July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Methods to improve and quantify retinal integration of transplanted stem cell-derived retinal ganglion cells for optic nerve regeneration
Author Affiliations & Notes
  • Thomas Vincent Johnson
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Caitlyn Tuffy
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Joseph L Mertz
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Harry A Quigley
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Donald J Zack
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Thomas Johnson, None; Caitlyn Tuffy, None; Joseph Mertz, None; Harry Quigley, None; Donald Zack, None
  • Footnotes
    Support  NIH Grant EY02120; NIH Grant EY01765; Maryland Stem Cell Research Fund; Research to Prevent Blindness; Bright Focus Foundation; and the Guerrieri Family Foundation
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 652. doi:
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    • Get Citation

      Thomas Vincent Johnson, Caitlyn Tuffy, Joseph L Mertz, Harry A Quigley, Donald J Zack; Methods to improve and quantify retinal integration of transplanted stem cell-derived retinal ganglion cells for optic nerve regeneration. Invest. Ophthalmol. Vis. Sci. 2019;60(9):652.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Retinal ganglion cell (RGC) replacement via cell transplantation holds promise for restoring vision lost to optic neuropathies, such as glaucoma. Initial steps include integration of transplanted cells into the recipient retina and extension of dendrites into the inner retinal layers. Previous work suggested that the inner limiting membrane (ILM) inhibits integration of transplanted cells. We demonstrate methods to overcome this barrier for human stem cell-derived RGCs.

Methods : Organotypic retinal explants from adult CD1 mice were cultured according to reported techniques for 1 week. RGC-like neurons were differentiated from a human embryonic stem (hES) cell line expressing murine THY1.2 and fluorescent tdTomato under BRN3B promoter control. Retinal explant ILM was digested with 1 of 3 proteolytic enzymes (pronase, papain, or collagenase) applied at varying concentrations to the vitreous surface. Immunopurified RGCs were then co-cultured on retinal explants. Transplanted RGC bodies and neurites were visualized within retinal tissue using confocal microscopy, then analyzed using NeuronJ.

Results : In retinal explants with intact ILM, transplanted RGCs remained external to the neural retina and extended processes that integrated minimally, except at the cut edges of explants. Pre-application of proteolytic enzymes digested ILM to varying degrees. Pronase proved most efficacious. It completely degraded ILM laminin and increased transplanted RGC neurite extension into the inner retina by 10-fold: total inner plexiform layer (IPL) neurite length measured 4118.6±279.1 vs 409.3±282.8 µm/cell/mm2 (p<0.01) and the number of neurites within the IPL measured 279.1±84.9 vs 28.8±22.1 neurites/cell/mm2 (p<0.01). Papain significantly compromised retinal explant viability, whereas collagenase least altered the histologic ILM appearance.

Conclusions : The ILM represents a barrier to transplanted hES-derived RGC integration that can be circumvented by proteolytic degradation, significantly increasing extension of transplanted RGC neurites into the inner layers of recipient retinas.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

hES-derived RGCs co-cultured on retinal explants pre-treated with BSS (control) or Pronase. Projections of 3D reconstructions showed similar cell morphology in superficial layers (magenta) but pronase significantly increased transplanted RGC neurite growth into the IPL (green). Scalebar = 50μm.

hES-derived RGCs co-cultured on retinal explants pre-treated with BSS (control) or Pronase. Projections of 3D reconstructions showed similar cell morphology in superficial layers (magenta) but pronase significantly increased transplanted RGC neurite growth into the IPL (green). Scalebar = 50μm.

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