RPE Transplantation Research Articles

Imaging the pediatric retina: An overview.

Recent decade has seen a shift in the causes of childhood blinding diseases from anterior segment to retinal disease in both developed and developing countries. The common retinal disorders are retinopathy of prematurity and vitreoretinal infections in neonates, congenital anomalies in infants, and vascular retinopathies including type 1 diabetes, tumors, and inherited retinal diseases in children (up to 12 years). Retinal imaging helps in diagnosis, management, follow up and prognostication in all these disorders. These imaging modalities include fundus photography, fluorescein angiography, ultrasonography, retinal vascular and structural studies, and electrodiagnosis. Over the decades there has been tremendous advances both in design (compact, multifunctional, tele-consult capable) and technology (wide- and ultra-wide field and noninvasive retinal angiography). These new advances have application in most of the pediatric retinal diseases though at most times the designs of new devices have remained confined to use in adults. Poor patient cooperation and insufficient attention span in children demand careful crafting of the devices. The newer attempts of hand-held retinal diagnostic devices are welcome additions in this direction. While much has been done, there is still much to do in the coming years. One of the compelling and immediate needs is the pediatric version of optical coherence tomography angiography. These needs and demands would increase many folds in future. A sound policy could be the simultaneous development of adult and pediatric version of all ophthalmic diagnostic devices, coupled with capacity building of trained medical personnel.

HLA-Matched Allogeneic iPS Cells-Derived RPE Transplantation for Macular Degeneration.

Immune attacks are key issues for cell transplantation. To assess the safety and the immune reactions after iPS cells-derived retinal pigment epithelium (iPS-RPE) transplantation, we transplanted HLA homozygote iPS-RPE cells established at an iPS bank in HLA-matched patients with exudative age-related macular degeneration. In addition, local steroids without immunosuppressive medications were administered. We monitored immune rejections by routine ocular examinations as well as by lymphocytes-graft cells immune reaction (LGIR) tests using graft RPE and the patient’s blood cells. In all five of the cases that underwent iPS-RPE transplantation, the presence of graft cells was indicated by clumps or an area of increased pigmentation at 6 months, which became stable with no further abnormal growth in the graft during the 1-year observation period. Adverse events observed included corneal erosion, epiretinal membrane, retinal edema due to epiretinal membrane, elevated intraocular pressure, endophthalmitis, and mild immune rejection in the eye. In the one case exhibiting positive LGIR tests along with a slight fluid recurrence, we administrated local steroid therapy that subsequently resolved the suspected immune attacks. Although the cell delivery strategy must be further optimized, the present results suggest that it is possible to achieve stable survival and safety of iPS-RPE cell transplantation for a year.

British Society for Gene and Cell Therapy Annual Conference Wednesday 19th June – Friday 21st June 2019 University of Sheffield, Sheffield, UK www.bsgct.org

British Society for Gene and Cell Therapy Annual Conference Wednesday 19th June – Friday 21st June 2019 University of Sheffield, Sheffield, UK www.bsgct.org

A new immunodeficient retinal dystrophic rat model for transplantation studies using human-derived cells.

To create new immunodeficient Royal College of Surgeons (RCS) rats by introducing the defective MerTK gene into athymic nude rats. Female homozygous RCS (RCS-p+/RCS-p+) and male nude rats (Hsd:RH-Foxn1mu, mutation in the foxn1 gene; no T cells) were crossed to produce heterozygous F1 progeny. Double homozygous F2 progeny obtained by crossing the F1 heterozygotes was identified phenotypically (hair loss) and genotypically (RCS-p+ gene determined by PCR). Retinal degenerative status was confirmed by optical coherence tomography (OCT) imaging, electroretinography (ERG), optokinetic (OKN) testing, superior colliculus (SC) electrophysiology, and by histology. The effect of xenografts was assessed by transplantation of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and human-induced pluripotent stem cell-derived RPE (iPS-RPE) into the eye. Morphological analysis was conducted based on hematoxylin and eosin (H&E) and immunostaining. Age-matched pigmented athymic nude rats were used as control. Approximately 6% of the F2 pups (11/172) were homozygous for RCS-p+ gene and Foxn1mu gene. Homozygous males crossed with heterozygous females resulted in 50% homozygous progeny for experimentation. OCT imaging demonstrated significant loss of retinal thickness in homozygous rats. H&E staining showed photoreceptor thickness reduced to 1-3 layers at 12weeks of age. Progressive loss of visual function was evidenced by OKN testing, ERG, and SC electrophysiology. Transplantation experiments demonstrated survival of human-derived cells and absence of apparent immune rejection. This new rat animal model developed by crossing RCS rats and athymic nude rats is suitable for conducting retinal transplantation experiments involving xenografts.

Evaluation of the Surgical Device and Procedure for Extracellular Matrix-Scaffold-Supported Human iPSC-Derived Retinal Pigment Epithelium Cell Sheet Transplantation.

To develop a clinically applicable transplantation device and surgical procedure for extracellular matrix-scaffold-supported human-induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) cell sheet transplantation for clinical use. The developed surgical device consisted of a custom-designed hand piece and a cannula. The subretinal transplantation of hiPSC-RPE cell sheets was performed in 12 rabbits. The results evaluated were the graft condition (damage or fold), side (front or back), position (center, near, far), and direction (anterior, posterior, right, left) immediately after surgery and the graft condition (shrinking or fold) 2 weeks after surgery. These results were evaluated by fundus photography and optical coherence tomography, followed by immersion-fixed histology. All grafts could be transplanted without obvious damage. The transplanted grafts included 2 of 12 folded grafts, 12 of 12 front side, 12 of 12 center position, 10 of 12 anterior direction, and 2 of 12 right direction immediately after surgery, whereas transplantation with a distance between an inlet and an outlet greater than graft and the coaxial direction of the flow paths and the insertion device posed the correct condition and direction. Two weeks after the surgery, the transplanted grafts included two folded grafts and four shrunken grafts; however, complete drainage of subretinal fluid for adhesion between the graft and the host prevented shrunken grafts. A developed surgical device and procedure allow grafts to be transplanted into the targeted transplantation site safely and reproducibly. This surgical method will provide additional information on the advancement of future RPE transplantation therapies.

A Step by Step Protocol for Subretinal Surgery in Rabbits.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision. Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology. The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Stem Cell Therapy in Nonneovascular Age-Related Macular Degeneration.

Age-related macular degeneration (ARMD) is the leading cause of blindness in subjects older than 50 years of age in the developed world. There are two types of ARMD, neovascular (NV) and nonneovascular (NN). While anti-VEGF-based therapies have significantly decreased the visual morbidity associated with NV-ARMD, there are no effective treatments for NN-ARMD. A detailed discussion of NV-ARMD and related therapies is the topic of another section of this special supplement. This review will focus mainly on NN-ARMD. Vision loss in nonneovascular ARMD is highly correlated with the loss of RPE cells and areas of geographic atrophy (GA). Pilot studies using subretinal transplantation of autologous or allogeneic RPE during the past 20 to 30 years have demonstrated that stem cell-derived RPE have the potential to rescue photoreceptor function and restore vision. New methods of differentiating RPE from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) have created a potentially unlimited supply of RPE cells to meet the demands of future commercially viable stem cell products. Thanks to fundamental advances in stem cell biology, vitreoretinal surgery, and noninvasive retinal imaging, stem cell-based therapies for NN-ARMD are emerging and several clinical trials are in progress. However, there are major regulatory, safety, and technical challenges that remain. This review will focus on summarizing the most promising aspects of stem cell-based therapy for NN-ARMD and highlighting areas that require further research.

Subretinal Transplantation of Embryonic Stem Cell-Derived Retinal Pigment Epithelium for the Treatment of Macular Degeneration: An Assessment at 4 Years.

Advanced macular degeneration is an important cause of vision loss in the United States with over 2 million people affected by the disease. Despite substantial progress in the development of new therapies for wet AMD, the severe visual impairment associated with geographic atrophy in dry AMD or Stargardt disease remains untreatable. Recently, two phase I/II studies involving 18 patients with these diseases have demonstrated that it is possible to safely implant human embryonic stem cell-derived RPE (hESC-RPE) in an attempt to rescue photoreceptors and visual function. The anatomical and functional results are encouraging, with more than half of treated patients experiencing sustained improvements in visual acuity and demonstrating evidence of possible cellular engraftment. However, any conclusions remain tempered by the relatively short follow-up time, lack of a formal control group, poor initial visual acuity, and small number of patients. Aside from an instance of postoperative infectious endophthalmitis, no adverse events related to the cell therapy, such as hyperproliferation, tumorigenicity, or rejection-related inflammation were noted in this initial cohort of 18 patients. These first-in-human safety studies have opened the door to future studies enrolling patients with less advanced disease, treating other diseases that result in RPE loss, employing shorter immunosuppressive regimens, and using alternative strategies for RPE transplantation such as sheets of cells with or without scaffolding to mimic Bruch's membrane. The ultimate goal of these initial safety studies is to promote continued translation of complex biological therapies into meaningful treatment strategies that may address unmet medical needs.

Human Adult Retinal Pigment Epithelial Stem Cell–Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue

We tested what native features have been preserved with a new culture protocol for adult human RPE. We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used. Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 Ω·cm2 (mean ± SEM, n = 31). Application of 100 μM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 μL·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01). The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.

Morphological versus functional photoreceptor viability of retinal pigment epithelium tears

Editor, Retinal pigment epithelium tears (RPE tears) are a serious complication in patients with age-related macular degeneration (AMD) leading to a loss of photoreceptors. Previously, we have shown that the photoreceptors can be rescued and function restored by autologous transplantation of RPE and choroid in these patients (Caramoy et al. 2011a). We have also described previously that the morphological signs of viable photoreceptors can be seen in the area of pigment epithelium tears up to 325 days (Caramoy et al. 2011b), however little is known about the function of these photoreceptors in the area without retinal pigment epithelium. In a retrospective study, we examined the morphology of RPE tears using spectral-domain optical coherence tomography (SD-OCT) (Spectralis HRA + OCT; Heidelberg Engineering, Heidelberg, Germany) in relation to its function in microperimetry (MP1; Nidek Technologies, Padova, Italy). Mean age was 76 ± 7.9 years (range 63–85 years) and mean best corrected visual acuity was 0.54 ± 0.37 LogMAR. Among the seven patients, three were men and four were women. Four patients showed unstable fixation. Relatively unstable and stable fixations were seen in two patients and in one patient, respectively. In the patient with stable fixation, the fovea was still supported by pigment epithelium. The fixation stability (percentage of fixation within the central 2° and 4°) was 42 ± 29.65% and 75 ± 19.61%, respectively. Among these patients, morphological signs of viable photoreceptors were seen in four of seven patients, that is junction of inner and outer photoreceptor segments, external limiting membrane and nonatrophic outer nuclear layer. Time from RPE tear to OCT and microperimetry in these patients was <30 days in two patients, 21 and 109 days in the other two. These signs were not seen in three of seven patients because of retinal atrophy. Time from RPE tear to OCT and microperimetry in these three patients was <30 days in two patients and 717 days in one patient. Subretinal fluid was seen in the RPE denuded area in five of seven patients. Independent of these morphological signs of viable photoreceptors and independent of subretinal fluid presence, all patients showed an absolute scotoma in the RPE denuded area (Fig. 1). Mean retinal sensitivity among this region was 0.07 ± 0.1 dB. Microperimetry and spectral-domain optical coherence tomography of a pigment epithelium tear. A dense absolute scotoma is seen in the region without retinal pigment epithelium support in microperimetry (A). However, the corresponding spectral-domain optical coherence tomography shows signs of viable photoreceptors (B and C). Identifying signs of viable photoreceptors in the wrinkled RPE region was challenging. The retina in this region was thin; its anatomical layers could not be visualized closely. In this region, mean retinal sensitivity was 0.89 ± 1.20 dB. Unlike the RPE denuded area, which exhibited a dense absolute scotoma, the region of wrinkled RPE was more heterogenous. It consisted of areas of relative and absolute scotoma. The results from our study underlined the importance of the RPE regarding visual function. In normal retina, the RPE is essential in the visual cycle towards regenerating visual pigments in cones and rods. Kühne (1878) found in bleached frog retina that rhodopsin could only regenerate partially, if the retina was detached from the retinal pigment epithelium. Weinstein et al. (1967) have also shown that no rhodopsin regeneration is observed in rat retina perfused with enriched medium detached from retinal pigment epithelium. Photoreceptors lose their functions after separation from the pigment epithelium as shown by microperimetry. But they still show signs of viability in SD-OCT. These morphological signs seem to be correct as shown by the above-mentioned recovery of photoreceptors after transplantation of RPE and choroid. These findings emphasize the importance of RPE for photoreceptors towards regenerating visual pigments, which are crucial for the visual function.

This issue of Acta, printed and electronic

The cover illustration shows a painting of Allvar Gullstrand painted in 1915, 4 years after he received the Nobel Prize for medicine. This issue is dedicated to the centennial of Allvar Gullstrand’s Nobel Prize. He is the only ophthalmologist ever to receive this prestigious price. Stefan Seregard writes an editorial commemorating this event. This is followed by an historical article by Ehinger and Grzybowski on the life and times of Allvar Gullstrand. Yilmaz et al. in USA, Spain and Ireland review the use of intravitreal bevacizumab for diabetic macular oedema. This is an extensive review of the literature for this new and important treatment modality. Lundström et al. in Sweden and Australia found the Catquest questionnaire useful for measuring self-assessed visual function before and after cataract surgery. Mönestam and Behndig found that light scattering and glistening in intraocular lenses does not affect visual acuity. Gazieva et al. in Denmark performed LASIK with two types of laser systems, both which effectively reduce high degrees of myopia with reasonable but slightly different predictability. Rijneveld et al. in the Netherlands found that emergency corneal grafts fail mostly due to host factors, not the quality of the donor tissue. Reinshagen et al. in Switzerland and Germany used mesenchymal stem cell in rabbits to treat total limbal stem cell deficiency. Heijl et al. in Sweden and USA report that intraocular pressure reduction with drug treatment depends on the baseline IOP level and suggest that the baseline IOP level should be documented when reporting treatment effect on intraocular pressure. Larsson et al. in Sweden used the Heidelberg retina tomography system to evaluate optic nerve heads in children. Lin et al. in Taiwan describe lacrimal canaliculitis in a series of patients. Mehlsen et al. in Denmark found that a calcium channel blocker and an angiotensin converting enzyme inhibitor did not influence retinal autoregulation in diabetic patients. Kook et al. in Germany found that intravitreal bevacizumab and/or triamcinolone do not provide long-term improvement for diabetic macular oedema. Kofoed et al. in Copenhagen found that hyperbaric oxygen exposure influenced electrophysiological components related to bipolar in Müller cells. It did not influence retinal thickness. Holton et al. in Denmark examined the influence of light source on contrast sensitivity in patients with AMD. They found that full spectral radiance incandescent light sources seem optimal. Available on http://www.actaophthalmologica.com Stalmans and a large international group review extensively the use of colour Doppler imaging in ocular blood flow research. Founti et al. in Greece and USA found good reproducibility between technicians in imaging retrobulbar blood flow in healthy volunteers. Frøen et al. in Norway and Hungary found that cells from the iris pigment epithelium have limited capabilities as retinal stem cells. Andjelić et al. in Slovenia found that lens capsule contraction with changing in calcium levels or mechanical stimulus. Sieving in USA explains Medline indexing and keywords. Bull in Bergen proposes that fireworks should be banned to reduce eye injuries. Basit et al. in Trondheim describe a case of Wernicke’s encephalopathy in a young boy. Russo et al. in Liverpool found scleral calcification in a case of recurrent melanoma after brachytherapy. Rama et al. in Italy report severe keratitis following corneal cross-linking for keratoconus. Jonas and Jonas in Germany compare peripapillary scleral thickness in glaucoma patients and normal subjects. Lim and An in Korea report good results after epiretinal membrane removal. Kernt et al. in Germany had good experiences with focal and panretinal photocoagulation with a navigated laser. Fielden et al. in Canada describe a cluster of cases with intraocular inflammation following bevacizumab injection. Bloch et al. in Denmark and Egypt describe neovascular membrane contraction and retinal pigment epithelial rupture following verteporfin and bevacizumab treatment. Caramoy et al. in Germany found recurrent choroidal neovascular membrane after autologous RPE transplantation. I wish all readers a Merry Christmas and a Happy New Year and hope that they enjoy reading ACTA during the holidays.

Polarized Secretion of PEDF from Human Embryonic Stem Cell–Derived RPE Promotes Retinal Progenitor Cell Survival

Human embryonic stem cell-derived RPE (hES-RPE) transplantation is a promising therapy for atrophic age-related macular degeneration (AMD); however, future therapeutic approaches may consider co-transplantation of hES-RPE with retinal progenitor cells (RPCs) as a replacement source for lost photoreceptors. The purpose of this study was to determine the effect of polarization of hES-RPE monolayers on their ability to promote survival of RPCs. The hES-3 cell line was used for derivation of RPE. Polarization of hES-RPE was achieved by prolonged growth on permeable inserts. RPCs were isolated from 16- to 18-week-gestation human fetal eyes. ELISA was performed to measure pigment epithelium-derived factor (PEDF) levels from conditioned media. Pigmented RPE-like cells appeared as early as 4 weeks in culture and were subcultured at 8 weeks. Differentiated hES-RPE had a normal chromosomal karyotype. Phenotypically polarized hES-RPE cells showed expression of RPE-specific genes. Polarized hES-RPE showed prominent expression of PEDF in apical cytoplasm and a marked increase in secretion of PEDF into the medium compared with nonpolarized culture. RPCs grown in the presence of supernatants from polarized hES-RPE showed enhanced survival, which was ablated by the presence of anti-PEDF antibody. hES-3 cells can be differentiated into functionally polarized hES-RPE cells that exhibit characteristics similar to those of native RPE. On polarization, hES-RPE cells secrete high levels of PEDF that can support RPC survival. These experiments suggest that polarization of hES-RPE would be an important feature for promotion of RPC survival in future cell therapy for atrophic AMD.

Longterm cultures of the aged human RPE do not maintain epithelial morphology and high transepithelial resistance

Dear Editor, Visual rehabilitation achieved with peripheral RPE autografts in exudative AMD is only modest. Moreover, complication rates using the popular RPE–choroid patch technique remain relatively high. Tissue-engineered transplants might improve the outcome of RPE transplantation. A culturing step could place cells on a substrate prior to transplantation, thereby optimizing delivery and long-term function [1]. This would require sufficient cellular plasticity (i.e., response to environmental stimuli) to achieve a well-differentiated, functional graft. While culture plasticity was confirmed for fetal cells or RPE cell lines [2, 3], only sparse information is available for aged RPE [4]. We studied the influence of culture conditions on select differentiation characteristics in long-term primary RPE cultures from ten human donors above age 55, and compared them to a 23-year-old and two fetal donors (20 and 21 weeks of gestation respectively). The main outcome measures at 6 weeks post-confluence (PC) were morphology and transepithelial resistance (TER), an electrophysiological method for assessing tight junctions build-up. Eyes were preserved within 6 hours of death. Adult primary RPE cultures (aRPE) were initiated from: (1) mechanically scraped RPE, or (2) collagenase pretreated (1 mg/ml for 40 minutes, CLS I, Worthington Biochemical Corp, Lakewood, NJ, USA) and mechanically scraped RPE, or (3) fetal (fRPE) and adult cultures started from floater passages [2]. Cells were grown on permeable polyester inserts (Transwells, Corning Inc., Corning, NY, USA) in modified Hu & Bok media [2], containing 2% calf serum, basic FGF (5 ng/ml), L-alanyl-L-glutamine (2 mM), Albumax (200 μg/ml), and antibiotics/antimycotics. RPE culture purity was confirmed by a uniform pancytokeratin staining. Experimental matrices analyzed the influence of above harvesting methods, seeding density, laminin coating (2 μg/cm), and media composition (2% vs 10% serum until confluence, or calcium switch at confluence, or 5 mM vs 25 mM glucose) on above outcome measures. Morphology was monitored weekly in all and transepithelial resistance (TER) in all conditions, except the laminin matrix (i.e., seven of ten aged donors). Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL, USA, April 30–May 4 2006.

Recurrence of choroidal neovascular membrane after autologous transplantation of RPE and choroid for neovascular AMD

Recurrence of choroidal neovascular membrane after autologous transplantation of RPE and choroid for neovascular AMD

Transplante autólogo do epitélio pigmentado da retina na degeneração macular relacionada com a idade

Retinal pigment epithelial dysfunction is believed to be the main cause of many debilitating retinal diseases of which age-related macular degeneration is the most common. In this disease, the retinal pigment epithelial dysfunction leads to photoreceptors damage causing severe vision loss. The retinal pigment epithelium and Bruch's membrane suffer cumulative damage over lifetime, which is thought to induce age-related macular degeneration in susceptible individuals. In the past 20 years, a huge amount of research has been conducted in the area of transplantation of RPE. This technique aims to restore the subretinal anatomy and reestablish the critical interaction between the retinal pigment epithelium and the photoreceptor, which is fundamental to sight. Retinal pigment epithelial transplantation has been performed with two different techniques: retinal pigment epithelial suspension and autologous full-thickness retinal pigment epithelial-choroid transplantation in some cases of the age-related macular degeneration (AMD). Despite the feasibility of this technique, search for a cell source to replace autologous retinal pigment epithelium such as embryonic stem cells, marrow-derived stem cells and umbilical cord-derived cells continues. The combination of cell transplantation with other modalities of treatment such as gene transfer remains an exciting future prospect.

Histological analysis of retinas sampled during translocation surgery: a comparison with normal and transplantation retinas

Aims:To carry out a histopathological analysis of retinal specimens of patients undergoing translocation surgery for age-related macular degeneration (ARMD).Methods:A histopathological analysis, using confocal microscopy, was performed on six retinal specimens....

Microperimetry : understanding what went wrong or predicting therapeutic routes

Abstract Purpose: To investigate macular sensitivity and fixation patterns with automatic fundus perimetry (microperimetry) in patients with early and late age‐related macular degeneration (ARMD). Methods: A hundred and twenty eyes of 101 consecutive patients with early ARMD or with subfoveal choroidal neovascularization (CNV) secondary to ARMD were evaluated. Best corrected visual acuity (ETDRS charts), fundus photography, and fluorescein angiography were performed. Microperimetry (MP1, Nidek Technologies, Padova, Italy) was used to quantify macular sensitivity and fixation patterns (location and stability). Results: Macular sensitivity was significantly decreased over large drusen and over pigment abnormalities (p&lt;0.0001) and in patients with CNV when compared to normal age‐related values (p&lt;0.0001). Fixation moved from central to eccentric and from stable to unstable in patients with active CNV, (p&lt;0.001 and p&lt;0.05) respectively. Angiographic classification of subfoveal CNV (occult versus classic) was not significantly related to fixation pattern (p&gt;0.05). In cases that responded to treatment retinal sensitivity and fixation characteristics improved significantly, (p&lt;0.05). Conclusions: Microperimetric quantification of macular sensitivity and fixation patterns in eyes with early and late ARMD offers new data about the impact of visual impairment in these eyes. In retinal areas affected by early ARMD retinal sensitivity deteriorates, despite good visual acuity. Microperimetry may allow early detection of functional impairment due to these lesions. Moreover, microperimetry may be of value in predicting the outcome of CNV when different treatments (laser, PDT, anti‐VEGF drugs, macular translocation and RPE transplants) are planned in current or future studies

Comparison of microinjection methods for subretinal RPE transplantation in the rat

To compare iatrogenic damage to the host eye caused by different injection techniques for transplantation RPE into the subretinal space of Lewis rats. A total of 45 eyes from Lewis rats were divided into three groups and immortalized hTERT-RPE1 cells (clontechniques) transfected with green fluorescent protein (GFP) was injected into the subretinal space. The injection was given by using Hamilton syringe, Eppendorf repeat micro-pipettor, or a pediatric syringe infusion pump in different groups respectively. Iatrogenic damage to the host eye was evaluated by light microscopy. No damage to the host eye was observed in 12 of 15 eyes injected with the syringe infusion pump. In contrast, severe or mild damage was detected in 14 of 15 eyes after injections using either the Hamilton syringe or the Eppendorf micro-pipettor. The syringe infusion pump minimizes iatrogenic damage to the host eye during RPE transplantation into the subretinal space of the rat. The slow and controlled fluid infusion provided by the low-geared electric motor of the pump is presumably beneficial during the surgical procedure.

Growth of cultured porcine retinal pigment epithelial cells.

To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation. Primary pRPE cell cultures were established. Cell morphology was assessed by phase contrast and electron microscopy. Growth was determined by the crystal violet dye uptake assay. DNA synthesis and content were measured by incorporation of 3H-thymidine and flow cytometry. This primary culture resulted in cells with well-preserved morphology that could be propagated in up to six passages. The deterioration observed over time in cultures was not due to a constant high rate of cell turnover as postconfluency cell proliferation was limited. However, a large fraction of the cells had a high DNA content despite a lack of active DNA synthesis. The present method yields pRPE cells of high purity and proliferative capacity with preserved epithelial phenotype. However, aberrant DNA profiles and the deterioration of cell morphology observed over time in this graft material represent serious problems in RPE transplantation.