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Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography. Visual function was accessed by optokinetic tests and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin and eosin; or processed for immunohistochemistry to label human donor cells, retinal cell types, and synaptic markers. After transplantation, ROs integrated into the host RD retina, formed functional photoreceptors, and improved vision in rats with advanced RD. The survival and vision improvement are comparable with our previous results of hESC-ROs without a long-distance delivery. Furthermore, for the first time in the stem cell transplantation field, we demonstrated that the response heatmap on the SC showed a similar shape to the location of the transplant in the host retina, which suggested the point-to-point projection of the transplant from the retina to SC. In conclusion, our results showed that using our special device and protocol, the hESC-derived ROs can be shipped over long distance and are capable of survival and visual improvement after transplantation into the RD rats. Our data provide a proof-of-concept for stem cell replacement as a therapy for RD patients.

Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however, their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study, we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles, which, with a capacity of up to 36 kb, can potentially accommodate all known retinal gene coding sequences. We utilized HC-AdVs based on the classical adenoviral type 5 (AdV5) and on a fiber-modified AdV5.F50 version, both engineered to deliver a 29.6 kb vector genome encoding a fluorescent reporter construct. The tropism of these HC-AdVs was evaluated in an induced pluripotent stem cell (iPSC)-derived human retinal organoid model. Both vector types demonstrated robust transduction efficiency, with sustained transgene expression observed for up to 110 days post-transduction. Moreover, we found efficient transduction of photoreceptors and Müller glial cells, without evidence of reactive gliosis or loss of photoreceptor cell nuclei. However, an increase in the thickness of the photoreceptor outer nuclear layer was observed at 110 days post-transduction, suggesting potential unfavorable effects on Müller glial or photoreceptor cells associated with HC-AdV transduction and/or long-term reporter overexpression. These findings suggest that while HC-AdVs show promise for large retinal gene delivery, further investigations are required to assess their long-term safety and efficacy. [ABSTRACT FROM AUTHOR]

Introduction: Considering the significant role played by both intrinsic and extrinsic electric fields in the growth and maturation of the central nervous system, the impact of short exposure to external electric fields on the development and differentiation of retinal organoids was investigated. Methods: Retinal organoids derived from human embryonic stem cells were used at day 80, a key stage in their differentiation. A single 60-minute exposure to a biphasic electrical field was administered to assess its influence on retinal cell populations and maturation markers. Immunohistochemistry, qPCR, and RNA sequencing were employed to evaluate cell type development and gene expression changes. Results: Electrical stimulation significantly enhanced neuronal development and increased the population of photoreceptors within the organoids. RNA sequencing data showed upregulated expression of genes related to rod photoreceptors, Müller cells, horizontal cells, and amacrine cells, while genes associated with retinal pigment epithelium and retinal ganglion cells were downregulated. Variations in development and maturation were observed depending on the specific parameters of the applied electric field. Discussion: These findings highlight the significant impact of extrinsic electrical fields on early retinal development and suggest that optimizing electrical field parameters could effectively address certain limitations in retinal organoid technology, potentially reducing the reliance on chemicals and small molecules. [ABSTRACT FROM AUTHOR]

In zebrafish and various mammalian species, HB-EGF has been shown to promote Müller glia proliferation and activation of repair mechanisms that have not been fully investigated in human retina. In the current study, 70- to 90-day-old human retinal organoids were treated with 20 μM 4-hydroxytamoxifen (4-OHT), and CRX, REC, NRL, PAX6, VIM, GFAP, and VSX2 gene and protein expression were assessed at various times points after treatment. Organoids with or without 4-OHT-induced damage were then cultured with HB-EGF for 7 days. We showed that 20 μM 4-OHT caused a reduction in the number of recoverin-positive cells; an increase in the number of TUNEL-positive cells; and downregulation of the photoreceptor gene markers CRX, NRL, and REC. Culture of organoids with HB-EGF for 7 days after 4-OHT-induced damage caused a marked reduction in the number of TUNEL-positive cells and small increases in the number of Ki67-positive cells and PAX6 and NOTCH1 gene expression. The current results suggest that treatment of human ESC-derived retinal organoids with 4-OHT may be used as a model of retinal degeneration in vitro. Furthermore, HB-EGF treatment of human retinal organoids increases proliferating Müller cells, but only after 4-OHT induced damage, and may be an indication of Muller reactivity in response to photoreceptor damage. Further studies will aim to identify factors that may induce Müller cell-mediated regeneration of the human retina, aiding in the development of therapies for retinal degeneration. [ABSTRACT FROM AUTHOR]

Retinal organoids are three-dimensional (3D) microscopic tissues that are induced and differentiated from stem cells or progenitor cells in vitro and have a highly similar structure to the retina. With the optimization and development of 3D retinal culture system and the improvement of induced differentiation technology, retinal organoids have broad application prospects in retinal development, regenerative medicine, biomaterial evaluation, disease mechanism investigation, and drug screening. In this review we summarize recent development of retinal organoids and their applications in ophthalmic regenerative medicine. In particular, we highlight the promise and challenges in the use of retinal organoids in disease modeling and drug discovery.

This study investigated the therapeutic effects of exosomes derived from human-induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) on corneal epithelial wound healing. Exosomes were isolated from the culture medium of the hiPSC-derived ROs (Exo-ROs) using ultracentrifugation, and then they were characterized by a nanoparticle tracking analysis and transmission electron microscopy. In a murine model of corneal epithelial wounds, these exosomes were topically applied to evaluate their healing efficacy. The results demonstrated that the exosome-treated eyes showed significantly enhanced wound closures compared with the controls at 24 h post-injury. The 5-ethyl-2′-deoxyuridine assay and quantitative reverse transcription polymerase chain reaction revealed a substantial increase in cell proliferation and a decrease in inflammatory marker contents in the exosome-treated group. The RNA sequencing and exosomal microRNA analysis revealed that the Exo-RO treatment targeted various pathways related to inflammation and cell proliferation, including the PI3K-Akt, TNF, MAPK, and IL-17 signaling pathways. Moreover, the upregulation of genes related to retinoic acid and eicosanoid metabolism may have enhanced corneal epithelial healing in the eyes treated with the Exo-ROs. These findings suggest that hiPSC-derived RO exosomes could be novel therapeutic agents for promoting corneal epithelial wound healing. [ABSTRACT FROM AUTHOR]

Human retinal organoids (ROs) have emerged as valuable tools for studying retinal development, modeling human retinal diseases, and screening drugs. However, their application is limited primarily due to time-intensive generation, high costs, and low reproducibility. Quality assessment of RO differentiation is crucial for their application in research. However, traditional methods such as morphological evaluation and immunohistochemical analysis have limitations due to their lack of precision and invasiveness, respectively. This study aims to identify non-invasive biomarkers for RO differentiation quality using exosomal microRNAs (miRNAs), which are known to reflect cell-specific functions and development in the retina. We differentiated ROs from human induced pluripotent stem cells (hiPSCs) and classified them into 'superior' and 'inferior' groups based on morphological and immunohistochemical criteria. Exosomes from the conditioned media were isolated and analyzed for miRNA content. Our findings revealed distinct miRNA profiles between superior and inferior ROs, with superior ROs exhibiting higher miRNA diversity and specifically up- or down-regulated miRNAs. Gene ontology and pathway enrichment analyses indicated that the target genes of these miRNAs are involved in neuron proliferation and differentiation. The study suggests the potential of exosomal hsa-miR-654-3p and hsa-miR-451a as non-invasive biomarkers for real-time monitoring of RO quality, facilitating the development of standardized, efficient, and cost-effective culture methods. [ABSTRACT FROM AUTHOR]

The proneural transcription factor atonal basic helix–loop–helix transcription factor 7 (ATOH7) is expressed in early progenitors in the developing neuroretina. In vertebrates, this is crucial for the development of retinal ganglion cells (RGCs), as mutant animals show an almost complete absence of RGCs, underdeveloped optic nerves, and aberrations in retinal vessel development. Human mutations are rare and result in autosomal recessive optic nerve hypoplasia (ONH) or severe vascular changes, diagnosed as autosomal recessive persistent hyperplasia of the primary vitreous (PHPVAR). To better understand the role of ATOH7 in neuroretinal development, we created ATOH7 knockout and eGFP-expressing ATOH7 reporter human induced pluripotent stem cells (hiPSCs), which were differentiated into early-stage retinal organoids. Target loci regulated by ATOH7 were identified by Cleavage Under Targets and Release Using Nuclease with sequencing (CUT&RUN-seq) and differential expression by RNA sequencing (RNA-seq) of wildtype and mutant organoid-derived reporter cells. Additionally, single-cell RNA sequencing (scRNA-seq) was performed on whole organoids to identify cell type-specific genes. Mutant organoids displayed substantial deficiency in axon sprouting, reduction in RGCs, and an increase in other cell types. We identified 469 differentially expressed target genes, with an overrepresentation of genes belonging to axon development/guidance and Notch signaling. Taken together, we consolidate the function of human ATOH7 in guiding progenitor competence by inducing RGC-specific genes while inhibiting other cell fates. Furthermore, we highlight candidate genes responsible for ATOH7-associated optic nerve and retinovascular anomalies, which sheds light to potential future therapy targets for related disorders. [ABSTRACT FROM AUTHOR]

Remodeling retinal Müller glial fate, including gliosis inhibition and pro-reprogramming, represents a crucial avenue for treating degenerative retinal diseases. Stem cell transplantation exerts effects on modulating retinal Müller glial fate. However, the optimized stem cell products and the underlying therapeutic mechanisms need to be investigated. In the present study, we found that retinal progenitor cells from human embryonic stem cell-derived retinal organoids (hERO-RPCs) transferred extracellular vesicles (EVs) into Müller cells following subretinal transplantation into RCS rats. Small EVs from hERO-RPCs (hERO-RPC-sEVs) were collected and were found to delay photoreceptor degeneration and protect retinal function in RCS rats. hERO-RPC-sEVs were taken up by Müller cells both in vivo and in vitro, and inhibited gliosis while promoting early dedifferentiation of Müller cells. We further explored the miRNA profiles of hERO-RPC-sEVs, which suggested a functional signature associated with neuroprotection and development, as well as the regulation of stem cell and glial fate. Mechanistically, hERO-RPC-sEVs might regulate the fate of Müller cells by miRNA-mediated nuclear factor I transcription factors B (NFIB) downregulation. Collectively, our findings offer novel mechanistic insights into stem cell therapy and promote the development of EV-centered therapeutic strategies. [Display omitted] • hERO-RPC-sEVs protect the retinal structure and visual function of RCS rats after transplantation. • hERO-RPC-sEVs are transferred into Müller cells, inhibiting gliosis and promoting dedifferentiation after transplantation. • miRNA profiles of hERO-RPC-sEVs indicate a functional signature linked to stem/glial cell fate regulation. • hERO-RPC-sEVs remodel Müller cell fate via miRNA-mediated NFIB downregulation. [ABSTRACT FROM AUTHOR]

The field of large numerical aperture microscopy has witnessed significant advancements in spatial and temporal resolution, as well as improvements in optical microscope imaging quality. However, these advancements have concurrently raised the demand for enhanced precision, extended range, and increased load-bearing capacity in objective motion carrier (OMC). To address this challenge, this study introduces an innovative OMC that employs a ball screw mechanism as its primary driving component. Furthermore, a robust nonlinear motion control strategy has been developed, which integrates fast nonsingular terminal sliding mode, experimental estimation techniques, and adaptive radial basis neural network, to mitigate the impact of nonlinear friction within the ball screw mechanism on motion precision. The stability of the closed-loop control system has been rigorously demonstrated through Lyapunov theory. Compared with other enhanced sliding mode control strategies, the maximum error and root mean square error of this controller are improved by 33% and 34% respectively. The implementation of the novel OMC has enabled the establishment of a high-resolution bio-optical microscope, which has proven its effectiveness in the microscopic imaging of retinal organoids. • Novel SMC Strategy: To address the limitations of traditional SMC, characterized by high gains and pronounced chattering, a novel SMC strategy is introduced. • Online Estimation and Compensation: The utilization of TDE techniques enables online estimation and real-time compensation of unknown terms. • Biophotonic Microscope Development: The research culminates in the development of a biophotonic microscope, wherein the objective motion carrier plays a crucial role in achieving focal plane movement. [ABSTRACT FROM AUTHOR]

IntroductionConsidering the significant role played by both intrinsic and extrinsic electric fields in the growth and maturation of the central nervous system, the impact of short exposure to external electric fields on the development and differentiation of retinal organoids was investigated.MethodsRetinal organoids derived from human embryonic stem cells were used at day 80, a key stage in their differentiation. A single 60-minute exposure to a biphasic electrical field was administered to assess its influence on retinal cell populations and maturation markers. Immunohistochemistry, qPCR, and RNA sequencing were employed to evaluate cell type development and gene expression changes.ResultsElectrical stimulation significantly enhanced neuronal development and increased the population of photoreceptors within the organoids. RNA sequencing data showed upregulated expression of genes related to rod photoreceptors, Müller cells, horizontal cells, and amacrine cells, while genes associated with retinal pigment epithelium and retinal ganglion cells were downregulated. Variations in development and maturation were observed depending on the specific parameters of the applied electric field.DiscussionThese findings highlight the significant impact of extrinsic electrical fields on early retinal development and suggest that optimizing electrical field parameters could effectively address certain limitations in retinal organoid technology, potentially reducing the reliance on chemicals and small molecules.

Zika virus (ZIKV) causes microcephaly and congenital eye disease. The cellular and molecular basis of congenital ZIKV infection are not well understood. Here, we utilized a biologically relevant cell-based system of human fetal retinal pigment epithelial cells (FRPEs), hiPSC-derived retinal stem cells (iRSCs), and retinal organoids to investigate ZIKV-mediated ocular cell injury processes. Our data show that FRPEs were highly susceptible to ZIKV infection exhibiting increased apoptosis, whereas iRSCs showed reduced susceptibility. Detailed transcriptomics and proteomics analyses of infected FRPEs were performed. Nucleoside analogue drug treatment inhibited ZIKV replication. Retinal organoids were susceptible to ZIKV infection. The Asian genotype ZIKV exhibited higher infectivity, induced profound inflammatory response, and dysregulated transcription factors involved in retinal organoid differentiation. Collectively, our study shows that ZIKV affects ocular cells at different developmental stages resulting in cellular injury and death, further providing molecular insight into the pathogenesis of congenital eye disease.

Abstract Background The CRX-associated autosomal dominant retinopathies suggest a possible pathogenic mechanism of gene haploinsufficiency. However, based on reported human patient cases and studies with mouse models, it is hard to confirm the specific weight of haploinsufficiency in pathogenesis due to the interspecies gaps between gene expression and function. Methods We created monoallelic CRX by replacing one allele with tdTomato in human embryonic stem cells (hESCs) and subsequently dissect pathogenesis in hESCs-derived retinal organoids. We used transcriptome and immunofluorescence analyses to dissect phenotypic differences between CRX-monoallelic knockout and control wildtype organoids. For location analysis of CRX + cells, a CRX-expression-tracing system was constructed in control hESCs. We implemented long-term live-cell imaging to describe the translocation of CRX+ cells between two groups in early organoid differentiation. The expression pattern of these dynamic differences was validated using RNA-seq and immunofluorescence assays. Results We identified delayed differentiation of outer nuclear layer (ONL) stratification along with thinner ONL, serious loss of photoreceptor outer segments, as well as downregulated expression of gene for phototransduction and inner/outer segment formation. By live-cell imaging and immunostaining, we observed the overtension of actomyosin network and the arrested translocation of monoallelic CRX + cells in the early stage of retinal differentiation. Conclusions We confirmed that gene haploinsufficiency is the mechanism for the dominant pathogenicity of CRX and discovered that CRX regulated postmitotic photoreceptor precursor translocation in addition to its specification of photoreceptor cell fates during human retinal development. These findings revealed a new underlying mechanism of CRX dominant pathogenesis and provided a new clue for the treatment of CRX-associated human retinopathies.

Retinal degeneration (RD) is a significant cause of incurable blindness worldwide. Photoreceptors and retinal pigmented epithelium are irreversibly damaged in advanced RD. Functional replacement of photoreceptors and/or retinal pigmented epithelium cells is a promising approach to restoring vision. This paper reviews the current status and explores future prospects of the transplantation therapy provided by pluripotent stem cell-derived retinal organoids (ROs). This review summarizes the status of rodent RD disease models and discusses RO culture and analytical tools to evaluate RO quality and function. Finally, we review and discuss the studies in which RO-derived cells or sheets were transplanted. In conclusion, methods to derive ROs from pluripotent stem cells have significantly improved and become more efficient in recent years. Meanwhile, more novel technologies are applied to characterize and validate RO quality. However, opportunity remains to optimize tissue differentiation protocols and achieve better RO reproducibility. In order to screen high-quality ROs for downstream applications, approaches such as noninvasive and label-free imaging and electrophysiological functional testing are promising and worth further investigation. Lastly, transplanted RO-derived tissues have allowed improvements in visual function in several RD models, showing promises for clinical applications in the future.

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGRORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGRORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial. [ABSTRACT FROM AUTHOR]

Within the last decade, a wide variety of protocols have emerged for the generation of retinal organoids. A subset of studies have compared protocols based on stem cell source, the physical features of the microenvironment, and both internal and external signals, all features that influence embryoid body and retinal organoid formation. Most of these comparisons have focused on the effect of signaling pathways on retinal organoid development. In this study, our aim is to understand whether starting cell conditions, specifically those involved in embryoid body formation, affect the development of retinal organoids in terms of differentiation capacity and reproducibility. To investigate this, we used the popular 3D floating culture method to generate retinal organoids from stem cells. This method starts with either small clumps of stem cells generated from larger clones (clumps protocol, CP) or with an aggregation of single cells (single cells protocol, SCP). Using histological analysis and gene-expression comparison, we found a retention of the pluripotency capacity on embryoid bodies generated through the SCP compared to the CP. Nonetheless, these early developmental differences seem not to impact the final retinal organoid formation, suggesting a potential compensatory mechanism during the neurosphere stage. This study not only facilitates an in-depth exploration of embryoid body development but also provides valuable insights for the selection of the most suitable protocol in order to study retinal development and to model inherited retinal disorders in vitro. [ABSTRACT FROM AUTHOR]

Retinal degeneration (RD), a group of diseases leading to irreversible vision loss, is characterised by retinal pigment epithelium (RPE) or retinal neuron damage and loss. With fewer risks of immune rejection and tumorigenesis, stem cell‐secreted extracellular vesicles (EVs) offer a new cell‐free therapeutic paradigm for RD, which remains to be investigated. Human retinal organoid‐derived retinal progenitor cells (hERO‐RPCs) are an easily accessible and advanced cell source for RD treatment. However, hERO‐RPCs‐derived EVs require further characterisation. Here, we compared the characteristics of EVs from hERO‐RPCs (hRPC‐EVs) with those of human embryonic stem cell (hESC)‐derived EVs (hESC‐EVs) as controls. Based on in‐depth proteomic analysis, we revealed remarkable differences between hRPC‐EVs and hESC‐EVs. A comparison between EVs and their respective cells of origin demonstrated that the protein loading of hRPC‐EVs was more selective than that of hESC‐EVs. In particular, hESC‐EVs were enriched with proteins related to angiogenesis and cell cycle, whereas hRPC‐EVs were enriched with proteins associated with immune modulation and retinal development. More importantly, compared with that of hESC‐EVs, hRPC‐EVs exhibited a lower correlation with cell proliferation and a unique capacity to regulate lipid metabolism. It was further confirmed that hRPC‐EVs potentially eliminated lipid deposits, inhibited lipotoxicity and oxidative stress, and enhanced phagocytosis and survival of oleic acid‐treated ARPE‐19 cells. Mechanistically, hRPC‐EVs are integrated into the mitochondrial network of oleic acid‐treated ARPE‐19 cells, and increased the level of mitochondrial fatty acid β‐oxidation‐related proteins. Thus, organoid‐derived hRPC‐EVs represent a promising source of cell‐free therapy for RD, especially for blinding diseases related to abnormal lipid metabolism in RPE cells. [ABSTRACT FROM AUTHOR]

Background: The CRX-associated autosomal dominant retinopathies suggest a possible pathogenic mechanism of gene haploinsufficiency. However, based on reported human patient cases and studies with mouse models, it is hard to confirm the specific weight of haploinsufficiency in pathogenesis due to the interspecies gaps between gene expression and function. Methods: We created monoallelic CRX by replacing one allele with tdTomato in human embryonic stem cells (hESCs) and subsequently dissect pathogenesis in hESCs-derived retinal organoids. We used transcriptome and immunofluorescence analyses to dissect phenotypic differences between CRX-monoallelic knockout and control wildtype organoids. For location analysis of CRX+ cells, a CRX-expression-tracing system was constructed in control hESCs. We implemented long-term live-cell imaging to describe the translocation of CRX+ cells between two groups in early organoid differentiation. The expression pattern of these dynamic differences was validated using RNA-seq and immunofluorescence assays. Results: We identified delayed differentiation of outer nuclear layer (ONL) stratification along with thinner ONL, serious loss of photoreceptor outer segments, as well as downregulated expression of gene for phototransduction and inner/outer segment formation. By live-cell imaging and immunostaining, we observed the overtension of actomyosin network and the arrested translocation of monoallelic CRX+ cells in the early stage of retinal differentiation. Conclusions: We confirmed that gene haploinsufficiency is the mechanism for the dominant pathogenicity of CRX and discovered that CRX regulated postmitotic photoreceptor precursor translocation in addition to its specification of photoreceptor cell fates during human retinal development. These findings revealed a new underlying mechanism of CRX dominant pathogenesis and provided a new clue for the treatment of CRX-associated human retinopathies. [ABSTRACT FROM AUTHOR]

The majority of patients with mutations in CRB1 develop either early-onset retinitis pigmentosa as young children or Leber congenital amaurosis as newborns. The cause for the phenotypic variability in CRB1-associated retinopathies is unknown, but might be linked to differences in CRB1 and CRB2 protein levels in Müller glial cells and photoreceptor cells. Here, CRB1KO and CRB1KOCRB2+/− differentiation day 210 retinal organoids showed a significant decrease in the number of photoreceptor nuclei in a row and a significant increase in the number of photoreceptor cell nuclei above the outer limiting membrane. This phenotype with outer retinal abnormalities is similar to CRB1 patient-derived retinal organoids and Crb1 or Crb2 mutant mouse retinal disease models. The CRB1KO and CRB1KOCRB2+/− retinal organoids develop an additional inner retinal phenotype due to the complete loss of CRB1 from Müller glial cells, suggesting an essential role for CRB1 in proper localization of neuronal cell types. Adeno-associated viral (AAV) transduction was explored at early and late stages of organoid development. Moreover, AAV-mediated gene augmentation therapy with AAV.hCRB2 improved the outer retinal phenotype in CRB1KO retinal organoids. Altogether, these data provide essential information for future gene therapy approaches for patients with CRB1-associated retinal dystrophies.

Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids' (RtOgs') long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.

Ongoing cell therapy trials have demonstrated the need for precision control of donor cell behavior within the recipient tissue. We present a methodology to guide stem cell-derived and endogenously regenerated neurons by engineering the microenvironment. Being an "approachable part of the brain," the eye provides a unique opportunity to study neuron fate and function within the central nervous system. Here, we focused on retinal ganglion cells (RGCs)--the neurons in the retina are irreversibly lost in glaucoma and other optic neuropathies but can potentially be replaced through transplantation or reprogramming. One of the significant barriers to successful RGC integration into the existing mature retinal circuitry is cell migration toward their natural position in the retina. Our in silico analysis of the single-cell transcriptome of the developing human retina identified six receptor-ligand candidates, which were tested in functional in vitro assays for their ability to guide human stem cell-derived RGCs. We used our lead molecule, SDF1, to engineer an artificial gradient in the retina, which led to a 2.7-fold increase in donor RGC migration into the ganglion cell layer (GCL) and a 3.3-fold increase in the displacement of newborn RGCs out of the inner nuclear layer. Only donor RGCs that migrated into the GCL were found to express mature RGC markers, indicating the importance of proper structure integration. Together, these results describe an "in silico-in vitro-in vivo" framework for identifying, selecting, and applying soluble ligands to control donor cell function after transplantation. [ABSTRACT FROM AUTHOR]

The human eye plays a critical role in vision perception, but various retinal degenerative diseases such as retinitis pigmentosa (RP), glaucoma, and age-related macular degeneration (AMD) can lead to vision loss or blindness. Although progress has been made in understanding retinal development and in clinical research, current treatments remain inadequate for curing or reversing these degenerative conditions. Animal models have limited relevance to humans, and obtaining human eye tissue samples is challenging due to ethical and legal considerations. Consequently, researchers have turned to stem cell-based approaches, specifically induced pluripotent stem cells (iPSCs), to generate distinct retinal cell populations and develop cell replacement therapies. iPSCs offer a novel platform for studying the key stages of human retinogenesis and disease-specific mechanisms. Stem cell technology has facilitated the production of diverse retinal cell types, including retinal ganglion cells (RGCs) and photoreceptors, and the development of retinal organoids has emerged as a valuable in vitro tool for investigating retinal neuron differentiation and modeling retinal diseases. This review focuses on the protocols, culture conditions, and techniques employed in differentiating retinal neurons from iPSCs. Furthermore, it emphasizes the significance of molecular and functional validation of the differentiated cells. [ABSTRACT FROM AUTHOR]

Prenatal alcohol exposure‐induced fetal alcohol syndrome (FAS) can lead to serious maldevelopment in many organ systems, including the eyes. In the present study, the effects of alcohol exposure on early development of the human retina and the therapeutic effects of resveratrol on alcohol‐induced neural retinal damage were observed for the first time in an in vitro retinal organoid model. We report that the number of proliferating and apoptotic cells decreased and increased, respectively, following ethanol treatment. In addition, the number of PAX6+ cells and migrating TUJ1+ cells decreased after ethanol exposure. However, pretreatment with resveratrol prevented all of these negative effects. Using RNA sequencing and immunofluorescence, we identified activation of the PI3K‐AKT signalling pathway as the possible mechanism through which resveratrol protects the retina from alcohol‐induced damage. These results suggest that while ethanol exposure can restrict the growth of the human retina and impede the development of specific retinal cells, pretreatment with resveratrol may be a feasible method for preventing these effects.

Stargardt disease type 1 (STGD1) is the most common hereditary form of maculopathy and remains untreatable. STGD1 is caused by biallelic variants in the ABCA4 gene, which encodes the ATP-binding cassette (type 4) protein (ABCA4) that clears toxic byproducts of the visual cycle. The c.5461-10T>C p.[Thr1821Aspfs∗6,Thr1821Valfs∗13] variant is the most common severe disease-associated variant, and leads to exon skipping and out-of-frame ABCA4 transcripts that prevent translation of functional ABCA4 protein. Homozygous individuals typically display early onset STGD1 and are legally blind by early adulthood. Here, we applied antisense oligonucleotides (AONs) to promote exon inclusion and restore wild-type RNA splicing of ABCA4 c.5461-10T>C. The effect of AONs was first investigated in vitro using an ABCA4 midigene model. Subsequently, the best performing AONs were administered to homozygous c.5461-10T>C 3D human retinal organoids. Isoform-specific digital polymerase chain reaction revealed a significant increase in correctly spliced transcripts after treatment with the lead AON, QR-1011, up to 53% correct transcripts at a 3 μM dose. Furthermore, western blot and immunohistochemistry analyses identified restoration of ABCA4 protein after treatment. Collectively, we identified QR-1011 as a potent splice-correcting AON and a possible therapeutic intervention for patients harboring the severe ABCA4 c.5461-10T>C variant.

In recent decades, many researchers have attempted to restore vision via transplantation of retina/retinal cells in eyes with retinal degeneration. The advent of induced pluripotent stem cells (iPSC) and retinal organoid induction technologies has boosted research on retinal regeneration therapy. Although the recognition of functional integration of graft photoreceptor cells in the host retina from 2006 has been disputed a decade later by the newly evidenced phenomenon denoted as “material transfer,” several reports support possible reconstruction of the host-graft network in the retinas of both end-stage degeneration and in progressing degeneration cases. Based on proof of concept (POC) studies in animal models, a clinical study was conducted in Kobe, Japan in 2020 and showed the feasibility of cell-based therapy using iPSC retinal organoid technology. Although the graft potency of human embryonic stem (ES)/iPS cell-derived retinal organoid/retinal cells has been suggested by previous studies, much is still unknown regarding host capability, that is, how long-standing human degenerating retinas are capable of rewiring with transplanted cells. This review summarizes past POC studies on photoreceptor replacement therapy and introduces some new challenges to maximize the possible efficacy in future human clinical studies of regenerative therapy.

Summary: Characterizing cell identity in complex tissues such as the human retina is essential for studying its development and disease. While retinal organoids derived from pluripotent stem cells have been widely used to model development and disease of the human retina, there is a lack of studies that have systematically evaluated the molecular and cellular fidelity of the organoids derived from various culture protocols in recapitulating their in vivo counterpart. To this end, we performed an extensive meta-atlas characterization of cellular identities of the human eye, covering a wide range of developmental stages. The resulting map uncovered previously unknown biomarkers of major retinal cell types and those associated with cell-type-specific maturation. Using our retinal-cell-identity map from the fetal and adult tissues, we systematically assessed the fidelity of the retinal organoids in mimicking the human eye, enabling us to comprehensively benchmark the current protocols for retinal organoid generation.

The demand for induced pluripotent stem cells (iPSC)‐derived retinal organoid and retinal pigment epithelium (RPE) models for the modelling of inherited retinopathies has increased significantly in the last decade. These models are comparable with foetal retinas up until the later stages of retinogenesis, expressing all of the key neuronal markers necessary for retinal function. These models have proven to be invaluable in the understanding of retinogenesis, particular in the context of patient‐specific diseases. Inherited retinopathies are infamously described as clinically and phenotypically heterogeneous, such that developing gene/mutation‐specific animal models in each instance of retinal disease is not financially or ethically feasible. Further to this, many animal models are insufficient in the study of disease pathogenesis due to anatomical differences and failure to recapitulate human disease phenotypes. In contrast, iPSC‐derived retinal models provide a high throughput platform which is physiologically relevant for studying human health and disease. They also serve as a platform for drug screening, gene therapy approaches and in vitro toxicology of novel therapeutics in pre‐clinical studies. One unique characteristic of stem cell‐derived retinal models is the ability to mimic in vivo retinogenesis, providing unparalleled insights into the effects of pathogenic mutations in cells of the developing retina, in a highly accessible way. This review aims to give the reader an overview of iPSC‐derived retinal organoids and/or RPE in the context of disease modelling of several inherited retinopathies including Retinitis Pigmentosa, Stargardt disease and Retinoblastoma. We describe the ability of each model to recapitulate in vivo disease phenotypes, validate previous findings from animal models and identify novel pathomechanisms that underpin individual IRDs. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that exosomes are involved in retinal cell degeneration, including their insufficient release; hence, they have become important indicators of retinopathies. The exosomal microRNA (miRNA), in particular, play important roles in regulating ocular and retinal cell functions, including photoreceptor maturation, maintenance, and visual function. Here, we generated retinal organoids (ROs) from human induced pluripotent stem cells that differentiated in a conditioned medium for 60 days, after which exosomes were extracted from ROs (Exo-ROs). Subsequently, we intravitreally injected the Exo-RO solution into the eyes of the Royal College of Surgeons (RCS) rats. Intravitreal Exo-RO administration reduced photoreceptor apoptosis, prevented outer nuclear layer thinning, and preserved visual function in RCS rats. RNA sequencing and miRNA profiling showed that exosomal miRNAs are mainly involved in the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, the expression of MAPK-related genes and proteins was significantly decreased in the Exo-RO-treated group. These results suggest that Exo-ROs may be a potentially novel strategy for delaying retinal degeneration by targeting the MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

In our daily life, we are exposed to numerous industrial chemicals that may be harmful to the retina, which is a delicate and sensitive part of our eyes. This could lead to irreversible changes and cause retinal diseases or blindness. Current retinal environmental health studies primarily utilize animal models, isolated mammalian retinas, animal- or human-derived retinal cells, and retinal organoids, to address both pre- and postnatal exposure. However, as there is limited toxicological information available for specific populations, human induced pluripotent stem cell (hiPSC)-induced models could be effective tools to supplement such data. In order to obtain more comprehensive and reliable toxicological information, we need more appropriate models, novel evaluation methods, and computational technologies to develop portable equipment. This review mainly focused on current toxicology models with particular emphasis on retinal organoids, and it looks forward to future models, analytical methods, and equipment that can efficiently and accurately evaluate retinal toxicity. Graphical Abstract [ABSTRACT FROM AUTHOR]

Stem cell therapies can potentially treat various retinal degenerative diseases, including age-related macular degeneration (AMD) and inherited retinal diseases like retinitis pigmentosa. For these diseases, transplanted cells may include stem cell-derived retinal pigmented epithelial (RPE) cells, photoreceptors, or a combination of both. Although stem cell-derived RPE cells have progressed to human clinical trials, therapies using photoreceptors and other retinal cell types are lagging. In this review, we discuss the potential use of human pluripotent stem cell (hPSC)-derived photoreceptors for the treatment of retinal degeneration and highlight the progress and challenges for their efficient production and clinical application in regenerative medicine.

Retinal organoid technology enables generation of an inexhaustible supply of three-dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid-derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage-reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation-competent) hPSC-derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid-derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.

Abstract Background Human-induced pluripotent stem cell-derived retinal organoids are a valuable tool for disease modelling and therapeutic development. Many efforts have been made over the last decade to optimise protocols for the generation of organoids that correctly mimic the human retina. Most protocols use common media supplements; however, protocol-dependent variability impacts data interpretation. To date, the lack of a systematic comparison of a given protocol with or without supplements makes it difficult to determine how they influence the differentiation process and morphology of the retinal organoids. Methods A 2D-3D differentiation method was used to generate retinal organoids, which were cultured with or without the most commonly used media supplements, notably retinoic acid. Gene expression was assayed using qPCR analysis, protein expression using immunofluorescence studies, ultrastructure using electron microscopy and 3D morphology using confocal and biphoton microscopy of whole organoids. Results Retinoic acid delayed the initial stages of differentiation by modulating photoreceptor gene expression. At later stages, the presence of retinoic acid led to the generation of mature retinal organoids with a well-structured stratified photoreceptor layer containing a predominant rod population. By contrast, the absence of retinoic acid led to cone-rich organoids with a less organised and non-stratified photoreceptor layer. Conclusions This study proves the importance of supplemented media for culturing retinal organoids. More importantly, we demonstrate for the first time that the role of retinoic acid goes beyond inducing a rod cell fate to enhancing the organisation of the photoreceptor layer of the mature organoid.

Rare DRAM2 coding variants cause retinal dystrophy with early macular involvement via unknown mechanisms. We found that DRAM2 is ubiquitously expressed in the human eye and expression changes were observed in eyes with more common maculopathy such as Age-related Macular Degeneration (AMD). To gain insights into pathogenicity of DRAM2-related retinopathy, we used a combination of in vitro and in vivo models. We found that DRAM2 loss in human pluripotent stem cell (hPSC)-derived retinal organoids caused the presence of additional mesenchymal cells. Interestingly, Dram2 loss in mice also caused increased proliferation of cells from the choroid in vitro and exacerbated choroidal neovascular lesions in vivo. Furthermore, we observed that DRAM2 loss in human retinal pigment epithelial (RPE) cells resulted in increased susceptibility to stress-induced cell death in vitro and that Dram2 loss in mice caused age-related photoreceptor degeneration. This highlights the complexity of DRAM2 function, as its loss in choroidal cells provided a proliferative advantage, whereas its loss in post-mitotic cells, such as photoreceptor and RPE cells, increased degeneration susceptibility. Different models such as human pluripotent stem cell-derived systems and mice can be leveraged to study and model human retinal dystrophies; however, cell type and species-specific expression must be taken into account when selecting relevant systems.

Prenatal alcohol exposure‐induced fetal alcohol syndrome (FAS) can lead to serious maldevelopment in many organ systems, including the eyes. In the present study, the effects of alcohol exposure on early development of the human retina and the therapeutic effects of resveratrol on alcohol‐induced neural retinal damage were observed for the first time in an in vitro retinal organoid model. We report that the number of proliferating and apoptotic cells decreased and increased, respectively, following ethanol treatment. In addition, the number of PAX6+ cells and migrating TUJ1+ cells decreased after ethanol exposure. However, pretreatment with resveratrol prevented all of these negative effects. Using RNA sequencing and immunofluorescence, we identified activation of the PI3K‐AKT signalling pathway as the possible mechanism through which resveratrol protects the retina from alcohol‐induced damage. These results suggest that while ethanol exposure can restrict the growth of the human retina and impede the development of specific retinal cells, pretreatment with resveratrol may be a feasible method for preventing these effects. [ABSTRACT FROM AUTHOR]

Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co‐culture system containing retinal organoids and microglia‐like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co‐cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co‐culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina. [ABSTRACT FROM AUTHOR]

Retinal organoids (ROs) are three-dimensional retinal tissues, which are differentiated in vitro from induced pluripotent stem cells (iPSC), ultimately forming all main retinal cell types under defined culture conditions. ROs show several highly specialized retinal features, including the outgrowth of photoreceptor outer segments (OSs). In vivo, the photoreceptor OSs are enveloped and maintained by protrusions of retinal pigment epithelium (RPE) cells, the so-called apical microvilli, while ROs fail to recapitulate this critical interaction in culture development. Here, we define specific co-culture conditions aiming to compensate for the missing physical proximity of RPE and OSs in RO development. Accordingly, functional RPE cells and ROs were differentiated simultaneously from the same iPSC clone, the former resulting in byproduct RPE or bRPE cells. While some co-culture approaches indicated a temporary functional interaction between bRPE and RO photoreceptors, they did not improve the photoreceptor histoarchitecture. In contrast, embedding ROs in a basement membrane extract without bRPE cells showed a robust improvement in the rate of photoreceptor OS retention. RO embedding is a quick and easy method that greatly enhances the preservation of photoreceptor OSs, an important structure for modelling retinal diseases with the involvement of photoreceptors. [ABSTRACT FROM AUTHOR]

Aniridia is a pan-ocular genetic developmental eye disorder characterized by complete or partial iris and foveal hypoplasia, for which there is no treatment currently. Progressive sight loss can arise from cataracts, glaucoma, and aniridia-related keratopathy, which can be managed conservatively or through surgical intervention. The vast majority of patients harbor heterozygous mutations involving the PAX6 gene, which is considered the master transcription factor of early eye development. Over the past decades, several disease models have been investigated to gain a better understanding of the molecular pathophysiology, including several mouse and zebrafish strains and, more recently, human-induced pluripotent stem cells (hiPSCs) derived from aniridia patients. The latter provides a more faithful cellular system to study early human eye development. This review outlines the main aniridia-related animal and cellular models used to study aniridia and highlights the key discoveries that are bringing us closer to a therapy for patients. [ABSTRACT FROM AUTHOR]

In two unrelated families with X-linked inherited retinal dystrophy, identification of the causative variants was elusive. Interrogation of the next-generation sequencing (NGS) data revealed a "dark" intergenic region on Xq27.1 with poor coverage. Long-range PCR and DNA walking across this region revealed different inter-chromosomal insertions into the human-specific palindrome on Xq27.1: a 58 kb insertion of 9p24.3 [der(X)dir ins(X;9)(q27.1;p24.3)] in family 1 and a 169 kb insertion of 3p14.2 [der(X)inv ins(X;3)(q27.1;p14.2)] in family 2. To explore the functional consequence of these structural variants in genomic and cellular contexts, induced pluripotent stem cells were derived from affected and control fibroblasts and differentiated to retinal organoids (ROs) and retinal pigment epithelium. Transcriptional dysregulation was evaluated using RNA sequencing (RNA-seq) and RT-qPCR. A downstream long non-coding RNA, LINC00632 (Xq27.1), was upregulated in ROs from both families compared to control samples. In contrast, the circular RNA CDR1as/ciRS-7 (circular RNA sponge for miR-7), spliced from linear LINC00632, was downregulated. To investigate this tissue-specific dysregulation, we interrogated the landscape of the locus using Hi-C and cleavage under targets and tagmentation sequencing (CUT&Tag). This revealed active retinal enhancers within the insertions within a topologically associated domain that also contained the upstream promoter of LINC00632, permitting ectopic contact. Furthermore, CDR1as/ciRS-7 acts as a "sponge" for miR-7, and target genes of miR-7 were also dysregulated in ROs derived from both families. We describe a new genomic mechanism for retinal dystrophy, and our data support a convergent tissue-specific mechanism of altered regulation of LINC00632 and CDR1as/ciRS-7 as a consequence of the insertions within the palindrome on Xq27.1., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Human induced pluripotent stem cell (hiPSC)-based disease modeling can be successfully recapitulated to mimic disease characteristics across various human pathologies. Glaucoma, a progressive optic neuropathy, primarily affects the retinal ganglion cells (RGCs). While multiple groups have successfully generated RGCs from non-diseased hiPSCs, producing RGCs from glaucomatous human samples holds significant promise for understanding disease pathology by revealing patient-specific disease signatures. Given that keratocytes originate from the neural crest and previous reports suggest that ocular fibroblasts from glaucomatous donors carry pathogenic signatures, it is highly plausible that these signatures imprinted within the keratocytes will also be present in the derived RGCs. Thus, we aimed to generate RGCs from both glaucomatous and non-glaucomatous donor keratocytes and validate disease-specific signatures in 3D retinal organoids and in isolated RGCs. Our protocol describes the generation of iPSCs from keratocytes of both glaucomatous and non-glaucomatous donors, followed by their differentiation into retinal organoids. Subsequent isolation and culturing of RGCs were performed. Disease signatures in the RGCs were validated in both 3D retinal organoids (ROs) and 2D RGC cultures, and glaucomatous RGCs in 3D and 2D cultures demonstrated increased cleaved CASP3 and significant RGC loss, indicating disease imprints in the hiPSC-derived RGCs. This model offers a venue and high throughput platform for studying glaucomatous disease pathology and holds significant potential for drug discovery using RGCs derived from human donors. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing of keratocytes from human cadaveric donors Basic Protocol 2: Reprogramming donor keratocytes into iPSCs Basic Protocol 3: Evaluation of chromosomal loss during reprogramming in iPSCs by karyotyping Basic Protocol 4: Generation of 3D ROs Basic Protocol 5: Dissociation and culturing of RGCs from 3D ROs Support Protocol 1: Immunostaining for phenotypic characterization of cells Support Protocol 2: Sectioning of 3D ROs and immunostaining Support Protocol 3: Western blotting for cleaved CASP3 and THY1., (© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Abstract Retinitis Pigmentosa (RP) is a common form of inherited degenerative disease that often leads to blindness. About 10% autosomal dominant RP cases have been associated with mutations in PRPF31 gene, which is involved in pre‐mRNA splicing. This commentary summarises the key findings of our recent publication ‘Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient‐induced pluripotent stem cell‐derived retinal pigment epithelium cells’ in the context of large cytoplasmic aggregates which accumulate progressive with time and impair cell function and survival. Understanding the pathomechanism of PRPF31‐RP provides invaluable information that can be used to understand other PRPF‐RPs, and help to design effective and appropriate therapeutic strategies for the treatment of RP patients with PRPF31 mutations.

Retinal organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that mimic the retina's spatial and temporal differentiation, making them useful as in vitro retinal development models. Retinal organoids can be assembled with brain organoids, the 3D self-assembled aggregates derived from hPSCs containing different cell types and cytoarchitectures that resemble the human embryonic brain. Recent studies have shown the development of optic cups in brain organoids. The cellular components of a developing optic vesicle-containing organoids include primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. The importance of retinal organoids in ocular diseases such as age-related macular degeneration, Stargardt disease, retinitis pigmentosa, and diabetic retinopathy are described in this review. This review highlights current developments in retinal organoid techniques, and their applications in ocular conditions such as disease modeling, gene therapy, drug screening and development. In addition, recent advancements in utilizing extracellular vesicles secreted by retinal organoids for ocular disease treatments are summarized. [ABSTRACT FROM AUTHOR]

Background: Human-induced pluripotent stem cell-derived retinal organoids are a valuable tool for disease modelling and therapeutic development. Many efforts have been made over the last decade to optimise protocols for the generation of organoids that correctly mimic the human retina. Most protocols use common media supplements; however, protocol-dependent variability impacts data interpretation. To date, the lack of a systematic comparison of a given protocol with or without supplements makes it difficult to determine how they influence the differentiation process and morphology of the retinal organoids. Methods: A 2D-3D differentiation method was used to generate retinal organoids, which were cultured with or without the most commonly used media supplements, notably retinoic acid. Gene expression was assayed using qPCR analysis, protein expression using immunofluorescence studies, ultrastructure using electron microscopy and 3D morphology using confocal and biphoton microscopy of whole organoids. Results: Retinoic acid delayed the initial stages of differentiation by modulating photoreceptor gene expression. At later stages, the presence of retinoic acid led to the generation of mature retinal organoids with a well-structured stratified photoreceptor layer containing a predominant rod population. By contrast, the absence of retinoic acid led to cone-rich organoids with a less organised and non-stratified photoreceptor layer. Conclusions: This study proves the importance of supplemented media for culturing retinal organoids. More importantly, we demonstrate for the first time that the role of retinoic acid goes beyond inducing a rod cell fate to enhancing the organisation of the photoreceptor layer of the mature organoid. [ABSTRACT FROM AUTHOR]

Cellular 3D structures, for example, organoids, are an excellent model for studying and developing treatments for various diseases, including hereditary ones. Therefore, they are increasingly being used in biomedical research. From the point of view of safety and efficacy, recombinant adeno‐associated viral (rAAV) vectors are currently most in demand for the delivery of various transgenes for gene replacement therapy or other applications. The delivery of transgenes using rAAV vectors to various types of organoids is an urgent task, however, it is associated with a number of problems that are discussed in this review. Cellular heterogeneity and specifics of cultivation of 3D structures determine the complexity of rAAV delivery and are sometimes associated with low transduction efficiency. This review surveys the main ways to solve emerging problems and increase the efficiency of transgene delivery using rAAVs to organoids. A clear understanding of the stage of development of the organoid, its cellular composition and the presence of surface receptors will allow obtaining high levels of organoid transduction with existing rAAV vectors. [ABSTRACT FROM AUTHOR]