Evaluation of photoreceptor features in retinitis pigmentosa with cystoid macular edema by using an adaptive optics fundus camera.

OBJECTIVE: Cystoid macular edema (CME) in retinitis pigmentosa (RP) is an important complication causing visual dysfunction. We investigated the effect of CME on photoreceptors in RP patients with previous or current CME, using an adaptive optics (AO) fundus camera. METHODS: We retrospectively observed the CME and ellipsoid zone (EZ) length (average of horizontal and vertical sections) by optical coherence tomography. The density and regularity of the arrangement of photoreceptor cells (Voronoi analysis) were examined at four points around 1.5° from superior to inferior and temporal to nasal. We also performed a multivariate analysis using CME duration, central macular thickness and transversal length of CME. RESULTS: We evaluated 18 patients with previous or current CME (18 eyes; age, 48.7 ± 15.6 years) and 24 patients without previous or current CME (24 eyes; age, 46.0 ± 14.5 years). There were no significant differences in age, logMAR visual acuity, or EZ length. In groups with and without CME, cell density was 11967 ± 3148 and 16239 ± 2935 cells/mm2, and sequence regularity was 85.5 ± 3.4% and 88.5 ± 2.8%, respectively; both parameters were significantly different. The correlation between photoreceptor density and age was more negative in group with CME. The CME group tended toward greater reductions in duration of CME. CONCLUSION: Complications of CME in RP patients may lead to a decrease in photoreceptor density and regularity. Additionally, a longer duration of CME may result in a greater reduction in photoreceptor density.

Label-free enrichment of human pluripotent stem cell-derived early retinal progenitor cells for cell-based regenerative therapies.

Pluripotent stem cell-based therapy for retinal degenerative diseases is a promising approach to restoring visual function. A clinical study using retinal organoid (RO) sheets was recently conducted in patients with retinitis pigmentosa. However, the graft preparation currently requires advanced skills to identify and excise suitable segments from the transplantable area of the limited number of suitable ROs. This remains a challenge for consistent clinical implementations. Herein, we enabled the enrichment of wild-type (non-reporter) retinal progenitor cells (RPCs) from dissociated ROs using a label-free ghost cytometry (LF-GC)-based sorting system, where a machine-based classifier was trained in advance with another RPC reporter line. The sorted cells reproducibly formed retinal spheroids large enough for transplantation and developed mature photoreceptors in the retinal degeneration rats. This method of enriching early RPCs with no specific surface antigens and without any reporters or chemical labeling is promising for robust preparation of graft tissues during cell-based therapy.

Investigation of the effectiveness of gelatin hydrolysate in human iPS-RPE cell suspension transplantation.

Introduction: The retinal pigment epithelium (RPE) plays essential roles in maintaining retinal functions as well as choroidal capillaries and can lead to visual disorders if dysfunctional. Transplantation of human-induced pluripotent stem cell-derived RPE (hiPSC-RPE) is a promising therapy for such RPE impaired conditions including age-related macular degeneration. The challenge with cell suspension transplantation is targeted delivery of graft cells and undesired cell reflux. Gelatin hydrolysate, a soluble variant with specific molecular weight distribution, is examined in this study for its potential use in hiPSC-RPE suspension transplantation, particularly in reducing cell reflux and enhancing RPE engraftment. Methods: A retinal bleb model was created using polydimethylsiloxane (PDMS) soft lithography to quantify cellular reflux. We examined the effects of gelatin hydrolysate on the hiPSC-RPE of various aspects of cell behavior and performance such as cell viability, hypoxia reaction, morphology, induction of inflammation and immune responses. Results: Gelatin hydrolysate at 5 % concentration effectively mitigated cell reflux in vitro mimic, improved cell viability, reduced cell aggregation, and had an inhibitory effect on hypoxic reactions due to cell deposition with hiPSC-RPE. Additionally, gelatin hydrolysate did not affect cell adhesion and morphology, and decreased the expression of major histocompatibility complex class II molecules, which suggests reduced immunogenicity of hiPSC-RPE. Conclusion: Gelatin hydrolysate is considered a valuable and useful candidate for future regenerative therapies in hiPSC-RPE suspension transplantation.

Safety and stable survival of stem-cell-derived retinal organoid for 2 years in patients with retinitis pigmentosa.

Transplantation of induced pluripotent stem cell (iPSC)-derived retinal organoids into retinal disease animal models has yielded promising results, and several clinical trials on iPSC-derived retinal pigment epithelial cell transplantation have confirmed its safety. In this study, we performed allogeneic iPSC-derived retinal organoid sheet transplantation in two subjects with advanced retinitis pigmentosa (jRCTa050200027). The primary endpoint was the survival and safety of the transplanted retinal organoid sheets in the first year post-transplantation. The secondary endpoints were the safety of the transplantation procedure and visual function evaluation. The grafts survived in a stable condition for 2 years, and the retinal thickness increased at the transplant site without serious adverse events in both subjects. Changes in visual function were less progressive than those of the untreated eye during the follow-up. Allogeneic iPSC-derived retinal organoid sheet transplantation is a potential therapeutic approach, and the treatment's safety and efficacy for visual function should be investigated further.

Detailed Evaluation of Chromatic Pupillometry and Full-Field Stimulus Testing to Assess Ultralow Vision in Retinitis Pigmentosa.

Purpose: Novel therapeutic options, such as regenerative medicine and gene therapy, are now emerging as viable treatment options for patients with severe visual impairments, such as retinitis pigmentosa (RP). Gradable assessment of patients' visual function is essential to consider treatment options and to evaluate treatment outcomes; however, evaluation of visual function in patients with advanced low vision is often challenging because of patients' poor and sometimes unpredictable responses. In this study, we attempted to accurately assess visual capabilities and disease stage in patients with RP with a visual acuity (VA) of ≤ 0.01. Design: Retrospective analysis of visual function indicators, including VA, retinal thickness, full-field stimulus testing (FST), and chromatic pupillometry. Subjects: Overall, 43 patients (84 eyes) with advanced RP with a VA of ≤ 0.01 visited Kobe City Eye Hospital from 2019 to 2021. Methods: Hierarchical (multilevel) Bayesian modeling was used to estimate individual eye's pupil response and FST threshold, taking into account the ambiguity and randomness often observed in patients with ultralow vision. Using the estimated ability obtained from each test, the correlation between each test and retinal thickness was further analyzed to make a comprehensive assessment of the data. Main Outcome Measures: Visual acuity, retinal thickness, FST threshold, and pupil diameter change to different light stimuli. Results: Full-field stimulus testing and pupillometry measurements were moderately correlated with VA but exhibited a wide range of values within the same VA groups. Full-field stimulus testing was not correlated with central retinal thickness at counting fingers/hand motion VA range and seemed to reflect overall remaining photoreceptor function, including peripheral retina. Pupillometry may be able to distinguish between different levels of inner retinal function. Conclusions: The combination of pupillometry and FST allowed for graded evaluation of visual function within patients grouped in the same VA groups in patients with advanced RP with ultralow vision. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Mouse embryonic stem cells embody organismal-level cold resistance.

During hibernation, some mammals show low body temperatures (<10°C). Tissues from hibernators exhibit cold resistance even when the animal is not hibernating. Mice can also enter hypothermic fasting-induced torpor (FIT), but the cold resistance of FIT has never been related to their tissues. Here, we show that an inbred mouse STM2 exhibits lower body temperature during FIT than C57BL/6J or MYS/Mz. Thus, STM2 resists the cold more than other strains. Analysis of strain-specific mouse embryonic stem (ES) cells shows that STM2 ES cells are more cold-resistant than others and rely on the oxidative phosphorylation (OXPHOS) pathway but respire independently of the electron transfer chain complex I in the cold. We also found that the liver of STM2 uses OXPHOS more in cold than other strains. This study demonstrates that an organismal phenotype associated with torpor can be effectively studied in an in vitro setup using mouse cells.

Estimation of Visual Function Using Deep Learning From Ultra-Widefield Fundus Images of Eyes With Retinitis Pigmentosa.

Importance: There is no widespread effective treatment to halt the progression of retinitis pigmentosa. Consequently, adequate assessment and estimation of residual visual function are important clinically. Objective: To examine whether deep learning can accurately estimate the visual function of patients with retinitis pigmentosa by using ultra-widefield fundus images obtained on concurrent visits. Design, Setting, and Participants: Data for this multicenter, retrospective, cross-sectional study were collected between January 1, 2012, and December 31, 2018. This study included 695 consecutive patients with retinitis pigmentosa who were examined at 5 institutions. Each of the 3 types of input images-ultra-widefield pseudocolor images, ultra-widefield fundus autofluorescence images, and both ultra-widefield pseudocolor and fundus autofluorescence images-was paired with 1 of the 31 types of ensemble models constructed from 5 deep learning models (Visual Geometry Group-16, Residual Network-50, InceptionV3, DenseNet121, and EfficientNetB0). We used 848, 212, and 214 images for the training, validation, and testing data, respectively. All data from 1 institution were used for the independent testing data. Data analysis was performed from June 7, 2021, to December 5, 2022. Main Outcomes and Measures: The mean deviation on the Humphrey field analyzer, central retinal sensitivity, and best-corrected visual acuity were estimated. The image type-ensemble model combination that yielded the smallest mean absolute error was defined as the model with the best estimation accuracy. After removal of the bias of including both eyes with the generalized linear mixed model, correlations between the actual values of the testing data and the estimated values by the best accuracy model were examined by calculating standardized regression coefficients and P values. Results: The study included 1274 eyes of 695 patients. A total of 385 patients were female (55.4%), and the mean (SD) age was 53.9 (17.2) years. Among the 3 types of images, the model using ultra-widefield fundus autofluorescence images alone provided the best estimation accuracy for mean deviation, central sensitivity, and visual acuity. Standardized regression coefficients were 0.684 (95% CI, 0.567-0.802) for the mean deviation estimation, 0.697 (95% CI, 0.590-0.804) for the central sensitivity estimation, and 0.309 (95% CI, 0.187-0.430) for the visual acuity estimation (all P < .001). Conclusions and Relevance: Results of this study suggest that the visual function estimation in patients with retinitis pigmentosa from ultra-widefield fundus autofluorescence images using deep learning might help assess disease progression objectively. Findings also suggest that deep learning models might monitor the progression of retinitis pigmentosa efficiently during follow-up.

Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy.

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

Genotype and phenotype characteristics of RHO-associated retinitis pigmentosa in the Japanese population.

PURPOSE: To identify the genotypic and phenotypic characteristics of rhodopsin (RHO)-associated retinitis pigmentosa (RP) in the Japanese population. STUDY DESIGN: Cross-sectional, single-center study METHODS: The medical records of 1336 patients with RP who underwent genetic testing at our clinic between November 2008 and September 2021 were reviewed, and patients with RHO variants were included. The patients were divided into class A and class B to assess genotype-phenotype correlations based on previous reports. The clinical findings, including best-corrected visual acuity (BCVA), OCT parameters (ellipsoid zone [EZ] width and central retinal thickness [CRT]), and presence of macular degeneration, of the 2 groups were compared. RESULTS: The study included 28 patients diagnosed with RHO-associated RP (class A, 19; class B, 9). The BCVA was significantly worse in class A patients than in class B patients (P = 0.045). Superior EZ width was significantly shorter in class A than in class B patients (P = 0.016). Class A patients tended to have thinner CRT and shorter inferior EZ width than those of class B patients, although this difference was not significant. Macular degeneration was observed in 61.5% of class A and 12.5% of class B patients, demonstrating that macular degeneration can be a common complication in class A variants. CONCLUSION: Patients with class A variants presented with a severer form of RP than that of patients with class B variants in the Japanese population. These results suggest that the phenotype of RHO-associated RP is linked to the location of the variants and that such a genotype-phenotype correlation is less affected by ethnicities with different genetic backgrounds.

iPSC-RPE in Retinal Degeneration: Recent Advancements and Future Perspectives.

Regenerative medicine is a great hope for patients suffering from diseases for which no effective treatment is available. With the creation of induced pluripotent stem cells (iPSCs) in 2006, research and development has accelerated expeditiously, reaching a practical stage worldwide. The iPSC-regenerative medicine in ophthalmology is one of the pioneers, which has kicked off clinical application ahead of other fields owing to its advantages. The clinical safety issues of iPSC-derived retinal pigment epithelial (iPSC-RPE) transplantation for exudative age-related macular degeneration have been addressed to a certain extent. Preparations are being made for the next clinical study based on the improvement of its therapeutic effects and expansion of indications globally. Steady progress toward the practical applications of regenerative medicine for the treatment of retinal disorders is expected in the future while strengthening global cooperation amid various research areas, clinical fields, and regulations.

Comparing the transcriptome of developing native and iPSC-derived mouse retinae by single cell RNA sequencing.

We report the generation and analysis of single-cell RNA-Seq data (> 38,000 cells) from mouse native retinae and induced pluripotent stem cell (iPSC)-derived retinal organoids at four matched stages of development spanning the emergence of the major retinal cell types. We combine information from temporal sampling, visualization of 3D UMAP manifolds, pseudo-time and RNA velocity analyses, to show that iPSC-derived 3D retinal organoids broadly recapitulate the native developmental trajectories. However, we observe relaxation of spatial and temporal transcriptome control, premature emergence and dominance of photoreceptor precursor cells, and susceptibility of dynamically regulated pathways and transcription factors to culture conditions in retinal organoids. We demonstrate that genes causing human retinopathies are enriched in cell-type specifying genes and identify a subset of disease-causing genes with expression profiles that are highly conserved between human retinae and murine retinal organoids. This study provides a resource to the community that will be useful to assess and further improve protocols for ex vivo recapitulation and study of retinal development.

Progress of iPS cell-based transplantation therapy for retinal diseases.

The discovery of induced Pluripotent Stem) (iPS) cells has instigated innovation in various fields, including ophthalmology. Cell therapy has shown tremendous progress in translational research on retinal diseases, including the first-in-human transplantation of autologous iPS cell-derived retinal pigment epithelium (RPE) cells for patients with age-related macular degeneration (AMD). Cell therapy for retinitis pigmentosa (RP) has also been developed. Retinal organoid and photoreceptor cell transplantation has been shown to incorporate into the degenerated host retina, forming synapses with host neurons and resulting in functional recovery. Based on preclinical data, first-in-human transplantation of iPS cell-derived retinal sheets has been conducted. In this review, we summarize the current progress in iPS cell-based retinal cell transplantation research for retinal diseases, addressing some remaining challenges and future prospects.

Performance of ChatGPT in Board Examinations for Specialists in the Japanese Ophthalmology Society.

We investigated the potential of ChatGPT in the ophthalmological field in the Japanese language using board examinations for specialists in the Japanese Ophthalmology Society. We tested GPT-3.5 and GPT-4-based ChatGPT on five sets of past board examination problems in July 2023. Japanese text was used as the prompt adopting two strategies: zero- and few-shot prompting. We compared the correct answer rate of ChatGPT with that of actual examinees, and the performance characteristics in 10 subspecialties were assessed. ChatGPT-3.5 and ChatGPT-4 correctly answered 112 (22.4%) and 229 (45.8%) out of 500 questions with simple zero-shot prompting, respectively, and ChatGPT-4 correctly answered 231 (46.2%) questions with few-shot prompting. The correct answer rates of ChatGPT-3.5 were approximately two to three times lower than those of the actual examinees for each examination set (p = 0.001). However, the correct answer rates for ChatGPT-4 were close to approximately 70% of those of the examinees. ChatGPT-4 had the highest correct answer rate (71.4% with zero-shot prompting and 61.9% with few-shot prompting) in "blepharoplasty, orbit, and ocular oncology," and the lowest answer rate (30.0% with zero-shot prompting and 23.3% with few-shot prompting) in "pediatric ophthalmology." We concluded that ChatGPT could be one of the advanced technologies for practical tools in Japanese ophthalmology.

Competency of iPSC-derived retinas in MHC-mismatched transplantation in non-human primates.

Transplantation of embryonic/induced pluripotent stem cell-derived retina (ESC/iPSC-retina) restores host retinal ganglion cell light responses in end-stage retinal degeneration models with host-graft synapse formation. We studied the immunological features of iPSC-retina transplantation using major histocompatibility complex (MHC)-homozygote monkey iPSC-retinas in monkeys with laser-induced retinal degeneration in MHC-matched and -mismatched transplantation. MHC-mismatched transplantation without immune suppression showed no evident clinical signs of rejection and histologically showed graft maturation without lymphocytic infiltration, although immunological tests using peripheral blood monocytes suggested subclinical rejection in three of four MHC-mismatched monkeys. Although extensive photoreceptor rosette formation was observed on histology, evaluation of functional integration using mouse models such as mouse ESC-retina (C57BL/6) transplanted into rd1(C3H/HeJ, MHC-mismatched model) elicited light responses in the host retinal ganglion cells after transplantation but with less responsiveness than that in rd1-2J mice (C57BL/6, MHC-matched model). These results suggest the reasonable use of ESC/iPSC-retina in MHC-mismatched transplantation, albeit with caution.

Robotic search for optimal cell culture in regenerative medicine.

Induced differentiation is one of the most experience- and skill-dependent experimental processes in regenerative medicine, and establishing optimal conditions often takes years. We developed a robotic AI system with a batch Bayesian optimization algorithm that autonomously induces the differentiation of induced pluripotent stem cell-derived retinal pigment epithelial (iPSC-RPE) cells. From 200 million possible parameter combinations, the system performed cell culture in 143 different conditions in 111 days, resulting in 88% better iPSC-RPE production than that obtained by the pre-optimized culture in terms of the pigmentation scores. Our work demonstrates that the use of autonomous robotic AI systems drastically accelerates systematic and unbiased exploration of experimental search space, suggesting immense use in medicine and research.

Retinal pigment epithelium melanin imaging using polarization-sensitive optical coherence tomography for patients with retinitis pigmentosa.

This study aimed to evaluate the distribution of retinal pigment epithelium (RPE) melanin in patients with retinitis pigmentosa (RP) using entropy measurements by custom-made polarization-sensitive optical coherence tomography (PS-OCT) images, and compare entropy with the intensity of short-wavelength (SW) and near-infrared (NIR) autofluorescence (AF). We retrospectively reviewed the retinal images, including PS-OCT, SW-AF, and NIR-AF of patients with RP who had a hyperautofluorescent ring on AF. A total of 12 eyes of 12 patients (8 women and 4 men; mean age: 37.9 years) were included. There was a strong positive correlation between entropy value and NIR-AF intensity (r = 0.626, p < 0.001), and there was a very weak negative correlation between entropy value and SW-AF (r = - 0.197, p = 0.001). The mean values of the entropy in the foveal, temporal (2 mm from the fovea), and nasal (2 mm from the fovea) sections were 0.41 (± 0.09), 0.29 (± 0.08), and 0.26 (± 0.08), respectively. The entropy was significantly higher in the foveal section than in the temporal and nasal sections (p = 0.002 and p = 0.003, respectively). There was no significant difference between the entropies values for the temporal and nasal sections (p = 0.157). Age, logMAR best-corrected visual acuity, ellipsoid zone width, and central retinal thickness were not correlated with foveal entropy. We presented RPE melanin imaging in patients with RP using PS-OCT for the first time. PS-OCT can be a useful tool for monitoring patients with RP.

Correlation Between Genetic Abnormalities in Induced Pluripotent Stem Cell-Derivatives and Abnormal Tissue Formation in Tumorigenicity Tests.

Cell therapy using induced pluripotent stem cell (iPSC) derivatives may result in abnormal tissue generation because the cells undergo numerous cycles of mitosis before clinical application, potentially increasing the accumulation of genetic abnormalities. Therefore, genetic tests may predict abnormal tissue formation after transplantation. Here, we administered iPSC derivatives with or without single-nucleotide variants (SNVs) and deletions in cancer-related genes with various genomic copy number variant (CNV) profiles into immunodeficient mice and examined the relationships between mutations and abnormal tissue formation after transplantation. No positive correlations were found between the presence of SNVs/deletions and the formation of abnormal tissues; the overall predictivity was 29%. However, a copy number higher than 3 was correlated, with an overall predictivity of 86%. Furthermore, we found CNV hotspots at 14q32.33 and 17q12 loci. Thus, CNV analysis may predict abnormal tissue formation after transplantation of iPSC derivatives and reduce the number of tumorigenicity tests.

Strategies of pluripotent stem cell-based therapy for retinal degeneration: update and challenges.

Stem cell-based therapy for retinal degeneration is transitioning from the research stage to the clinical stage and is being developed as a treatment across the globe. In clinical studies on induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) transplantation, the safety of the technique has started to clarify, and clinical study on further advances such as the long-desired transplantation of iPSC-derived retina to treat retinitis pigmentosa (RP) has begun. Ophthalmologists are now working closely with basic researchers to incorporate new technology areas with a synergy that is anticipated to realize the practical application of stem cell-based therapy for retinal degeneration.

Perception of genetic testing among patients with inherited retinal disease: Benefits and challenges in a Japanese population.

Inherited retinal disease (IRD) is clinically and genetically heterogeneous. Awareness of the importance of genetic testing for IRD in the clinical setting is increasing with the recent development of new therapeutic strategies, such as gene therapy. Here, the perception of genetic testing, including its benefits and potential challenges, among patients with IRD was investigated to establish strategies for IRD genetic testing and counseling practices that can meet the requirements of the patients in Japan. An anonymous self-administered questionnaire was distributed to 275 patients with IRD who underwent genetic testing after clinical consultation and genetic counseling to investigate the motivations for genetic testing, benefits, challenges, status of communication of results to family, and attitude to timing of genetic testing. In total, 228 (82.9%) responses were analyzed. Several major motivations for genetic testing were identified, including gaining information on future treatment options and clarification of the inheritance pattern, among others. No association was found between the sharing of results with family members and the results of genetic testing. Moreover, according to patients who received positive results, the benefits of genetic testing included information on the inheritance pattern, additional information on the diagnosis, and mental preparation for the future. Even patients who received negative or inconclusive (variant of uncertain significance) results reported certain informative and psychological benefits. Altogether, these findings suggest that provisions for genetic testing and genetic counseling are necessary within a certain period after clinical diagnosis and it is necessary to facilitate appropriate family communication about genetic testing results while paying attention to the background of family relationships. Moreover, the benefits of genetic testing can be influenced by the careful interpretation and information provided on the test results during genetic counseling and consultation.