Electrophysiological Characterization of Photoreceptor-Like Cells in Human Inducible Pluripotent Stem Cell-Derived Retinal Organoids During in Vitro Maturation

Abstract

Retinal organoids (ROs) derived from human inducible pluripotent stem cells (hiPSCs) exhibit considerable therapeutic potential. However, current quality control of ROs during in vitro differentiation is largely limited to the detection of molecular markers, often by immunostaining, polymerase chain reaction (PCR) assays and sequencing, often without proper functional assessments. As such, in the current study, we systemically characterized the physiological maturation of photoreceptor-like cells in hiPSC-derived ROs. By performing patch-clamp recordings from photoreceptor-like cells in ROs at distinct differentiation stages (ie, Differentiation Day [D]90, D150, and D200), we determined the electrophysiological properties of the plasma membrane and several characteristic ion channels closely associated with the physiological functions of the photoreceptors. Ionic hallmarks, such as hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and cyclic nucleotide-gated (CNG) channels, matured progressively during differentiation. After D200 in culture, these characteristic currents closely resembled those in macaque or human native photoreceptors. Furthermore, we demonstrated that the hyperpolarization-activated inward current/depolarization-activated outward current ratio (I−120/I+40), termed as the inward-outward current (IOC) ratio hereon, accurately represented the maturity of photoreceptors and could serve as a sensitive indicator of pathological state. Thus, this study provides a comprehensive dataset describing the electrophysiological maturation of photoreceptor-like cells in hiPSC-derived ROs for precise and sensitive quality control during RO differentiation.

Increasing cell culture density during a developmental window prevents fated rod precursors derailment toward hybrid rod-glia cells

“…3a, we selected the day of birth (PN0) for cell isolation to ensure enough cells for subsequent analysis and a timespan to adapt to the in vitro conditions and develop a response to cell culture density. As a preliminary step, we evaluated whether isolated rod precursors maintain their electrophysiological identity in culture by monitoring the Cs-sensitive hyperpolarisation-activated current (I HYP ), a prominent inward current of adult rods expressed by both mice 39 and human rod precursors 40 . A substantial fraction of I HYP flows through ion channels coded by Hcn1, a gene already expressed at an early developmental time and whose expression progressively builds up till PN28 (adult).…”

“…Although Hcn1 is not a rod-specific gene and is not among the genes most upregulated between PN4 and PN8, it is expressed by most primary sensory neurons 26 , and I HYP measurement may indicate whether rod precursors maintain their sensory neuron phenotype in culture. Furthermore, recent evidence in human rod precursors of iPSC-derived retinal organoids indicates that the current carried by HCN1 channels is sensitive to 4-hydroxy tamoxifen 40 , a blocker of estrogen-related receptor beta (ERRβ) critical for rod viability 41 coded by the DEG Esrrb.…”

Recent Advances in Electrophysiological Recording Platforms for Brain and Heart Organoids

“…Furthermore, patch-clamp technology is being extended further through methods such as outside-out recording and interface patch clamping. [65][66][67][68] Paşca et al recorded human cortical neurons using a patchclamp, and the results showed a temporal inward current following depolarization. [15] Tetrodotoxin (TTX) is a poison that strongly inhibits voltage-gated Na þ channels, and it has been used experimentally to silence neuron firing.…”

“…Furthermore, patch‐clamp technology is being extended further through methods such as outside‐out recording and interface patch clamping. [ 65–68 ]…”

The Prospects for Retinal Organoids in Treatment of Retinal Diseases

“…Another important aspect is to evaluate the functionalities of ROs in advanced stages for light sensitivity and synapses generation. Common methods for RO electrophysiological functional analysis include patch-clamp, 64,83 fluorescent calcium imaging, [84][85][86] 2-photon microscopy 87 and microelectrode arrays (MEAs), 88 reviewed by Afanasyeva et al 89 In more recent studies, Li et al 90 systematically characterized the electrophysiology of ROs at different stages (D90, D150, and D200) using patch-clamp recording and found that PR cells in ROs after D200 showed similar characteristic currents as those in human retina. Cowan et al 91 compared ROs with human retina in transcriptomes, and they further characterized the functionality of ROs by measuring the light responsiveness, imaging synaptic layers, and functional synapses.…”