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 (Ih) 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 Ih 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.

1. Introduction

Retinal degenerative diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are among the primary contributors to irreversible vision loss globally [1–3]. A hallmark of these diseases is the progressive dysfunction or loss of photoreceptors, which critically impairs the fundamental phototransduction and synaptic transmission processes, representing the primary cause of blindness in AMD and RP [4–6]. Current treatments are limited, primarily focusing on slowing disease progression rather than restoring lost vision [7, 8]. Stem cell-derived photoreceptors offer a promising avenue for retinal repair [9]; yet their therapeutic potential hinges critically on the ability to exhibit native electrophysiological functionality [10, 11].

Pluripotent stem cell-derived retinal organoids (ROs) provide powerful in vitro models for studying retinal development and disease, recapitulating the 3D structure, cellular diversity, and developmental progression of the human retina [11–15]. Single-cell transcriptomics confirms their trajectory closely mirrors native retina development, with transcriptomes stabilizing and converging with mature peripheral retinal cell types around 30–38 weeks [16]. However, transcriptomic similarity does not equate to functional maturity. Neuronal integration critically depends on ion channel expression and electrophysiological network formation [17–19]. Notably, while markers like recoverin are expressed early in differentiation and rhodopsin emerges at mid-stage [20, 21], the developmental phase defining functionally mature photoreceptors remains unresolved. This underscores the necessity of staged electrophysiological assessment to evaluate functional maturity in ROs.