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.

A CASE OF RAPIDLY PROGRESSIVE RETINOPATHY ASSOCIATED WITH PEMBROLIZUMAB IMMUNOTHERAPY FOR METASTATIC UROTHELIAL CARCINOMA.

PURPOSE: To describe a novel case of bilateral rapidly progressive retinopathy after immunotherapy with pembrolizumab for metastatic urothelial carcinoma. METHODS: Case report. RESULTS: A 64-year-old man undergoing pembrolizumab immunotherapy was referred to our hospital because of bilateral acute vision loss. His best-corrected visual acuity was 20/30 in the right eye and 20/320 in the left eye, and a visual field test revealed central and paracentral scotomas in the right eye and central scotoma in the left eye. We suspected immune-related retinopathy based on the progressive photoreceptor damage with abnormal electroretinogram findings, absence of overt intraocular inflammation, and presence of malignancy. Cessation of pembrolizumab and steroid pulse therapy followed by decreasing oral prednisolone improved visual function and photoreceptor damage, although there was recurrence after pembrolizumab was restarted. CONCLUSION: We reported a case of rapidly progressive retinopathy that may have been triggered by pembrolizumab immunotherapy for metastatic urothelial carcinoma. High-dose corticosteroid pulse therapy was effective in reversing photoreceptor damage.

Role of Recent PCR Tests for Infectious Ocular Diseases: From Laboratory-Based Studies to the Clinic.

Infectious uveitis is a vision-threatening condition that requires prompt clinical diagnosis and proper treatment. However, rapid and proper diagnosis in infectious uveitis remains challenging. Several examination tests, including polymerase chain reaction (PCR) tests, are transitioning from laboratory-based basic research-level tests to bedside clinical tests, and recently tests have changed to where they can be performed right next to clinicians. In this review, we introduce an updated overview of recent studies that are representative of the current trends in clinical microbiological techniques including PCR tests for infectious uveitis.

Genetic Ablation of Nrf2 Exacerbates Neuroinflammation in Ocular Autoimmunity.

Experimental autoimmune uveoretinitis (EAU) is an animal model of non-infectious uveitis and is developed by immunization with retinal antigen, interphotoreceptor retinoid-binding protein (IRBP). Nuclear factor erythroid 2- (NF-E2-) related factor 2 (Nrf2) is responsible for regulating antioxidant and inflammatory responses. In this study, we investigated the role of Nrf2 on the development of EAU. Clinical and pathological examination demonstrated that retinal inflammation was exacerbated in Nrf2 knockout (Nrf2 KO) mice compared to wild type (WT) mice, and the expression of inflammatory cytokines (IFN-γ, IL-6, and IL-17) in the retina was significantly elevated in Nrf2 KO mice. GFAP positive cells (astrocytes) and Iba-1 positive cells (microglia cells) in the retina were more numerous in Nrf2 KO mice compared to WT mice. Furthermore, we examined the suppressive effect of the Nrf2 activator CDDO-Im (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline) on the development of EAU. The treatment with CDDO-Im significantly reduced the clinical and pathological score of EAU compared to those of vehicle-treated mice. These findings suggest that Nrf2 plays a regulatory role in the pathogenesis of autoimmune uveoretinitis and the activation of the Nrf2 system may have therapeutic potential for protecting vision from autoimmune neuroinflammation.

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.

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.

Detection of Mycoplasma Contamination in Transplanted Retinal Cells by Rapid and Sensitive Polymerase Chain Reaction Test.

Contamination of cells/tissues by infectious pathogens (e.g., fungi, viruses, or bacteria, including mycoplasma) is a major problem in cell-based transplantation. In this study, we tested a polymerase chain reaction (PCR) method to provide rapid, simple, and sensitive detection of mycoplasma contamination in laboratory cultures for clinical use. This mycoplasma PCR system covers the Mycoplasma species (spp.) listed for testing in the 17th revision of the Japanese Pharmacopoeia, and we designed it for use in transplantable retinal cells. Here, we analyzed mycoplasma contamination in induced pluripotent stem cell (iPS cell)-derived transplantable retinal pigment epithelium (RPE) cells. In the spike tests to RPE cells with nine species of class Mollicutes bacteria, including seven Mycoplasma spp. and one of each Acholeplasma spp. and Ureaplasma spp., contamination at the concentration of 100 and 10 CFU/mL were detected with 100% probability in all cases, while 1 CFU/mL had a detection rate of 0-75%. DNA prepared from bacteria species other than class Mollicutes species was not detectable, indicating the specificity of this PCR. While iPS cells and iPS-RPE cells established in our laboratory were all negative by this PCR, some of the commercially available cell lines were positive. Cells for transplantation should never have infection, as once pathogens are implanted into the eyes, they can cause severe intraocular inflammation. Thus, it is imperative to monitor for infections in the transplants, although generally, mycoplasma infection is difficult to detect.

Dynamics of Cyclooxygenase-1 Positive Microglia/Macrophage in the Retina of Pathological Model Mice as a Biomarker of the Retinal Inflammatory Diseases.

In an intraocular inflammatory state, microglia residing in the retina become active and migrate inside the retina. In this study, we investigated whether cyclooxygenase-1 (COX-1) expressed by retinal microglia/macrophage can be a biomarker for the diagnosis of retinal diseases. COX-1 was immunopositive in microglia/macrophage and neutrophils, while COX-2 was immunopositive in astrocytes and neurons in the inner layer of normal retina. The number of COX-1 positive cells per section of the retinal tissue was 14 ± 2.8 (mean ± standard deviation) in normal mice, which showed significant increase in the lipopolysaccharide (LPS)-administrated model (62 ± 5.0, p = 8.7 × 10-9). In addition to microglia, we found neutrophils that were positive for COX-1. In the early stage of inflammation in the experimental autoimmune uveoretinitis (EAU), COX-1 positive cells, infiltrating from the ciliary body into the retinal outer nuclear layer, were observed. The number of infiltrating COX-1 positive cells correlated with the severity of EAU. Taken together, the increased number of COX-1 positive microglia/macrophage with morphological changes were observed in the retinas of retinal inflammatory disease models. This suggests that COX-1 can be a marker of disease-related activities of microglia/macrophage, which should be useful for the diagnosis of retinal diseases.

CTLA-2 Alpha Is a Potent Inhibitor of Angiogenesis in Murine Ocular Tissue.

Cytotoxic T lymphocyte antigen-2 (CTLA-2) alpha has been reported to suppress the activities of cathepsin L (Cath L), which is deeply involved in angiogenesis. Therefore, we assessed whether CTLA-2 alpha plays a role in angiogenesis in ocular tissue. To establish models of corneal inflammation and experimental choroidal neovascularization (CNV), male C57BL/6J mice (n = 5) underwent corneal suture placement or laser-induced CNV, respectively. Mice were then injected with recombinant CTLA-2 alpha (1 µg) into the peritoneal cavity at day 0 and every 2 days after operation. In vitro experiments were performed to assess the inflammatory response by measuring TNF-alpha secretion in peritoneal cavity exudate cells (PECs) or the proliferation of mouse vascular endothelial cells (mVECs). CTLA-2 alpha treatment dramatically suppressed corneal angiogenesis, as well as laser-induced CNV. Moreover, CTLA-2 alpha inhibited the proliferation of mVECs in vitro, while CTLA-2 alpha abolishment was able to rescue proliferation. However, CTLA-2 alpha could not suppress cytokine secretion from inflammatory cells such as PECs. In summary, CTLA-2 alpha was able to suppress angiogenesis by suppressing endothelial cell proliferation. Further studies are needed to investigate its usefulness as a new antiangiogenic treatment for a variety of conditions, including age-related macular degeneration.

Trends of Stem Cell Therapies in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a highly prevalent irreversible impairment in the elderly population worldwide. Stem cell therapies have been considered potentially viable for treating AMD through the direct replacement of degenerated cells or secretion of trophic factors that facilitate the survival of existing cells. Among them, the safety of pluripotent stem cell-derived retinal pigment epithelial (RPE) cell transplantation against AMD, and some hereditary retinal degenerative diseases, has been discussed to a certain extent in clinical studies of RPE cell transplantation. Preparations are in progress for its clinical application. On the other hand, clinical trials using somatic stem cells are also being conducted, though these had controversial outcomes. Retinal regenerative medicine using stem cells is expected to make steady progress toward practical use while new technologies are incorporated from various fields, thereby making the role of ophthalmologists in this field increasingly important.

A ROCK Inhibitor Promotes Graft Survival during Transplantation of iPS-Cell-Derived Retinal Cells.

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.

Low Immunogenicity and Immunosuppressive Properties of Human ESC- and iPSC-Derived Retinas.

ESC- and iPSC-derived retinal transplantation is a promising therapeutic approach for disease with end-stage retinal degeneration, such as retinitis pigmentosa and age-related macular degeneration. We previously showed medium- to long-term survival, maturation, and light response of transplanted human ESC- and iPSC-retina in mouse, rat, and monkey models of end-stage retinal degeneration. Because the use of patient hiPSC-derived retina with a disease-causing gene mutation is not appropriate for therapeutic use, allogeneic transplantation using retinal tissue/cells differentiated from a stocked hESC and iPSC line would be most practical. Here, we characterize the immunological properties of hESC- and iPSC-retina and present their three major advantages: (1) hESC- and iPSC-retina expressed low levels of human leukocyte antigen (HLA) class I and little HLA class II in vitro, (2) hESC- and iPSC-retina greatly suppressed immune activation of lymphocytes in co-culture, and (3) hESC- and iPSC-retina suppressed activated immune cells partially via transforming growth factor ß signaling. These results support the use of allogeneic hESC- and iPSC-retina in future clinical application.

Practical use of multiplex and broad-range PCR in ophthalmology.

BACKGROUND: Polymerase chain reaction (PCR) can be used to confirm or deny infectious ocular inflammation such as uveitis. The purpose of this article is to review the current practical use of PCR examination in ophthalmology, especially multiplex and broad-range PCR, and a novel PCR, termed Strip PCR. At first, in the Introduction, we show the development of the PCR examination in ophthalmology. We next show the clinical applications of multiplex PCR and broad-range PCR. These advances in PCR continue to contribute greatly to the ophthalmology field. We also show how the sample for PCR is collected. Recently, we established a novel examination, a multiplex real-time PCR (Strip PCR) prototype for detecting 24 pathogens responsible for ocular infectious diseases. Moreover, we developed the Direct Strip PCR method, which skips the DNA extraction step in the procedure. This PCR is anticipated to ease etiologic evaluation, increasing pathogen detection in the intraocular fluids of uveitis patients even by general ophthalmologists. We also describe the following: (1) representative cases in which PCR is useful, (2) representative cases in which PCR can exclude a diagnosis, (3) the current status of PCR in the diagnosis of infectious uveitis and advanced medical service, and (4) the prospects for clinical PCR in ophthalmology. CONCLUSION: We have established and developed the multiplex PCR, broad-range PCR, Strip PCR, and Direct Strip PCR methods and have reported the efficacy of such tests in multicenter studies. Our goal is "rapid and simple comprehensive PCR diagnosis anywhere and by anyone" for ocular infections.

Immunological aspects of RPE cell transplantation.

Retinal pigment epithelial (RPE) cells have several functions, including support of the neural retina and choroid in the eye and immunosuppression. Cultured human RPE cells directly suppress inflammatory immune cells. For instance, they directly suppress the activation of T cells in vitro. In contrast, transplanted allogeneic human RPE cells are rejected by bystander immune cells such as T cells in vivo. Recently, human embryonic stem cell-derived RPE cells have been used in several clinical trials, and human induced pluripotent stem cell (iPSC)-RPE cells have also been tested in our clinical study in patients with retinal degeneration. Major safety concerns after stem cell-based transplantation surgery include hyper-proliferation, tumorigenicity, or ectopic tissue formation, but these events have currently not been seen in any of these patients. However, if RPE cells are allogeneic, there are concerns about immune rejection issues that have been raised in previous clinical trials. We therefore performed a preclinical study of allogeneic iPSC-RPE cell transplantation in animal rejection models. We then conducted autogenic or allogeneic iPSC-RPE cell transplantation in clinical studies of patients with age-related macular degeneration. In this review, we focus on immunological studies of RPE cells, including iPSC-derived cells. iPSC-RPE cells have unique inflammatory (immunosuppressive and immunogenic) characteristics like primary cultured RPE cells. The purpose of this review is to summarize the current findings obtained from preclinical (basic research) and clinical studies in iPSC-RPE cell transplantation, especially the immunological aspects.

Multiplex Solid-Phase Real-Time Polymerase Chain Reaction without DNA Extraction: A Rapid Intraoperative Diagnosis Using Microvolumes.

PURPOSE: Current polymerase chain reaction (PCR) methods for the diagnosis of infections are time consuming and require large sample volume and skilled technicians. We developed a novel, easy-to-use, and rapid (processing time, 1 minute; total time, 33 minutes) multiplex real-time PCR test (Direct Strip PCR) that did not require DNA extraction to detect 9 pathogens that could cause uveitis in 20-µl samples. DESIGN: Multicenter prospective evaluation of a diagnostic PCR test. PARTICIPANTS: A total of 511 participants (patients with infectious uveitis and controls) were examined at 18 institutes worldwide. METHODS: After validation, intraocular fluid samples were subjected to etiologic or exclusive diagnosis, including intraoperative rapid diagnosis. MAIN OUTCOME MEASURES: The concordance and correlations between Direct Strip PCR and quantitative PCR (qPCR) results. RESULTS: Direct Strip PCR exhibited rapid detection, good repeatability and specificity, long storage stability, and detection ability equal to that of qPCR. It also showed low interinstitutional variability compared with qPCR, even when PCR beginners used various real-time PCR machines. The Direct Strip PCR for 9 pathogens exhibited high concordance against the qPCR (positive concordance rate, 98.8%-100%; negative concordance rate, 99.8%-100%; κ coefficient, 0.969-1.000; P < 0.001-0.031). Additionally, results obtained using Direct Strip PCR and qPCR were highly correlated (ρ = 0.748; P < 0.001). This assay was used for rapid intraoperative diagnosis. CONCLUSIONS: The Direct Strip PCR test may improve the prognosis of various infectious diseases because it facilitates rapid etiologic evaluation at the first hospital visit and can be used for intraoperative diagnosis.

Capacity of Retinal Ganglion Cells Derived from Human Induced Pluripotent Stem Cells to Suppress T-Cells.

Retinal ganglion cells (RGCs) are impaired in patients such as those with glaucoma and optic neuritis, resulting in permanent vision loss. To restore visual function, development of RGC transplantation therapy is now underway. Induced pluripotent stem cells (iPSCs) are an important source of RGCs for human allogeneic transplantation. We therefore analyzed the immunological characteristics of iPSC-derived RGCs (iPSC-RGCs) to evaluate the possibility of rejection after RGC transplantation. We first assessed the expression of human leukocyte antigen (HLA) molecules on iPSC-RGCs using immunostaining, and then evaluated the effects of iPSC-RGCs to activate lymphocytes using the mixed lymphocyte reaction (MLR) and iPSC-RGC co-cultures. We observed low expression of HLA class I and no expression of HLA class II molecules on iPSC-RGCs. We also found that iPSC-RGCs strongly suppressed various inflammatory immune cells including activated T-cells in the MLR assay and that transforming growth factor-ß2 produced by iPSC-RGCs played a critical role in suppression of inflammatory cells in vitro. Our data suggest that iPSC-RGCs have low immunogenicity, and immunosuppressive capacity on lymphocytes. Our study will contribute to predicting immune attacks after RGC transplantation.

Microglia dynamics in retinitis pigmentosa model: formation of fundus whitening and autofluorescence as an indicator of activity of retinal degeneration.

In patients with retinitis pigmentosa (RP), color fundus photography and fundus autofluorescence (FAF) have been used to estimate the disease progression. To understand the origin and the diagnostic interpretation of the fundus color and FAF, we performed in vivo imaging of fundus color and FAF together with histological analyses of the retinal degeneration process using the RP model mice, rd10. FAF partly represented the accumulation of microglia in the photoreceptor outer segments. Fundus whitening suggested the presence of apoptotic cells, which spatiotemporally preceded increase in FAF. We observed two patterns of FAF localization, arcuate and diffuse, each indicating different pattern of apoptosis, wavy and diffuse, respectively. Diffuse pattern of apoptosis was suppressed in dark-raised rd10 mice, in which outer nuclear layer (ONL) loss was significantly suppressed. The occupancy of FAF correlated with the thinning rate of the ONL. Fractalkine, a microglia chemotactic factor, was detected in apoptotic photoreceptors, suggesting chemokine-induced recruitment of microglia into the ONL, which paralleled with accelerated ONL loss and increased FAF occupancy. Thus, we propose that the degree of photoreceptor apoptosis and the rate of ONL thinning in RP patients might be read from the fundus color and the FAF.