Itzhak Hemo

Directed Differentiation of Human Embryonic Stem Cells into Functional Retinal Pigment Epithelium Cells

Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.

Vascular Endothelial Growth Factor Upregulation in Human Central Retinal Vein Occlusion

BACKGROUND AND OBJECTIVE Vascular endothelial growth factor (VEGF), a key mediator of intraocular neovascularization, is triggered by hypoxia and has been shown in the eyes of animal models of central retinal vein occlusion (CRVO). However, there is little information on CRVO in humans, in particular, the identity of VEGF-producing cells. STUDY DESIGN The study design was molecular localization of the site of VEGF production in the eyes of patients with CRVO. PARTICIPANTS Ten formaldehyde solution-fixed and paraffin-embedded eyes removed surgically from patients with CRVO and neovascular glaucoma were studied. Five eyes with uveal melanoma and no neovascularization served as control specimens. METHODS Thin whole-eye sections were hybridized in situ with a VEGF-specific probe to identify cells producing VEGF messenger RNA (mRNA). RESULTS All ten eyes with CRVO showed evidence of intraretinal expression of VEGF mRNA. In all eyes, the inner nuclear layer showed VEGF-upregulated expression. Upregulation of VEGF mRNA was identified in four eyes in the ganglion cell layer and in two eyes with retinal detachment in the outer nuclear layer as well. CONCLUSIONS The population of VEGF-producing retinal cells in each eye is likely to represent cells residing in ischemic regions of the retina. Hypoxia-induced VEGF is, most likely, the linking factor between retinal ischemia and iris and retinal neovascularization in CRVO.

Retinal Incorporation and Differentiation of Neural Precursors Derived from Human Embryonic Stem Cells

Retinal and macular degenerations are a major cause of blindness. Cell transplantation is a possible therapeutic approach for the replacement of degenerating retinal cells. Here, we studied the potential of human embryonic stem cells (hESCs) to survive, integrate, and differentiate into retinal cells after intraocular transplantation. Highly enriched cultures of neural precursors (NPs) expressing transcripts of key regulatory genes of retinal development were developed from the hESCs. After spontaneous differentiation in vitro, the NPs gave rise to progeny expressing markers of retinal progenitors and photoreceptor development, though this was uncommon and cells expressing markers of mature photoreceptors were not observed. After transplantation into rat eyes, the NPs survived for 16 weeks, migrated large distances, and integrated in the host retina. Teratoma tumors were not observed. Human cells expressing rhodopsin, blue cone opsin, and neural retina leucine zipper transcription factor were observed in subretinal grafts, but not within vitreal and inner retinal grafts. The results suggest that hESCs have the potential to differentiate into retinal cells and that the subretinal microenvironment supports their differentiation toward a photoreceptor fate. This may be the first step toward further developments that eventually may allow the use of hESCs for transplantation in retinal degenerations.

Upregulated expression of vascular endothelial growth factor in proliferative diabetic retinopathy.

AIMS/BACKGROUND: Vascular endothelial growth factor (VEGF) is a hypoxia induced angiogenic factor. Recent studies have shown that high levels of VEGF accumulate in the vitreous of patients with proliferative diabetic retinopathy (PDR). The purpose of the present study was to identify the retinal cells that upregulate VEGF expression in human PDR patients representing progressive stages of retina deterioration. METHODS: Thirteen formalin fixed and paraffin embedded enucleated eyes with PDR were used (eyes were enucleated because of being blind and painful as a result of neovascular glaucoma). Thin retina sections were hybridised in situ with a VEGF specific probe, to identify cells producing VEGF mRNA. RESULTS: All eyes with PDR showed upregulated expression of VEGF mRNA, specifically in the cells of the neurosensory retina. VEGF expression was upregulated in all three nuclear layers--namely, the ganglion cell layer, the inner nuclear layer, and the outer nuclear layer. However, in each patient, VEGF producing cells were mostly distributed in a different layer, or even confined to a specific region in that layer. For example, expression by the outer nuclear layer was mostly detected in detached (presumably hypoxic) regions of the retina. CONCLUSIONS: Progression of PDR is distinguished by a sustained, upregulated expression of VEGF by the neurosensory retina. Cells in all retina layers can potentially contribute to augmented VEGF production. The restricted population of VEGF producing cells in each case is likely to represent cells residing in ischaemic regions of the retina. Thus, VEGF may function as a linking factor between retinal ischaemia and PDR associated neovascularisation.

Molecular Anthropology Meets Genetic Medicine to Treat Blindness in the North African Jewish Population: Human Gene Therapy Initiated in Israel

The history of the North African Jewish community is ancient and complicated with a number of immigration waves and persecutions dramatically affecting its population size. A decade-long process in Israel of clinical-molecular screening of North African Jews with incurable autosomal recessive blindness led to the identification of a homozygous splicing mutation (c.95-2A > T; IVS2-2A > T) in RPE65, the gene encoding the isomerase that catalyzes a key step in the retinoid-visual cycle, in patients from 10 unrelated families. A total of 33 patients (four now deceased) had the severe childhood blindness known as Leber congenital amaurosis (LCA), making it the most common cause of retinal degeneration in this population. Haplotype analysis in seven of the patients revealed a shared homozygous region, indicating a population-specific founder mutation. The age of the RPE65 founder mutation was estimated to have emerged 100-230 (mean, 153) generations ago, suggesting it originated before the establishment of the Jewish community in North Africa. Individuals with this RPE65 mutation were characterized with retinal studies to determine if they were candidates for gene replacement, the recent and only therapy to date for this otherwise incurable blindness. The step from molecular anthropological studies to application of genetic medicine was then taken, and a representative of this patient subgroup was treated with subretinal rAAV2-RPE65 gene therapy. An increase in vision was present in the treated area as early as 15 days after the intervention. This process of genetically analyzing affected isolated populations as a screen for gene-based therapy suggests a new paradigm for disease diagnosis and treatment.

Application of the Standard Photodynamic Treatment Protocol for Symptomatic Circumscribed Choroidal Hemangioma

Purpose: To describe the results of photodynamic therapy (PDT) using a standard protocol, developed for treating choroidal neovascularization (CNV), for the treatment of circumscribed choroidal hemangioma (CCH). Methods: A prospective, uncontrolled, consecutive case series of patients with symptomatic CCH that were treated using the standard PDT protocol was evaluated periodically with ophthalmic exams and echography. Results: Nine CCH patients were included in the study. Mean tumor height decreased from 2.7 mm before treatment to 0.8 mm at the end of the follow-up period. Mean visual acuity improved from 6/15 to 6/12. Six patients required one PDT session, two patients two sessions, and one patient three sessions. Side effects included transient visual disturbances in two patients. One patient, who concomitantly presented with age-related macular degeneration, developed CNV. Conclusions: The standard PDT protocol is effective for the treatment of CCH. Complications are uncommon. Further studies should assess the optimal PDT protocol for the treatment of CCH.

Controlled lens formation with unapertured excimer lasers : use with organic polymers and corneal tissues

A theoretical model is developed which allows for well-defined lenses to be formed with different dioptric properties using lasers that are known to ablate a variety of materials. With the theoretical development as a guide an experimental arrangement is defined for the ArF excimer laser and this arrangement is used to form lenses with highly smooth surfaces in three polymeric materials (polymethyl methacrylate, polycarbonate, and polyvinyl chloride) to test the validity of the theory. As part of the procedure to obtain these lenses, ablation curves (etch depth per pulse vs log of the laser fluence) were measured for each of these polymers and these curves are also reported. The experiments were extended to the rabbit cornea in which the corneal button was reshaped as part of keratomileusis surgery. This preliminary experiment resulted in a clear cornea with a smooth corneal surface and a measured change of 3 diopters in the treated eye.

Bevacizumab for choroidal neovascularization related to inflammatory diseases.

Purpose:The purpose of this study was to report our experience with intravitreal bevacizumab for inflammation-related choroidal neovascularization in two tertiary centers. Methods:This study was a retrospective analysis of patients with choroidal neovascularization related to inflammatory diseases, treated with intravitreal bevacizumab injections (1.25 mg/0.05 mL). Results:Ten eyes of 10 patients (range, 14-78 years; mean age, 44 years) with underlying uveitis were treated with intravitreal bevacizumab for inflammation-related choroidal neovascularization from 2006 to 2008. Mean follow-up time was 13 ± 8 months, and the mean number of injections was 2.7 ± 2. Resolved leakage on fluorescein angiography and resolution of subretinal fluid on optical coherence tomography occurred in all patients, with improvement in visual acuity in 9 of 10 eyes and no change in visual acuity in 1 of 10 eyes. Seven patients received additional treatment based on the underlying condition. Mean macular thickness on optical coherence tomography decreased from 394 ± 116 μm to 254 ± 52 μm (P < 0.01). Mean visual acuity improved from 0.87 ± 0.74 logarithm of the minimum angle of resolution to 0.38 ± 0.63 (P = 0.005). Seven patients reached a visual acuity of 0.2 logarithm of the minimum angle of resolution (Snellen 6/9) or better. Conclusion:Intravitreal bevacizumab is an effective treatment for choroidal neovascularization related to inflammatory diseases when inflammation is controlled.

Dynamics of cavitation bubble induced by 193 nm ArF excimer laser in concentrated sodium chloride solutions

Cavitation bubbles were generated by the 193 nm argon fluoride excimer laser delivered with a specially designed tip into absorbing NaCl solution. The dynamics of bubble formation and collapse have been studied using fast flash photomicrography. The bubble dimensions were measured at different time delays as a function of the tip exit diameter, the energy fluence of the laser, and the NaCl concentration. The dynamics of the cavitation bubble created on the tip is compared with the well‐studied dynamics of bubbles resulting from dielectric breakdown near a boundary.

Intravitreal bevacizumab therapy for neovascular age-related macular degeneration associated with poor initial visual acuity

Aim: The aim of the study was to assess the efficacy of intravitreal bevacizumab injections for eyes with neovascular age-related macular degeneration (NVAMD) and poor initial visual acuity (VA). Methods: A retrospective study of 44 consecutive treatment-naïve eyes with NVAMD who had initial VA of 0.1 decimal or worse, and that were treated with intravitreal bevacizumab injections, was undertaken. Charts, optical coherence tomography (OCT) and fluorescein angiograms (FAs) were reviewed for the purpose of the study. Results: Mean lesion size was 3375 (SD 2116) μm, all lesions showed sub- or intra-retinal fluid in OCT, and active neovascularisation comprised 41.6 (SD 17.7)% (range 10–90%) of the lesion area according to FA. The mean follow-up time was 3.9 (SD 5.8) (range 1–21) months. Patients received a mean of 2.6 (SD 2.4) bevacizumab injections (range 1–14), and mean VA improved from 1.85 (SD 0.64) to 1.52 (SD 0.77) LogMAR (p = 0.002). At final examination, nine eyes (20%) had reduced VA, ten eyes (23%) had stable VA and 25 eyes (57%) had improved VA compared with baseline. Following treatment, mean macular thickness was reduced from 332 (SD 98) to 248 (SD 79) μm (p<0.0001). Conclusions: Poor initial VA should not prevent use of bevacizumab in eyes with NVAMD. Selection of patients with signs of active neovascularisation based on ophthalmoscopy, OCT and FA may increase the likelihood of a favourable response to treatment.