June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Neuroprotection of transplanted human stem cell derived retinal ganglion cells: advancing cell replacement strategies for optic nerve regeneration
Author Affiliations & Notes
  • Kevin Y Zhang
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Aru Nagalingam
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Pingwu Zhang
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Xiaoli Chang
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Harry A Quigley
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Derek S Welsbie
    University of California San Diego, La Jolla, California, United States
  • Donald J Zack
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Thomas Vincent Johnson
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Kevin Zhang, None; Aru Nagalingam, None; Pingwu Zhang, None; Xiaoli Chang, None; Harry Quigley, None; Derek Welsbie, Oriole Therapeutics (I), Oriole Therapeutics (C), Oriole Therapeutics (P); Donald Zack, Oriole Therapeutics (I), Oriole Therapeutics (C), Oriole Therapeutics (P); Thomas Johnson, None
  • Footnotes
    Support  NIH K08EY031801; NIH P30EY001765; RBP Career Development Award (TVJ); RPB Unrestricted Grant Support to Wilmer Eye Institute
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 2376. doi:
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      Kevin Y Zhang, Aru Nagalingam, Pingwu Zhang, Xiaoli Chang, Harry A Quigley, Derek S Welsbie, Donald J Zack, Thomas Vincent Johnson; Neuroprotection of transplanted human stem cell derived retinal ganglion cells: advancing cell replacement strategies for optic nerve regeneration. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2376.

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

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Abstract

Purpose : Neuronal transplantation has emerged as a potential therapy to restore vision in advanced optic neuropathies by repopulating RGCs. However, successful engraftment is limited by poor donor survival. Dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) signaling pathways regulate endogenous RGC apoptosis. Here, we examined the neuroprotective effects of DLK and LZK inhibition on human embryonic stem cell (hES) derived RGC transplantation by using transgenic and pharmacologic approaches.

Methods : Organotypic retinal explants from 8-week-old C57BL/6J mice were co-cultured with hES-RGCs on the vitreous surface for 1 week. We compared transplanted RGC survival in wildtype (WT) hES-RGCs versus CRISPR-Cas9 engineered DLK-/-and LZK-/- hES-RGCs. In addition, we assessed survival and neurite integration of WT hES-RGCs on pronase-treated explants co-cultured in regular media or media containing 1µM VX-680 (a kinase inhibitor with strong activity against DLK). Cell survival was quantified using tiled retinal wholemount confocal images. Donor neurite integration, normalized to the number of RGCs in area measured, was assessed in high resolution 3D confocal reconstructions using Imaris software.

Results : DLK-/-hES-RGC survival was significantly greater than WT hES-RGCs (16.1±2.8% vs 9.2±3.3%, p<0.001); LZK-/-hES-RGCs did not show neuroprotection over WT cells (10.0±1.6%, p>0.05). On pronase-treated retinas, VX-680 enrichment conferred significantly increased survival of donor RGCs compared to control (20.20±4.66% vs 16.04±2.14%, p<0.05). The percentage of donor RGCs with neurite integration was similar between groups (1.69±0.38% in VX-680 vs 1.76±0.89% in control, p>0.05). However, the total length and dendritic complexity of integrated neurites localized to the recipient inner plexiform layer were significantly higher in the VX-680 treated group (98.9±37.7 μm/mm2/RGC vs 41.83±27.8 μm/mm2/RGC, p<0.005; 2.81±0.34 branches/mm2/RGC vs 1.25±0.86 branches/mm2/RGC, p<0.001).

Conclusions : Transgenic DLK deletion and pharmacologic kinase inhibition with VX-680 promoted survival of transplanted hES-RGCs on organotypic retinal explants. DLK inhibition also demonstrated significantly higher donor neurite integration and dendritic complexity. Ongoing studies aim to examine the neuroprotective effects of DLK inhibition on transplanted hES-RGC survival in vivo.

This is a 2021 ARVO Annual Meeting abstract.

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