June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Masking the “eat me” signal drastically increases the short-term survival of donor human retinal ganglion cells after xenotransplantation
Author Affiliations & Notes
  • Monichan Hayes Phay
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
    Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
  • Sophia Grace Bauer
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
    Northeastern University, Boston, Massachusetts, United States
  • Petr Y Baranov
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
    Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Monichan Phay None; Sophia Bauer None; Petr Baranov None
  • Footnotes
    Support  NIH/NEI U24 EY029893, NIH/NEI core gant P30EY003790, Iraty Award, Gilbert Family Foundation
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1117. doi:
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    • Get Citation

      Monichan Hayes Phay, Sophia Grace Bauer, Petr Y Baranov; Masking the “eat me” signal drastically increases the short-term survival of donor human retinal ganglion cells after xenotransplantation. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1117.

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

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Abstract

Purpose : A key challenge in retinal ganglion cell (RGC) replacement therapy is low donor cell survival. Phosphatidylserine (PS) is a ubiquitous “eat me” signal that is externalized and permanently displayed on the surface of apoptotic cells to facilitate their engulfment and clearance by tissue macrophages. Under stress, viable neurons also expose PS to the outer membrane in a reversible fashion. Since donor RGCs experience a significant stress during the isolation and transplantation process, we hypothesize that PS-mediated neurophagy by the host microglia/macrophages is a major contributor to donor cell death.

Methods : Donor human RGCs were derived from Brn3b-Tdtomato-Thy1.2 embryonic stem cells through retinal organoid approach with gentle dissociation and sorting for Thy1.2. To mask the externalized PS from macrophage/microglia recognition, donor cells were pretreated with annexin V (5mg/mL) for at least 20 minutes. We transplanted 20,000 viable donor RGCs subretinally into wild-type mice and processed whole mount retinas at day 1 and day 4 for immunostaining and confocal imaging to carry out subsequent analysis.

Results : Annexin V pretreatment led to the increase in transplant success: 92% (11 out of 12 recipients) had >1% (200) donor cells at day 4, compared to only 55% (6 out of 11) in the control group. We also observed ~2.5 fold-change in number of donor RGCs per eye: 1,280 cells in pretreated vs. 520 cells in control. In both groups, grafted donor RGCs displayed extensive neurite outgrowth (>150mm) and axon projections (>1000mm). Furthermore, donor cells axon fasciculation and entry into the optic nerve head were observed in the successful grafts: 33% vs. 9% in the Annexin V group vs. control. The recruitment of Iba1+ and CD45+ cells to the delivery site and their co-localization with donor RGCs was lower in the annexin V pretreated group at day 1 (19% vs. 40%) and was not different between groups at day 4.

Conclusions : Blocking externalized PS “eat me” signal with annexin V resulted in a drastic improvement in donor RGC survival and transplant success. However, persistent recruitment of Iba1+ and CD45+ to the delivery site and their co-localization with donor RGCs indicates that annexin V pretreatment only offers temporary protection. Our findings suggest that neurophagy by host microglia plays a significant role in eliminating viable donor RGCs.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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