Investigating cone photoreceptor migration using stem cell derived retinal organoids

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Inherited retinal dystrophies, featuring the progressive degeneration of the rod and cone photoreceptors, lead to irreversible blindness. Collectively, they represent the most frequent inherited forms of human visual handicap, with an estimated prevalence of 1 in 3000 worldwide. While rods enable vision in dim lighting conditions, human high acuity and colour vision depends on cone photoreceptors. Even though cone photoreceptors are a rare population, forming only 2-4% of total retinal cells, diseases affecting them, either directly or through the loss of rod derived trophic signalling, have an incapacitating impact on the lives of those affected. Unfortunately, current treatments are very limited and can only delay the onset of sight-loss, but not stop or reverse it. Thus, there is a great impetus to develop new and more effective treatment options. Retinal cell therapy, aiming to replace lost photoreceptors, using pluripotent stem cell (hPSC) derived sources, is one of the most promising future treatment options. In particular, hPSC derived retinal organoids hold great promise for the development of novel therapeutic interventions, including donor cell production for cell therapy, disease modelling and drug screens. However, while production methods for photoreceptor donor cells have advanced significantly in recent years, cell transplantation outcomes remain poor with few cells integrating into the remaining retinal circuitry after sub-retinal transplantation. This is in large part due to a lack of a robust understanding of photoreceptor development and the means to leverage it for therapeutic applications. In this context, the details of the signalling network controlling human cone photoreceptor migration, the process cell therapy efforts aim to harness for the clinical setting, remain to be elucidated. Here, we propose a research programme, which combines stem cell and genome editing technology to conduct functional high-content screens to elucidate components of the signalling cascade involved in controlling cone migration and identify pharmacological tools to manipulate this process. To achieve this, we will take advantage of a cone specific human pluripotent stem cell reporter line to produce 3D retinal organoids and track, as well as isolate cones at different phases of migration. First, with the goal of advancing our understanding of the differential transcriptional landscape before, during and after migration, we will establish the gene expression signatures of purified cone photoreceptors. Second, using a large scale, CRISPR mediated, genetic loss-of function approach, we will determine the involvement of all expressed components of the G-protein signalling machinery in the stereotypical process of apico-basal cone migration that occurs following exit from cell cycle. Lastly, using target genes identified in the screen, we will identify and test chemical compounds, which can activate them and thus stimulate migratory behaviour of cones. We expect that this work will greatly advance our understanding of human cone photoreceptor development, but also reveal common principles guiding the assembly of the complex retinal architecture as a whole. Furthermore, the pharmacological reagents identified here may facilitate migration and synaptogenesis of transplanted donor cells in the cell replacement therapy setting.