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Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor-associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants of IRAK3 , which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD. Irak3 -knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)-expressing human IRAK3 rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in Irak3 -knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.

Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)-derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more. We report successful delivery and survival of the RPE patch by biomicroscopy and optical coherence tomography, and a visual acuity gain of 29 and 21 letters in the two patients, respectively, over 12 months. Only local immunosuppression was used long-term. We also present the preclinical surgical, cell safety and tumorigenicity studies leading to trial approval. This work supports the feasibility and safety of hESC-RPE patch transplantation as a regenerative strategy for AMD.

Age-related macular degeneration remains the most common cause of blindness in the western world, severely comprising patients' and carers' quality of life and presenting a great cost to the healthcare system. As the disease progresses, the retinal pigmented epithelium (RPE) layer at the back of the eye degenerates, contributing to a series of events resulting in visual impairment. The easy accessibility of the eye has allowed for in-depth study of disease progression in patients, while in vivo studies have facilitated investigations into healthy and diseased RPE. Consequently, a number of research groups are examining different approaches for the replacement of RPE cells in age-related macular degeneration (AMD) patients. This chapter examines some of these initial proof-of-principle studies and goes on to review the use of pluripotent stem cells as a source for RPE replacement in a number of current AMD clinical trials. Finally, we consider just some of the regulatory and manufacturing challenges presented in taking a promising AMD treatment from the research bench into clinical trials in patients, and how to mitigate potential risks early in process development., (© 2017 Elsevier B.V. All rights reserved.)

Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study, we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519C>T (p.R120X) into induced pluripotent stem cells (iPSC), and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells, suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization, Golgi cohesion and Gβ1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren), we were able to restore up to 20% of endogenous, full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients., (© The Author 2014. Published by Oxford University Press.)

Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-β pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture., (©AlphaMed Press.)

Controlling the differentiation of human pluripotent stem cells is the goal of many laboratories, both to study normal human development and to generate cells for transplantation. One important cell type under investigation is the retinal pigmented epithelium (RPE). Age-related macular degeneration (AMD), the leading cause of blindness in the Western world, is caused by dysfunction and death of the RPE. Currently, RPE derived from human embryonic stem cells are in clinical trials for the treatment of AMD. Although protocols to generate RPE from human pluripotent stem cells have become more efficient since the first report in 2004, they are still time-consuming and relatively inefficient. We have found that the addition of defined factors at specific times leads to conversion of approximately 80% of the cells to an RPE phenotype in only 14 days. This protocol should be useful for rapidly generating RPE for transplantation as well as for studying RPE development in vitro.

Purpose: Cultured retinal pigment epithelium (RPE) may become a therapeutic option for transplantation in retinal disease. However maintaining a native RPE phenotype in vitro has proven challenging. The human RPE cell-line ARPE-19 is used widely as an alternative to primary RPE. It is grown in DMEM/F12 medium as standard, but its phenotype is dependent on culture conditions, and many differentiation markers are usually absent. The purpose of this study was to examine how this sensitive phenotype of ARPE-19 can be modulated by growth media with or without the metabolite pyruvate to elucidate better RPE growth conditions., Methods: ARPE-19 cells at passages p22 to p28 were cultured on filters for up to 3 months in DMEM/F12 or DMEM media with or without pyruvate and 1% fetal calf serum. Assessment of differentiation was performed using pigmentation, immunocytochemistry, protein/mRNA expression, transepithelial resistance, VEGF secretion, and ultrastructure., Results: Pyruvate, in combination with DMEM, induced dark pigmentation and promoted differentiation markers such as CRALBP and MerTK. Importantly, RPE65 protein was detected by Western blotting and was enhanced by pyruvate, high glucose, and DMEM. ARPE-19 cells maintained in this medium could also phagocytose human photoreceptor outer segments (POS). VEGF secretion was greater in DMEM cultures and was affected by glucose but not by pyruvate. Pigmentation never occurred in DMEM/F12., Conclusions: This study demonstrated important differentiation markers, including pigmentation and Western blots of RPE65 protein, and showed human POS phagocytosis in ARPE-19 cultures using a simple differentiation protocol. The results favor the use of high-glucose DMEM with pyruvate for future RPE differentiation studies.

Background: To describe the 2- to 4-year visual and microperimetry outcomes of autologous retinal pigment epithelium (RPE)-choroid graft in patients with neovascular age-related macular degeneration (AMD)., Methods: In this retrospective cohort study, 12 patients with subfoveal neovascular AMD who had undergone autologous RPE-choroid graft between August 2004 and June 2005 were reviewed. Change in visual acuity (VA), contrast sensitivity (CS), fixation stability and retinal sensitivity on microperimetry after 2-3 years and the rates of late postoperative complications were examined., Results: Patients were followed for 26-48 months (mean, 39). Median preoperative VA (logMAR) was 0.87 but declined to 1.43 (1 year), 1.46 (2 years) and 1.38 (3 years), P = 0.001. Median CS (logCS) was 0.75 preoperatively but declined to 0.45 at 2 years. Six patients had serial microperimetry. Fixation stability declined in 1 but improved in 2 patients. All 6 had decline in retinal sensitivity over the graft during follow up. Retinal detachment did not occur after 12 months but 8 developed epiretinal membrane, 12 had cystic retinal change over the graft and 4 developed recurrent choroidal neovascularization. However, 10 grafts retained autofluorescence signal at 18-48 months of follow up., Conclusions: Autologous RPE-choroid graft can maintain VA, stable fixation and retinal sensitivity in some patients for over 3 years. The spatial correlation between graft autofluorescence, outer retinal structures on optical coherence tomography and retinal sensitivity are consistent with photoreceptor cell rescue. However, we caution the use of this technique as there is high complication rate and delayed loss of retinal function.

Purpose: To compare the long-term outcomes of macular translocation (MT) and autologous RPE-choroid patch graft (PG) in patients with neovascular age-related macular degeneration (AMD)., Methods: This is a retrospective review of the first 12 patients who underwent MT and the first 12 patients who underwent PG. Visual acuity (VA), contrast sensitivity (CS), clinical findings, and complications were recorded. Microperimetry and fundus imaging were reviewed. Outcome measures were the change in VA and CS over 3 years in each group and rates of complication. Microperimetry and fixation in three best cases from each group were described., Results: The two groups were matched for age and VA. Median follow-up durations were 41 (MT) and 38 (PG) months. Median VA (logMAR) was maintained in the MT group: 0.90 at baseline and 0.69 at 3 years (P=0.09) whereas in the PG group, median VA declined from 0.87 to 1.38 at 3 years (P<0.001). Both surgical modalities had high rates of detachment and macular edema. Although more extensive RPE damage occurred in PG, the graft resisted growth of recurrent choroidal neovascularization toward the fovea. Near normal VA was achievable by each technique but macular sensitivity and fixation stability were superior in the MT group., Conclusions: In the present cohort, MT maintained VA for 3 years but PG did not. This outcome may be related to the differences in surgical approach, source of RPE, and choroidal perfusion. The authors recommend MT in preference to PG for treatment of patients with the second eye affected by neovascular AMD unsuitable for other treatment.

Purpose: To examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells., Methods: HESC-RPE cells were derived from a super-confluent preparation of the Shef1 HESC line. Pigmented colonies were isolated and expanded into pigmented monolayers on Matrigel matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer segments isolated from the porcine eye and assessed for phagocytic activity at regular intervals. Expression of molecules associated with RPE phagocytosis was analyzed by RT-PCR, immunocytochemistry, and western blot. The role of Mer Tyrosine Kinase (MERTK) in the phagocytosis of outer segments was investigated using antibodies directed against MERTK to block function. In a novel approach, cells were also exposed to fresh human neural retina tissue then examined by electron microscopy for evidence of phagocytosis and changes in cell morphology., Results: HESC-derived RPE cells are capable of phagocytosing isolated porcine outer segments and express molecules associated with RPE-specific phagocytosis, including MERTK. Pre-incubation with antibodies against MERTK blocked phagocytosis of photoreceptor outer segments, but not polystyrene beads. HESC-RPE cells also phagocytosed outer segments in a novel human retinal explant system. Furthermore co-culture adjacent to human retina tissue in this preparation resulted in the appearance of features in HESC-derived RPE cells normally observed only as the RPE matures., Conclusions: The ingestion of photoreceptor outer segments from an isolated population and an artificial ex vivo human retina system demonstrates HESC-derived RPE cells are functional. HESC-derived RPE possess the relevant molecules required for phagocytosis, including MERTK, which is essential for the phagocytosis of outer segments but not latex beads. Furthermore, some changes observed in cell morphology after co-culture with human retina may have implications for understanding the full development and differentiation of RPE cells.

Healthy Retinal Pigment Epithelium (RPE) cells are required for proper visual function and the phenomenon of RPE derivation from Human Embryonic Stem Cells (HESC) holds great potential for the treatment of retinal diseases. However, little is known about formation, expansion and expression profile of RPE-like cells derived from HESC (HESC-RPE). By studying the genesis of pigmented foci we identified OTX1/2-positive cell types as potential HESC-RPE precursors. When pigmented foci were excised from culture, HESC-RPE expanded to form extensive monolayers, with pigmented cells at the leading edge assuming a precursor role: de-pigmenting, proliferating, expressing keratin 8 and subsequently re-differentiating. As they expanded and differentiated in vitro, HESC-RPE expressed markers of both developing and mature RPE cells which included OTX1/2, Pax6, PMEL17 and at low levels, RPE65. In vitro, without signals from a developing retinal environment, HESC-RPE could produce regular, polarised monolayers with developmentally important apical and basal features. Following transplantation of HESC-RPE into the degenerating retinal environment of Royal College of Surgeons (RCS) dystrophic rats, the cells survived in the subretinal space, where they maintained low levels of RPE65 expression and remained out of the cell cycle. The HESC-RPE cells responded to the in vivo environment by downregulating Pax6, while maintaining expression of other markers. The presence of rhodopsin-positive material within grafted HESC-RPE indicates that in the future, homogenous transplants of this cell type may be capable of supporting visual function following retinal dystrophy.

Inherited retinal dystrophies are an important cause of blindness, for which currently there are no effective treatments. In order to study this heterogeneous group of diseases, adequate disease models are required in order to better understand pathology and to test potential therapies. Induced pluripotent stem cells offer a new way to recapitulate patient specific diseases in vitro, providing an almost limitless amount of material to study. We used fibroblast-derived induced pluripotent stem cells to generate retinal pigment epithelium (RPE) from an individual suffering from retinitis pigmentosa associated with biallelic variants in MERTK. MERTK has an essential role in phagocytosis, one of the major functions of the RPE. The MERTK deficiency in this individual results from a nonsense variant and so the MERTK-RPE cells were subsequently treated with two translational readthrough inducing drugs (G418 & PTC124) to investigate potential restoration of expression of the affected gene and production of a full-length protein. The data show that PTC124 was able to reinstate phagocytosis of labeled photoreceptor outer segments at a reduced, but significant level. These findings represent a confirmation of the usefulness of iPSC derived disease specific models in investigating the pathogenesis and screening potential treatments for these rare blinding disorders.

This article discusses the safety, structure, and function of two patients who underwent human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) patch transplantation for acute neovascular age-related macular degeneration (AMD) with submacular hemorrhage. The study followed the patients for five years and found no evidence of uncontrolled RPE proliferation, tumor formation, or major ocular immune reactions. The patients experienced improved best corrected visual acuity (BCVA) at two years, although the BCVA decreased for one patient at five years. The study also observed patch pigmentation and areas of continuous hyper-reflective lines consistent with the hESC-RPE on imaging. The authors note the need for further studies to address remaining questions in the field. [Extracted from the article]

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention.

(1) Background: We reviewed a stem cell-derived therapeutic strategy for advanced neovascular age-related macular degeneration (nAMD) using a human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) monolayer delivered on a coated, synthetic basement membrane (BM)—the patch—and assessed the presence and distribution of hESC-RPE over 5 years following transplantation, as well as functional outcomes. (2) Methods: Two subjects with acute vision loss due to sub-macular haemorrhage in advanced nAMD received the hESC-RPE patch. Systematic immunosuppression was used peri-operatively followed by local depot immunosuppression. The subjects were monitored for five years with observation of RPE patch pigmentation, extension beyond the patch boundary into surrounding retina, thickness of hESC-RPE and synthetic BM and review for migration and proliferation of hESC-RPE. Visual function was also assessed. (3) Results: The two study participants showed clear RPE characteristics of the patch, preservation of some retinal ultrastructure with signs of remodelling, fibrosis and thinning on optical coherence tomography over the 5-year period. For both participants, there was evidence of pigment extension beyond the patch continuing until 12 months post-operatively, which stabilised and was preserved until 5 years post-operatively. Measurement of hESC-RPE and BM thickness over time for both cases were consistent with predefined histological measurements of these two layers. There was no evidence of distant RPE migration or proliferation in either case beyond the monolayer. Sustained visual acuity improvement was apparent for 2 years in both subjects, with one subject maintaining the improvement for 5 years. Both subjects demonstrated initial improvement in fixation and microperimetry compared to baseline, at year 1, although only one maintained this at 4 years post-intervention. (4) Conclusions: hESC-RPE patches show evidence of continued pigmentation, with extension, to cover bare host basement membrane for up to 5 years post-implantation. There is evidence that this represents functional RPE on the patch and at the patch border where host RPE is absent. The measurements for thickness of hESC-RPE and BM suggest persistence of both layers at 5 years. No safety concerns were raised for the hypothetical risk of RPE migration, proliferation or tumour formation. Visual function also showed sustained improvement for 2 years in one subject and 5 years in the other subject. [ABSTRACT FROM AUTHOR]

Highlights: [•] Age-related macular degeneration (AMD) is the leading cause of vision loss in older adults. Recent research for treating AMD has focused on replacing the retinal pigment epithelium (RPE), a monolayer of cells vital to photoreceptor cell health [•] Various methods are used to differentiate and purify RPE from human embryonic stem cells, and their efficacy as treatments are being tested in existing and forthcoming clinical trials. [ABSTRACT FROM AUTHOR]