Functional development of photoreceptors in human retinal organoids

Abstract Background Retinal organoids (ROs) derived from human pluripotent stem cells are crucial for modeling retinal development and disease. However, the functional electrophysiological maturation of photoreceptors within ROs remains poorly characterized. This study aimed to define the functional maturation timeline of photoreceptors in human embryonic stem cell (hESC)-derived ROs. Methods H9 hESC-derived ROs which included a CRX-tdTomato reporter line for specific photoreceptor identification were utilized. An integrated approach of RNA-sequencing analysis, immunofluorescence staining, and whole-cell patch-clamp recordings was employed to systematically assess photoreceptor maturation over 300 days of differentiation. Results Transcriptional and protein analysis revealed progressive upregulation of key ion channels. Patch-clamp recordings demonstrated stage-dependent maturation of membrane properties, which stabilized by D120–125. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents (I h ) increased progressively, peaking at D240, with amplitudes comparable to mature primate photoreceptors. Voltage-gated sodium (Nav) currents also showed significant developmental upregulation, reaching a maximum, stable plateau from D210–215 onward. Pharmacological blockade confirmed the identity of HCN and Nav currents. Critically, the capacity for action potential (AP) generation increased developmentally, with the proportion of photoreceptors capable of firing APs rising from 16.7% at D90–95 to a peak of 90.2% by D240–245. Conclusions This study defines a comprehensive electrophysiological maturation timeline for photoreceptors in human ROs and establishes D240 as a key benchmark for functional maturity, characterized by peak I h currents and AP generation capacity equivalent to mature native photoreceptors. These findings provide essential physiological criteria for standardizing RO quality control, enhancing their utility for modeling retinal degenerative diseases and developing cell replacement therapies.