Isolating and characterising human developing cone photoreceptors towards a cell replacement therapy for retinal dystrophies

Inherited retinal dystrophies resulting in the death of the light sensitive photoreceptor cells are a major cause of incurable blindness. Loss of the cone photoreceptor cells, which are crucial for colour detection, daylight vision and high visual acuity, has the greatest impact on sight. Retinal differentiation of human pluripotent stem cells (hPSCs) provides a potential renewable source of cone photoreceptors for cell replacement therapy. However many critical challenges remain to be addressed, including i) assessing the similarity between the transcriptomes of bona fide human foetal and in vitro hPSC-derived cone cells and ii) developing strategies to purify cones prior to transplantation. The work in this thesis addresses these questions in relation to L/M-opsin photoreceptors, which form 90% of the total cone population in the human retina. The onset of cone photoreceptor markers in the developing human retina was characterised, before using an adeno-associated virus (AAV2/9.pR2.1:GFP) reporter to specifically label and isolate a foetal L/M-opsin cone population. Total mRNA sequencing revealed the transcriptome of this human foetal cone population for the first time and by performing differential gene expression analysis at early and late foetal timepoints, a novel cone-enriched gene signature was defined. Additionally, single cell transcriptome analysis revealed a subtle heterogeneity of these cone cells, which deviate based on maturity. Putative cell surface marker genes and cluster of differentiation (CD) marker proteins expressed by L/M-opsin cones were profiled, which led to a CD marker combination allowing for the enrichment of foetal L/M-opsin cones compared to the unsorted cell population via fluorescence activated cell sorting. Translating these tools into an hPSC-derived 3D retinal differentiation culture system revealed similarities in cone gene expression and cone cell enrichment post sorting to human foetal cone cells. Overall, these data provides vital pre-clinical steps towards generating a cone cell replacement therapy for retinal dystrophies