Jones, Owen John Rhys;
(2025)
An investigation into small molecule enhancement of stem cell-derived photoreceptor genesis and neurite formation, for retinal repair.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Stem cell-derived retinal cell replacement therapies offer promise to treat sight loss of millions worldwide suffering from retinal degeneration. However, challenges including low efficiency of photoreceptor generation from stem cells and limited synaptic integration following transplant currently limit their translational application. The work in this thesis aims to provide new insight to better inform these challenges by identifying small molecules that enhance photoreceptor genesis and neurite formation in vitro. 3D retinal organoids (ROs) were differentiated from embryonic stem cells (ESC), mimicking retinal development, and generating photoreceptors and inner retinal neurons. High content image analysis of RECOVERIN+ immunolabelled photoreceptors in two dimensional RO cultures and fetal retina cultures was carried out to screen 19 small molecules for effects on neurite outgrowth. Significantly enhanced RECOVERIN+ neurite outgrowth was observed following treatment with Rho-kinase inhibitor Y-27632 or myosin II ATPase inhibitor blebbistatin. Timelapse imaging of retinal organoid cultures derived from a CRISPR-engineered dual fluorescent reporter ESC line revealed that solitary rod and cone photoreceptors elongated processes over time. Photoreceptors also migrated and contacted nonphotoreceptor cells and cone photoreceptors extended long axon-like neurites. To enhance the generation of rods and cones within ROs, the timing of retinoic acid and triiodothyronine (T3) supplementation was manipulated throughout differentiation and relative photoreceptor proportions were evaluated by flow cytometry. Elevated proportions of rods at week 29, and cones at week 18 of differentiation were observed within ROs when supplementation with retinoic acid was delayed or T3 was provided, respectively. In summary, small molecules targeting actomyosin can promote neurite elongation of dissociated stem cell derived photoreceptors. Furthermore, rod and cone photoreceptor production within ROs can be enhanced by supplementing differentiation cultures with small molecules produced endogenously during retinal development. Together these results will contribute to improved design of future photoreceptor replacement therapies for conditions of blindness.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | An investigation into small molecule enhancement of stem cell-derived photoreceptor genesis and neurite formation, for retinal repair |
| Language: | English |
| Additional information: | Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10205786 |
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