Human retinal organoids derived from pluripotent stem cells represent a robust in vitro model for investigating retinal development and disease mechanisms of retinal disorders. However, achieving structural maturation that faithfully recapitulates the intricate architecture of photoreceptors within a feasible and cost-efficient culture timeframe remains a significant challenge. Here, we present an optimized differentiation protocol that enables the generation of retinal organoids exhibiting advanced photoreceptor maturation within 140 days. Photoreceptors in the retinal organoids displayed compartmentalized architecture, including distinct inner and outer segments and connecting cilia. Notably, we observed the emergence of budding calyceal process-like structures—a feature not previously emphasized in photoreceptors derived from pluripotent stem cells. These results suggest that our protocol may promote advanced photoreceptor maturation within a relatively shortened culture period. Thus, this method could serve as a useful model for investigating retinal development and related pathologies, building upon previous protocols.

BackgroundThe lack of understanding of the molecular and cellular characteristics of human retinal progenitor cells (RPCs) has hindered their application in cell therapy for retinal degenerative diseases. This study aims to employ retinal organoids (ROs) derived from a VSX2-enhanced green fluorescent protein (eGFP) reporter human induced pluripotent stem cell (hiPSC) line for positive selection of human RPCs, investigate their features, and facilitate their applications.MethodshiPSCs were differentiated into three-dimensional ROs following established protocols. The fidelity of the VSX2-eGFP reporter was confirmed through immunostaining. Fluorescence-activated cell sorting was employed to select VSX2-eGFP-positive (+) cells at distinct developmental stages, followed by bulk RNA sequencing (RNA-seq) analysis to assess their transcriptome profile. Immunostaining and flow cytometry were utilized to validate the identity of VSX2-eGFP+ cells and potential cluster of differentiation (CD) biomarkers for identifying human RPCs.ResultshiPSCs were successfully differentiated into ROs containing abundant RPCs. The spatiotemporal activity of the VSX2-eGFP reporter recapitulated the dynamic expression of endogenous VSX2 protein. Compared to VSX2-eGFP-negative (-) cells, VSX2-eGFP+ cells mainly exhibited characteristics of RPCs at early stages of retinal development and of bipolar cells at late stages. RNA-seq analysis revealed transcriptional heterogeneity within VSX2-eGFP+ cells across four distinct developmental stages. Moreover, the dynamic expression of 394 known CD biomarkers in VSX2-eGFP+ cells at distinct developmental stages was analyzed herein for the first time. One CD biomarker, TNFRSF1B, which has never been reported to be expressed in RPCs, was found to be highly expressed in RPCs at the early stages and might serve as a candidate CD biomarker for sorting RPCs.ConclusionsThis study provides valuable insights into the molecular and cellular characteristics of human RPCs, especially their expression profiles of CD biomarkers, laying a foundation for research on retinal development and the clinical translation of hiPSC-derived RPCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04700-z.

The mechanisms governing the generation of neuronal subtypes at distinct times and proportions during human retinal development are poorly understood. While thyroid hormone (TH) signaling specifies cone photoreceptor subtypes, how this regulation changes over time remains unclear. To address this question, we studied the expression and function of type 3 iodothyronine deiodinase (DIO3), an enzyme that degrades TH, in human retinal organoids. We show that DIO3 is a master regulator of human photoreceptor developmental timing and cell fate stability. DIO3 is highly expressed in retinal progenitor cells (RPCs) and decreases as these cells asynchronously differentiate into neurons, progressively reducing TH degradation and increasing TH signaling. DIO3 mutant organoids display precocious development of S cones, L/M cones, and rods; increased photoreceptor density; and subpopulations of photoreceptors that coexpress different opsin proteins. Our multiomics and chimeric organoid experiments show that cell-autonomous and non-cell-autonomous mechanisms locally coordinate and maintain DIO3 expression and TH signaling levels among cells. Computational modeling reveals a mechanism that couples TH levels and fate specification, providing robustness to photoreceptor development as compared with a probabilistic, cell-intrinsic mechanism. Based on our findings, we propose an hourglass-like mechanism in which the proportion of progenitors to neurons decreases over time to relieve TH degradation, triggering development of photoreceptor subtypes at specific times. Our study identifies how local regulation of thyroid hormone signaling influences neural cell fate specification, which may be a consideration for designing regenerative therapies.

Human macula formation involves two waves of retinoic acid suppression via CYP26A1 that modulate cell cycle exit and cone subtype specification.

Chronic low-dose cadmium exposure induces neurodevelopmental impairment in hESC-derived retinal organoids.

MYCN amplification without concurrent RB1 mutations characterizes a rare yet highly aggressive subtype of retinoblastoma; however, its precise developmental origins and therapeutic vulnerabilities remain incompletely understood. Here, we modeled this subtype by lentiviral-mediated MYCN overexpression in human pluripotent stem cell-derived retinal organoids, revealing a discrete developmental window (days 70–120) during which retinal progenitors showed heightened susceptibility to transformation. Tumors arising in this period exhibited robust proliferation, expressed SOX2, and lacked CRX, consistent with origin from primitive retinal progenitors. MYCN-overexpressing organoids generated stable cell lines that reproducibly gave rise to MYCN-driven tumors when xenografted into immunodeficient mice. Transcriptomic profiling demonstrated that MYCN-overexpressing organoids closely recapitulated molecular features of patient-derived MYCN-amplified retinoblastomas, particularly through activation of MYC/E2F and mTORC1 signaling pathways. Pharmacological screening further identified distinct therapeutic vulnerabilities, demonstrating distinct subtype-specific sensitivity of MYCN-driven cells to transcriptional inhibitors (THZ1, Flavopiridol) and the cell-cycle inhibitor Volasertib, indicative of a unique oncogene-addicted state compared to RB1-deficient retinoblastoma cells. Collectively, our study elucidates the developmental and molecular mechanisms underpinning MYCN-driven retinoblastoma, establishes a robust and clinically relevant human retinal organoid platform, and highlights targeted transcriptional inhibition as a promising therapeutic approach for this aggressive pediatric cancer subtype.

ABCA4-mutant human retinal organoids sequencing reveals organoids application in inherited retinal diseases.

AAV2.7m8 transduction of stage 2 human retinal organoids induces highly variable responses in innate and inflammatory gene expression and cytokine secretion.

Retinal organoids (ROs) represent a promising regenerative strategy for restoring vision in retinal degenerative diseases, but whether host cone bipolar cells (BCs) in the primate macula can rewire with transplanted photoreceptors remains unresolved. Here, we transplanted genome-edited human retinal organoids lacking ON-BCs (Islet-1−/− ROs) into a non-human primate macular degeneration model. Remarkably, host rod and cone BCs extended dendrites toward grafted photoreceptors, forming functional synapses confirmed by immunohistochemistry, ultrastructural imaging, and focal macular electroretinography. Both ON- and OFF-pathway connectivity was rebuilt, providing the first demonstration of host–graft synaptic integration in the primate macula. These results establish that primate cone circuits retain a surprising capacity for rewiring and highlight genome-edited ROs as a powerful platform for vision restoration. Our findings represent a critical translational step toward stem cell–based therapies capable of repairing central vision in patients with advanced macular degeneration.

The developing mammalian retina initially contains undifferentiated cells, providing a model for investigating the mechanisms of differentiation. Notch signaling, mediated by four Notch receptors (Notch 1-4) in mammals, has been studied in the differentiation of neural progenitor cells. Among the four Notch receptors, the frequency, rather than the peak level, of Notch1-mediated signaling has been suggested to promote the activation of neural progenitor cells. In contrast to Notch1, the involvement of Notch3 in this process is poorly documented, although Notch3 is known for its role in vascular integrity. By re-analyzing publicly available single-cell RNA-seq data from one mouse retinal dataset, two human retinal organoid datasets and two human embryonic retinal datasets, we found that, along with Notch1, Notch3 is expressed in neural progenitor cells in the retina. In addition, the results of the co-expression profile analyses varied among the datasets, leaving uncertainty regarding the regulatory mechanisms of Notch1 and Notch3. Our findings shed light on Notch3 in neurogenic progenitor cells of the developing mammalian retina. Since Notch3 has been suggested to cause ligand-independent signaling, Notch3 expression might antagonize Notch1-mediated signaling oscillations, maintaining the quiescent state of neurogenic progenitor cells.

To explore the changes in early retinal development after the occurrence of ischemia. Human retinal organoids (hROs) of day 18 or day 30 were treated with oxygen-glucose deprivation and reperfusion (OGD/R) to simulate the retinal ischemia. All hROs were maintained normally until day 60 to evaluate changes in ischemic injuries during retinal development. Paraffin section staining was used for detecting changes in organoid structure and cell number. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB) analyses were used to observe the change in the expression of retinal cell markers. In hROs, OGD/R induced the decrease of proliferating cells, inhibited the expression of proliferated marker Ki67 and promoted early apoptosis of retinal cells (P<0.05). Under OGD/R condition, the progenitor cell layer and ganglion cell layer of hROs lost normal structure, and the number of neural stem cells (SOX2+), retinal progenitor cells (CHX10+) and retinal ganglion cells (TUJ1+/BRN3+/ATOH7+) decreased (P<0.05). The expression of corresponding retinal cell markers also decreased (P<0.05). Organoids treated with OGD/R on day 30 had similar injuries in retinal structure and retinal cell markers to those on day 18. Long-term observations revealed that day 18-treated organoids remained disorganized progenitor and ganglion cell layers by day 60, with no recovery in proliferating cell nuclear antigen (PCNA) protein expression. RT-qPCR showed persistently low Ki67 transcription levels (P<0.001), while other retinal cell markers recovered or exceeded normal levels, indicating a limited self-repair happened in the development of hROs. In contrast, day 30-treated organoids exhibited normal structure and marker expression by day 60, with transcription levels of retinal cell markers returning to normal (P>0.05), demonstrating complete recovery from OGD/R damage. Retinal ischemia damage the retinal development in the short-term. After the restoration of retinal blood supply, the retinal ischemic damage can be recovered during subsequent development. However, retinal ischemic injuries at different developmental stages exhibit varying degrees of reversibility. The earlier ischemic injury occurs, the more difficult it is to repair retinal cell and structure damage.

The delicate and complex structure of the neural retina that enables proper visual function is achieved during embryonic development through a precise balance of proliferation, differentiation, and cell death. Retinal ganglion cells (RGC), the only output neurons of the retina, show a steady increase in numbers during development except for two waves of cell death that are highly conserved in vertebrates. However, the mechanisms responsible for these phenomena and their conservation in the human retina are incompletely understood. In this work we took advantage of human induced pluripotent stem cell (hiPSC)-derived retinal organoids to explore these questions. Using different markers and quantitative techniques in three different hiPSC lines, we found a consistent decrease in RGC numbers at week 8 of differentiation, a developmental stage that is equivalent to that of the first wave of RGC death in other species. This decrease coincided with a peak in caspase 3 activation and TUNEL(+) staining, suggesting an apoptotic mechanism. Notably, this was accompanied by a decrease in the BAX/BCL2 ratio and a lack of caspase 9 activation. However, we observed a marked increase in caspase 8 activation at this stage, suggesting the involvement of the extrinsic apoptotic pathway. Together, these results show for the first time the intrinsic ability of the human retina to regulate RGC numbers through programmed cell death mechanisms, which could lead to new insights regarding congenital retinal abnormalities. Moreover, this work has implications for experimental design in basic and translational research using human stem cell-derived retinal organoid models.

While the etiology of Alzheimer’s disease remains unknown, there is growing support for the amyloid-β antimicrobial hypothesis. Amyloid-β, the main component of amyloid plaques in Alzheimer’s disease, has been shown to be generated in the presence of microbes. Entrapment of microbes by aggregated amyloid-β may serve as an innate immune response to pathogenic infections. To understand the association of amyloid-β plaques and pathogenic infections in the central nervous system, we obtained viable short-interval postmortem human retinal tissue and generated human retinal organoids that contain electrophysiologically active neurons. Here, we demonstrate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces amyloid-β extracellular protein aggregates in human retinal explants and retinal organoids. Last, pharmacological inhibition of neuropilin-1 resulted in reduced amyloid-β deposition in human retinal explants treated with SARS-CoV-2 Spike 1 protein. These results suggest that Spike 1 protein, during infection with SARS-CoV-2, can induce amyloid-β aggregation, which may be associated with the neurological symptoms experienced in COVID-19.

Hypoxia-induced activation of calpain and oxidative stress in mitochondria leads to RGC death in human iPSC-derived retinal organoids.

Single-cell RNA sequencing in the study of human retinal organoids.

The development of therapeutics builds on testing their efficiency in vitro. To optimize gene therapies, for example, fluorescent reporters expressed by treated cells are typically utilized as readouts. Traditionally, their global fluorescence signal has been used as an estimate of transduction efficiency. However, analysis in individual cells within a living 3D tissue remains a challenge. Readout on a single-cell level can be realized via fluorescence-based flow cytometry at the cost of tissue dissociation and loss of spatial information. Complementary, spatial information is accessible via immunofluorescence of fixed samples. Both approaches impede time-dependent studies on the delivery of the vector to the cells. Here, quantitative 3D characterization of viral transduction efficiencies in living retinal organoids is introduced. The approach combines quantification of gene delivery efficiency in space and time, leveraging human retinal organoids, engineered adeno-associated virus (AAV) vectors, confocal live imaging, and deep learning-based image segmentation. The integration of these tools in an organoid imaging and analysis pipeline allows quantitative testing of future treatments and other gene delivery methods. It has the potential to guide the development of therapies in biomedical applications.

hRetOrgs offer an unprecedented platform for functional genetic studies of human retinal development and disease. However, existing methods for gene manipulation in hRetOrg are limited by low throughput, inefficiency, and lack of scalability, hindering systematic analysis of gene function and regulatory elements. To address these limitations, we developed a streamlined, high-efficiency pipeline that enables spatially targeted electroporation of hRetOrg during early retinogenesis, combined with fast, high-resolution imaging of whole organoids using two-photon microscopy, allowing studies at both tissue and subcellular scales.

Human embryonic stem cells (hESC)-derived retinal organoids are sophisticated in vitro systems for dissecting the complex dynamics of human retinal development. The formation of the human retina is a precisely organized process that depends on the regulated differentiation of retinal progenitor cells; however, many of the basic mechanisms remain to be explored. Here, using hESC-derived retinal organoids, we elucidated the temporal contribution of RAX2 to retinal development, with an emphasis on photoreceptor cells (PC) formation. The results were corroborated using human fetal retinal tissue at various gestational ages. Using CRISPR/Cas9-mediated gene knockout, we delineated the essential role of RAX2 in modulating PC specifications. RAX2 deficiency significantly altered the expression of PAX6 and SOX2, two essential regulators of retinogenesis. Our results suggested that RAX2 is significant in retinal development, underpinning its potential as a therapeutic target in related retinal disorders.

CRISPR/Cas9 gene editing for the retina: transient delivery using RNP in vivo and development of human retinal organoids as a model

Antisense oligonucleotide (ASO) therapy holds promise in gene therapy but faces challenges due to poor delivery efficiency and limited evaluation models. This investigation employs magnetic nanoparticles (MNPs) to augment the delivery efficiency of ASOs. It assesses their distribution and therapeutic efficacy across various models, including retinal explants from mice and macaques or human retinal and inner ear organoids. Retinal explants from both mice and monkeys are methodically arranged to expose the ganglion cell layer (GCL) or the photoreceptor layer (PL). MNPs markedly enhanced the penetration and targeting of ASOs, resulting in a 60% accumulation in the GCL or 72% in the photoreceptors. Furthermore, an in vitro biomimetic model of the neuroretina‐RPE/choroid‐sclera complex is developed to examine ASO distribution under dynamic flow conditions. Moreover, the utilization of MNP‐assisted ASO‐Cy3 markedly enhanced transfection efficiency within human retinal and inner ear organoids, resulting in an increase in positively transfected cells to 60% and 70%, respectively. Here, for the first time, an MNP‐explant‐organoid platform is carried out for the promotion of ASO transfection efficiency, therapeutic screening and targeted delivery. This development paves the way for investigating novel gene therapy strategies targeting retinal diseases.