Abstract
Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), result in irreversible vision loss due to the destruction of photoreceptor (PhR) cells, which lack the capacity to regenerate. Human pluripotent stem cells (hPSCs) present a promising avenue for regenerative therapies aimed at restoring vision by replacing lost PhRs. This thesis details the development, characterisation, and validation of an innovative, xeno-free protocol for the directed differentiation of human-induced pluripotent stem cells (hIPSCs) into PhR-like cells using a 2D monolayer culture system.The protocol was specifically designed to address key limitations of conventional 3D culture systems, such as protracted culture times and inefficient PhR subtype generation. By leveraging a targeted combination of small molecules and cytokines, the protocol directs hIPSCs through defined developmental stages, from eyefield progenitors to mature PhR-like cells, with high efficiency. Time-dependent expression of key PhR-specific markers was observed, effectively recapitulating the natural ontogeny of retinal development.
Validation experiments demonstrated the reproducibility of this protocol across independent biological replicates, consistently yielding PhR-like cells with robust expression of lineage-specific markers. The potential for the protocol’s adaptation to 3D culture systems was also evaluated, enhancing its applicability for drug testing and disease modelling.
Comprehensive characterisation of the differentiated cells revealed critical insights into gene expression dynamics and the transcriptional networks governing PhR differentiation. Transcriptomic analysis corroborated experimental findings, identifying key regulatory pathways and validating the protocol's effectiveness. A comparative analysis with conventional 3D culture methods indicated that the 2D system efficiently generates near-homogeneous populations of specific PhR subtypes, with earlier expression of differentiation markers compared to the 3D culture systems.
The translational potential of the developed protocol was further explored through a pilot study involving the subretinal transplantation of hIPSCs derived PhR-like cells into a small animal model of AMD. The proof-of-concept experiment demonstrated the precise surgical placement of cells near the lesioned retinal area, highlighting the feasibility of this approach for retinal cell replacement therapies pending further analysis.
In conclusion, this thesis presents a reproducible, xeno-free and cost-effective method for generating PhR-like cells from hIPSCs, offering advancements for stem cell-based therapeutic strategies in retinal degeneration and positioning the 2D monolayer system as a powerful tool for both research and clinical applications.
| Date of Award | 2025 |
|---|---|
| Original language | English |
| Supervisor | Jason Limnios (Supervisor) & Helen O'Neill (Supervisor) |