Developing novel methods for the generation of 3D retinal organoids from rat and macaque pluripotent stem cells

Abstract

Pluripotent stem cells (PSCs) can be differentiated toward certain lineages using chemical, spatial and temporal cues. They can form 3D structures, termed organoids, which replicate the cellular diversity and organisation of native tissue along the timeline of embryological development. Retinal organoids (ROs) can represent the stratified complexity of native retina in vitro and therefore have huge potential for pre-clinical studies, biomedical research and the development of therapeutics for retinal disease. The generation of ROs from animal models such as macaque and rat is relevant for drug development as these are commonly used species for the safety assessment of candidate therapeutic agents. By using a method combining generation of retinal progenitor cells, through an intermediate stage of adherent culture, and suspension culture in media used for the maturation of mouse ROs, rat embryonic stem cells (ESCs) were differentiated into 3D organoids. Using morphological characterisation, protein and gene expression assays, rat organoids developed phase-bright neural retina, and cells expressing retinal progenitor (Pax6/CHX10), retinal ganglion (SNCG/RBPMS) and photoreceptor (RCVRN) cell markers. However, the stratified cellular organisation typical of the retina was lacking after 28 days in culture. Comparison with mouse ESC-derived ROs showed variable responses to the same culture conditions indicating species-specific differences. By adapting the timeline of human RO differentiation protocols to the shorter gestation of the macaque, induced pluripotent stem cell (iPSC)-derived organoids were generated which maintained typical retinal morphological features and cell marker expression at day 120. Macaque organoids show the sequential expression of neural-epithelia (Rax, Pax6, Sox2), photoreceptor precursor (Crx), photoreceptor (OPN1SW, OPN1LW/MW, RHO), interneuron (AP2α, PRKCα) and Müller glia (CRALBP) cell markers indicating some retinal development and maturation. Macaque iPSC lines showed variable responses with RPE conditioned media and IGF1-based protocols improving morphology and photoreceptor marker expression in one cell line. These results develop the understanding of mammalian in vitro retinal differentiation by showing the capacity of rat and macaque PSCs to differentiate using known retinal stimulating factors. Further research should optimise cellular organisation to better replicate the retinal structure.