Over the past few decades, the prevalence of visual impairments has increased substantially, and some are incurable. This study aimed to present a co-culture method that promotes the differentiation of retinal progenitor cells (RPCs) in the presence of retinal pigment epithelium (RPE) cells.
Methods
We implemented a three-dimensional culture system using a modified alginate substrate to culture human embryonic stem cell-derived RPCs on top of an RPE layer, mimicking an in vivo environment. The characteristics of RPCs in experimental and control groups were analyzed at the RNA and protein levels, and the 3D retinal-like structures were examined by H&E staining. Statistical analysis was conducted in GraphPad Prism (v5.1) using unpaired Student’s t-tests and one-way ANOVA.
Results
Hypoxic culture conditions better maintained naïve RPC characteristics than normoxic conditions, with significantly higher expression of RAX, LHX2, and PAX6. The addition of Matrigel to simulate the subretinal space supported RPE survival. In this 3D coculture model, RPCs differentiated into neural tube–like structures—potentially due to RPE-secreted inductive factors—and exhibited higher expression of photoreceptor markers than RPCs cultured without RPE.
Conclusion
Our results demonstrate that a 3D coculture system can be used to investigate the cellular and molecular mechanisms underlying retinal development and to support the formation of 3D retinal structures for future preclinical and clinical applications.
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