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bioRxiv · 10.1101/2021.10.10.463810

Human pluripotent stem cell-derived photoreceptors switch from cell autonomous axon extension to non-cell autonomous process pulling during synaptic marker redistribution.

Rempel, S. K.·Welch, M. J.·Ludwig, A. L.·Phillips, M. J.·Kancherla, Y.·Zack, D.·Gamm, D. M.·Gomez, T. M.
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Abstract

Photoreceptors (PRs) are the primary visual sensory cells, and their loss leads to blindness that is currently incurable. Cell replacement therapy holds promise as a therapeutic approach to restore vision to those who have lost PRs through damage or disease. While PR transplant research is ongoing in animal models, success is hindered by our limited understanding of PR axon growth during development and regeneration. Using a human pluripotent stem cell (hPSC) reporter line that labels PRs (WA09 CRX+/tdTomato), we generated retinal organoids in order to study mechanisms of PR process extension. We found that the earliest born PRs exhibit autonomous axon extension from dynamic terminals that appear similar to projection neuron growth cones. However, as hPSC-derived PRs age from 40 to 80 days of differentiation, they lose dynamic terminals in 2D plated cultures and within 3D retinal organoids, which does not correlate with cell birth date. Using a rod-specific hPSC reporter line (WA09 NRL+/eGFP), we further determined that rod PRs never form motile growth cones. Interestingly, PRs without motile terminals are still capable of extending axons, but neurites are generated from process stretching via their attachment to motile non-PR cells, which underlies the observed differences in PR neurite lengths on different substrata. While immobile PR terminals express actin, it is less polymerized and less organized than actin present in motile terminals. However, immobile PRs do localize synaptic proteins to their terminals, suggesting a normal developmental progression. These findings help inform the development of PR transplant therapies to treat blinding diseases and provide a platform to test treatments that restore autonomous PR axon extension. Significance StatementLoss of photoreceptors (PRs) in the retina through damage or disease causes irreversible vision loss and blindness. One treatment approach is to replace lost cells with transplanted human stem cell-derived PRs, but this requires PR axons to integrate into the host retina to restore the required neural connections. For this strategy to succeed, we need to understand how PRs extend processes to their targets during development in situ, and whether dissociated human stem cell (hPSC)-derived PRs behave in a similar fashion. In this paper, we show that hPSC-PRs have only a short window during which they are capable of autonomous axon extension, which has implications for PR transplant efforts and for our basic understanding of human retinal development.

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Rempel, S. K., Welch, M. J., Ludwig, A. L., Phillips, M. J., Kancherla, Y., Zack, D., Gamm, D. M., Gomez, T. M.. 2021-10-10. Human pluripotent stem cell-derived photoreceptors switch from cell autonomous axon extension to non-cell autonomous process pulling during synaptic marker redistribution.. https://doi.org/10.1101/2021.10.10.463810

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