Antisense Therapy for a Common Corneal Dystrophy Ameliorates TCF4 Repeat Expansion-Mediated Toxicity.

Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.

A role for the ciliary marginal zone in the melanopsin-dependent intrinsic pupillary light reflex.

Maintenance of pupillary constriction in light-adapted rodents has traditionally been thought to involve a reflex between retina, brain and iris, with recent work identifying the melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) as the major conduits for retinal input to the brain. There is also a less well-understood phenomenon whereby the iris of some mammals, including mice, will constrict to light when either the eye, or the iris itself is physically isolated from the brain. The intrinsic pupillary light reflex (iPLR) is the term given to pupil constriction in the absence of retinal input to the brain. Here, using an intraocular axotomy approach, we show that the iPLR in conscious mice spans a dynamic range over 3 log units of irradiance. This iPLR response is absent in melanopsin knockout (MKO) mice and can be significantly inhibited by atropine. Immunohistochemistry for cfos and melanopsin, in combination with light exposure revealed a population of small ipRGCs in the retinal ciliary marginal zone (CMZ), which remain responsive to light in axotomised mice. We report that damage to the CMZ in a novel in vitro preparation removes a significant component of the iPLR response, while a detailed immunohistochemical analysis of the CMZ in wildtype mice revealed a melanopsin-rich plexus, which was consistently most intense in nasal retina. There were clear examples of melanopsin-positive, direct retino-ciliary projections, which appear to emanate from Brn3b negative, M1 type ipRGCs. These cells are clustered along the melanopsin-rich plexus nasally and may channel ipRGC signals from retina into the iris via ciliary body. Comparison between wildtype and MKO mice reveals that the ciliary body is also weakly stained for melanopsin. Our results show that the full extent of iPLR in mice requires cholinergic neurotransmission and intact signalling at the CMZ/ciliary body. This response may be mediated to some extent by ipRGCs, which send direct projections from the retina into ciliary body. In addition to the melanopsin-mediated iris sphincter constriction suggested by others, we propose a new mechanism, which may involve constriction of the ciliary body and ipRGC-mediated relaxation of the iris dilator muscle.

Melanopsin contributions to irradiance coding in the thalamo-cortical visual system.

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a subset of retinal ganglion cells expressing the photopigment melanopsin (mRGCs). These mRGCs are known to drive such reflex light responses as circadian photoentrainment and pupillomotor movements. By contrast, until now there has been no direct assessment of their contribution to conventional visual pathways. Here, we address this deficit. Using new reporter lines, we show that mRGC projections are much more extensive than previously thought and extend across the dorsal lateral geniculate nucleus (dLGN), origin of thalamo-cortical projection neurons. We continue to show that this input supports extensive physiological light responses in the dLGN and visual cortex in mice lacking rods+cones (a model of advanced retinal degeneration). Moreover, using chromatic stimuli to isolate melanopsin-derived responses in mice with an intact visual system, we reveal strong melanopsin input to the ∼40% of neurons in the LGN that show sustained activation to a light step. We demonstrate that this melanopsin input supports irradiance-dependent increases in the firing rate of these neurons. The implication that melanopsin is required to accurately encode stimulus irradiance is confirmed using melanopsin knockout mice. Our data establish melanopsin-based photoreception as a significant source of sensory input to the thalamo-cortical visual system, providing unique irradiance information and allowing visual responses to be retained even in the absence of rods+cones. These findings identify mRGCs as a potential origin for aspects of visual perception and indicate that they may support vision in people suffering retinal degeneration.

Rodent models in translational circadian photobiology.

Over the last decades remarkable advances have been made in the understanding of the photobiology of circadian rhythms. The identification of a third photoreceptive system in the mammalian eye, in addition to the rods and cones that mediate vision, has transformed our appreciation of the role of light in regulating physiology and behavior. These photosensitive retinal ganglion cells (pRGCs) express the blue-light sensitive photopigment melanopsin and project to the suprachiasmatic nuclei (SCN)-the master circadian pacemaker-as well as many other brain regions. Much of our understanding of the fundamental mechanisms of the pRGCs, and the processes that they regulate, comes from mouse and other rodent models. Here we describe the contribution of rodent models to circadian photobiology, including both their strengths and limitations. In addition, we discuss how an appreciation of both rodent and human data is important for translational circadian photobiology. Such an approach enables a bi-directional flow of information whereby an understanding of basic mechanisms derived from mice can be integrated with studies from humans. Progress in this field is being driven forward at several levels of analysis, not least by the use of personalized light measurements and photoreceptor specific stimuli in human studies, and by studying the impact of environmental, rather than laboratory, lighting on different rodent models.

Developmental dynamics of cone photoreceptors in the eel.

BACKGROUND: Many fish alter their expressed visual pigments during development. The number of retinal opsins expressed and their type is normally related to the environment in which they live. Eels are known to change the expression of their rod opsins as they mature, but might they also change the expression of their cone opsins? RESULTS: The Rh2 and Sws2 opsin sequences from the European Eel were isolated, sequenced and expressed in vitro for an accurate measurement of their lambdamax values. In situ hybridisation revealed that glass eels express only rh2 opsin in their cone photoreceptors, while larger yellow eels continue to express rh2 opsin in the majority of their cones, but also have <5% of cones which express sws2 opsin. Silver eels showed the same expression pattern as the larger yellow eels. This observation was confirmed by qPCR (quantitative polymerase chain reaction). CONCLUSIONS: Larger yellow and silver European eels express two different cone opsins, rh2 and sws2. This work demonstrates that only the Rh2 cone opsin is present in younger fish (smaller yellow and glass), the sws2 opsin being expressed additionally only by older fish and only in <5% of cone cells.

Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay.

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.

Embryonic stem cells and retinal repair.

In this review we examine the potential of embryonic stem cells (ESCs) for use in the treatment of retinal diseases involving photoreceptors and retinal pigment epithelium (RPE). We outline the ontogenesis of target retinal cell types (RPE, rods and cones) and discuss how an understanding of developmental processes can inform our manipulation of ESCs in vitro. Due to their potential for cellular therapy, special emphasis is placed upon the derivation and culture of human embryonic stem cells (HESCs) and their differentiation towards a retinal phenotype. In terms of achieving this goal, we suggest that much of the success to date reflects permissive in vitro environments provided by established protocols for HESC derivation, propagation and neural differentiation. In addition, we summarise key factors that may be important for enhancing efficiency of retinal cell-type derivation from HESCs. The retina is an amenable component of the central nervous system (CNS) and as such, diseases of this structure provide a realistic target for the application of HESC-derived cellular therapy to the CNS. In order to further this goal, the second component of our review focuses on the cellular and molecular cues within retinal environments that may influence the survival and behaviour of transplanted cells. Our analysis considers both the potential barriers to transplant integration in the retina itself together with the remodelling in host visual centres that is known to accompany retinal dystrophy.

Mature retinal pigment epithelium cells are retained in the cell cycle and proliferate in vivo.

PURPOSE: To investigate the capacity of mature retinal pigment epithelium (RPE) cells to enter the cell cycle in vivo using a range of RPE-specific and proliferative specific markers in both pigmented and albino rats. METHODS: Whole-mounted retinas of both Dark Agouti and albino rats were immunolabeled with cell cycle markers Ki67 or PCNA and double labeled with RPE cell marker RPE65 or CRALBP. The number and distribution of these cells was mapped. An additional group of Dark Agouti rats were given repeated intraperitoneal injections of Bromodeoxyuridine (BrdU )for 20 days and then sacrificed 30 days later. The retinas were then processed for BrdU detection and Otx, a RPE cell-specific marker. For comparison, human RPE tissue from a postmortem donor was also labeled for Ki67. RESULTS: In both pigmentation phenotypes, a subpopulation of mature RPE cells in the periphery were positive for both cell cycle markers. These cells were negative for Caspase 3, hence were not apoptotic. Ki67-positive cells were also seen in human RPE. Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate. There was a ten fold increase in the number of RPE cells positive for cell cycle markers in albino (approximately 200 cells) compared to pigmented rats (approximately 20 cells). CONCLUSIONS: Peripheral RPE cells in rats have the capacity to enter the cell cycle and complete cellular division.

Survival and remodeling of melanopsin cells during retinal dystrophy.

The melanopsin positive, intrinsically photosensitive retinal ganglion cells (ipRGCs) of the inner retina have been shown to send wide-ranging projections throughout the brain. To investigate the response of this important cell type during retinal dystrophy, we use the Royal College of Surgeons (RCS) dystrophic rat, a major model of retinal degeneration. We find that ipRGCs exhibit a distinctive molecular profile that remains unaltered during early stages of outer retinal pathology (15 weeks of age). In particular, these cells express betaIII tubulin, alpha-acetylated tubulin, and microtubule-associated proteins (MAPs), while remaining negative for other RGC markers such as neurofilaments, calretinin, and parvalbumin. By 14 months of age, melanopsin positive fibers invade ectopic locations in the dystrophic retina and ipRGC axons/dendrites become distorted (a process that may involve vascular remodeling). The morphological abnormalities in melanopsin processes are associated with elevated immunoreactivity for MAP1b and a reduction in alpha-acetylated tubulin. Quantification of ipRGCs in whole mounts reveals reduced melanopsin cell number with increasing age. Focusing on the retinal periphery, we find a significant decline in melanopsin cell density contrasted by a stability of melanopsin positive processes. In addition to these findings, we describe for the first time, a distinct plexus of melanopsin processes in the far peripheral retina, a structure that is coincident with a short wavelength opsin cone-enriched rim. We conclude that some ipRGCs are lost in RCS dystrophic rats as the disease progresses and that this loss may involve vascular remodeling. However, a significant number of melanopsin positive cells survive into advanced stages of retinal degeneration and show indications of remodeling in response to pathology. Our findings underline the importance of early intervention in human retinal disease in order to preserve integrity of the inner retinal photoreceptive network.

Eel visual pigments revisited: the fate of retinal cones during metamorphosis.

During their complex life history, anguilliform eels go through a major metamorphosis when developing from a fresh water yellow eel into a deep-sea silver eel. In addition to major changes in body morphology, the visual system also adapts from a fresh water teleost duplex retina with rods and cones, to a specialized deep-sea retina containing only rods. The history of the rods is well documented with an initial switch from a porphyropsin to a rhodopsin (P523(2) to P501(1)) and then a total change in gene expression with the down regulation of a "freshwater" opsin and its concomitant replacement by the expression of a typical "deep-sea" opsin (P501(1) to P482(1)). Yellow eels possess only two spectral classes of single cones, one sensitive in the green presumably expressing an RH2 opsin gene and the second sensitive in the blue expressing an SWS2 opsin gene. In immature glass eels, entering into rivers from the sea, the cones contain mixtures of rhodopsins and porphyropsins, whereas the fully freshwater yellow eels have cone pigments that are almost pure porphyropsins with peak sensitivities at about 540-545 nm and 435-440 nm, respectively. However, during the early stages of metamorphosis, the pigments switch to rhodopsins with the maximum sensitivity of the "green"-sensitive cone shifting to about 525 nm, somewhat paralleling, but preceding the change in rods. During metamorphosis, the cones are almost completely lost.

Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models.

Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing.

Retrograde Melanopsin Signaling Increases With Age in Retinal Degenerate Mice Lacking Rods and the Majority of Cones.

PURPOSE: Following on from reports of retrograde retinal signaling in mice, we sought to investigate the influence of age and retinal location on this phenomenon using mice that lack rods and the majority of cones. METHODS: We used functional anatomy for c-fos (Fos) and tyrosine hydroxylase (TH) to measure light-driven activation of dopamine neurons along a dorsal-ventral transect in C3H/He wild-type and rodless-coneless rd/rd cl (rdcl) mice aged 3, 5, and >14 months. A parallel series of retinae from 3-month-old mice was also stained for cone opsins and melanopsin. RESULTS: Analysis by confocal microscopy revealed light-driven Fos activation in TH cells residing in the middorsal retina of the youngest rdcl mice. This region was largely devoid of residual cones but contained a large number of intrinsically photosensitive retinal ganglion cells (ipRGCs) and the highest density of melanopsin neurites. With advancing age, there was a paradoxical increase in retrograde signaling from ∼3% Fos-positive (Fos+) TH cells at 3 months to ∼36% in rdcl mice >14 months. This increased activation occurred in more central and peripheral retinal regions. CONCLUSIONS: Our data provide new insights into the anatomy and plasticity of retrograde melanopsin signaling in mice with severe rod/cone dystrophy. The increased retrograde signaling we detect may result from either an increased potency of melanopsin signaling with advancing age and/or postsynaptic modification to dopaminergic neurons.

Efficacy and Safety of Human Retinal Progenitor Cells.

PURPOSE: We assessed the long-term efficacy and safety of human retinal progenitor cells (hRPC) using established rodent models. METHODS: Efficacy of hRPC was tested initially in Royal College of Surgeons (RCS) dystrophic rats immunosuppressed with cyclosporine/dexamethasone. Due to adverse effects of dexamethasone, this drug was omitted from a subsequent dose-ranging study, where different hRPC doses were tested for their ability to preserve visual function (measured by optokinetic head tracking) and retinal structure in RCS rats at 3 to 6 months after grafting. Safety of hRPC was assessed by subretinal transplantation into wild type (WT) rats and NIH-III nude mice, with analysis at 3 to 6 and 9 months after grafting, respectively. RESULTS: The optimal dose of hRPC for preserving visual function/retinal structure in dystrophic rats was 50,000 to 100,000 cells. Human retinal progenitor cells integrated/survived in dystrophic and WT rat retina up to 6 months after grafting and expressed nestin, vimentin, GFAP, and ßIII tubulin. Vision and retinal structure remained normal in WT rats injected with hRPC and there was no evidence of tumors. A comparison between dexamethasone-treated and untreated dystrophic rats at 3 months after grafting revealed an unexpected reduction in the baseline visual acuity of dexamethasone-treated animals. CONCLUSIONS: Human retinal progenitor cells appear safe and efficacious in the preclinical models used here. TRANSLATIONAL RELEVANCE: Human retinal progenitor cells could be deployed during early stages of retinal degeneration or in regions of intact retina, without adverse effects on visual function. The ability of dexamethasone to reduce baseline visual acuity in RCS dystrophic rats has important implications for the interpretation of preclinical and clinical cell transplant studies.

Impact of age and retinal degeneration on the light input to circadian brain structures.

Aging causes anatomical and functional changes in visual and circadian systems. In wild type mice rods, cones, and photosensitive retinal ganglion cells (pRGCs) decline with age. In rd/rd cl mice, the early loss of rods and cones is followed by protracted transneuronal loss of inner retinal neurons as well as the pRGCs. Here we use Fos induction to study the light input pathway to the suprachiasmatic nuclei (SCN), the intergeniculate leaflets (IGL) and ventral lateral geniculate nuclei (vLGN) of old (∼700 days) and young (∼150 days) wild type and rd/rd cl mice. Cholera toxin tracing was used in parallel to study the anatomy of this pathway. We find that aging rather than retinal degeneration is a more important factor in reducing light input to the SCN, causing both a reduction in Fos expression and retinal afferents. Furthermore, we show light-induced Fos within the vLGN and IGL is predominantly subserved by rods and cones, and once again aging reduces the amplitude of this response.

Degeneration of cortical function in the Royal College of Surgeons rat.

The purpose of the current study was to determine the progress of cortical functional degeneration in the Royal College of Surgeons (RCS) rat. Cortical responses were measured with optical imaging of intrinsic signals using gratings of various spatial frequencies. Subsequently, electrophysiological recordings were also taken across cortical layers in response to a pulse of broad-spectrum light. We found significant degeneration in the cortical processing of visual information as early as 4 weeks of age. These results show that degeneration in the cortical response of the RCS rat starts before development has been properly completed.

Induction of differentiation by pyruvate and DMEM in the human retinal pigment epithelium cell line ARPE-19.

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.

Dissecting a role for melanopsin in behavioural light aversion reveals a response independent of conventional photoreception.

Melanopsin photoreception plays a vital role in irradiance detection for non-image forming responses to light. However, little is known about the involvement of melanopsin in emotional processing of luminance. When confronted with a gradient in light, organisms exhibit spatial movements relative to this stimulus. In rodents, behavioural light aversion (BLA) is a well-documented but poorly understood phenomenon during which animals attribute salience to light and remove themselves from it. Here, using genetically modified mice and an open field behavioural paradigm, we investigate the role of melanopsin in BLA. While wildtype (WT), melanopsin knockout (Opn4(-/-)) and rd/rd cl (melanopsin only (MO)) mice all exhibit BLA, our novel methodology reveals that isolated melanopsin photoreception produces a slow, potentiating response to light. In order to control for the involvement of pupillary constriction in BLA we eliminated this variable with topical atropine application. This manipulation enhanced BLA in WT and MO mice, but most remarkably, revealed light aversion in triple knockout (TKO) mice, lacking three elements deemed essential for conventional photoreception (Opn4(-/-) Gnat1(-/-) Cnga3(-/-)). Using a number of complementary strategies, we determined this response to be generated at the level of the retina. Our findings have significant implications for the understanding of how melanopsin signalling may modulate aversive responses to light in mice and humans. In addition, we also reveal a clear potential for light perception in TKO mice.

Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat.

Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.

Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation.

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.

Paradoxical opsin expressing cells in the inner retina that are augmented following retinal degeneration.

Here we reveal a population of cells that express cone photoreceptor opsins that are located in the inner retina, distant from outer retinal photoreceptors. These cells are present in rodents and human. They also express a range of key proteins critical in the cone phototransduction cascade and make contact with other retinal neurons. Their opsins are not generally confined to cellular specialized regions but are present throughout the plasma membrane, although their nuclear configurations are similar to those of outer retinal cones. This population is distinct from the ganglion cells that contain melanopsin and which are known to be inner retinal irradiance detectors regulating circadian behaviour. Surprisingly, the size of the population of short wavelength opsin positive cells in the ganglion cell layer is plastic. In normal animals their number declines with age. However, their numbers increase significantly in response to outer retinal photoreceptor loss, probably by drawing on a pool of inner retinal cells that express cone specific markers, but not opsins.