Bioengineered Retinal Organoids: Incorporating dECM-PEG Hybrid Scaffolds to Advance Retinal Ganglion Cell Layer Maturation and Physiological Functionality

Retinal ganglion cells (RGCs) are the core cellular targets damaged in neurodegenerative blindness such as advanced glaucoma and traumatic optic neuropathies. Although modern human pluripotent stem cell (hPSC)-derived ۳D retinal organoids successfully mirror outer neuroepithelial architectures (including early-stage photoreceptors), modeling the prolonged structural preservation and functional maturation of the innermost retinal ganglion cell layer (GCL) has remained highly constrained due to stochastic cell shedding, internal nutrient starvation, and immediate developmental arrest under standard culture environments. This investigation addresses these engineering bottlenecks by manufacturing an advanced biomimetic platform that interfaces hPSCs with customized dECM-PEG hybrid hydrogels tuned to an embryonic retinogenesis-mimicking stiffness profile of ۱۳.۵ kPa. By systematically combining native biological signaling motifs extracted from tissue-specific decellularized extracellular matrix (dECM)—specifically rich in laminin and fibronectin complexes—with synthetic, mechanically stable polyethylene glycol-vinyl sulfone (PEG-VS), we generated an inductive microenvironmental niche that promotes localized RGC specification, polar spatial arrangement, and axonal projection bundling. Long-term single-cell transcriptomics, deep confocal immunofluorescence profiling, and multielectrode array (MEA) functional microcircuit evaluations confirmed that these specialized hybrid frameworks significantly accelerate the upregulation of core RGC transcriptional networks (BRN۳B, ISL۱) and definitive GCL structural biomarkers (Thy-۱, MAP۲, and RBPMS) by Day ۴۰. Mechanistically, hydrogel-mediated activation of the mechanosensitive YAP/TAZ signaling cascade successfully suppressed interior tissue apoptosis, decreasing the overall apoptotic index down to ۲.۸% ± ۰.۶% compared to ۲۸.۴% ± ۳.۹% in standard scaffold-free controls. The resulting bioengineered retinal tissue demonstrates robust, coordinated spontaneous network activity and multi-channel bursting, establishing an robust, scalable, high-fidelity platform for personalized regenerative medicine, precision drug screening, and functional visual pathway disease modeling