June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Enzymatic disruption of retinal extracellular matrix affects surface topology of transplanted human retinal ganglion cells
Author Affiliations & Notes
  • Kevin Y Zhang
    Wilmer Eye Institute, Johns Hopkins University, Maryland, United States
  • Caitlyn Tuffy
    Wilmer Eye Institute, Johns Hopkins University, Maryland, United States
  • Joseph L Mertz
    Wilmer Eye Institute, Johns Hopkins University, Maryland, United States
  • Harry A Quigley
    Wilmer Eye Institute, Johns Hopkins University, Maryland, United States
  • Donald J Zack
    Wilmer Eye Institute, Johns Hopkins University, Maryland, United States
  • Thomas Vincent Johnson
    Wilmer Eye Institute, Johns Hopkins University, Maryland, United States
  • Footnotes
    Commercial Relationships   Kevin Zhang, None; Caitlyn Tuffy, None; Joseph Mertz, None; Harry Quigley, None; Donald Zack, None; Thomas Johnson, None
  • Footnotes
    Support  2019 ARVO David L Epstein Award; Wilmer Core Grant P30EY001765; Research to Prevent Blindness
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 635. doi:
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    • Get Citation

      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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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).

Conclusions : 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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