June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Human stem cell-derived photoreceptors switch from cell autonomous to non-cell autonomous axon extension with age
Author Affiliations & Notes
  • Sarah Rempel
    Neuroscience, University of Wisconsin - Madison, Madison, Wisconsin, United States
  • Yochana Kancherla
    Neuroscience, University of Wisconsin - Madison, Madison, Wisconsin, United States
  • David M Gamm
    Department of Ophthalmology and Visual Science, University of Wisconsin - Madison, Madison, Wisconsin, United States
  • Timothy Gomez
    Neuroscience, University of Wisconsin - Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Sarah Rempel, None; Yochana Kancherla, None; David Gamm, Opsis Therapeutics LLC (S), US Patent No US9752119B2 (P); Timothy Gomez, None
  • Footnotes
    Support  NIH T32 EY027721
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4016. doi:
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    • Get Citation

      Sarah Rempel, Yochana Kancherla, David M Gamm, Timothy Gomez; Human stem cell-derived photoreceptors switch from cell autonomous to non-cell autonomous axon extension with age. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4016.

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

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Abstract

Purpose : Human stem cell derived photoreceptors (PRs) are a candidate source for cell transplant therapy to cure blindness caused by photoreceptor loss, but current strategies presuppose transplanted PRs would cell autonomously extend axons to connect to target bipolar cells. We tested the hypothesis that PR axon extension competence changes with age using high resolution live imaging of PRs in 2D dissociated cultures and in 3D whole retinal organoids.

Methods : PRs differentiated from a PR reporter line (WA09 Crx+/tdTomato) within retinal organoids were imaged with multi-photon microscopy or dissociated for 2D culture and confocal microscopy. Growth cone (GC) dynamicity ({[Area of Extension] + [Area of Retraction]}/[GC Area]) was determined from time lapse imaging of dissociated organoid cultures. Day 40-50 or day 80-90 PRs were dissociated and co-cultured with motile Muller Glia (MG) and imaged live to measure cell body and GC displacement of PRs that either interacted with MG or were isolated.

Results : Growth cone dynamicity decreases with PR age, with higher dynamicity in day 40 PRs (mean=0.19) than day 80 PRs (0.03, p<0.0001). When co-cultured with motile MG, day 80-90 PRs had greater cell body (CB) and growth cone (GC) displacement when touching MG (T) (CB:10.31, GC:11.26) than when alone (A) (CB:0.54, GC:1.21), with no difference between CB and GC displacement (A,T: p>0.999), which is not consistent with GC-mediated, cell autonomous axon growth. In contrast, while day 40-50 PRs had higher CB (p<0.0001) and GC (p<0.0001) displacement when touching MG (CB: 8.64, GC: 12.66) compared to alone (CB: 1.10, GC: 6.23), they also had higher GC displacement than CB displacement in both conditions (A: p=0.017, T: p=0.007), consistent with cell autonomous axon extension. PRs with motile growth cones were also observed in whole retinal organoids at days 44 and 54 of differentiation.

Conclusions : Our results demonstrate that day 40 PRs have the ability to make motile GCs that are capable of cell autonomous displacement, but they lose this ability by 80 days of differentiation. These findings suggest a developmental switch that may be important for human photoreceptor axon development in vivo. These data also provide important information about the developmental window in which PRs may be capable of cell autonomous axon growth for the purposes of cell transplant therapies to restore vision.

This is a 2020 ARVO Annual Meeting abstract.

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