5±1.7%) compared to control (12.3±2.6%); collagenase treatment significantly lowered RGC survival (5.7±0.9%, p<0.05).RGCs on control retinas appeared to form clusters whereas they appeared more dispersed on enzyme treated explants. Average NND of RGCs in controls was 16.9±1.8mm, significantly lower than that of pronase treated (21.3±2.8mm) and collagenase treated (39.3±4.0mm) groups (p<0.05 for both). DRP-derived average local density was significantly higher in control retinas (490±163cells/mm2) compared to pronase treated (255±84cells/mm2) and collagenase treated (83±26cells/mm2) explants (p<0.05 for both).
Enzymatic disruption of retinal ECM components reduced clustering and more uniformly distributed RGCs on retinal explants. Improving spatial distribution of transplanted RGCs may be desirable to maximize retinotopic coverage.
This is a 2020 ARVO Annual Meeting abstract.
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