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.

An Innovative Surgical Technique for Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigmented Epithelium in Yucatan Mini Pigs: Preliminary Results.

BACKGROUND AND OBJECTIVE: To develop a safe and efficient surgical procedure for subretinal implantation into porcine eyes of a human embryonic stem cell-derived retinal pigmented epithelium (hESC-RPE) monolayer seeded onto a Parylene-C scaffold. This implant is referred to as CPCB-RPE1. MATERIALS AND METHODS: Ultrathin Parylene-C scaffolds were seeded with hESC-RPE and surgically implanted into the subretinal space of Yucatan mini pigs (n = 8). The surgery consisted of pars plana vitrectomy, induction of a limited retinal detachment, and peripheral retinotomy for insertion of the monolayer using a novel tissue injector, followed by silicone oil tamponade injection, laser photocoagulation around the retinotomy site, and inferior iridectomy. Oral cyclosporine was administered from day 1 and during the entire follow-up period. Three months later, the animals were euthanized and the eyes and major organs were submitted for histological analysis. Adjacent sections underwent immunohistochemical analysis to detect human cells using anti-TRA-1-85 (human blood group antigen) antibody and DAPI antibodies. RESULTS: The cell monolayer was immunopositive for TRA-1-85 3 months after implantation and migration from the Parylene-C scaffold was not detected. One eye had a mild inflammatory reaction around the implant that was negative for human biomarkers. No intraocular or systemic tumors were detected. CONCLUSION: The hESC-RPE cells survived for 3 months in this animal model. The surgical procedure for subretinal implantation of CPCB-RPE1 is feasible and safe, without cell migration off the scaffold or development of ocular or systemic tumors.

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.

Subretinal implantation of retinal pigment epithelial cells derived from human embryonic stem cells: improved survival when implanted as a monolayer.

PURPOSE: To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM). METHODS: Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage), and anti-cytokeratin (epithelial marker). RESULTS: The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P < 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group. CONCLUSIONS: hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects.

Acute variations in retinal vascular oxygen content in a rabbit model of retinal venous occlusion.

PURPOSE: To study the variation in intravascular oxygen saturation (oximetry) during an acute retinal vein occlusion (RVO) using hyperspectral computed tomographic spectroscopy based oximetry measurements. METHODS: Thirty rabbits were dilated and anesthetized for experiments. Baseline oximetry measurements were made using a custom-made hyperspectral computed tomographic imaging spectrometer coupled to a fundus camera. RVO were induced using argon green laser following an intravenous injection of Rose Bengal. RVO induction was confirmed by fluorescein angiography. Retinal oximetry measurements were repeated in arterial and venous branches one hour after RVO induction and up to 4 weeks afterwards. Comparison of retinal oximetry before and after vein occlusion was made using the Student T-test. RESULTS: One hour after RVO induction, we observed statistically significant reductions in the intravascular oxygen saturation in temporal retinal arteries (85.1 ± 6.1% vs. 80.6 ± 6.6%; p<0.0001) and veins (71.4 ± 5.5% vs. 64.0 ± 4.7%; p<0.0001). This decrease was reversible in animals that spontaneously recannulated the vein occlusion. There were no statistically significant differences in oxygen saturation in the nasal control arteries and veins before and after temporal vein RVO induction. CONCLUSIONS: We demonstrate, for the first time, acute changes in the intravascular oxygen content of retinal vessels 1 hour after RVO. These changes are reversible upon spontaneous recannulation of retinal vessels. This study demonstrates that hyperspectral computer tomographic spectroscopy based oximetry can detect physiological variations in intravascular retinal oxygen saturation. The study also provides the first qualitative and quantitative evidence of the variation in retinal vascular oxygen content directly attributable to an acute retinal vein occlusion.

Evaluation of ultrasound-assisted thrombolysis using custom liposomes in a model of retinal vein occlusion.

PURPOSE: To study the potential efficacy of ultrasound (US) assisted by custom liposome (CLP) destruction as an innovative thrombolytic tool for the treatment of retinal vein occlusion (RVO). METHODS: Experimental RVO was induced in the right eyes of 40 rabbits using laser photothrombosis; the US experiment took place 48 hours later. Rabbits were randomly divided into four equal groups: US+CLP group, US+saline group, CLP+sham US group, and no treatment group. The latter three groups acted as controls. Fundus fluorescein angiography and Doppler US were used to evaluate retinal blood flow. RESULTS: CLP-assisted US thrombolysis resulted in restoration of flow in seven rabbits (70%). None of the control groups showed significant restoration of retinal venous blood flow. CONCLUSIONS: US-assisted thrombolysis using liposomes resulted in a statistically significant reperfusion of retinal vessels in the rabbit experimental model of RVO. This approach might be promising in the treatment of RVO in humans. Further studies are needed to evaluate this approach in patients with RVO. Ultrasound assisted thrombolysis can be an innovative tool in management of retinal vein occlusion.

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.

Morphometric analysis of optic nerves and retina from an end-stage retinitis pigmentosa patient with an implanted active epiretinal array.

PURPOSE: To characterize optic nerve and retinal changes in a patient with end-stage retinitis pigmentosa (RP) with an implanted active epiretinal array. METHODS: A 74-year-old man with end-stage X-linked RP underwent implantation of an epiretinal array over the macula in the right eye and subsequent stimulation until his death at 5 years and 3 months after implantation. The optic nerves from this study patient, as well as those from two age-matched normal patients and two age-matched RP patients, were morphometrically analyzed against two different sets of criteria and compared. The retina underlying the array in the study patient was also morphometrically analyzed and compared with corresponding regions of the retina in the age-matched RP patients. RESULTS: Optic nerve total axon counts were significantly lower in the study patient and RP patients than in normal patients. However, there was no significant difference when comparing total axon counts from the optic nerve corresponding to the patient's implanted right eye versus the optic nerves from the RP patients (P = 0.59 and P = 0.61 using the two different criteria). Degenerated axon data quantified damage and did not show increased damage in the optic nerve quadrant that retinotopically corresponded to the site of epiretinal array implantation and stimulation. Except for the tack site, there was no significant difference when comparing the retina underlying the array and the corresponding perimacular regions of two RP patients. CONCLUSIONS: Long-term implantation and electrical stimulation with an epiretinal array did not result in damage that could be appreciated in a morphometric analysis of the optic nerve and retina.

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.