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Joe Phillips, Allison Ludwig, Patrick Barney, Katherine Barlow, Lindsey Jager, Elizabeth E Capowski, David M Gamm; Comprehensive in vivo assessment of human pluripotent stem cell-derived photoreceptor survival and differentiation potential in the immune-compromised S334ter rat. Invest. Ophthalmol. Vis. Sci. 2018;59(9):543.
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© ARVO (1962-2015); The Authors (2016-present)
To rigorously assess donor photoreceptor (PR) precursor survival, differentiation and integration following transplantation of human pluripotent stem cell-derived optic vesicles (hPSC-OVs) in the Foxn1-S334ter rat model of PR degeneration.
The WA09 hPSC-CRX+/tdTomato reporter line was used to track PR production in vitro and identify transplanted PRs in vivo. hPSC-OVs were generated as previously described and harvested at day 70 for transplantation. At this stage, PR precursor production has peaked, but few have yet committed to a rod or cone fate. To allow passage through a 29G needle, hPSC-OVs were gently triturated, and then approximately 300,000 cells were injected into the subretinal space of adult immune-compromised S334ter (SD-Foxn1 Tg[S334ter]3LavRrrc) rats. Rat eyes were processed for histology at 1, 3 and 6 months post-transplant (n=4 for each) and immunofluorescence was performed with PR and multiple neural retina markers. Transplanted cells were identified by tdTomato fluorescence and human nuclear antibody labeling. Single plane confocal images were captured and loaded into Columbus software for cell counting, and statistical analysis was performed with GraphPad Prism.
At 1 month post-transplant, many hPSC-PR precursors differentiated into rods based upon NRL expression, and by 3 months, extensive rod and cone opsin expression was noted. Differentiated rods and cones survived until at least 6 months post-transplant, the latest time point examined. While a large percentage of proliferative (Ki-67+) neural retina progenitor cells were noted at 1 month post-transplant, they were significantly reduced at 3 months, and were nearly absent by 6 months. At each time point examined, the majority of transplanted cells present were CRX-tdTomato+ (>60%). In addition, hPSC-PRs expressed presynaptic proteins and host bipolar cells extended dendrites into the transplanted cell mass, indicative of anatomic integration in this model that lacks an outer nuclear layer (ONL).
hPSC-PR precursors dominate the OV-derived donor cell population post-transplantation in the Foxn1-S334ter rat subretinal space and most hPSC-PRs express mature rod and cone markers over time. As such, transplantation of whole or partial hPSC-OVs offers potential to reconstruct an ONL-like region following severe host PR degeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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