A rabbit model for outer retinal atrophy caused by surgical RPE removal.

PURPOSE: We aimed to establish a rabbit model with retinal atrophy induced by an iatrogenic retinal pigment epithelium (RPE) removal, for future testing of the efficacy and safety of cell therapy strategies. METHODS: A localized detachment of the retina from the RPE/choroid layer was created in 18 pigmented rabbits. The RPE was removed by scraping with a custom-made extendable loop instrument. The resulting RPE wound was observed over a time course of 12 weeks with optical coherence tomography and angiography. After 4 days (group 1) and 12 weeks (group 2), histology was done and staining with hematoxylin and eosin, as well as immunofluorescence performed to further investigate the effects of debridement on the RPE and the overlying retina. RESULTS: Already after 4 days, we observed a closure of the RPE wound by proliferating RPE and microglia/macrophage cells forming a multilayered clump. This pattern continued over the observation time course of 12 weeks, whereby the inner and outer nuclear layer of the retina became atrophic. No neovascularization was observed in the angiograms or histology. The observed changes were limited to the site of the former RPE wound. CONCLUSIONS: Localized surgical RPE removal induced an adjacent progressive retinal atrophy. Altering the natural course of this model may serve as a basis to test RPE cell therapeutics.

Sodium-Iodate Injection Can Replicate Retinal Degenerative Disease Stages in Pigmented Mice and Rats: Non-Invasive Follow-Up Using OCT and ERG.

PURPOSE: The lack of suitable animal models for (dry) age-related macular degeneration (AMD) has hampered therapeutic research into the disease, so far. In this study, pigmented rats and mice were systematically injected with various doses of sodium iodate (SI). After injection, the retinal structure and visual function were non-invasively characterized over time to obtain in-depth data on the suitability of these models for studying experimental therapies for retinal degenerative diseases, such as dry AMD. METHODS: SI was injected into the tail vein (i.v.) using a series of doses (0-70 mg/kg) in adolescent C57BL/6J mice and Brown Norway rats. The retinal structure and function were assessed non-invasively at baseline (day 1) and at several time points (1-3, 5, and 10-weeks) post-injection by scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), and electroretinography (ERG). RESULTS: After the SI injection, retinal degeneration in mice and rats yielded similar results. The lowest dose (10 mg/kg) resulted in non-detectable structural or functional effects. An injection with 20 mg/kg SI did not result in an evident retinal degeneration as judged from the OCT data. In contrast, the ERG responses were temporarily decreased but returned to baseline within two-weeks. Higher doses (30, 40, 50, and 70 mg/kg) resulted in moderate to severe structural RPE and retinal injury and decreased the ERG amplitudes, indicating visual impairment in both mice and rat strains. CONCLUSIONS: After the SI injections, we observed dose-dependent structural and functional pathological effects on the retinal pigment epithelium (RPE) and retina in the pigmented mouse and rat strains that were used in this study. Similar effects were observed in both species. In particular, a dose of 30 mg/kg seems to be suitable for future studies on developing experimental therapies. These relatively easily induced non-inherited models may serve as useful tools for evaluating novel therapies for RPE-related retinal degenerations, such as AMD.

Persistent macular holes - what is the best strategy for revision?

PURPOSE: This study aims to analyze the success rate and functional outcome after revision surgery of persistent idiopathic full-thickness macular holes in a large patient cohort and to identify the optimal tamponade strategy and the value of new adjunctive manipulation techniques for persistent macular hole (pMH) closure. METHODS: Retrospective, comparative, non-consecutive case series of all revisional surgeries for idiopathic pMH between 2011 and 2019 at the Eye Clinic Sulzbach were identified. Of 1163 idiopathic MH surgeries, 74 eyes of 74 patients had pMH. Of those, group 1 (n = 38) had vitrectomy with tamponade alone (20% sulfur hexafluoride gas, 15% hexafluoroethane gas, silicone oil 5000, Densiron®), while group 2 (n = 36) included tamponade with adjuvant manipulation (internal limiting membrane (ILM) translocation, subretinal fluid injection, epiretinal amniotic membrane, free retina graft, or autologous blood). Main statistical outcomes were anatomic closure rate, visual acuity (VA), minimum linear diameter (MLD), and base diameter (BD). RESULTS: Overall total anatomical success rate was 81.1% and mean VA improved 3.5 lines from LogMAR 1.03 ± 0.30 to 0.68 ± 0.38 (p < .001). Preoperative MLD or BD had no effect on total anatomic success (p = 0.074, p = 0.134, respectively). When comparing the two groups, slightly better anatomic success rates were achieved in group 1 (84.2%) compared to that in group 2 (77.8%) (p = 0.68). Final VA in group 1 (LogMAR 0.67 ± 0.39) outperformed group 2 (LogMAR 0.86 ± 0.38) (p = 0.03). CONCLUSIONS: Revisional surgery for persistent idiopathic MH with tamponade alone had comparable anatomical closure but better VA outcomes, compared to tamponade with adjuvant manipulation.

Publisher Correction: The choice of biopolymer is crucial to trigger angiogenesis with vascular endothelial growth factor releasing coatings.

A Correction to this paper has been published: https://doi.org/10.1007/s10856-020-06463-w.

The choice of biopolymer is crucial to trigger angiogenesis with vascular endothelial growth factor releasing coatings.

Bio-based coatings and release systems for pro-angiogenic growth factors are of interest to overcome insufficient vascularization and bio-integration of implants. This study compares different biopolymer-based coatings on polyethylene terephthalate (PET) membranes in terms of coating homogeneity and stability, coating thickness in the swollen state, endothelial cell adhesion, vascular endothelial growth factor (VEGF) release and pro-angiogenic properties. Coatings consisted of carbodiimide cross-linked gelatin type A (GelA), type B (GelB) or albumin (Alb), and heparin (Hep), or they consisted of radically cross-linked gelatin methacryloyl-acetyl (GM5A5) and heparin methacrylate (HepM5). We prepared films with thicknesses of 8-10 µm and found that all coatings were homogeneous after washing. All gelatin-based coatings enhanced the adhesion of primary human endothelial cells compared to the uncoated membrane. The VEGF release was tunable with the loading concentration and dependent on the isoelectric points and hydrophilicities of the biopolymers used for coating: GelA-Hep showed the highest releases, while releases were indistinguishable for GelB-Hep and Alb-Hep, and lowest for GM5A5-HepM5. Interestingly, not only the amount of VEGF released from the coatings determined whether angiogenesis was induced, but a combination of VEGF release, metabolic activity and adhesion of endothelial cells. VEGF releasing GelA-Hep and GelB-Hep coatings induced angiogenesis in a chorioallantoic membrane assay, so that these coatings should be considered for further in vivo testing.

In-depth mass spectrometric mapping of the human vitreous proteome.

Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from "surrogate normal patients" with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.

Effect of novel vital dyes on outer blood-retina barrier function in cultured human retinal pigment epithelium.

PURPOSE: To assess tight junction (TJ) integrity in cultured human fetal retinal pigment epithelium (HFRPE) after exposure to clinically relevant novel vital dyes. METHODS: HFRPE floater cells were harvested from RPE primary cultures of 4 donor eyes and seeded on polyester Transwell® for 4-6 weeks. The apical compartments of well-differentiated cultures were exposed to 0.005 mg/ml Coomassie violet R200 (CVR), methyl 2B (M2B) or Orange II. Periods of 30-300 s were chosen to mimic surgical exposure times, while 3 h was used for toxicity assays, with subsequent washout. Cell-cell junctions were studied by immunofluorescence (zonula occludens-1, ZO-1). Transepithelial electrical resistance (TER) was measured regarding blood-retina barrier (BRB) function. RESULTS: At 4-6 weeks after confluence, HFRPE had grown into pigmented hexagonal monolayers with stable TER values (451-1,520 Ω·cm(2)). After 300-second dye treatments, a continuous ZO-1 signal was detected in all vital dye-treated groups 1.5 h after exposure, whereas trypsin controls showed patchy loss of the TJ stain. TER of CVR-, M2B- and Orange-II-treated groups had dropped 1.5 h after exposure to 148 ± 58.4, 162 ± 23.7 and 164 ± 18.5 Ω·cm(2), respectively, compared to 73 ± 44.9 Ω·cm(2) in positive controls. After 3 h of exposure to 0.005 mg/ml vital dyes in thick drops, TER maintained similar levels to those prior to exposure (90.8 ± 4.7% of the original values, 93.8 ± 6.5 and 91.9 ± 3.6%, respectively), together with no difference from the vehicle controls (94.8 ± 6.6%). TER values recovered in all groups to prior levels within 3 days. CONCLUSION: Novel vital dyes (CVR, M2B and Orange II) caused no outer BRB function alteration.

Safety and effectiveness of pre-emptive diabetic vitrectomy in patients with severe, non-fibrotic retinal neovascularisation despite panretinal photocoagulation.

OBJECTIVES: To investigate the safety and effectiveness of pre-emptive vitrectomy in eyes with severe non-fibrotic proliferative diabetic retinopathy. METHODS: A multi-centre, retrospective, observational study. Pre-emptive vitrectomy was performed in non-fibrotic diabetic eyes with a visual acuity (VA) of 20/50 or better, where there was extensive persistent neovascularisation despite prior panretinal photocoagulation, and where the fellow eye had established sight loss despite vitrectomy for tractional complications. The primary outcome measure was the VA at last visit. RESULTS: Twenty patients were included. The mean age was 39 ± 14 years. Fifteen patients were Type 1 diabetic. The median baseline VA was 20/30 and remained stable at 20/28 at last visit (median follow-up period: 24 months). Eight eyes (40.0%) developed post-operative vitreous cavity haemorrhage; 4 of which required a vitreous cavity washout procedure. There were no post-operative retinal detachments. The index eye remained the significantly better eye at all time points bar one month post-surgery. Regression of retinopathy grading was observed in all eyes. CONCLUSION: In this pilot study, we found no sight loss with pre-emptive diabetic vitrectomy. Better eye status was maintained in this high-risk group. Further study with larger number of patients and longer-term follow-up is indicated.

Release and velocity of micronized dexamethasone implants with an intravitreal drug delivery system: kinematic analysis with a high-speed camera.

PURPOSE: Ozurdex, a novel dexamethasone (DEX) implant, is released by a drug delivery system into the vitreous cavity. We analyzed the mechanical release aperture of the novel applicator, obtained real-time recordings using a high-speed camera system and performed kinematic analysis of the DEX application. DESIGN: Experimental study. METHODS: : The application of intravitreal DEX implants (6 mm length, 0.46 mm diameter; 700 µg DEX mass, 0.0012 g total implant mass) was recorded by a high-speed camera (500 frames per second) in water (Group A: n = 7) or vitreous (Group B: n = 7) filled tanks. Kinematic analysis calculated the initial muzzle velocity as well as the impact on the retinal surface at approximately 15 mm of the injected drug delivery system implant in both groups. A series of drug delivery system implant positions was obtained and graphically plotted over time. RESULTS: High-speed real-time recordings revealed that the entire movement of the DEX implant lasted between 28 milliseconds and 55 milliseconds in Group A and 1 millisecond and 7 milliseconds in Group B. The implants moved with a mean muzzle velocity of 820 ± 350 mm/s (±SD, range, 326-1,349 mm/s) in Group A and 817 ± 307 mm/s (±SD, range, 373-1,185 mm/s) in Group B. In both groups, the implant gradually decelerated because of drag force. With greater distances, the velocity of the DEX implant decreased exponentially to a complete stop at 13.9 mm to 24.7 mm in Group A and at 6.4 mm to 8.0 mm in Group B. Five DEX implants in Group A reached a total distance of more than 15 mm, and their calculated mean velocity at a retinal impact of 15 mm was 408 ± 145 mm/s (±SD, range, 322-667 mm/s), and the consecutive normalized energy was 0.55 ± 0.44 J/m (±SD). In Group B, none of the DEX implants reached a total distance of 6 mm or more. An accidental application at an angle of 30 grade and consecutively reduced distance of approximately 6 mm may result in a mean velocity of 844 and mean normalized energy of 0.15 J/m (SD ± 0.47) in a water-filled eye. CONCLUSION: The muzzle velocity of DEX implants is approximately 0.8 m/s and decreases exponentially over distance. The drag over time in vitreous is faster than in water. The calculated retinal impact energy does not reach reported damage levels for direct foreign bodies or other projectiles.

A nanofibrillar surface promotes superior growth characteristics in cultured human retinal pigment epithelium.

BACKGROUND: To evaluate the influence of surface topography on the proliferation of the retinal pigment epithelium (RPE) by comparing nanofibrillar and smooth substrates. METHODS: Electrospun polyamide nanofibers (EPN) are an engineered surface mimicking native basement membranes. Commonly used plastic (polystyrene, PS) and glass substrates have a smooth topography. All were analyzed by scanning electron microscopy. RPE cultures were established from fetal and adult donors. Growth curves were established on the above substrates. Cell cycle and growth fractions were analyzed with 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI). RESULTS: At a magnification of ×5,000, EPN showed randomly overlapping fibers and pores. The surface of glass was slightly studded yet regular, in contrast to ideally smooth PS. Polygonal cells grew on nanofibers in a colony-like distribution, while randomly spread spindle-shaped cell morphologies were seen on smooth surfaces. This was observed at all donor ages. Initial proliferation rates were higher on EPN, and similar final cell densities were reached in all age groups, compared to an age-related decline on PS. EdU/DAPI revealed faster cell cycles on EPN. Growth fractions were higher and maintained longer on EPN. Observed substrate differences in growth behavior were statistically significant. CONCLUSION: Surface topography appears to induce distinct RPE proliferation characteristics.

Hints for Gentle Submacular Injection in Non-Human Primates Based on Intraoperative OCT Guidance.

Purpose: Delivery of Advanced Therapy Medicinal Products to the submacular space is increasingly evolving into a therapeutic modality. Cell replacement for age-related macular degeneration (AMD) and gene therapy for RPE65 are recent successful examples. Herein, a nonhuman primate (NHP) model was used to investigate surgical means to detach the macula. Methods: Sixteen eyes of 13 healthy macaques underwent a 25-gauge vitrectomy and subretinal injection of balanced salt solution monitored by microscope-integrated intraoperative optical coherence tomography (miOCT). The animals were followed with OCT and histology. Results: The miOCT monitoring allowed a more precise definition of surgical trauma ranging from an initial full-thickness foveal tear, or induction of a cystoid macular edema (CME), until no foveal defect was discernible, as the technique improved. However, as the subretinal fluid wave detached the fovea, the aforementioned lesions formed, whereas persistent retinal adhesion reproducibly proved to remain in the distal parafoveal semi-annulus. Measures to reduce foveal trauma during submacular fluid injection included reducing intraocular pressure, injection volume, and velocity, as well as the retinal location for bleb initiation, use of a vitreous tamponade, and a dual-bore subretinal cannula. Conclusions: A stable very low intraocular pressure and careful subretinal injection may avoid tangential macular stretching or mechanical CME formation, while vitreous tamponade may facilitate a more lamellar subretinal flow, all thereby reducing foveal trauma during submacular injection in NHP. Translational Relevance: These results can be relevant to any submacular surgery procedure used today, as they synergistically reduce the risk of compromising foveal integrity.

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy. This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

Submacular integration of hESC-RPE monolayer xenografts in a surgical non-human primate model.

BACKGROUND: Human pluripotent stem cells (hPSCs) provide a promising cell source for retinal cell replacement therapy but often lack standardized cell production and live-cell shipment logistics as well as rigorous analyses of surgical procedures for cell transplantation in the delicate macula area. We have previously established a xeno- and feeder cell-free production system for hPSC differentiated retinal pigment epithelial (RPE) cells, and herein, a novel immunosuppressed non-human primate (NHP) model with a disrupted ocular immune privilege is presented for transplanting human embryonic stem cell (hESC)-derived RPE on a scaffold, and the safety and submacular graft integration are assessed. Furthermore, the feasibility of intercontinental shipment of live hESC-RPE is examined. METHODS: Cynomolgus monkeys were systemically immunosuppressed and implanted with a hESC-RPE monolayer on a permeable polyester-terephthalate (PET) scaffold. Microscope-integrated intraoperative optical coherence tomography (miOCT)-guided surgery, postoperative follow-up incorporated scanning laser ophthalmoscopy, spectral domain (SD-) OCT, and full-field electroretinography (ERG) were used as outcome measures. In addition, histology was performed after a 28-day follow-up. RESULTS: Intercontinental cell shipment, which took >30 h from the manufacturing to the transplantation site, did not alter the hESC-RPE quality. The submacular hESC-RPE xenotransplantation was performed in 11 macaques. The miOCT typically revealed foveal disruption. ERG showed amplitude and peak time preservation in cases with favorable surgical outcomes. Histology confirmed photoreceptor preservation above the grafts and in vivo phagocytosis by hESC-RPE, albeit evidence of cytoplasmic redistribution of opsin in photoreceptors and glia hypertrophy. The immunosuppression protocol efficiently suppressed retinal T cell infiltration and microglia activation. CONCLUSION: These results suggest both structural and functional submacular integrations of hESC-RPE xenografts. It is anticipated that surgical technique refinement will further improve the engraftment of macular cell therapeutics with significant translational relevance to improve future clinical trials.

Surgical Transplantation of Human RPE Stem Cell-Derived RPE Monolayers into Non-Human Primates with Immunosuppression.

Recent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month. The present study placed hRPESC-RPE monolayers under the macula of a non-human primate model for 3 months. The transplant was able to recover in vivo and maintained healthy photoreceptors. Importantly, there was no evidence that subretinally transplanted monolayers underwent an epithelial-mesenchymal transition. Neither gliosis in adjacent retina nor epiretinal membranes were observed. These findings suggest that hRPESC-RPE monolayers are safe and may be a useful source for RPE cell replacement therapy.

[Acute macular neuroretinopathy in a 13-year-old female patient with focal choroidal excavation and pachychoroid disease]. / Akute makuläre Neuroretinopathie bei einer 13-jährigen Patientin mit fokaler choroidaler Exkavation und Pachychoroid.

Acute macular neuroretinopathy (AMN) is a rare disease that causes sudden onset of paracentral scotomas. The current case report describes a 13-year-old girl with AMN adjacent to a focal choroidal excavation (FCE) with pachychoroidal features. The patient was followed for 20 months and examined with multimodal imaging. During follow-up the AMN area showed an incomplete atrophy of the outer nuclear layer, while the FCE changed along with decreasing choroidal thickness from a non-conforming into a conforming type. This case provides an interesting association of AMN, FCE and pachychoroid disease.

Age-Related Loss of Human Vitreal Viscoelasticity.

PURPOSE: To determine the viscoelasticity of human vitreous bodies and its changes with age in order to benefit the understanding and therapy of vitreoretinal diseases. METHODS: In a postmortem study, 190 human vitreous bodies were extracted from 33- to 92-year-old donors, analyzed with regard to their viscoelastic properties via dynamic mechanical analyses, and compared with bovine and porcine vitreous. Postmortem intervals and donor-related parameters were examined as potential parameters influencing vitreous viscoelasticity. Dynamic moduli of different hyaluronic acid (HA) solutions as well as human vitreous treated with HA injections were determined by frequency sweep tests. RESULTS: With age the viscoelasticity of human vitreous bodies decreased significantly and independently of postmortem intervals, diabetes, and the donor's sex. The storage modulus G' and loss modulus G″ correlated strongly with the donor's age with r = -0.789 and r = -0.764, respectively. Bovine and porcine vitreous bodies exhibited dynamic moduli comparable only to the viscoelastic properties of aged human vitreous and are thus limited models for the simulation of the human vitreous. The viscoelasticity of aged human vitreous bodies was found to be increased after intravitreal injections of highly concentrated HA. CONCLUSIONS: The present postmortem study is the first to show a significant age-related reduction in the viscoelasticity of entire human vitreous bodies. Highly concentrated HA injections may serve as a possible therapeutic approach for restoring the viscoelasticity of aged vitreous bodies. TRANSLATIONAL RELEVANCE: These findings improve the understanding and therapy of the vitreous liquefaction with age and the associated vitreoretinal diseases.

Survival and functionality of xeno-free human embryonic stem cell-derived retinal pigment epithelial cells on polyester substrate after transplantation in rabbits.

PURPOSE: To study immunogenic properties of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and to evaluate subretinal xenotransplantation of hESC-RPE on porous polyethylene terephthalate (PET) in rabbits. METHODS: Human ESC-RPE cells were characterized by morphology, transepithelial electrical resistance (TER), protein expression and photoreceptor outer segment phagocytosis in vitro. Expression of major histocompatibility complex (MHC) proteins was assessed in conventionally or xeno-free produced hESC-RPE ± interferon-gamma (IFN-γ) stimulation (n = 1). Xeno-free hESC-RPE on PET with TER < 200 Ω·cm2  > or PET alone were transplanted into 18 rabbits with short-term triamcinolone ± extended tacrolimus immunosuppression. Rabbits were monitored by spectral domain optical coherence tomography. After 4 weeks, the eyes were processed for histology and transmission electron microscopy. RESULTS: Upon in vitro IFN-γ stimulation, xeno-free hESC-RPE expressed lower level of MHC-II proteins compared to the conventional cells. Outer nuclear layer (ONL) atrophy was observed over the graft in most cases 4 weeks post-transplantation. In 3/4 animals with high TER hESC-RPE, but only in 1/3 animals with low TER hESC-RPE, ONL atrophy was observed already within 1 week. Retinal cell infiltrations were more frequent in animals with high TER hESC-RPE. However, the difference was not statistically significant. In three animals, preservation of ONL was observed. Weekly intravitreal tacrolimus did not affect ONL preservation. In all animals, hESC-RPE cells survived for 4 weeks, but without tacrolimus, enlarged vacuoles accumulated in hESC-RPE (n = 1). CONCLUSIONS: Xenografted xeno-free hESC-RPE monolayers can survive and retain some functionality for 4 weeks following short-term immunosuppression. The preliminary findings of this study suggest that further investigations to improve transplantation success of hESC-RPE xenografts in rabbits should be addressed especially toward the roles of hESC-RPE maturation stage and extended intravitreal immunosuppression.

Transplantation of the RPE in AMD.

The retinal pigment epithelium (RPE) maintains retinal function as the metabolic gatekeeper between photoreceptors (PRs) and the choriocapillaries. The RPE and Bruch's membrane (BM) suffer cumulative damage over lifetime, which is thought to induce age-related macular degeneration (AMD) in susceptible individuals. Unlike palliative pharmacologic treatments, replacement of the RPE has a curative potential for AMD. This article reviews mechanisms leading to RPE dysfunction in aging and AMD, laboratory studies on RPE transplantation, and surgical techniques used in AMD patients. Future strategies using ex vivo steps prior to transplantation, BM prosthetics, and stem cell applications are discussed. The functional peculiarity of the macular region, epigenetic phenomena leading to an age-related shift in protein expression, along with the accumulation of lipofuscin may affect the metabolism in the central RPE. Thickening of BM with age decreases its hydraulic conductivity. Drusen are deposits of extracellular material and formed in part by activation of the alternative complement pathway in individuals carrying a mutant allele of complement factor H. AMD likely represents an umbrella term for a disease entity with multifactorial etiology and manifestations. Presently, a slow progressing (dry) non-neovascular atrophic form and a rapidly blinding neovascular (wet) form are discerned. No therapy is currently available for the former, while RPE transplantation and promising (albeit non-causal) anti-angiogenic therapies are available for the latter. The potential of RPE transplantation was demonstrated in animal models. Rejection of allogeneic homologous transplants in patients focused further studies on autologous sources. In vitro studies elucidated cell adhesion and wound healing mechanisms on aged human BM. Currently, autologous RPE, harvested from the midperiphery, is being transplanted as a cell suspension or a patch of RPE and choroid in AMD patients. These techniques have been evaluated from several groups. Autologous RPE transplants may have the disadvantage of carrying the same genetic information that may have led to AMD manifestation. An intermittent culturing step would allow for in vitro therapy of the RPE, its rejuvenation and prosthesis of BM to improve the success RPE transplants. Recent advances in stem cell biology when combined with lessons learned from studies of RPE transplantation are intriguing future therapeutic modalities for AMD patients.