Diabetic retinopathy (DR) is a leading cause of vision impairment worldwide, driven by chronic hyperglycaemia and its complex metabolic consequences. While animal models have been widely used to study DR, they often fail to replicate the physiology of the human retina. Here, we employed human retinal organoids to investigate the effects of incremental hyperglycaemic stress-a modest increase from a standard high-glucose baseline (17.5 mM) to 25 mM D-glucose-across different stages of retinal differentiation. Early-stage organoids demonstrated resilience to high-glucose levels, maintaining normal morphology, viability, and gene expression. In contrast, late-stage organoids exhibited marked photoreceptor vulnerability, including downregulation of outer segment (OS)-specific genes, shortened OSs, increased oxidative stress, astrocyte activation, and significantly higher levels of apoptosis. Transcriptomic analysis revealed substantial changes in pathways related to vision, including the G protein-coupled receptor signalling pathway, response to light stimulus, and visual perception. While photoreceptors were particularly vulnerable, other retinal cell types, including bipolar cells, ganglion cells, and Müller glia, showed greater resilience. Additionally, glial activation, evidenced by increased expression of astrocyte markers, suggested an adaptive response to hyperglycaemia. To validate our findings, we compared our dataset with publicly available transcriptomic datasets from human retinas with DR, confirming key overlaps in pathways related to photoreceptor dysfunction, gliogenesis, and oxidative stress responses. While this non-vascularised model does not replicate the onset of DR from physiological glucose levels, it provides a human-specific platform for dissecting the molecular mechanisms of neurodegeneration associated with incremental hyperglycaemic stress.

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Ophthalmology University Hospital Brno and Faculty of Medicine Brno Czech Republic

Wellcome Wolfson Institute for Experimental Medicine Queen's University Belfast Belfast United Kingdom

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