July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells Display Extensive Neurite Outgrowth With Target Recognition
Author Affiliations & Notes
  • Clarisse Fligor
    Biology, IUPUI, Indianapolis, Indiana, United States
  • Priya K Shields
    Biology, IUPUI, Indianapolis, Indiana, United States
  • Kirstin Brooke VanderWall
    Biology, IUPUI, Indianapolis, Indiana, United States
  • Jason S Meyer
    Biology, IUPUI, Indianapolis, Indiana, United States
    Stark Neurosciences Research Institute, Indiana University, Indianapolis, Indiana, United States
  • Footnotes
    Commercial Relationships   Clarisse Fligor, None; Priya Shields, None; Kirstin VanderWall, None; Jason Meyer, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 3340. doi:
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      Clarisse Fligor, Priya K Shields, Kirstin Brooke VanderWall, Jason S Meyer; Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells Display Extensive Neurite Outgrowth With Target Recognition. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3340.

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

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Abstract

Purpose : Numerous degenerative disorders adversely affect retinal ganglion cells (RGCs), with injury to their axons resulting in vision loss or blindness. However, there has been a lack of success in the development of replacement strategies for RGCs due to obstacles such as the long distance outgrowth of RGC axons and the formation of functional synapses with post-synaptic targets. The current study establishes human pluripotent stem cells (hPSCs) as a reliable tool to test the ability of RGCs to project long-distance neurites and display target recognition with appropriate brain regions.

Methods : Retinal organoids were generated following established protocols from CRISPR-engineered hPSCs which allowed for the identification and isolation of RGCs. Neurite outgrowth of hPSC-derived RGCs was assessed via immunocytochemistry (ICC) and quantified using ImageJ. Additionally, as the lateral geniculate nucleus (LGN) is a primary post-synaptic target of RGCs, the LGN was extracted from P0-P3 mice and plated for co-culture with hPSC-derived RGCs. After one week, samples were analyzed for the extent of RGC outgrowth, including neurite length, number and directionality of outgrowth. Additionally, samples were collected for protein analysis via western blot or ICC.

Results : RGC neurites identified by tdTomato expression reached lengths of over 1 mm in the first 24 hours of growth. This outgrowth was enhanced in the presence of LGN explants, with the average neurite length of RGCs co-cultured with LGN explants significantly increased compared to controls. Additionally, RGCs displayed recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Furthermore, while hPSC-derived RGCs were found to express synaptic proteins in culture, this expression was significantly enhanced in the presence of LGN explants.

Conclusions : While hPSC-derived RGCs provide an unlimited source of cells for cell replacement strategies, a number of obstacles remain, particularly the long distance extension of neurites and formation of synaptic contacts. Results of this study demonstrate that the in vivo environment likely modulates RGC neurite outgrowth. As such, these results will facilitate the eventual use of hPSC-derived RGCs for cellular replacement, in vitro disease modeling and pharmaceutical screening.

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

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