Retinal organoids (ROs) derived from human embryonic stem cells (hESCs) hold immense potential for modeling retinal development and diseases. However, current differentiation protocols often overlook the physiological hypoxic microenvironment of early embryogenesis, potentially compromising developmental fidelity. In this study, we investigated how staged oxygen modulation-hypoxic priming followed by normoxic transition-optimizes RO development by mimicking in vivo oxygen dynamics. The H9 hESC line was differentiated into ROs using a modified serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) protocol under four oxygen regimens: constant normoxia (20% O 2 ), chronic hypoxia (5% O 2 ), hypoxia-to-normoxia transition (5% → 20% O 2 ), and normoxia-to-hypoxia transition (20% → 5% O 2 ). RO morphology, retinal progenitor cell (RPC) and retinal ganglion cell (RGC) marker expression using immunofluorescence, and transcriptomic profiles of ROs were assessed at key developmental stages. Early hypoxia (5% O 2 , Days 0-6) significantly increased embryoid body volume (+ 55%, P < 0.001) and antigen Kiel 67 (Ki67)-positive proliferating RPCs (2.78-fold, P < 0.001) compared with normoxia. Early hypoxia also delayed class Ⅲ β-tubulin (TUJ1) expression but enhanced atonal homolog 7 (ATOH7)-positive RGC precursors (2.46-fold, P < 0.001). Upon transition to normoxia (Days 6-60), RPC expansion, indicated by a higher ratio of Ki67-positive proliferating cells, was maintained, and robust RGC differentiation was induced, yielding 38% larger ROs than those formed under chronic hypoxia ( P < 0.001). Normoxic conditions also reduced the decline in the ratio of outer-layer CHX10-positive cells and increased the mature TUJ1-positive neurite density of RGC. In contrast, chronic hypoxia markedly impeded paired box 6 (PAX6)-positive RGC differentiation. Transcriptomic analyses showed significant enrichment of sensory and visual system development pathways ( P < 0.01) in hypoxia-to-normoxia ROs, supporting distinct developmental patterns influenced by staged oxygen exposure. Staged oxygen modulation-hypoxic priming followed by normoxic transition-synergistically enhanced RO development by expanding progenitor reservoirs and promoting RGC maturation. This protocol offers a physiologically relevant framework for generating high-fidelity ROs for disease modeling and regenerative applications.
Gao et al. (Mon,) studied this question.