Retinal Organoids from Induced Pluripotent Stem Cells of Patients with Inherited Retinal Diseases: A Systematic Review

Retinitis pigmentosa (RP) comprises a genetically heterogeneous group of retinopathies characterized by the loss of photoreceptors and the presence of retinal pigment deposits, ultimately leading to blindness [108]. As the most common IRD, RP affects approximately 1 in 4000 individuals worldwide [56, 109]. Mutations in approximately 70 genes, including RP2, RPGR, RHO, USH2A, CRB1, and PRPF31, are known to cause RP [110].

Studies have successfully generated ROs from patient-derived iPSCs for 14 RP-associated genes, demonstrating the potential of this approach for disease modeling. These ROs generally exhibited proper lamination and contained identifiable photoreceptors and other retina-specific structures, while also manifesting structural abnormalities corresponding to individual genetic predispositions.

USH2A mutations, one of the most common causes of autosomal recessive RP, have been modeled using patient-derived ROs [111]. These models revealed early developmental abnormalities, including delayed self-organization, disrupted arrangement of retinal cells, abnormal retinal neuroepithelium differentiation, and defects in photoreceptor differentiation and cilium formation [48, 49]. These findings provide insights into the early pathogenesis of USH2A-associated RP.

ROs modeling RPGR mutations, primarily responsible for X-linked RP, demonstrated shortened cilia and defective photoreceptor development [39, 40]. RPGR-ROs exhibited increased expression of glial fibrillary acidic protein (GFAP), indicative of reactive gliosis, a phenomenon commonly observed in various forms of retinal degeneration [39, 42].

Patient-specific ROs with RHO mutations, a major cause of autosomal dominant RP, showed disrupted photoreceptor outer segment development and increased endoplasmic reticulum stress-induced apoptosis in rod cells, and gradual loss of rod photoreceptors [45, 46]. A recent study using ROs derived from patients with RHO copy number variations (CNVs) demonstrated photoreceptor dysgenesis, increased RHO mRNA expression, and elevated levels and mislocalization of rhodopsin protein within rod photoreceptors over an extended culture period [47].

Studies on PRPF31 mutations using ROs and RPE cells revealed disrupted alternative splicing in genes crucial for splicing and ciliogenesis [34, 36]. PRPF31-mutant ROs showed splicing defects early in development, leading to subsequent photoreceptor degeneration.

ROs derived from patients with CRB1 compound heterozygous mutations have been used to validate new iPSC lines [26, 28, 30]. A recent study on patient-derived CRB1-ROs demonstrated that a specific variant caused exon 11 skipping, thus providing evidence for the underlying pathogenic mechanism [32].

ROs with NR2E3 mutations exhibited atypical rod photoreceptor development, characterized by a novel cell population expressing both rod and cone-specific genes, highlighting the gene’s critical function in photoreceptor fate determination and phototransduction pathway regulation [82]. Patient-derived RP2-ROs exhibited progressive rod photoreceptor degeneration, with peak cell death at day 150 and subsequent outer nuclear layer thinning by day 180, mirroring the clinical progression of X-linked RP [57]. TBC1D32 and DYNC2H1 variants both disrupted retinal ciliogenesis [51, 58]. IMPG2-mutant ROs showed photoreceptor outer segment abnormalities [52], while MYO7A-deficient ROs, associated with Usher syndrome type 1B, did not exhibit overt photoreceptor death [53]. PDE6B-mutant ROs recapitulated features of late-onset RP, including progressive photoreceptor degeneration [55]. TRNT1-associated RP ROs showed deficits in autophagy and inefficient expression of full-length TRNT1 protein [59].

Chronological events during defective retinogenesis have been observed in several models. For instance, USH2A-ROs showed early developmental abnormalities in the formation of the retinal neuroepithelium, preceding photoreceptor-specific defects [49]. In PRPF31-ROs, splicing defects were observed early in development, leading to subsequent photoreceptor degeneration [34].

These RO models have also been used to investigate obscure disease mechanisms. For example, Hi-C analysis of RP17-ROs demonstrated that structural variants can generate new topologically associating domains, leading to aberrant interactions between retinal enhancers and GDPD1, thus elucidating a potential gain-of-function mechanism in autosomal dominant RP [56]. Studies on PRPF31-associated RP using ROs have provided insights into the non-penetrance mechanism through dominant mutation carrier-derived ROs [35].

To validate their efficacy in representing the clinical phenotypes of RP as in vitro prototypes, these diverse RO models have been subjected to various experimental techniques. These include transcriptome analysis, immunofluorescence, western blotting, and morphological assessment, collectively providing a comprehensive approach for investigating the molecular and cellular basis of RP pathogenesis.

Leber congenital amaurosis (LCA) constitutes a family of congenital retinal dystrophies with serious clinical manifestations that cause vision loss during infancy [112]. It is the second most common IRD following RP, with a prevalence of 1 in 80,000 [113, 114]. Although not as common as RP, LCA is heterogeneous, with mutations in 26 genes known to cause LCA [110].

Five studies [60–64], comprising approximately 42% of the total studies, focused on generating ROs from iPSCs of patients with CEP290-associated LCA, the most common genetic cause of LCA. These studies successfully developed patient-specific optic cups with proper lamination, containing photoreceptors identified by specific markers. The ROs effectively recapitulated LCA pathogenesis, exhibiting defective ciliogenesis, with shorter and fewer cilia in photoreceptors compared to control ROs [60]. This finding was corroborated by observations of distinct cilia dysfunctions in CEP290-mutant ROs, including reduced cilia length and altered protein localization within the cilia [62]. These ciliary defects were consistent with the retinal degeneration and vision loss observed in patients with LCA, providing a potential mechanistic link between the cellular phenotype and clinical manifestations. Furthermore, CEP290-LCA ROs exhibited proteostasis imbalance, providing insights into the cellular mechanisms underlying the disease [64]. Collectively, these studies highlight the utility of CEP290-LCA ROs in modeling the cellular and molecular aspects of LCA, particularly in recapitulating the ciliary defects characteristic of the disease.

AIPL1-associated LCA-ROs closely resemble healthy control ROs but exhibit key molecular alterations characteristic of AIPL1-LCA [65–67]. These include decreased expression and mislocalization of AIPL1 and its interacting protein PDE6 in photoreceptor inner segments, as well as elevated cyclic guanosine monophosphate (cGMP) levels due to the absence of PDE6. Notably, despite these changes, AIPL1-LCA ROs do not display overt photoreceptor degeneration, contrasting with the severe early-onset degeneration observed in patients. This discrepancy suggests that while AIPL1-LCA ROs accurately model certain aspects of LCA pathogenesis, they may require further development, such as prolonged culture or interaction with RPE, to fully recapitulate the degenerative phenotype. These findings highlight both the potential and limitations of ROs in modeling complex retinal diseases.

RPE65-LCA ROs, generated from urine cell-derived iPSCs, demonstrated differentiation capabilities comparable to those of healthy controls [71]. These organoids exhibited typical retinal ultrastructure, including the outer limiting membrane, and photoreceptors with rudimentary outer segments in long-term culture, suggesting the potential of mutation-bearing iPSCs to differentiate into ROs and serve as disease models. CRX-LCA ROs exhibited reduced opsin expression in photoreceptors [69], whereas NPHP5-LCA ROs demonstrated impaired ciliogenesis, abnormal outer segment structures, and decreased CEP290 protein levels [70]. ROs from patients with LCA8 harboring CRB1 mutations have been used to characterize the pathophysiology and disease phenotypes [68].

Retinoblastoma (RB) is the most common intraocular tumor occurring in children, with a global incidence of 1 in 15,000–20,000 live births [115]. A mutation in the RB1 gene, the translated protein of which is a key regulator of the cell cycle, is the sole cause of inherited retinoblastoma [116, 117]. Despite its relative genetic simplicity, the life-threatening aspects of retinoblastoma emphasize its clinical significance in IRD research.

RB-iPSC-ROs have been generated from patients and carriers of RB1 germline mutations to assess the influence of patient context on tumor formation. The ROs displayed features of RB, rendering them an appropriate disease model [73]. ROs injected orthotopically into immunocompromised mice exhibited an in vivo RB formation indistinguishable from those of patient RBs [72]. Single-cell RNA sequencing (scRNA-seq) of RB-RO cells revealed retinal progenitor cells as the most common cell type and the co-expression of normally mutually exclusive genes such as HES6 and AIPL1 [72]. The results of sequencing could not distinguish RB-ROs from RBs of patients and orthotopic patient-derived xenografts in terms of cell type. In addition, a second hit mutation in patient iPSCs with a heterozygous mutation led to RB tumorigenesis in ROs, verifying Knudson’s “two-hit” hypothesis [74]. These outcomes imply that tumor tissues can be acquired without enucleation from multiple patients, and that RB can be derived from RB1 mutation carriers who have never developed RB. In addition, ROs derived from iPSCs with heterozygous RB1 mutations exhibited elevated levels of ATP and pyruvate, indicating significant alterations in energy metabolism without overt morphological changes during early retinal development [75]. These findings suggest that metabolic dysregulation may precede the onset of RB, highlighting the potential role of energy metabolism in the early stages of disease progression.

Several studies have reported the manipulation of healthy iPSCs and embryonic stem cells (ESCs) to retain RB1 deletions using CRISPR/Cas9-mediated genome editing. Research on iPSC-RB-ROs contributed to the discovery that RB1 deletion does not affect the maturation and proliferation of iPSCs, and that cell survival is unaffected despite the known function of RB1 in inhibiting apoptosis [118]. Dysregulation of oncogenic pathways has been revealed via disease modeling using human ESC (hESC)-derived genetically engineered ROs [119, 120]. ROs have served as valuable models for studying RB, providing insights into the disease mechanisms [121, 122].

X-linked juvenile retinoschisis (XLRS) is an IRD characterized by schisis (splitting) of the retina, primarily affecting males due to mutations in the RS1 gene [76–78]. The RS1 gene encodes retinoschisin, a protein crucial for maintaining retinal structural integrity and synaptic function.

ROs derived from patients with XLRS have been successfully generated, recapitulating key features of the disease and providing valuable insights into its pathophysiology. Interestingly, macroscopic morphological features of the diseased retina were also observed in ROs, further validating their relevance as disease models. These XLRS-ROs exhibited retinal splitting and defective retinoschisin production, mirroring the clinical manifestations of XLRS [76]. Molecular analyses of XLRS-ROs revealed dysregulation of Na/K-ATPase and increased ERK signaling pathway activity, potentially contributing to the disease mechanism [77]. Transcriptomic studies of XLRS-ROs demonstrated decreased expression of retinal cell markers, particularly those associated with photoreceptors, suggesting impaired retinal development [77, 78].

RNA sequencing of XLRS-ROs uncovered downregulation of genes involved in synapsis, nervous system development, and rhodopsin-like receptors, indicating delayed photoreceptor development [78]. Interestingly, co-culture experiments with control ROs during early differentiation stages showed partial recovery of the XLRS phenotype, highlighting the potential for cell-based therapeutic approaches [77].

ROs are effective disease models for recapitulating retinal degeneration in various IRDs. Achromatopsia, an IRD caused by ATF6 mutations, has been modeled using patient-derived ROs. These models failed to form regular cone photoreceptors, highlighting ATF6’s crucial role in human cone development [79]. A subsequent study investigated mitochondrial and endoplasmic reticulum function in achromatopsia-ROs, revealing dysfunctions that may contribute to the cellular pathology of this condition [80].

In Stargardt disease, a relatively common IRD with a prevalence of 1 in 8,000–10,000 [123], ROs derived from patient iPSCs with intronic ABCA4 variants demonstrated splicing defects leading to retinal degeneration [84]. A study on ABCA4-associated retinal disease utilized ROs to investigate the c.4539+1321A>G variant, revealing its potential pathogenicity through mis-splicing in photoreceptor cells, resulting in a truncated protein. This variant was found to be potentially disease-causing when paired with a deleterious allele on the opposing chromosome [85].

ROs have also been valuable in studying less common IRDs. For instance, ROs modeling CLN3-associated non-syndromic retinopathy revealed novel transcripts inducing faulty CLN3 synthesis, toxic substance accumulation, and morphological defects in photoreceptor progenitor cells [87]. In a study of choroideremia using ROs, no significant differences were observed during early developmental stages. However, these ROs served as a valuable platform for validating isogenic iPSC lines, providing a foundation for future disease modeling studies [92].

Enhanced S-cone syndrome, caused by NR2E3 or NRL mutations, has been effectively modeled using ROs [81–83]. scRNA-seq of these ROs revealed a unique branch point in the rod photoreceptor developmental lineage specific to the disease state. This study demonstrated NR2E3’s critical role in proper expression of phototransduction genes and identified regulatory sites influencing photoreceptor development [82].

In DRAM2-associated cone-rod dystrophy, ROs exhibited abnormalities in lipid metabolism, autophagic flux defects, and reduced lysosomal enzyme activity. These findings suggest DRAM2’s function in maintaining photoreceptor and RPE cell integrity through regulation of lysosomal function and autophagy [91]. A recent study established an iPSC line from a patient with early-onset pattern dystrophy carrying an OTX2 c.259G>A variant, exploring the developmental capabilities of these mutation-bearing iPSCs in the context of retinal differentiation [93].

ROs can also function as a platform for evaluating the pathogenicity of certain mutations. In autosomal recessive rod-cone dystrophy, ROs facilitated the validation of in silico predictions regarding an UBAP1L intronic variant. RT-PCR experiments on 150-day-old ROs demonstrated that this variant leads to mis-splicing, likely resulting in the production of an elongated protein with altered function [90]. Similarly, ROs derived from patients with autosomal recessive adult-onset rod-cone dystrophy revealed the pathogenicity of two CRB1 variants: c.1892A>G demonstrating exon skipping and c.2548G>A resulting in a missense mutation [88]. For X-linked rod-cone dystrophy, ROs have been employed to model RPGR mutations causing abnormal splicing [89]. In EYS-associated retinal dystrophy, RO studies uncovered the mislocalization of key proteins and heightened photoreceptor sensitivity to light stimuli, particularly blue light [94]. Interestingly, in the case of CLCN2-associated retinopathy, comparative analysis of patient-derived RPE and ROs indicated that RPE dysfunction, rather than photoreceptor degeneration, was the primary causative factor [86]. These diverse applications highlight the versatility of ROs in elucidating disease mechanisms and validating the pathogenicity of genetic variants across various IRDs.

Collectively, these findings underscore the ability of ROs derived from iPSCs of patients to facilitate a range of experiments using patient-derived tissues. This feature establishes a foundation for personalized medicine, in which the unique context of each patient is considered in the development of novel therapies. To maximize the utility of these models, researchers have increasingly employed advanced analytical techniques to delve deeper into the molecular and cellular dynamics within ROs. One such powerful tool is scRNA-seq, which allows for the high-resolution analysis of individual cellular profiles by capturing mRNA transcripts that reflect functional states [124]. This technology provides invaluable insights into the cellular heterogeneity of ROs by monitoring changes at a single-cell resolution, enabling the detection of subtle pathological changes and the evaluation of therapeutic interventions with unprecedented detail [53, 125]. By capturing the transcriptomes of individual cells at different stages of development, this technique provides a detailed view of cellular composition over time. Analyzing the transcripts of individual cells at the spatial and temporal levels provides insights into differentiation trajectories and allows for the identification of key signaling cues [126, 127]. Furthermore, scRNA-seq contributes to the enhancement of RO production protocols by offering novel perspectives on signaling pathways and cell type-specific markers [127].

Genome editing technologies, particularly the CRISPR/Cas9 system, offer the potential to directly modify pathogenic genes at the DNA level. This approach can effectively halt the production of mutant proteins in the edited cells by altering the underlying genetic sequence responsible for the disease phenotype. Genome-editing approaches using CRISPR/Cas9 have rescued the disease phenotype in iPSCs from patients with RP, XLRS, LCA4, LCA5, enhanced S-cone syndrome, or nonsyndromic retinopathy harboring CLN3 mutations, by revealing differentiation into ROs [34, 38–40, 48, 54, 66, 76, 87, 128, 129]. In addition, an RPE-specific CRISPR/Cas9 system was directly applied to human ROs, demonstrating its potential for targeted cell therapy [130]. Furthermore, Cas9-directed base editing was tested on iPSCs derived from patients with XLRS, which confirmed the high repair efficiency of the base editors [76].

Alternatively, protein deficiencies resulting from genetic mutations can be addressed through the introduction of wild-type DNA sequences via adeno-associated virus (AAV) vectors into ROs. This approach, known as gene augmentation therapy, aims to restore normal protein expression and function in affected cells. RP phenotypes have been rescued by AAV-mediated RPGR and CRB1 gene augmentation, respectively [42, 125]. In AIPL1-associated LCA4 ROs, AAV-mediated gene augmentation therapy effectively rescued molecular features, including the restoration of retinal PDE6 and reduction of elevated cGMP levels, with effects persisting for at least 70 days [67]. Furthermore, LCA-ROs ameliorated disease phenotypes via AAV-mediated CRX and IQCB1/NPHP5 gene augmentation, respectively [69, 70]. ROs from RP2 knockout iPSCs have been transduced with AAV2/5, which improved photoreceptor survival, thickened the outer nuclear layer, and restored rhodopsin expression, eventually reversing the RP phenotype [57]. AAV-driven PRPF31 gene therapy could rescue photoreceptor cells in patient-derived ROs [38]. In XLRS-ROs, AAV2-mediated RS1 gene augmentation partially restored delayed photoreceptor development and improved rod photoreceptor morphology [78]. For EYS-associated retinal dystrophy, a high-capacity helper-dependent adenoviral vector (HDAdV) was employed to deliver the full-length EYS gene, successfully rescuing protein mislocalization in organoids [94]. Notably, a study on enhanced S-cone syndrome ROs demonstrated that the timing of gene therapy is crucial, as most divergent rods became refractory to NR2E3 supplementation by 130 days of differentiation, highlighting the importance of early intervention [82].

Optogenetics is an approach that introduces light-sensitive opsins, such as channelrhodopsins, into the eye so that the remaining retinal neurons can bear photosensitive properties. The technique can be especially applied for advanced-stage retinal degeneration in cases where other gene therapies are unavailable. Unlike genome editing or gene augmentation, which can only be utilized in patients with known genetic mutations, optogenetics is applicable regardless of the primary genetic defect. This is meaningful in IRDs, where genetic causes have been identified in only approximately 75% of cases [131]. To date, no studies have used patient-derived ROs to screen for optogenetic therapy, while normal human ROs have been used as screening tools to evaluate the membrane trafficking efficacy of microbial opsins [132]. Optogenetics in patient-derived ROs should be explored to evaluate the induction of opsins and the effectiveness of the therapy for further clinical applications, especially because transduction efficacy and photic responses vary depending on the condition of the cells. Eleven active/recruiting/completed clinical trials on the application of optogenetics in IRDs have been identified using the keyword “optogenetics” in ClinicalTrials.gov, with one showing encouraging results [133]. In this study, a patient with RP who underwent optogenetic therapy demonstrated partial visual recovery when using specialized light-transducing goggles. The patient exhibited improved ability to perceive, localize, count, and interact with various objects, representing a significant advancement in the field of optogenetic interventions for visual restoration.

Antisense oligonucleotides (ASOs) target pre-mRNAs or mRNAs to regulate their splicing and protein expression [134]. ROs derived from iPSCs of patients have been tested for several IRDs with ASO therapy. ROs derived from patients with Stargardt disease have been targeted for preclinical research on ASO-based medications to alleviate splicing defects in the ABCA4 gene [84, 135]. Additionally, ROs derived from patients with CEP290-mutant LCA were treated with antisense morpholino (CEP290-MO) and ASO (QR-110) to reduce aberrant splicing and restore ciliogenesis [60, 61]. This treatment led to clinical trials with QR-110, in which patients showed vision improvements at three months, suggesting the potential of ROs as adequate pre-clinical research tools [136–138].

Nonsense suppression therapy using translational readthrough-inducing drugs (TRIDs) can also induce the production of full-length proteins by enhancing the insertion of cognate/near-cognate tRNAs. PTC124 (ataluren), the only TRID approved for clinical application, partially rescued nonsense mutations in AIPL1-LCA ROs [66]. Although their therapeutic effects are insufficient to reverse the disease phenotype, ROs have shown potential as testing platforms for novel drugs.

Small-molecule drugs are reliable options for the treatment of IRDs. ROs derived from patients with achromatopsia were tested using a small-molecule ATF6 agonist (AA147). This small molecule reversed the transcriptional activity of the ATF6 variant, encouraging cone cell growth in ROs, suggesting a potential pharmacological approach [79]. In addition, RB organoids have been treated with chemotherapeutic agents (melphalan and topotecan), SYK inhibitors, and a small-molecule inhibitor of Bcl-2, resulting in decreased cell proliferation [73, 119]. ROs derived from patients with CEP290 LCA10 demonstrated improved cilia formation and restored photoreceptor survival after treatment with eupatilin and resperine, respectively [63, 64]. A recent study demonstrated that Photoregulin3, a small molecule targeting NR2E3, partially rescued rhodopsin protein localization in ROs derived from patients with RHO-CNV-associated RP, suggesting a potential therapeutic approach for modifying disease progression [47].

Thus, patient-specific ROs have the potential to screen novel therapies or test existing drug combinations in individual patients. However, research on delivery modalities for these treatments is essential because therapeutic outcomes depend strongly on delivery efficacy. Notably, AAV is a promising vector that can be used to convey genome editing tools, such as CRISPR/Cas9 and base editors; optogenes; or normal genes that require augmentation. In this context, ROs have been utilized to assess various properties of AAV vectors, including their tropism, transduction efficiency, and safety profiles [139–141]. Notably, AAV2 and AAV8, serotypes commonly employed in clinical trials for IRDs, have been specifically evaluated using RO models [142, 143]. Furthermore, AAV5 exhibited greater transduction efficiency in iPSC-derived ROs than AAV9 [27]. However, the limited packaging capacity of AAV vectors (typically less than 5 kb) poses challenges for delivering larger genes. To address this limitation, mRNA trans-splicing dual AAV vectors have been applied to ROs for evaluating the delivery of large genes, including CRISPR/Cas9 and base or prime editors [144]. For genes exceeding AAV capacity, HDAdVs have been employed, as demonstrated in a study using HDAdVs to deliver the 9.4 kb full-length EYS gene to ROs, utilizing their 36 kb packaging capacity for long-term transgene expression [94]. Additionally, lipoplexes that can co-encapsulate different mRNAs have been verified using mouse ROs [145].

Photoreceptor cells, that is, rod and cone cells, are frequently disrupted in IRDs such as RP, LCA, and Stargardt disease. Recent advancements in photoreceptor transplantation have shown promising results in preclinical studies.

Photoreceptor cell sheets derived from hESC-ROs have been successfully engrafted onto Royal College of Surgeons (RCS) rats, demonstrating functional integration with host retinal cells [154]. Furthermore, single-cell suspensions of human photoreceptor cells obtained from ROs have been transplanted into murine models of retinal degeneration, forming synapses with host cells and eliciting light responses [155–157]. Cell purification from ROs has been achieved using cell-surface marker panels or photoreceptor cell reporter human iPSC (hiPSC) lines [158, 159].

Several studies have focused on enriching specific photoreceptor subtypes, particularly cone cells, by enhancing cone signals using cone-specific markers and subsequently isolating these cells using FACS [160, 161]. Similarly, cone photoreceptors from hiPSC-ROs have been manipulated to express microbial opsins and fluorescent reporter genes, facilitating subsequent isolation [162]. Upon transplantation into rd1 mice, these cone cells exhibited heightened sensitivity to light, potentially improving temporal resolution. Efforts to increase photoreceptor yields have employed various methods, including the utilization of bioreactors, ultimately yielding an ample supply of mature photoreceptors [178].

Subretinal transplantation of photoreceptor precursor cells derived from hPSC-ROs into animal models of retinal degeneration has shown success [147, 165]. A notable study demonstrated that hESC-derived RO sheets, even after overnight shipping, could successfully integrate into host retinas of RD nude rats, forming functional photoreceptors and improving vision [153]. Importantly, the response heatmap in the superior colliculus corresponded to the transplant location in the host retina, suggesting point-to-point projection. Recent investigations into photoreceptor transplantation have revealed species-specific limitations in material transfer between transplanted and host cells, emphasizing the importance of considering species compatibility in developing effective transplantation strategies [163]. A novel study using 13-lined ground squirrels as a model for cone-rich retinal regions has shown successful long-term survival of transplanted hiPSC-derived photoreceptors [164]. This research employed advanced imaging techniques, including adaptive optics scanning light ophthalmoscopy, to non-invasively monitor transplanted cells for up to four months. This model offers new opportunities for developing cone replacement therapies, addressing a gap in commonly used rodent models that lack cone-rich regions.

Watari et al. devised a quality control method for hiPSC-derived retinal sheets for subretinal transplantation [95]. The authors dissected the neuroepithelium into an inner “cap” for transplantation and an outer “ring” for qPCR analysis, effectively excluding corresponding off-target tissues. While the peripheral “ring” tissues were used for qPCR, a non-freezing preservation method was used to successfully store the retinal sheet at 17 ± 5 °C in Optisol-GS for up to 4 days. Subsequent transplantation of these sheets into rats with retinal degeneration resulted in the long-term survival of mature and functional photoreceptors.

A recent groundbreaking clinical trial (jRCTa050200027) has demonstrated the long-term safety and stability of allogeneic iPSC-derived RO sheet transplantation in patients with advanced RP [179]. In this first-in-human study involving ROs, RO sheets were successfully transplanted into two subjects, with the grafts surviving without immune-mediated rejection or tumor formation for two years. The transplantation procedure increased retinal thickness at the graft site without serious adverse events, and changes in visual function were less pronounced in the treated eye compared to the untreated eye. This study provides evidence for the potential of iPSC-derived RO transplantation as a therapeutic approach for retinal degeneration, paving the way for further investigations into its safety and efficacy for visual function restoration.

Retinal ganglion cells (RGCs) are commonly injured in glaucoma, optic neuritis, and Leber’s hereditary optic neuropathy. Considering that substantial RGC loss occurs prior to the diagnosis of blindness related to RGC degeneration, cell transplantation has emerged as a potential treatment option [180].

Recent studies have demonstrated the successful generation, purification, and transplantation of RGCs derived from hESC-ROs. These transplanted cells have shown survival and integration in the host retina for up to one-week post-transplantation, highlighting the potential of this approach for RGC replacement therapies [168, 170]. RGCs generated and purified from ROs expressed RGC markers, with those harvested at day 90 exhibiting optimal survival and neurite extension [171]. Upon transplantation into mice with optic neuropathy, these isolated RGCs have shown the capacity to survive for more than one-month post-treatment and incorporate into the host ganglion cell layer [167].

A novel technique employing subretinal injection combined with chemokine-induced migration has shown promise in enhancing RGC integration. This method resulted in a 2.7-fold increase in RGC migration into the ganglion cell layer and retinal nerve fiber layer, with migrated cells expressing mature RGC markers. This technique addresses the limitations associated with intravitreal injections, where donor neurons often fail to integrate beyond the internal limiting membrane [169].

For successful RGC replacement, axon regeneration and the establishment of appropriate synaptic connections to the brain are crucial [181]. However, these processes are challenging because axons need to stretch several centimeters to extend into the brain while simultaneously establishing specific rewiring of targets [181, 182]. Alternative approaches include supporting endogenous RGCs with Müller glial or neural progenitor cells derived from ROs [96]. Notably, Müller glial cells isolated from ROs have demonstrated the ability to enhance RGC function and visual response [177].

The RPE is commonly damaged in IRDs such as RP and Stargardt disease. While RPE cells can be generated directly from hPSCs, they can also be obtained concurrently with ROs as adjacent structures to the optic cup. Historically, direct induction methods have been favored for RPE differentiation from PSCs. However, recent advancements have facilitated the generation of functionally mature, polarized RPE monolayers from iPSC-derived ROs. This novel approach involves isolating RPE spheroids from the NR component of organoids, followed by enzymatic dissociation and seeding onto Matrigel-coated transwell filters. Under these conditions, the dissociated RPE cells exhibit behavior similar to primary human RPE cells, forming characteristic pigmented cobblestone monolayers [183].

The potential of RO-derived RPE for transplantation has been demonstrated in animal models. For instance, transplantation of hESC-derived 3D RPE cells into RCS rats proved safe and effectively rescued retinal degeneration, demonstrating the potential of ROs for RPE transplantation [172]. A meta-analysis of RPE transplantation in animal models of retinal degeneration revealed significant improvements in electroretinogram responses and vision-based behaviors [184]. Efforts to enhance RPE transplantation efficacy have led to innovative approaches, such as using acellularized uveal hydrogel as a vehicle for subretinal RPE cell injection. This method has shown promising results in RCS rats, enhancing cell integration, reducing cell death and retinal gliosis, and restoring long-term vision [173].

Translating these preclinical findings to clinical applications, several clinical trials have been conducted using hESC- and hiPSC-derived RPE cells, transplanted either as sheets or cell suspensions. These studies have demonstrated short-term safety and improvements in visual function for patients with advanced-stage Stargardt disease and age-related macular degeneration [185–187].

Retinal progenitor cells (RPCs) represent a promising source for cell-based therapies in retinal degeneration. Various approaches have been developed to isolate RPCs from ROs. One method involves generating RPC lines from ROs using fluorescent reporter iPSC lines [174]. When RPCs isolated from hESC-derived ROs were transplanted subretinally into RCS rats, improvements in visual function and outer nuclear layer thickness were observed, along with the promotion of Müller glia dedifferentiation [175]. A recent advancement in RPC isolation is the label-free ghost cytometry (LF-GC) approach. This machine learning-optimized method efficiently produces transplantable retinal spheroids without requiring specific surface antigens or fluorescent labeling. LF-GC-sorted RPCs have demonstrated high graft indices and potential for structural maturation and synaptic integration in vivo [176]. This technique represents a significant step towards streamlining the preparation of clinical-grade grafts for retinal cell therapies, potentially enhancing the feasibility and efficacy of treatments for IRDs.

For clinical translation of hPSC-derived retinal cells, good manufacturing practice (cGMP)-compliant protocols should be developed to ensure immunological safety and meet the regulations of federal agencies [81]. Xeno-free and feeder-free culture methods have substituted undefined conditions of non-human derivatives; for example, FBS and Matrigel were replaced with human platelet lysate and recombinant laminin, respectively [37, 188]. Wiley et al. developed a cGMP-compliant RO differentiation protocol that utilizes a cGMP facility throughout the derivation of ROs [81]. Further research is necessary to develop an efficient and optimized cGMP-compliant protocol for the clinical translation of ROs. To serve as a potential option for a definitive cure for IRDs, optimization of transplantation conditions, including the transplantation site, method, and transplanted cell type, should precede clinical translation.