Abstract
Purpose :
Photoreceptor outer segments (POS) play a crucial role in the process of vision, and their dysfunction is often implicated in retinal degeneration. Retinal organoids (RO) have become a valuable tool for studying disease mechanisms and screening potential drug candidates pertaining to their ability to recapitulate aspects of the human retina. However, ROs have long developmental timelines and to accelerate drug discovery, off-the-shelf availability of ROs in large quantities is crucial. In this study we have assessed the dynamics, efficiency and batch to batch reproducibility of POS development across three batches of wildtype human iPSC-derived retinal organoids produced at large scale.
Methods :
Per batch, 100 ROs were selected based on the presence of phase-bright neural retina at D120 of differentiation and scored for POS development across maturing timepoints (D120, D150, D180, D210, D240, D270). Additional ROs with the highest appearance of POS at each timepoint, by brightfield imaging, were selected for immunofluorescence analysis. Markers of rod (RHO, ROM1), cones (OPNMLW), cilia (ARL13B), photoreceptor inner segments (TOM20) and outer limiting membrane (ZO1) were used to assess POS presence, development, organisation and morphology over time.
Results :
ROs start exhibiting POS by brightfield imaging between D120 (2%) and D150 (8%) of differentiation. Presence of POS dramatically increased by D180 (40%) and D210 (70%) peaking and plateauing at D240 (>80%). POS expression of RHO, ROM1 and OPNMLW increase in presence and length throughout RO differentiation, crossing above the RO outer limiting membrane marked by ZO1. Throughout later stages of differentiation ARL13B localises with TOM20 marker of inner segments, as they protrude from the outer nuclear layer. As expected for a highly complex model, some batch to batch variability was observed (up to ±10% of total ROs with POS), with the ROs following the same timeline of development across all three batches.
Conclusions :
Here we have determined the timeline of POS development in wildtype iPSC derived ROs by brightfield appearance and protein expression profiles across multiple batches produced at scale. We observed that ROs that develop POS will do so by D240 allowing these more mature ROs to be a suitable model for pre clinical studies involving disease modelling, drug discovery and gene therapy where POS formation is relevant.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.