Anthony Vugler

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.

Melanopsin-Based Brightness Discrimination in Mice and Humans

Summary Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photoreceptive retinal ganglion cells (ipRGCs), expressing the photopigment melanopsin [1–4]. ipRGCs are known to support various accessory visual functions including circadian photoentrainment and pupillary reflexes. However, despite anatomical and physiological evidence that they contribute to the thalamocortical visual projection [5–7], no aspect of visual discrimination has been shown to rely upon ipRGCs. Based on their currently known roles, we hypothesized that ipRGCs may contribute to distinguishing brightness. This percept is related to an objects luminance—a photometric measure of light intensity relevant for cone photoreceptors. However, the perceived brightness of different sources is not always predicted by their respective luminance [8–12]. Here, we used parallel behavioral and electrophysiological experiments to first show that melanopsin contributes to brightness discrimination in both retinally degenerate and fully sighted mice. We continued to use comparable paradigms in psychophysical experiments to provide evidence for a similar role in healthy human subjects. These data represent the first direct evidence that an aspect of visual discrimination in normally sighted subjects can be supported by inner retinal photoreceptors.

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.

Dopamine neurones form a discrete plexus with melanopsin cells in normal and degenerating retina.

In addition to rods and cones of the outer retina, a third class of photoreceptive cell has recently been described in the inner retina of mammals. These intrinsically photosensitive retinal ganglion cells (ipRGCs) have been shown to relay luminance information to the mammalian brain. In addition to their intrinsic photosensitivity, the function of ipRGCs may also be modulated by signals from within the retina itself. Such signals may emanate from classical photoreceptors in the outer retina or from the circadian activity of adjacent inner retinal neurones. Prime candidates for the latter are the retinal dopamine neurones which ramify at the border of the inner plexiform and inner nuclear layers. In order to investigate the nature of any interaction between dopamine and ipRGC populations in normal retina and to assess the impact of outer retinal degeneration on this interrelationship, we examined the retinae of normal and RCS dystrophic rats. We report a direct interaction between the dendrites of ipRGCs and dopaminergic neurones which is conserved across species. Triple immunolabelling using synaptic markers provides evidence for the unidirectionality of information transfer between the two cell types, with processes of ipRGCs being directly adjacent to sites of dopamine release. This fundamental architectural feature of the mammalian retina appears resistant to degeneration of classical photoreceptors and may provide the anatomical substrate by which dopamine cells influence the physiology of central circadian targets in the brain.

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.

Real-Time In Vivo Imaging of Retinal Cell Apoptosis after Laser Exposure

PURPOSE To investigate whether the detection of apoptosing retinal cells (DARC) could detect cells undergoing apoptosis in a laser model of retinal damage. METHODS Laser lesions were placed, with the use of a frequency-doubled Nd:YAG laser, on the retina in 34 eyes of anesthetized Dark Agouti rats. Lesion size and laser-induced retinal elevation were analyzed using in vivo reflectance imaging. Development of retinal cell apoptosis was assessed using intravitreal fluorescence-labeled annexin 5 in vivo with DARC technology from baseline until 90 minutes after laser application. Histologic analysis of retinal flat mounts and cross-sections was performed. RESULTS The lateral and anteroposterior depth extension of the zone of laser damage was significantly larger for higher exposure settings. A strong diffuse signal, concentrated at the outer retina, was seen with DARC for low exposures (<300 ms and <300 mW). In comparison, higher exposures (>300 ms and >300 mW) resulted in detectable hyperfluorescent spots, mainly at the level of the inner retinal layers. Dose-dependent effects on spot density and positive correlation of spot density between lesion size (P < 0.0001) and retinal elevation (P < 0.0001) were demonstrated. Histology confirmed the presence of apoptosing retinal cells in the inner nuclear and the ganglion cell layers. CONCLUSIONS This is the first time that DARC has been used to determine apoptotic effects in the inner nuclear layer. The ability to monitor changes spatially and temporally in vivo promises to be a major advance in the real-time assessment of retinal diseases and treatment effects.

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.

Light regulation of retinal dopamine that is independent of melanopsin phototransduction

Light‐dependent release of dopamine (DA) in the retina is an important component of light‐adaptation mechanisms. Melanopsin‐containing inner retinal photoreceptors have been shown to make physical contacts with DA amacrine cells, and have been implicated in the regulation of the local retinal environment in both physiological and anatomical studies. Here we determined whether they contribute to photic regulation of DA in the retina as assayed by the ratio of DA with its primary metabolite, 3,4‐dihydroxyphenylacetic acid (DOPAC), and by c‐fos induction in tyrosine hydroxylase (TH)‐labelled DA amacrine cells. Light treatment (∼0.7 log W/m2 for 90 min) resulted in a substantial increase in DA release (as revealed by an increase in the DOPAC : DA ratio), as well as widespread induction of nuclear c‐fos in DA amacrine cells in wild‐type mice and in mice lacking melanopsin (Opn4−/−). Light‐induced DA release was also retained in mice lacking rod phototransduction (Gnat1−/−), although the magnitude of this response was substantially reduced compared with wild‐types, as was the incidence of light‐dependent nuclear c‐fos in DAergic amacrines. By contrast, the DAergic system of mice lacking both rods and cones (rd/rd cl) showed no detectable light response. Our data suggest that light regulation of DA, a pivotal retinal neuromodulator, originates primarily with rods and cones, and that melanopsin is neither necessary nor sufficient for this photoresponse.

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.