Preface: Sight Restoration Through Stem Cell Therapy.

This publication presents chapters based on a meeting entitled "Sight Restoration Through Stem Cell Therapy" held on June 13, 2015, in Santa Monica, CA, sponsored by the Ocular Research Symposia Foundation (ORSF). It was chaired by Michael Young, PhD, Harvard Medical School, and Gerald Chader, PhD, University of Southern California. The mission of this publication and of the ORSF in general is to focus attention on unmet medical needs and current research opportunities in eye research with the objective of accelerating translation of research findings to effective clinical care. In the meeting, new research advances on stem cells and opportunities for their clinical application were highlighted and are recounted in the following chapters of this publication. By identifying "low-hanging fruit" (i.e., the best opportunities for successful transition of laboratory research to prevention and new treatments and cures for ocular diseases), we seek to spur funding at both the basic research and clinical levels, resulting in sight-saving and sight-restoration measures in the near future.

Stem cell based therapies for age-related macular degeneration: The promises and the challenges.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries. AMD is classified as either neovascular (NV-AMD) or non-neovascular (NNV-AMD). Cumulative damage to the retinal pigment epithelium, Bruch's membrane, and choriocapillaris leads to dysfunction and loss of RPE cells. This causes degeneration of the overlying photoreceptors and consequential vision loss in advanced NNV-AMD (Geographic Atrophy). In NV-AMD, abnormal growth of capillaries under the retina and RPE, which leads to hemorrhage and fluid leakage, is the main cause of photoreceptor damage. Although a number of drugs (e.g., anti-VEGF) are in use for NV-AMD, there is currently no treatment for advanced NNV-AMD. However, replacing dead or dysfunctional RPE with healthy RPE has been shown to rescue dying photoreceptors and improve vision in animal models of retinal degeneration and possibly in AMD patients. Differentiation of RPE from human embryonic stem cells (hESC-RPE) and from induced pluripotent stem cells (iPSC-RPE) has created a potentially unlimited source for replacing dead or dying RPE. Such cells have been shown to incorporate into the degenerating retina and result in anatomic and functional improvement. However, major ethical, regulatory, safety, and technical challenges have yet to be overcome before stem cell-based therapies can be used in standard treatments. This review outlines the current knowledge surrounding the application of hESC-RPE and iPSC-RPE in AMD. Following an introduction on the pathogenesis and available treatments of AMD, methods to generate stem cell-derived RPE, immune reaction against such cells, and approaches to deliver desired cells into the eye will be explored along with broader issues of efficacy and safety. Lastly, strategies to improve these stem cell-based treatments will be discussed.

Preface: The aging eye: normal changes, age-related diseases, and sight-saving approaches.

This volume presents articles based on a workshop held June 14 to 16, 2013 in Rancho Palos Verde, CA sponsored by the Ocular Research Symposia Foundation (ORSF). The mission of the ORSF is to focus attention on unmet needs and current research opportunities in eye research with the objective of accelerating translation of research findings to effective clinical care. In this workshop, the subject of the "The Aging Eye" was addressed, including the prevalence of eye diseases in aging and the economic burden imposed by these diseases. New research work was highlighted on the genetics, biology, biochemistry, neurochemistry, and the impact of nutrition and the environment on function in the older eye. By identifying "low-hanging fruit" (i.e., the best opportunities for successful transition of laboratory research for the prevention of and new treatments and cures for ocular diseases), we seek to spur funding at both the basic research and clinical levels, resulting in sight-saving and sight-restoration measures in the near future.

Screening of a large cohort of leber congenital amaurosis and retinitis pigmentosa patients identifies novel LCA5 mutations and new genotype-phenotype correlations.

This study was undertaken to investigate the prevalence of sequence variants in LCA5 in patients with Leber congenital amaurosis (LCA), early-onset retinal dystrophy (EORD), and autosomal recessive retinitis pigmentosa (arRP); to delineate the ocular phenotypes; and to provide an overview of all published LCA5 variants in an online database. Patients underwent standard ophthalmic evaluations after providing informed consent. In selected patients, optical coherence tomography (OCT) and fundus autofluorescence imaging were possible. DNA samples from 797 unrelated patients with LCA and 211 with the various types of retinitis pigmentosa (RP) were screened by Sanger sequence analysis of all LCA5 exons and intron/exon junctions. Some LCA patients were prescreened by APEX technology or selected based on homozygosity mapping. In silico analyses were performed to assess the pathogenicity of the variants. Segregation analysis was performed where possible. Published and novel LCA5 variants were collected, amended for their correct nomenclature, and listed in a Leiden Open Variation Database (LOVD). Sequence analysis identified 18 new probands with 19 different LCA5 variants. Seventeen of the 19 LCA5 variants were novel. Except for two missense variants and one splice site variant, all variants were protein-truncating mutations. Most patients expressed a severe phenotype, typical of LCA. However, some LCA subjects had better vision and intact inner segment/outer segment (IS/OS) junctions on OCT imaging. In two families with LCA5 variants, the phenotype was more compatible with EORD with affected individuals displaying preserved islands of retinal pigment epithelium. One of the families with a milder phenotype harbored a homozygous splice site mutation; a second family was found to have a combination of a stop mutation and a missense mutation. This is the largest LCA5 study to date. We sequenced 1,008 patients (797 with LCA, 211 with arRP) and identified 18 probands with LCA5 mutations. Mutations in LCA5 are a rare cause of childhood retinal dystrophy accounting for ∼2% of disease in this cohort, and the majority of LCA5 mutations are likely null. The LCA5 protein truncating mutations are predominantly associated with LCA. However, in two families with the milder EORD, the LCA5 gene analysis revealed a homozygous splice site mutation in one and a stop mutation in combination with a missense mutation in a second family, suggesting that this milder phenotype is due to residual function of lebercilin and expanding the currently known phenotypic spectrum to include the milder early onset RP. Some patients have remaining foveal cone structures (intact IS/OS junctions on OCT imaging) and remaining visual acuities, which may bode well for upcoming treatment trials.

In vivo detection of hESC-RPE cells via confocal near-infrared fundus reflectance.

BACKGROUND AND OBJECTIVE: To investigate whether the confocal near-infrared reflectance (NIR) imaging modality could detect the in vivo presence of retinal pigment epithelium cells derived from embryonic human stem cells (hESC-RPE) implanted into the subretinal space of the Royal College of Surgeons (RCS) rat. MATERIALS AND METHODS: Monthly NIR images were obtained from RCS rats implanted with either hESC-RPE seeded on a parylene membrane (n = 14) or parylene membrane without cells (n = 14). Two independent, masked investigators graded the images. Histology and immunohistochemistry were performed at different time points (150, 210, and 270 postnatal days of age). RESULTS: NIR images revealed that an average of 20.53% of the parylene membrane area was covered by hESC-RPE. RPE-65 and TRA-1-85 confirmed the presence of human-specific RPE cells in those animals. No areas corresponding to cells were found in the group implanted with membrane only. Intergrader agreement was high (r = 0.89-0.92). CONCLUSION: The NIR mode was suitable to detect the presence of hESC-RPE seeded on a membrane and implanted into the subretinal space of the RCS rat.

Fluorescein angiography, optical coherence tomography, and histopathologic findings in a VEGF(165) animal model of retinal angiogenesis.

BACKGROUND: To establish an animal model of retinal neovascularization using vascular endothelial growth factor (VEGF165) and analyze the model using optical coherence tomography (OCT), fluorescein angiography (FA), and histopathologic evaluation. METHODS: Twelve rabbits were divided into groups as follows: group 1 (n = 3), sham intravitreous injections of 0.1 ml of balanced saline; group 2 (n = 6), one 10-µg intravitreal injection of VEGF165 on day 0; and group 3 (n = 3), two 10-µg intravitreal injections of VEGF165, one on day 0 and one on day 7. Follow-up evaluations (days 0, 3, 7, 14, 21, 28) included obtaining fundus color photographs and FA, OCT, and histopathologic examinations. Eyes were enucleated and stained with hematoxylin and eosin (H&E). RESULTS: One injection of VEGF (group 2) was associated with dilatation and tortuosity of the retinal blood vessels that developed within 72 h. Retinal neovascularization was present by day 7 and regressed by day 14. However, even on day 28, the capillaries were still tortuous. Two VEGF injections (group 3) caused increased leakage and neovascularization up to day 14; severe capillary nonperfusion was seen during week 4. At the end of the follow-up period, OCT and histopathologic examination of group 3 showed peripapillary tractional retinal detachments. By day 7, the differences between the retinal thickness seen on OCT in groups 2 and 3 and the group 1 control group were significant (p < 0.001). The histologic findings showed increased vessel size in groups 2 and 3 by days 14 and 28 compared with the controls. CONCLUSIONS: FA, OCT, and histopathologic findings showed that this retinal neovascularization model is efficient, sustainable, and reliable. One injection of VEGF165 created neovascularization that peaked after 1 week; two injections created more intense neovascularization that evolved to retinal detachments after 4 weeks.

High-resolution OCT: an innovative tool for posterior segment imaging.

Optimal management of posterior segment disorders requires a high-resolution and preferably noninvasive imaging tool for better definition of diseases. High-resolution optical coherence tomography can provide noninvasive, high-definition imaging of the posterior segment, allowing earlier diagnosis, better follow-up of chronic cases, and more accurate and timely monitoring of the effect of therapeutic agents. Recent findings suggest an individualized approach to vitreoretinal and choroidal diseases is possible based not only on traditional ophthalmic investigations, but also on high-resolution optical coherence tomography. This innovative tool has the combined advantages of high speed, high resolution, and safe use.

Vitreal oxygenation in retinal ischemia reperfusion.

PURPOSE: To study the feasibility of anterior vitreal oxygenation for the treatment of acute retinal ischemia. METHODS: Twenty rabbits were randomized into an oxygenation group, a sham treatment group, and a no treatment group. Baseline electroretinography (ERG) and preretinal oxygen (Po(2)) measurements were obtained 3 to 5 days before surgery. Intraocular pressure was raised to 100 mm Hg for 90 minutes and then normalized. The oxygenation group underwent vitreal oxygenation for 30 minutes using intravitreal electrodes. The sham treatment group received inactive electrodes for 30 minutes while there was no intervention for the no treatment group. Preretinal Po(2) in the posterior vitreous was measured 30 minutes after intervention or 30 minutes after reperfusion (no treatment group) and on postoperative days (d) 3, 6, 9, and 12. On d14, rabbits underwent ERG and were euthanatized. RESULTS: Mean final (d12) Po(2) was 10.64 ± 0.77 mm Hg for the oxygenation group, 2.14 ± 0.61 mm Hg for the sham group, and 1.98 ± 0.63 mm Hg for the no treatment group. On ERG, scotopic b-wave amplitude was significantly preserved in the oxygenation group compared with the other two groups. Superoxide dismutase assay showed higher activity in the operated eyes than in the nonoperated control eyes in the sham treatment group and no treatment group only. Histopathology showed preservation of retinal architecture and choroidal vasculature in the oxygenation group, whereas the sham-treated and nontreated groups showed retinal thinning and choroidal atrophy. CONCLUSIONS: In severe total ocular ischemia, anterior vitreal oxygenation supplies enough oxygen to penetrate the retinal thickness, resulting in rescue of the RPE/choriocapillaris that continues to perfuse, hence sparing the retinal tissue from damage.

Effect of multiple injections of small divided doses vs single injection of intravitreal bevacizumab on retinal neovascular model in rabbits.

BACKGROUND: To compare effects of multiple injections of small divided doses of intravitreal bevacizumab vs a single injection using a retinal neovascular model in rabbits. METHODS: We assigned 12 pigmented rabbits to four groups of three each. All groups received an intravitreal injection of vascular endothelial growth factor (VEGF, 10 microg) on the first day. Group A received an intravitreal loading dose of bevacizumab (0.5 mg) on day 3, followed by five smaller injections (0.15 mg), one every third day. Those in groups B and C received a single intravitreal injection of bevacizumab (1.25 mg) on day 3, followed by five injections of sham, one every third day in group C. Group D received only intravitreal VEGF. Follow-up examinations were performed for 26 days. RESULTS: In groups A and B, vascular changes associated with VEGF injection decreased substantially in the first 3 days, and continued to show gradual regression during each follow-up interval. No statistically significant differences were found between the changes of mean retinal thicknesses in groups A and B in both areas. In group C, the extra sham injections did not lead to any further vascular changes. The mean retinal thickness in groups B and C did not have a statistically significant difference during the follow-up period. In group D, vascular changes resolved more gradually than in other groups. The difference in retinal thickness between group D and the other groups was statistically significant on day 6 in both groups (medullary and inferior part; p = 0.0003) and in medullary wing on day 12 (p = 0.03). CONCLUSIONS: Frequent smaller doses of bevacizumab can control VEGF-induced vascular changes as well as the currently utilized model of single large monthly injections. Dividing of currently used single injection (1.25 mg) of bevacizumab to multiple small doses can control VEGF-induced vascular changes as effectively as one large injection.

The effects of intravitreous bevacizumab on retinal neovascular membrane and normal capillaries in rabbits.

PURPOSE: To evaluate the effects of intravitreous bevacizumab in a rabbit retinal neovascularization model. METHODS: Twenty-four rabbits were divided into five groups. Group A included four rabbits; all other groups included five rabbits each. Group A received intravitreous VEGF only, and group E received intravitreous bevacizumab only. In groups B, C, and D, bevacizumab was injected at the same time, at day 2 and at week 1 after VEGF injection, respectively. Follow-up evaluations continued for 3 weeks and included color fundus photography, fluorescein angiography (FA), and optical coherence tomography (OCT). Enucleated eyes were processed for hematoxylin and eosin (H&E) staining. RESULTS: Intravitreous VEGF was associated with disc hyperemia, vascular dilatation and tortuosity, and fluorescein leakage at the disc and in the anterior chamber (AC) at day 2 and with formation of retinal neovascular membranes (NVM) by week 1. At weeks 2 and 3, the NVM was replaced by a fibrotic membrane and mild to moderate capillary nonperfusion. In groups B and C, injection of bevacizumab was very effective in preventing or stopping fluorescein leakage but was not able to prevent or reverse vascular dilatation and tortuosity completely. In group D, bevacizumab injection resulted in severe capillary nonperfusion at week 2. CONCLUSIONS: Intravitreous injection of VEGF in rabbits results in florid retinal neovascularization within the first week, followed by closure of normal capillaries by week 2. Early intravitreous injection of bevacizumab can prevent these effects, whereas late injection may be associated with more significant closure of normal capillaries. A sudden drop in effective VEGF concentration may be responsible for the closure of the normal capillaries.

An estrogen receptor repressor induces cataract formation in transgenic mice.

Despite the high prevalence of age-related cataracts, there are currently no known therapies to delay or prevent their occurrence. Studies in humans and rodent models suggest that estrogen may provide protection against age-related cataracts. The discovery of ocular estrogen receptors (ERs) indicates that estrogen protection may result from direct interactions with its receptors in the eye, instead an indirect consequence from effects on another tissue. Studies in our transgenic mouse model validate the concept that estrogen is beneficial for the eye. These mice express ER Delta , a dominant-negative form of ER alpha that inhibits ER alpha function. In the ER Delta 3 transgenic mice, cortical cataracts spontaneously form in ER Delta 3 females after puberty and progress with age. The cataracts initiate in the equatorial region of the lens where the epithelial cells differentiate into elongating fiber cells. Cataract formation can be prevented if the females are ovariectomized before, but not after, sexual maturity. Both male and female ER Delta 3 mice develop cataracts after neonatal treatment with the potent estrogen diethylstilbestrol (DES). The incidence of spontaneous and DES-induced cataracts in ER Delta 3 mice is 100%, yet these cataracts are absent from the wild-type mice. These data suggest that repression of estrogen action induces cortical cataract formation because estrogen is required to activate the ER Delta 3 repressor. Evidence of DES-induced cataracts in the ER Delta 3 males as well as the females suggests that estrogen is important in lens physiology in both sexes. The ER Delta 3 mice provide a powerful model for assessing the role of estrogen in maintaining the transparency of the lens.