Single-cell transcriptomics reveals maturation of transplanted stem cell-derived retinal pigment epithelial cells toward native state.

Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.

Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources.

Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications.

ACUTE SYPHILITIC POSTERIOR PLACOID CHORIORETINITIS: MULTIMODAL IMAGING AND ELECTROPHYSIOLOGIC FINDINGS BEFORE AND AFTER TREATMENT.

PURPOSE: To describe the clinical course of acute syphilitic posterior placoid chorioretinitis (ASPPC) in the preplacoid stage, placoid stage, and after treatment with penicillin. METHOD: A retrospective case report of serial multimodal imaging and electrophysiology studies of a patient with ASPPC, with 18 months of follow-up. RESULTS: A 47-year-old man presented with bilateral panuveitis. The patient defaulted follow-up and returned when his vision deteriorated. Tests for neurosyphilis and retroviral disease were positive, and treatment was initiated. The earliest change on serial optical coherence tomography was loss of the signal from the reflective band corresponding to the ellipsoid zone. In the placoid stage, there was nodular thickening of the retinal pigment epithelium. The ellipsoid zone signals reappeared after treatment. Fundus fluorescein angiogram at presentation showed peripapillary vasculitis and disk leakage; indocyanine green angiography revealed multiple hypofluorescent spots in the peripapillary region and posterior pole that was not visible clinically. The angiographic abnormalities resolved after treatment. Electrophysiology demonstrated bilateral maculopathy and reduction of both a- and b-waves from dark-adapted and light-adapted responses at presentation. The b-waves (inner retina) recovered partially with treatment. CONCLUSION: To the best of our knowledge, this is the first case report of the multimodal imaging and electrophysiology findings in a patient with acute syphilitic posterior placoid chorioretinitis, before the development of the classic placoid lesion. Improvement of structural and functional pathology after systemic treatment is demonstrated.

A Pilot Study on MicroRNA Profile in Tear Fluid to Predict Response to Anti-VEGF Treatments for Diabetic Macular Edema.

(1) Background: Intravitreal anti-vascular endothelial growth factor (anti-VEGF) is an established treatment for center-involving diabetic macular edema (ci-DME). However, the clinical response is heterogeneous. This study investigated miRNAs as a biomarker to predict treatment response to anti-VEGF in DME. (2) Methods: Tear fluid, aqueous, and blood were collected from patients with treatment-naïve DME for miRNA expression profiling with quantitative polymerase chain reaction. Differentially expressed miRNAs between good and poor responders were identified from tear fluid. Bioinformatics analysis with the miEAA tool, miRTarBase Annotations, Gene Ontology categories, KEGG, and miRWalk pathways identified interactions between enriched miRNAs and biological pathways. (3) Results: Of 24 participants, 28 eyes received bevacizumab (15 eyes) or aflibercept (13 eyes). Tear fluid had the most detectable miRNA species (N = 315), followed by serum (N = 309), then aqueous humor (N = 134). MiRNAs that correlated with change in macular thickness were miR-214-3p, miR-320d, and hsa-miR-874-3p in good responders; and miR-98-5p, miR-196b-5p, and miR-454-3p in poor responders. VEGF-related pathways and the angiogenin-PRI complex were enriched in good responders, while transforming growth factor-ß and insulin-like growth factor pathways were enriched in poor responders. (4) Conclusions: We reported a panel of novel miRNAs that provide insight into biological pathways in DME. Validation in larger independent cohorts is needed to determine the predictive performance of these miRNA candidate biomarkers.

Systemic Factors Associated with Treatment Response in Diabetic Macular Edema.

PURPOSE: To identify systemic factors that may influence the response to anti-VEGF therapy in patients with diabetic macular edema (DME). METHODS: 35 patients undergoing anti-VEGF injections for centre-involving DME were studied in this prospective observational study. The primary outcome was change in macular thickness one month after treatment, measured using spectral-domain optical coherence tomography (OCT). At baseline, information on various systemic factors was collected including glycosylated hemoglobin (HbA1c), serum VEGF levels, lipid profile and markers of renal function, and blood pressure. Thirty-three of the 35 patients were included in this study. Nonparametric statistical tests were used for the analysis of the data in view of the nonnormal distribution of the outcome variables. Multivariate analysis was performed using logistic regression. Stata 12.1 software was used for the analysis. Main Outcome Measures. Reduction in macular central subfield thickness (on spectral-domain OCT) and change in logMAR visual acuity at one month after injection. RESULTS: Lower HbA1c levels (7% or less) were significantly associated with greater reduction in central macular subfield thickness at one month after injection of bevacizumab or ranibizumab on both univariate analysis (p=0.012) and multivariate analysis (p=0.012) and multivariate analysis (. CONCLUSIONS: Better glycemic control is associated with a greater reduction in central macular thickness after the first injection of bevacizumab or ranibizumab in diabetic macular edema. Patients with high levels of HbA1c and poor response to anti-VEGF may benefit from strict control of their blood glucose.

Six-year incidence and progression of diabetic retinopathy in Indian adults: the Singapore Indian Eye study.

AIMS: Diabetes is a major public health problem in migrants and ethnic minorities worldwide. We determined the incidence and risk factors of diabetic retinopathy (DR) in migrant Indians living in Singapore. METHODS: We included data from 759 Indian adults with diabetes, who participated in the baseline (aged 40-80 years, 2007-2009) and 6-year follow-up 2012-2015 of the Singapore Indian Eye Study. Retinal photographs were graded for the presence and severity of DR using modified Airlie House Classification. Incidence was assessed in participants who were free of DR at baseline visit (n=501), while progression in those with DR but free of proliferative DR at baseline visit (n=189). Risk factors included demographic, lifestyle, socioeconomic, family history, genes, duration of diabetes, glycaemic control, insulin use, ocular and clinical factors. RESULTS: The 6-year age-standardised DR incidence and progression were 21.89% and 33.45%, respectively. HbA1c (risk ratio (RR) 1.41, 95% CI 1.28 to 1.55 per unit increase), current smoking (RR 1.63, 95% CI 1.02 to 2.62) and insulin use (RR 2.63, 95% CI 1.44 to 4.82) were associated with higher incidence, whereas estimated cerebrospinal fluid pressure (RR 0.90, 95% CI 0.82 to 0.98) and body mass index (BMI) (RR 0.74, 95% CI 0.60 to 0.93) were associated with lower incidence of DR. Higher HbA1c (RR 1.26, 95% CI 1.13 to 1.42), BMI (RR 1.26, 95% CI 1.02 to 1.56) and estimated cerebrospinal fluid pressure (RR 1.11, 95% CI 1.02 to 1.21) were associated with DR progression. The population attributable risk of HbA1c >8% was 41.29% and 49.63% for DR incidence and progression. CONCLUSION: DR incidence and progression in migrant Indians living in Singapore was more than double that reported in Indians living in urban India. Consistent with past studies, poor glycaemic control was an important predictor for incidence and progression of DR.

Peripheral retinal changes in highly myopic young Asian eyes.

PURPOSE: To determine the type and prevalence of peripheral retinal changes and its relationship with axial length (AL) in a population of young Asian adult males. METHODS: This was a cross-sectional study of male subjects aged between 19 and 25 years old. High myopes [spherical equivalent refraction, (SER) < -6.00 D] were recruited and underwent further ocular investigations including dilated retinal photography and binocular indirect ophthalmoscopy by two trained ophthalmologists. The frequency of peripheral retinal changes within high myopes was correlated with axial length and also compared with a group of emmetropes (SER between -0.50 D and +1.00 D). RESULTS: A total of 593 high myopes and 156 emmetropes were recruited. White without pressure (WWOP; n = 458, 46.5%) and lattice degeneration (LD; n = 109, 14.6%) were the commonest findings, and their prevalence was significantly higher among high myopes (LD, 16.9% versus 5.8%; WWOP, 57.2% versus 5.8%; both p < 0.001). Among high myopes, temporal LD was more common (71%) and 35% had more than one area in the same eye. Increasing AL was associated with LD (odds ratio 1.28, p = 0.01) and retinal holes (odd's ratio 1.44, p = 0.02) on multivariate analysis. CONCLUSION: White without pressure (WWOP) and LD were the commonest peripheral retinal changes. One-third of high myopes with LD had more than one area in the same eye. Increasing AL was associated with LD and retinal holes. Studies in older adults should be conducted to develop clinical guidelines for the management of high myopes.

RANIBIZUMAB AND AFLIBERCEPT FOR THE TREATMENT OF PIGMENT EPITHELIAL DETACHMENT IN NEOVASCULAR AGE-RELATED MACULAR DEGENERATION: Data from an Observational Study.

PURPOSE: To assess the effect of intravitreal ranibizumab and aflibercept on retinal pigment epithelial detachment (RPED) in patients with neovascular age-related macular degeneration. METHODS: This was a retrospective analysis of data from a prospectively designed and implemented clinical audit. Analysis included change in RPED dimensions and visual acuity in 92/233 treatment-naive eyes with neovascular age-related macular degeneration and RPED 6 months after treatment with either aflibercept or ranibizumab. RESULTS: There was no significant between-group difference in the adjusted mean change for maximum RPED height (P = 0.195), diameter (P = 0.522) or visual acuity (P = 0.836) at 6 months. Injection frequency was the only clinical variable that affected RPED height (P = 0.050) and visual acuity change for both treatment groups (P = 0.004). Around 30% of eyes in each group had complete resolution of RPED at 6 months. CONCLUSION: Eyes with neovascular age-related macular degeneration and RPED showed significant functional and anatomical responses after 6 months of intravitreal anti-vascular endothelial growth factor injections. However, we found no significant difference in anatomical response or change in visual acuity between eyes treated with ranibizumab compared with aflibercept. Larger, prospectively designed, randomized studies with longer term follow-up may identify a difference between the two drugs that we did not detect.

Quantitative Changes in Pigment Epithelial Detachment Area and Volume Predict Retreatment in Polypoidal Choroidal Vasculopathy.

PURPOSE: To determine if changes in pigment epithelial detachment (PED) area and volume predict retreatment in polypoidal choroidal vasculopathy (PCV). DESIGN: Retrospective case-control study. METHODS: PCV patients on pro re nata (PRN) anti-vascular endothelial growth factor (VEGF) therapy with >1 year follow-up at an academic retina service were included. Monthly anti-VEGF injections were given until a dry macula was achieved, and treatment deferred. Retreatment indication was recurrence of intraretinal or subretinal fluid or new hemorrhage. PED area and volume changes between visits with a dry macula ("D") and immediate preceding visits ("D-1") were analyzed with an automated optical coherence tomography-based software. Univariate and multivariate analyses were conducted to determine associations between changes in PED parameters and retreatment need at immediate subsequent visits ("D+1"). RESULTS: Twenty-two PCV patients (mean age 69.6 years) were included. Of 46 visits D, 11 (23.9%) were followed by retreatment at D+1. An increase in PED area (>0.43 mm2) and volume (>0.0245 mm3) from D-1 to D was associated with 18.2 (95% CI, 3.7-125.6; P < .001) and 101.9 (95% CI, 9.5-14 308.0; P < .001) higher retreatment odds at D+1, respectively. These associations remained significant after multivariate analyses adjusting for baseline PED area or volume, greatest linear dimension, and type of anti-VEGF agent. CONCLUSION: In PCV on PRN anti-VEGF therapy, increases in PED area and volume at one visit, despite achievement of a dry macula, are associated with retreatment at the next visit. Retreatment criteria relying on intraretinal or subretinal fluid or new hemorrhages may be expanded to include PED changes. Studies are needed to determine if using PED parameters in treatment decisions reduces recurrences.

Peripapillary choroidal thickness in young Asians with high myopia.

PURPOSE: To describe the topography and predictors of peripapillary choroidal thickness (PPCT) in highly myopic eyes of young, healthy, Asian subjects. METHODS: A total of 870 young male subjects aged 21.63 ± 1.15 years were recruited from the Singapore military. Choroidal imaging was performed using enhanced depth imaging (EDI) spectral-domain optical coherence tomography (SD-OCT). Peripapillary choroidal thickness was manually measured at eight locations around the optic disc. RESULTS: We analyzed 448 subjects with high myopia (defined as spherical equivalent [SE] worse than -6.0 diopters [D]) and 116 with emmetropia (SE > -0.5 and < 0.5 D). The mean SE was -8.52 ± 1.20 D for the high-myopic group, and 0.11 ± 0.24 D for the emmetropic group. The mean peripapillary choroid was significantly thinner (142.62 ± 43.84 µm) in high myopes compared with emmetropes (181.90 ± 46.43 µm, P < 0.001). Likewise, PPCT showed further decrease with increase in degree of myopic refractive error. Distribution of PPCT showed a markedly different pattern in high-myopic eyes (thickest superiorly) and emmetropic eyes (thickest temporally). However, peripapillary choroid in both the groups was thinnest at the inferior location. Among the ocular factors studied, axial length, IOP, presence of posterior staphyloma, and chorioretinal atrophy were the factors significantly associated with PPCT. CONCLUSIONS: Highly myopic eyes have significantly thinner peripapillary choroid and showed different distribution of thickness, compared with emmetropes. Axial length, IOP, and presence of posterior staphyloma and chorioretinal atrophy significantly influence PPCT and should be taken into consideration during clinical interpretation of PPCT measurement.

Choroidal thickness and high myopia: a case-control study of young Chinese men in Singapore.

PURPOSE: To determine the distribution of choroidal thickness (CT) and ocular factors associated with CT in high myopic eyes in comparison with emmetropic eyes of young healthy adults. METHODS: A case-control study of 648 young, male subjects, including 520 high myopes and 128 emmetropes. Choroidal imaging was performed using enhanced depth imaging spectral domain optical coherence tomography. Images were postprocessed using adaptive compensation for quality enhancement. CT was measured at nine locations, including subfovea and 1.5 and 3 mm nasal, temporal, superior and inferior to fovea. RESULTS: The CT at the subfovea was significantly thinner (mean ± standard error: 225.87 ± 5.51 µm) for high myopes compared to emmetropes (375.15 ± 6.58 µm, p < 0.001). Likewise, CT in high myopic group was significantly thinner than emmetropic control group at all locations (p for trend <0.001 for all locations). Distribution of CT showed a markedly different pattern in high myopic eyes (thickest superiorly at 3 mm, 265.97 ± 5.97 µm) and emmetropic eyes (thickest subfoveally, 375.15 ± 6.58 µm). Choroid was thinnest at nasal 3 mm location in both the myopic (108.85 ± 3.97 µm) and emmetropic (238.25 ± 6.72 µm) groups. Among the ocular factors studied, axial length, posterior staphyloma and chorio-retinal atrophy were the significant predictors of CT. CONCLUSIONS: Highly myopic eyes have significantly thinner choroid and showed different distribution pattern, compared to emmetropes. Axial length, posterior staphyloma and chorio-retinal atrophy are the strongest determinants of CT.

Prevalence and risk factors for retinopathy in persons without diabetes: the Singapore Indian Eye Study.

PURPOSE: To describe prevalence and risk factors for retinopathy in an Asian Indian population without diabetes. METHODS: A population-based cross-sectional study of 3400 Indians aged 40-80 years residing in Singapore was conducted. Retinopathy was assessed from retinal photographs by trained graders using modified Airlie House Classification System. Risk factors were assessed from standardized interviews, clinical examinations and laboratory investigations. Diabetes mellitus was defined as glycosylated haemoglobin ≥6.5%, use of diabetic medication or physician diagnosis of diabetes. RESULTS: Among the 1900 individuals without diabetes, mean HbA1c was 5.7% and mean systolic blood pressure was 132.4 mmHg. Age-standardized prevalence of retinopathy was 5.05% (n = 98; 95% confidence interval [CI], 4.07-6.21), with no significant difference in retinopathy prevalence between males (6.15%) and females (4.13%). Among non-diabetic persons with retinopathy, 96.9% (n = 95) had signs of minimal-to-mild retinopathy while 3.06% (n = 3) had moderate-to-severe retinopathy. After adjusting for multiple covariables, retinopathy signs were associated with higher levels of HbA1c (odds ratio [OR], 2.4; 95% CI, 1.3-4.5; per% increase), systolic blood pressure (OR, 1.02; 95% CI, 1.01-1.03; per mmHg increase) and serum creatinine (OR, 1.005; 95% CI, 1.002-1.009; per mm increase), but not C-reactive protein, cigarette smoking or lipid levels. CONCLUSION: One in 20 Asian Indian persons without diabetes had retinopathy signs. Risk factors for these signs include higher glycosylated haemoglobin, systolic blood pressure and serum creatinine.

Six-month visual prognosis in eyes with submacular hemorrhage secondary to age-related macular degeneration or polypoidal choroidal vasculopathy.

BACKGROUND: To determine clinical or imaging prognostic features for visual outcome in eyes with submacular hemorrhage secondary to age-related macular degeneration (AMD) or polypoidal choroidal vasculopathy (PCV). METHODS: A prospective case series of 11 eyes from 11 patients with submacular hemorrhage secondary to AMD or PCV. All participants had measurement of clinical characteristics, fundus angiogram, and indocyanine green angiography, spectral domain optical coherence tomography (OCT, Cirrus, Zeiss) at baseline and 6 months. RESULTS: Median visual acuity improved from 20/132 to 20/63 at month 6. The median improvement in vision was 0.20 LogMAR units. Proportion of eyes with best-corrected visual acuity (BCVA) ≥ 1.0 increased from 6/11 (54.5 %) at baseline to 8/11 (72.7 %) at month 6. Eyes with BCVA > 1.0 were more likely to have larger area of hemorrhage and thinner subfoveal neurosensory retinal thickness at baseline and at month 6. CONCLUSIONS: Thinner neurosensory retina demonstrated on OCT at baseline may be a useful prognostic sign for limited visual recovery.

Accuracy of diabetic retinopathy screening by trained non-physician graders using non-mydriatic fundus camera.

INTRODUCTION: We compared the agreement of diabetic retinopathy (DR) assessment between trained non-physician graders (NPGs) and family physicians (FPs) in a primary healthcare setting. METHODS: This was a cross-sectional study conducted retrospectively over a period of one month. The participants were diabetic patients from two primary healthcare clinics (polyclinics) in Singapore. Single-field digital retinal images were obtained using a non-mydriatic 45-degree fundus camera. Retinal images were graded for the presence or absence of DR by FPs at the polyclinics and by NPGs at a central ocular grading centre. The FPs' and NPGs' assessments of DR were compared with readings by a single retinal specialist (reference standard). RESULTS: A total of 367 diabetic patients (706 eyes) were included in the study. The mean age of the patients was 63 years, and the majority were Chinese (83.8%). For DR assessment, the agreement between NPGs and the retinal specialist was substantial (ĸ = 0.66), while the agreement between FPs and the retinal specialist was only fair (ĸ = 0.40). NPGs' assessment showed higher sensitivity (70% vs. 45%) and comparable specificity (94% vs. 92%) as compared to FPs' assessment. The area under the receiver operating characteristic curve of NPGs' assessment of DR was greater than that of the FPs' (0.82 vs. 0.69, p < 0.001). CONCLUSION: This study has demonstrated that trained NPGs are able to provide good detection of DR and maculopathy from fundus photographs. Our findings suggest that DR screening by trained NPGs may provide a costeffective alternative to FPs.

A truly knotless technique for scleral fixation of intraocular lenses: two-year results.

Scleral fixated intraocular lens (SFIOL) is a safe and effective option for managing optical aphakia. Suture related complications like suture erosion, suture breakage, endophthalmitis, etc. are unique to SFIOL. The knots can be covered by partial thickness flaps or they can be rotated into scleral tissues without flaps to reduce the complications. We performed a recently described novel technique which obviates the need for knot and scleral flaps in securing the SFIOL. This novel 2-point Ab externo knotless technique may reduce the knot related problems. Twenty-three eyes undergoing this knotless SFIOL procedure were analyzed for intraoperative and postoperative complications. Twenty-two eyes either maintained or improved on their preoperative vision. All patients had a minimum follow-up of 24 months.

Asian age-related macular degeneration phenotyping study: rationale, design and protocol of a prospective cohort study.

BACKGROUND: Current knowledge of the phenotypic characteristics (e.g. clinical features, risk factors, natural history and treatment response) of age-related macular degeneration (AMD) in Asians remains limited. This report summarizes the rationale and study design of a prospective observational study of Asian neovascular AMD, including polypoidal choroidovasculopathy variant. DESIGN: The Asian AMD phenotyping study is a prospective, observational clinical study of Asian patients with neovascular AMD or polypoidal choroidovasculopathy in three tertiary eye centres in Singapore. PARTICIPANTS: The study aims to recruit 500 consecutive patients from the retinal clinics of three tertiary eye centres in Singapore. METHODS: Standardized examination procedures include interviews, a comprehensive eye examination, digital photography of the retina, fundus fluorescein and indocyanine green angiography and spectral domain optical coherence tomography using a standardized protocol. Blood samples were collected for biochemical analyses and stored for genetic and proteomic studies. MAIN OUTCOME MEASURES: The aim of the study was to build a comprehensive database of clinical, angiographic, functional and natural history data of Asian AMD over a 12-month follow-up period. RESULTS: This article discusses the methodology and design of this prospective multi-centred study. CONCLUSION: This study will provide in-depth longitudinal data of the evolution of clinical features, risk factors, natural history and treatment pattern and response of Asian AMD and polypoidal choroidovasculopathy, allowing unique insights into pathogenesis and the design of new treatment strategies.