Photostimulation Promotes Maturation of Human Stem Cell-Derived Photoreceptors

Human retinal organoids derived from stem cells have become powerful tools to model retinal development and disease, but they often remain immature and lack key features required for full function. Light is not only the sensory target of photoreceptors but also an important developmental signal in vivo. However, light has rarely been used as a deliberate stimulus during in vitro differentiation. Here we show that exposing retinal organoids to rhythmic light flicker at a specific frequency enhances photoreceptor maturation across multiple levels. Overall design: Bulk RNA-seq: Organoids were exposed to four distinct light conditions in a 12-hour light / 12-hour dark cycle for 7 days: Dark (no light exposure – control), Constant light, Random-frequency flickering light (1–100 Hz), and 40 Hz flickering light (gamma-frequency stimulation). The aim was to identify global transcriptional changes in response to various light stimulation paradigms. Single-cell RNA-seq (scRNA-seq): Performed on organoids cultured under either Dark or 40 Hz flickering light conditions, using matched protocols. This allowed assessment of cell-type specific transcriptional responses and changes in cell composition. Thirty organoids were pooled per condition, with dissociation and scRNA-seq performed using the 10x Genomics Chromium platform. Together, the samples encompass both global and cell-type resolved transcriptomic responses to photostimulation, with experimental variables including light regime and biological replication.