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X-linked retinoschisis (XLRS) is a common retinal genetic disease that occurs in juvenile males and causes progressive visual impairment. This presents a schisis in the macula or peripheral retina of bilateral eyes, which has no effective treatment. Here, we introduced the RS1 (c.C304T, p.R102W) mutation into a normal induced pluripotent stem (iPS) cell line using CRISPR/Cas9 technology. This missense mutation was consistent with that observed in the XLRS patient-derived iPS cell line (CSUASOi001-A). Conclusively, establishing a directed gene mutation cell line (CSUi007-A) provides a useful cell resource to investigate XLRS pathogenesis.

Purpose: To investigate the risk factors for patients with focal choroidal excavation (FCE) and their correlation with chorioretinal diseases. Design: Retrospective cross-sectional study. Subjects: Patients with FCE were enrolled, while healthy subjects were recruited for the control group. Methods: The study collected demographic information, clinical features, and multimodal images. Parameters of FCE identified using spectral-domain OCT (SD-OCT) were manually measured using built-in software and subsequently analyzed statistically. Main Outcome Measures: Subfoveal choroidal thickness (SFCT), subexcavation choroidal thickness (SECT), and the greatest depth and width of each excavation were manually measured using built-in calipers in OCT software. Results: Twenty-one patients (13/8, male/female) with FCE were included in this study. The average age was 45.2 years, and their best-corrected visual acuity (BCVA) was 0.4 logarithm of the minimum angle of resolution (Snellen equivalent, 20/50). Focal choroidal excavation was present in 28 eyes of 21 patients, including isolated FCE (12 eyes) and complicated FCE (16 eyes) with choroidal neovascularization (sCNV), central serous chorioretinopathy, and other conditions. Patients with complicated FCE were significantly older than those isolated FCE (P = 0.015). The SFCT of the healthy subjects was significantly less than that of the fellow eyes of the patients with FCE (P

Abstract Background X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS. Methods hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy. Results ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation. Conclusions The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.

Abstract Microglia-mediated neuroinflammatory responses are recognized as a predominant factor during high intraocular pressure (IOP)-induced retinal and optic nerve injury along with potential therapeutic targets for the disease. Our previous research indicated that mesenchymal stem cell (MSC) treatment could reduce high IOP-induced neuroinflammatory responses through the TLR4 pathway in a rat model without apparent cell replacement and differentiation, suggesting that the anti-neuroinflammatory properties of MSCs are potentially mediated by paracrine signaling. This study aimed to evaluate the anti-neuroinflammatory effect of human adipose tissue-derived extracellular vesicles (ADSC-EVs) in microbead-induced ocular hypertension (OHT) animals and to explore the underlying mechanism since extracellular vesicles (EVs) are the primary transporters for cell secretory action. The anti-neuroinflammatory effect of ADSC-EVs on LPS-stimulated BV-2 cells in vitro and OHT-induced retinal and optic nerve injury in vivo was investigated. According to the in vitro research, ADSC-EV treatment reduced LPS-induced microglial activation and the TLR4/NF-κB proinflammatory cascade response axis in BV-2 cells, such as CD68, iNOS, TNF-α, IL-6, and IL-1β, TLR4, p-38 MAPK, NF-κB. According to the in vivo data, intravitreal injection of ADSC-EVs promoted RGC survival and function, reduced microglial activation, microglial-derived neuroinflammatory responses, and TLR4/MAPK/NF-κB proinflammatory cascade response axis in the OHT mice. Our findings provide preliminary evidence for the RGC protective and microglia-associated neuroinflammatory reduction effects of ADSC-EVs by inhibiting the TLR4/MAPK/NF-κB proinflammatory cascade response in OHT mice, indicating the therapeutic potential ADSC-EVs or adjunctive therapy for glaucoma.

Photoreceptor cell degeneration leads to blindness, for which there is currently no effective treatment. Our previous studies have shown that Lycium barbarum (L. barbarum) polysaccharide (LBP) protects degenerated photoreceptors in rd1, a transgenic mouse model of retinitis pigmentosa. L. barbarum glycopeptide (LbGP) is an immunoreactive glycoprotein extracted from LBP. In this study, we investigated the potential protective effect of LbGP on a chemically induced photoreceptor-degenerative mouse model. Wild-type mice received the following: oral administration of LbGP as a protective pre-treatment on days 1–7; intraperitoneal administration of 40 mg/kg N-methyl-N-nitrosourea to induce photoreceptor injury on day 7; and continuation of orally administered LbGP on days 8–14. Treatment with LbGP increased photoreceptor survival and improved the structure of photoreceptors, retinal photoresponse, and visual behaviors of mice with photoreceptor degeneration. LbGP was also found to partially inhibit the activation of microglia in N-methyl-N-nitrosourea-injured retinas and significantly decreased the expression of two pro-inflammatory cytokines. In conclusion, LbGP effectively slowed the rate of photoreceptor degeneration in N-methyl-N-nitrosourea-injured mice, possibly through an anti-inflammatory mechanism, and has potential as a candidate drug for the clinical treatment of photoreceptor degeneration.

Diabetic eye disease refers to a group of eye complications that occur in diabetic patients and include diabetic retinopathy, diabetic macular edema, diabetic cataracts, and diabetic glaucoma. However, the global epidemiology of these conditions has not been well characterized. In this study, we collected information on diabetic eye disease-related research grants from seven representative countries––the United States, China, Japan, the United Kingdom, Spain, Germany, and France––by searching for all global diabetic eye disease journal articles in the Web of Science and PubMed databases, all global registered clinical trials in the ClinicalTrials database, and new drugs approved by the United States, China, Japan, and EU agencies from 2012 to 2021. During this time period, diabetic retinopathy accounted for the vast majority (89.53%) of the 2288 government research grants that were funded to investigate diabetic eye disease, followed by diabetic macular edema (9.27%). The United States granted the most research funding for diabetic eye disease out of the seven countries assessed. The research objectives of grants focusing on diabetic retinopathy and diabetic macular edema differed by country. Additionally, the United States was dominant in terms of research output, publishing 17.53% of global papers about diabetic eye disease and receiving 22.58% of total citations. The United States and the United Kingdom led international collaborations in research into diabetic eye disease. Of the 415 clinical trials that we identified, diabetic macular edema was the major disease that was targeted for drug development (58.19%). Approximately half of the trials (49.13%) pertained to angiogenesis. However, few drugs were approved for ophthalmic (40 out of 1830; 2.19%) and diabetic eye disease (3 out of 1830; 0.02%) applications. Our findings show that basic and translational research related to diabetic eye disease in the past decade has not been highly active, and has yielded few new treatment methods and newly approved drugs.

Abstract Inherited retinal diseases (IRDs) can induce severe sight-threatening retinal degeneration and impose a considerable economic burden on patients and society, making efforts to cure blindness imperative. Transgenic animals mimicking human genetic diseases have long been used as a primary research tool to decipher the underlying pathogenesis, but there are still some obvious limitations. As an alternative strategy, patient-derived induced pluripotent stem cells (iPSCs), particularly three-dimensional (3D) organoid technology, are considered a promising platform for modeling different forms of IRDs, including retinitis pigmentosa, Leber congenital amaurosis, X-linked recessive retinoschisis, Batten disease, achromatopsia, and best vitelliform macular dystrophy. Here, this paper focuses on the status of patient-derived iPSCs and organoids in IRDs in recent years concerning disease modeling and therapeutic exploration, along with potential challenges for translating laboratory research to clinical application. Finally, the importance of human iPSCs and organoids in combination with emerging technologies such as multi-omics integration analysis, 3D bioprinting, or microfluidic chip platform are highlighted. Patient-derived retinal organoids may be a preferred choice for more accurately uncovering the mechanisms of human retinal diseases and will contribute to clinical practice.

Abstract Background Diabetic retinopathy (DR) development is associated with disturbances in the gut microbiota and related metabolites. Butyric acid is one of the short-chain fatty acids (SCFAs), which has been found to possess a potential antidiabetic effect. However, whether butyrate has a role in DR remains elusive. This study aimed to investigate the effect and mechanism of sodium butyrate supplementation on DR. Methods C57BL/6J mice were divided into three groups: Control group, diabetic group, and diabetic with butyrate supplementation group. Type 1 diabetic mouse model was induced by streptozotocin. Sodium butyrate was administered by gavage to the experimental group daily for 12 weeks. Optic coherence tomography, hematoxylin–eosin, and immunostaining of whole-mount retina were used to value the changes in retinal structure. Electroretinography was performed to assess the retinal visual function. The tight junction proteins in intestinal tissue were evaluated using immunohistochemistry. 16S rRNA sequencing and LC–MS/MS were performed to determine the alteration and correlation of the gut microbiota and systemic SCFAs. Results Butyrate decreased blood glucose, food, and water consumption. Meanwhile, it alleviated retinal thinning and activated microglial cells but improved electroretinography visual function. Additionally, butyrate effectively enhanced the expression of ZO-1 and Occludin proteins in the small intestine. Crucially, only butyric acid, 4-methylvaleric acid, and caproic acid were significantly decreased in the plasma of diabetic mice and improved after butyrate supplementation. The deeper correlation analysis revealed nine genera strongly positively or negatively correlated with the above three SCFAs. Of note, all three positively correlated genera, including norank_f_Muribaculaceae, Ileibacterium, and Dubosiella, were significantly decreased in the diabetic mice with or without butyrate treatment. Interestingly, among the six negatively correlated genera, Escherichia-Shigella and Enterococcus were increased, while Lactobacillus, Bifidobacterium, Lachnospiraceae_NK4A136_group, and unclassified_f_Lachnospiraceae were decreased after butyrate supplementation. Conclusion Together, these findings demonstrate the microbiota regulating and diabetic therapeutic effects of butyrate, which can be used as a potential food supplement alternative to DR medicine.

X-linked retinoschisis (XLRS) is one of the most common retinal genetic diseases with progressive visual impairment in childhood affecting males. It is manifested with macular and/or peripheral schisis in neural retinas with no effective treatment. Previously, we successfully generated a human-induced pluripotent stem cell (iPSC) line from an XLRS patient carrying the hemizygous RS1 c. 304C > T (p.R102W) mutation. Here, we corrected the c.304C > T mutation in the RS1 gene using CRISPR/Cas9 technology to generate an isogenic control. This cell line is valuable for the study of XLRS.

Tissue injury is a common clinical problem, which may cause great burden on patients' life. It is important to develop functional scaffolds to promote tissue repair and regeneration. Due to their unique composition and structure, microneedles have attracted extensive attention in various tissues regeneration, including skin wound, corneal injury, myocardial infarction, endometrial injury, and spinal cord injury et al. Microneedles with micro-needle structure can effectively penetrate the barriers of necrotic tissue or biofilm, therefore improving the bioavailability of drugs. The use of microneedles to deliver bioactive molecules, mesenchymal stem cells, and growth factors in situ allows for targeted tissue and better spatial distribution. At the same time, microneedles can also provide mechanical support or directional traction for tissue, thus accelerating tissue repair. This review summarized the research progress of microneedles for in situ tissue regeneration over the past decade. At the same time, the shortcomings of existing researches, future research direction and clinical application prospect were also discussed.

The scale of cities is increasing with continuous urban development. Effective methods, such as the source term estimation (STE) method, must be established for identifying the sources of air pollution in cities to prevent economic losses and casualties caused by pollutant leakage. Herein, methods for optimizing sensor configuration and identifying pollution sources are discussed, and an STE method based on the regularized minimum residual method is proposed. Urban wind environments were simulated using a computational fluid dynamics (CFD) model, and the results were compared with experimental data pertaining to the wind tunnel of an architectural ensemble to verify the model’s accuracy. The sensor layout was optimized using the simulated annealing (SA) algorithm and adjoint entropy, and the relationship between sensor responses and potential pollution sources was established using the CFD model. Pollutant concentrations measured using sensors were combined with the regularization method to extrapolate the pollution source strength, and the regularized minimum residual method was used to obtain the locations of the real pollution sources. The results show that compared with the Bayesian methods, the proposed method can more accurately identify pollution sources (100%), with a smaller source strength error of 2.01% for constant sources and one of 2.62% for attenuation sources.

Retinitis pigmentosa (RP) is one of the most common inherited retinal diseases characterized by nyctalopia, progressive vision loss and visual field contraction. we previously generated an induced pluripotent stem cell line (CSUASOi004-A) from a RP patient with heterozygous PRPF6 c.2699 G>A (p.R900H) mutation. Here we corrected the PRPF6 c.2699 G>A mutation genetically using CRISPR/Cas9 technology to generate an isogenic control (CSUASOi004-A-1), which can provide a valuable resource in the research of the disease.

Retinitis pigmentosa (RP) is a leading cause of vision impairment and blindness worldwide, with limited medical treatment options. USH2A mutations are one of the most common causes of non-syndromic RP. In this study, we developed retinal organoids (ROs) and retinal pigment epithelium (RPE) cells from induced pluripotent stem cells (iPSCs) of RP patient to establish a sustainable in vitro RP disease model. RT-qPCR, western blot, and immunofluorescent staining assessments showed that USH2A mutations induced apoptosis of iPSCs and ROs, and deficiency of the extracellular matrix (ECM) components. Transcriptomics and proteomics findings suggested that abnormal ECM-receptor interactions could result in apoptosis of ROs with USH2A mutations via the PI3K-Akt pathway. To optimize the culture conditions of ROs, we fabricated a microfluidic chip to co-culture the ROs with RPE cells. Our results showed that this perfusion system could efficiently improve the survival rate of ROs. Further, ECM components such as laminin and collagen IV of ROs in the RP group were upregulated compared with those maintained in static culture. These findings illustrate the potential of microfluidic chip combined with ROs technology in disease modelling for RP.

Type 2 diabetes mellitus (T2DM) is a major caused by insulin resistance with a relative deficiency in insulin secretion. Statistically, T2DM accounts for 90% of diabetes cases worldwide. We report the patient-specific human induced pluripotent stem cell line (iPSC) CSUASOi010-A by using Peripheral blood mononuclear cells (PBMCs) of a 62-year-old female from Type 2 diabetes mellitus (T2DM). Patient blood-derived cells were reprogrammed using the Sendai virus.

Hardware Trojans are intended malicious design modifications to integrated circuits, which can be used to launch powerful low-level attacks after being activated.A new security threat of lightweight stealthy hardware Trojans leveraging discrete satisfiability don't care signals was demonstrated.These don't care could not be satisfied under normal operation and thus the circuit design with Trojan is functionally equivalent to the Trojan-free baseline.The attacker could activate the Trojan through simple yet effective fault injection.Experimental results on a 1024-bit RSA cryptographic core show that the proposed hardware Trojan can escape from logic synthesis optimization, and that the RSA private key can be retrieved by simply over-clocking the design.A defense technique that can effectively detect such stealthy Trojan design was provided.

Diabetic retinopathy (DR) is one of the most common and severe microvascular complications of diabetes, and the leading cause of preventable blindness in working-aged people. Here, we generated an induced pluripotent stem (iPS) cell line using blood-derived cells from a patient with DR. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with Sendai virus.

Angiographically silent cystoid macular edema (CME) is a rare complication from nab-paclitaxel. Here we report a 45-year-old woman with breast cancer who developed CME after several months of treatment with albumin-bound paclitaxel (nab-paclitaxel). Her visual acuity did not improve significantly with the cessation of nab-paclitaxel and intravitreal ranibizumab treatment. Then, brinzolamide eye drops were prescribed. One month later, her vision improved, with the macular edema significantly subsided. Finally, we reviewed other cases of CME induced by nab-paclitaxel that have been reported in the literature and discussed the underlying pathogenesis of nab-paclitaxel-induced CME.

The pathogenesis of type 2 diabetes mellitus (T2DM) is commonly associated with altered gut bacteria. However, whether the microbial dysbiosis that exists in human diabetic patients with or without retinopathy is different remains largely unknown. Here, we collected clinical information and fecal samples from 75 participants, including 25 diabetic patients without retinopathy (DM), 25 diabetic patients with retinopathy (DR), and 25 healthy controls (HC). The gut microbial composition in the three groups was analyzed using 16S ribosomal RNA (rRNA) gene sequencing. Microbial structure and composition differed in the three groups. The α and β diversities in both the DM and DR groups were reduced compared with those in the HC group. Blautia was the most abundant genus, especially in the DM group. In addition, increased levels of Bifidobacterium and Lactobacillus and decreased levels of Escherichia-Shigella, Faecalibacterium, Eubacterium_hallii_group and Clostridium genera were observed in the DM and DR groups compared with the HC group. Furthermore, a biomarker set of 25 bacterial families, which could distinguish patients in the DR group from those in the DM and HC groups was identified, with the area under the curve values ranging from 0.69 to 0.85. Of note, Pasteurellaceae, which was increased in DM and decreased in DR compared with HC, generated a high AUC (0.74) as an individual predictive biomarker. Moreover, 14 family biomarkers were associated with fasting blood glucose levels or diabetes, with most of them being negatively correlated. In summary, our study establishes compositional alterations of gut microbiota in DM and DR, suggesting the potential use of gut microbiota as a non-invasive biomarker for clinical and differential diagnosis, as well as identifying potential therapeutic targets of diabetic retinopathy.

Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by the progressive photoreceptors and pigment epithelial cells dysfunction. Here, we report the human induced pluripotent stem cell line (iPSC) CSUASOi006-A, generated from urine-derived cells (UCs) of a 17-year-old male patient with clinically diagnosed RP carrying point mutation (c.C5792T) in the pre-mRNA processing factor 8 gene (PRPF8). The newly derived CSUASOi006-A cell line has the patient’s same mutation (c.C5792T) and could provide useful resources for studying the pathogenic mechanism of PRPF8-related RP.

We have established the patient-specific induced pluripotent stem (iPS) cell line CSUASOi004-A by using peripheral blood mononuclear cells (PBMCs) of a retinitis pigmentosa (RP) patient with a PRPF6 gene mutation (c.G2699A:p.R900H). CSUASOi004-A was established by a non-integrative method with four episomal plasmids containing the Yamanaka factors. The cell line with the specific point mutation had the typical features of normal iPS cells. For instance, the cells expressed pluripotency markers, generated all three germ layers and had a normal karyotype, and they can serve as a model for unravelling the pathogenic mechanisms underlying PRPF6-associated retinal degeneration.

Abstract:: Crumbs homologue 1 (CRB1) mutations have been found in retinitis pigmentosa (RP) patients lead to severe retinal dystrophies. The human induced pluripotent stem (iPS) cell line CSUASOi003-A derived from peripheral blood mononuclear cells (PBMCs) of a patient carrying two heterozygous mutations (2249G>A p.G750D and c.2809G>A p.A937T) in CRB1 gene was generated by non-integrative reprogramming technology. Pluripotency and differentiation capacity were assessed by immunocytochemistry and quantitative polymerase chain reaction (qPCR). The RP patient-specific iPS cell line provide a powerful model for evaluating the pathological phenotypes of the disease.

X-linked retinoschisis (XLRS) is a one of most common retinal genetic diseases of juvenile progressive vitreoretinal degeneration in males, which caused by the mutation of RS1 gene. In this study, an induced pluripotent stem cell (iPSC) line was generated from human peripheral blood mononuclear cells (PBMC) of a 13-year-old male patient with X-linked juvenile retinoschisis carrying a novel mutation in RS1 gene. The iPSCs exhibited iPSC morphology, expression of the pluripotency markers and in vitro differentiation potential, and the CSUASOi005-A iPSC line retained the original mutation (c.527T > A) of RS1 with a normal karyotype.

BackgroundDiabetic retinopathy (DR) is a main complication of diabetes mellitus (DM). Recent studies have implicated microRNAs in human retinal microvascular endothelial cell (HRMEC) dysfunction. In this study, we aim to investigate the apoptotic promotion of miR-29b-3p by blocking SIRT1 in HRMEC for DR situation.MethodBlood samples were obtained from DR patients and controls. Dual-luciferase reporter assay using HEK-293T cells was performed to show the direct interaction of miR-29b-3p and the 3′UTR of SIRT1. HRMECs were exposed to 5.5 mmol/L of glucose (normal control), 5.5 mmol/L of glucose and 24.5 mmol/L of mannitol (osmotic pressure control), 30 mmol/L of glucose [hyperglycemia (HG)], 150 μmol/L of CoCl2 (hypoxia), and 30 mmol/L of glucose plus 150 μmol/L of CoCl2 (HG-CoCl2). To identify the regulating relationship between miR-29b-3p and SIRT1, HRMECs were transfected with miR-29b-3p mimics/inhibitors or their negative controls. SRT1720 was used as a SIRT1 agonist. Cell viability was assessed with the cell counting kit-8 (CCK-8) assay, and apoptotic cells were stained by one-step terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit. Gene and protein expression were assayed by quantitative real-time reverse transcriptase-PCR (RT-qPCR) and western blotting separately.ResultMiR-29b-3p was upregulated to 3.2-fold, and SIRT1 protein was downregulated to 65% in DR patients. Dual-luciferase reporter assay showed the direct interaction of miR-29b-3p and SIRT1. HRMECs were identified as >95% positive for CD31 and von Willebrand factor (vWF). MiR-29b-3p and Bax/Bcl-2 ratio was upregulated, whereas SIRT1 was downregulated in HRMECs in the HG-CoCl2 condition. Decreased cell viability and upregulated apoptosis were also found in HRMECs of the HG-CoCl2 condition. Upregulated miR-29b-3p decreased the expression of SIRT1 and increased the ratio of Bax/Bcl-2, whereas downregulated miR-29b-3p increased the expression of SIRT1 protein and downregulated the ratio of Bax/Bcl-2. SRT1720 rescued miR-29b-3p-induced HRMEC apoptosis via upregulating the expression of SIRT1 protein.ConclusionThe dysregulation of miR-29b-3p/SIRT1 is a potential mechanism of HRMEC apoptosis in DR. MiR-29b-3p/SIRT1 may be a potential therapeutic target for DR.

We have established an induced pluripotent stem (iPS) cell line using urine-derived cells from a 27-year-old male patient with retinitis pigmentosa associated with point mutations in the USH2A gene. Feeder-free culture conditions and the integration-free CytoTune™-iPS 2.0 Sendai Reprogramming Kit were used.

We report the human induced pluripotent stem cell line (iPSC) CSUASOi002-A, generated from urine-derived cells (UCs) from a 51-year-old female patient carrying compound heterozygous mutations (c.62_63delTinsGA and c.C892T) in the carbohydrate sulfotransferase 6 gene (CHST6). This patient was from a Chinese family of three siblings with macular corneal dystrophy (MCD). Patient UCs were reprogrammed by electroporation using the episomal plasmids (OCT4, SOX2, KLF4, l-MYC, LIN28 and shP53). The human MCD-UiPS cell line CSUASOi002-A retained the disease-associated genotype, while expressed pluripotent stem cell markers and could be differentiated into cells of all three germ layers.

Retinitis pigmentosa (RP) represents a group of inherited retinopathies with early-onset nyctalopia followed by progressive photoreceptor degeneration causing irreversible vision loss. Mutations in USH2A are the most common cause of non-syndromic RP. Here, we reprogrammed induced pluripotent stem cells (iPSCs) from a RP patient with a mutation in USH2A (c.8559-2A > G/c.9127_9129delTCC). Then, multilayer retinal organoids including neural retina (NR) and retinal pigment epithelium (RPE) were generated by three-step “induction-reversal culture.” The early retinal organoids derived from the RP patient with the USH2A mutation exhibited significant defects in terms of morphology, immunofluorescence staining and transcriptional profiling. To the best of our knowledge, the pathogenic mutation (c.9127_9129delTCC) in USH2A has not been reported previously among RP patients. Notably, the expression of laminin in the USH2A mutation organoids was significantly lower than in the iPSCs derived from healthy, age- and sex-matched controls during the retinal organogenesis. We also observed that abnormal retinal neuroepithelium differentiation and polarization caused defective retinal progenitor cell development and retinal layer formation, disordered organization of NRs in the presence of the USH2A mutation. Furthermore, the USH2A mutation bearing RPE cells presented abnormal morphology, lacking pigmented foci and showing an apoptotic trend and reduced expression of specific makers, such as MITF, PEDF, and RPE65. In addition, the USH2A mutation organoids had lower expression of cilium-associated (especially CFAP43, PIFO) and dopaminergic synapse-related genes (including DLGAP1, GRIK1, SLC17A7, and SLC17A8), while there was higher expression of neuron apoptotic process-related genes (especially HIF1A, ADARB1, and CASP3). This study may provide essential assistance in the molecular diagnosis and screening of RP. This work recapitulates the pathogenesis of USH2A using patient-specific organoids and demonstrated that alterations in USH2A function due to mutations may lead to cellular and molecular abnormalities.

Previous research has shown that CXCR5−/− mice develop retinal degeneration (RD) with age, a characteristic related to age macular degeneration (AMD). RD in these mice is not well-understood, and in this study, we sought to characterize further the RD phenotype and to gain mechanistic insights into the function of CXCR5 in the retina. CXCR5−/− and WT control mice were used. Fundus images demonstrated a significant (p < 0.001) increase of hypo-pigmented spots in the retina of aged CXCR5−/− mice compared with WT control mice. PAS staining indicated localization of deposits in the sub-retinal pigment epithelia (RPE) layer. AMD-associated proteins Cryab, amyloid beta, and C3d were detected within the RPE/sub-RPE tissues by immunofluorescence (IF). In addition, western blot analysis of COX-2, Arg1, and VEGF-a revealed an increase in the signaling of these molecules within the RPE/choroid complex. Transmission electron microscopy (TEM) indicated a drusen-like structure of sub-RPE deposits with an accumulation of vacuolated cellular debris. Loss of photoreceptors was detected by peanut lectin staining and was corroborated by a reduction in MAP2 signaling. Loss of blood-retinal barrier integrity was demonstrated by a reduction of ZO-1 expression. Inflammatory cells were detected in the sub-RPE space, with an increase in IBA-1 positive microglia cells on the surface of the RPE. Mass spectrometry analysis of CXCR5−/− mouse RPE/choroid proteins extracts, separated by SDS-page and incubated with autologous serum, identified autoantibodies against AMD-associated proteins: Cryaa, Cryab, and Anxa2. In vitro evaluations in BV-2 cell culture indicated a significant increase in production of Arg-1 (p < 0.001) and COX-2 (p < 0.01) in the presence of anti-CXCR5 antibody when compared with Igg-treated control BV-2 cells stimulated with IL-4 and TNFα/IFNγ, respectively. Anti-CXCR5 antibody treatment without stimulating agents did not affect Arg-1 and COX-2 expression; this suggests that CXCR5 may have a regulatory role in microglia cells activation. These results indicate that with age, CXCR5−/− mice develop RD characterized by microglia dysfunction, increased production of CXCL13 in the RPE progressive photoreceptor, neuronal loss, and sub-RPE deposition of cellular debris, resulting in the production of immunogenic proteins and autoimmune-mediated RD.

X-linked juvenile retinoschisis (XLRS) is one of the most severely affected genetic causes of irreversible retinal degeneration diseases in young males, especially school-age boys. Here, we generated induced pluripotent stem cells (iPSCs) from a Chinese 11-year-old male with clinically diagnosed XLRS. Urine sample was collected with appropriate cooperation, then isolated cells were expanded for subsequent reprogramming procedure using integration-free Sendai virus. The newly derived CSUASOi001-A iPS cell line harboring the c.304C > T mutation in the RS1 gene (p.R102W) provides a useful resource to investigate pathogenic mechanisms in XLRS.

Glaucoma is characterized by progressive, irreversible damage to the retinal ganglion cells (RGCs) and their axons. Our previous study has shown that the intravitreal transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) reveals a neuroprotective role in microsphere injection-induced ocular hypertension (OHT) rat models. The protection is related to the modulation of glial cells, but the mechanisms are still unknown. The purpose of the present study is to clarify the potential neuroinflammatory mechanisms involved in the neuroprotective role of hUC-MSCs. OHT models were established with SD rats through intracameral injection of polystyrene microbeads. The animals were randomly divided into three groups: the normal group, the OHT+phosphate-buffered saline (PBS) group, and the OHT+hUC-MSC group. Retinal morphology was evaluated by measuring the inner retinal thickness via optical coherence tomography (OCT). Retinal cell apoptosis was examined by TUNEL staining and Bax expression 14 days following hUC-MSC transplantation. The expression levels of glial fibrillary acidic protein (GFAP), ionized calcium binding adapter molecule 1 (iba-1), and toll-like receptor 4 (TLR4) were assessed via immunohistochemistry, real-time quantitative PCR, and Western blot. RNA and proteins were extracted 14 days following transplantation, and the expression levels of the TLR4 signaling pathways and proinflammatory cytokines—myeloid differentiation factor 88 (MyD88), IL-1β, IL-6, and TNF-α—were determined. OCT showed that the intravitreal transplantation of hUC-MSCs significantly increased the inner thickness of the retina. A TUNEL assay and the expression of Bax suggested that the apoptosis of retinal cells was decreased by hUC-MSCs 14 days following transplantation. Intravitreal hUC-MSC transplantation resulted in a decreased expression of GFAP, iba-1, TLR4, MyD88, IL-1β, IL-6, and TNF-α 14 days following transplantation. In addition, via in vitro experiments, we found that the increased expression of the TLR4 signaling pathway induced by lipopolysaccharide (LPS) was markedly decreased after hUC-MSCs were cocultured with rMC-1 and BV2 cells. These findings indicate that hUC-MSC transplantation attenuates OHT-induced retinal neuroinflammation via the TLR4 pathway.