The new paradigm: retinal pigment epithelium cells generated from embryonic or induced pluripotent stem cells

Compared with neural crest‐derived melanocytes, retinal pigment epithelium (RPE) cells in the back of the eye are pigment cells of a different kind. They are a part of the brain, form an epithelial monolayer, respond to distinct extracellular signals, and provide functions that far exceed those of a light‐absorbing screen. For instance, they control nutrient and metabolite flow to and from the retina, replenish 11‐cis‐retinal by re‐isomerizing all‐trans‐retinal generated during photoconversion, phagocytose daily a portion of the photoreceptors’ outer segments, and secrete cytokines that locally control the innate and adaptive immune systems. Not surprisingly, RPE cell damage is a major cause of human blindness worldwide, with age‐related macular degeneration a prevalent example. RPE replacement therapies using RPE cells generated from embryonic or induced pluripotent stem cells provide a novel approach to a rational treatment of such forms of blindness. In fact, RPE‐like cells can be obtained relatively easily when stem cells are subjected to a two‐step induction protocol, a first step that leads to a neuroectodermal fate and a second to RPE differentiation. Here, we discuss the characteristics of such cells, propose criteria they should fulfill in order to be considered authentic RPE cells, and point out the challenges one faces when using such cells in attempts to restore vision.     

MEK-ERK and heparin-susceptible signaling pathways are involved in cell-cycle entry of the wound edge retinal pigment epithelium cells in the adult newt

The onset mechanism of proliferation in mitotically quiescent retinal pigment epithelium (RPE) cells is still obscure in humans and newts, although it can be a clinical target for manipulating both retinal diseases and regeneration. To address this issue, we investigated factors or signaling pathways involved in the first cell-cycle entry of RPE cells upon retinal injury using a newt retina-less eye-cup culture system in which the cells around the wound edge of the RPE exclusively enter the cell cycle. We found that MEK-ERK signaling is necessary for their cell-cycle entry, and signaling pathways whose activities can be modulated by heparin, such as Wnt-, Shh-, and thrombin-mediated pathways, are capable of regulating the cell-cycle entry. Furthermore, we found that the cells inside the RPE have low proliferation competence even in the presence of serum, suggesting inversely that a loss of cell-to-cell contact would allow the cells to enter the cell cycle.     

Retinal pigmented epithelium does not transdifferentiate in adult goldfish

The neural retina of adult goldfish can regenerate from an intrinsic source of proliferative neuronal progenitor cells, but it is not known whether the retina can regenerate by transdifferentiation of the retinal pigmented epithelium (RPE), a phenomenon demonstrated in adult newts. In this study, we asked whether following surgical removal of the neural retina in adult goldfish the RPE was capable of autonomously transdifferentiating and generating new neural retina. The retina was prelabeled by injecting the fluorescent dye Fluoro-Gold (FG) into the eye prior to surgical removal; this procedure ensured that residual retina was labeled with FG and could therefore be distinguished from unlabeled, regenerated retina. To examine the time course of retinal regeneration, and to identify regenerated retinal neurons, the thymidine analogue bromodeoxyuridine was injected intraocularly, and retinas were examined up to 2 months later. We found that the RPE did not transdifferentiate; instead, retinas regenerated only when pieces of residual neural retina were left intact. Under these circumstances, newly regenerated cells derived from proliferating cells intrinsic to the residual neural retina. When retinas were completely removed, as was evident from a lack of FG labeling, there was no retinal regeneration. © 1995 John Wiley & Sons, Inc.     

MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: Is FGF2 an induction factor?

Adult newts can regenerate their entire retinas through transdifferentiation of the retinal pigment epithelium (RPE) cells. As yet, however, underlying molecular mechanisms remain virtually unknown. On the other hand, in embryonic/larval vertebrates, an MEK [mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) kinase] pathway activated by fibroblast growth factor‐2 (FGF2) is suggested to be involved in the induction of transdifferentiation of the RPE into a neural retina. Therefore, we examined using culture systems whether the FGF2/MEK pathway is also involved in the adult newt RPE transdifferentiation. Here we show that the adult newt RPE cells can switch to neural cells expressing pan‐retinal‐neuron (PRN) markers such as acetylated tubulin, and that an MEK pathway is essential for the induction of this process, whereas FGF2 seems an unlikely primary induction factor. In addition, we show by immunohistochemistry that the PRN markers are not expressed until the 1–3 cells thick regenerating retina, which contains retinal progenitor cells, appears. Our current results suggest that the activation of an MEK pathway in RPE cells might be involved in the induction process of retinal regeneration in the adult newt, however if this is the case, we must assume complementary mechanisms that repress the MEK‐mediated misexpression of PRN markers in the initial process of transdifferentiation.     

Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: A study of retinal regeneration in a novel animal model

Our research group has extensively studied retinal regeneration in adult Xenopus laevis. However, X. laevis does not represent a suitable model for multigenerational genetics and genomic approaches. Instead, Xenopus tropicalis is considered as the ideal model for these studies, although little is known about retinal regeneration in X. tropicalis. In the present study, we showed that a complete retina regenerates at approximately 30 days after whole retinal removal. The regenerating retina was derived from the stem/progenitor cells in the ciliary marginal zone (CMZ), indicating a novel mode of vertebrate retinal regeneration, which has not been previously reported. In a previous study, we showed that in X. laevis, retinal regeneration occurs primarily through the transdifferentiation of retinal pigmented epithelial (RPE) cells. RPE cells migrate to the retinal vascular membrane and reform a new epithelium, which then differentiates into the retina. In X. tropicalis, RPE cells also migrated to the vascular membrane, but transdifferentiation was not evident. Using two tissue culture models of RPE tissues, it was shown that in X. laevis RPE culture neuronal differentiation and reconstruction of the retinal three‐dimensional (3‐D) structure were clearly observed, while in X. tropicalis RPE culture neither ßIII tubulin‐positive cells nor 3‐D retinal structure were seen. These results indicate that the two Xenopus species are excellent models to clarify the cellular and molecular mechanisms of retinal regeneration, as these animals have contrasting modes of regeneration; one mode primarily involves RPE cells and the other mode involves stem/progenitor cells in the CMZ. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 739–756, 2014     

Glycyrrhizin protects against sodium iodate‐induced RPE and retinal injury though activation of AKT and Nrf2/HO‐1 pathway

Glycyrrhizin is a bioactive triterpenoid saponin extracted from a traditional Chinese medicinal herb, glycyrrhiza, and has been reported to protect the organs such as liver and heart from injuries. However, there is no report about the effects of glycyrrhizin on atrophic age‐related macular degeneration (AMD). This study investigated the effects of glycyrrhizin on retinal pigment epithelium (RPE) in vitro and retina of mice in vivo treated with sodium iodate (SI). Glycyrrhizin significantly inhibited SI‐induced reactive oxygen species (ROS), and decreased apoptosis of RPE in vitro. The underlying mechanisms included increased phosphorylation of Akt, and increased expression of nuclear factor erythroid 2‐related factor2 (Nrf‐2) and HO‐1, thereby protecting RPE from SI‐induced ROS and apoptosis. Furthermore, glycyrrhizin significantly decreased the apoptosis of retinal cells in vivo, resulting in the inhibition of thinning of retina, decreasing the number of drusen and improving the function of retina. These findings suggested that glycyrrhizin may be a potential candidate for the treatment of atrophic AMD in clinical practice.     

Tsg101 Is Necessary for the Establishment and Maintenance of Mouse Retinal Pigment Epithelial Cell Polarity

The retinal pigment epithelium (RPE) forms a monolayer sheet separating the retina and choroid in vertebrate eyes. The polarized nature of RPE is maintained by distributing membrane proteins differentially along apico-basal axis. We found the distributions of these proteins differ in embryonic, post-natal, and mature mouse RPE, suggesting developmental regulation of protein trafficking. Thus, we deleted tumor susceptibility gene 101 (Tsg101), a key component of endosomal sorting complexes required for transport (ESCRT), in embryonic and mature RPE to determine whether ESCRT-mediated endocytic protein trafficking correlated with the establishment and maintenance of RPE polarity. Loss of Tsg101 severely disturbed the polarity of RPE, which forms irregular aggregates exhibiting non-polarized distribution of cell adhesion proteins and activation of epidermal growth factor receptor signaling. These findings suggest that ESCRT-mediated protein trafficking is essential for the development and maintenance of RPE cell polarity.     

Neural cell differentiation from retinal pigment epithelial cells of the newt: An organ culture model for the urodele retinal regeneration

Transdifferentiation from retinal pigment epithelium (RPE) to neural retina (NR) was studied under a new culture system as an experimental model for newt retinal regeneration. Adult newt RPEs were organ cultured with surrounding connective tissues, such as the choroid and sclera, on a filter membrane. Around day 7 in vitro, lightly pigmented “neuron‐like cells” with neuritic processes were found migrating out from the explant onto the filter membrane. Their number gradually increased day by day. BrdU‐labeling study showed that RPE cells initiated to proliferate under the culture condition on day 4 in vitro, temporally correlating to the time course of retinal regeneration in vivo. Histological observations of cultured explants showed that proliferating RPE cells did not form the stratified structure typically observed in the NR but they rather migrated out from the explants. Neuronal differentiation was examined by immunohistochemical detection of various neuron‐specific proteins; HPC‐1 (syntaxin), GABA, serotonin, rhodopsin, and acetylated tubulin. Immunoreactive cells for these proteins always possessed fine and long neurite‐like processes. Numerous lightly pigmented cells with neuron‐like morphology showed HPC‐1 immunoreactivity. Fibroblast growth factor‐2 (FGF‐2), known as a potent factor for the transdifferentiation of ocular tissues in various vertebrates, substantially increased the numbers of both neuron‐like cells and HPC‐1‐like immunoreactive cells in a dose‐dependent manner. These results indicate that our culture method ensures neural differentiation of newt RPE cells in vitro and provides, for the first time, a suitable in vitro experimental model system for studying tissue‐intrinsic factors responsible for newt retinal regeneration. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 209–220, 2002; DOI 10.1002/neu.10031     

Patterning the optic neuroepithelium by FGF signaling and Ras activation.

During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. We report that FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina.     

An in vitro model of the back of the eye for studying retinal pigment epithelial-choroidal endothelial interactions

At the back of the eye, the outermost cell layer of the retina, the pigmented epithelium, lies against a basement membrane that is adjacent to the choroidal vessels that supply the outer sensory retina. During pathogenesis, these interfaces become damaged, and the homeostatic balance between the retinal pigment epithelium (RPE) and the choroidal vessels becomes disrupted, leading to choroidal neovascularization and blindness. To study the cell interactions at the back of the eye, we have used a coculture system in which a stable RPE monolayer has been cultured on a transwell insert and placed over a collagen gel sandwich into which choroidal endothelial cells (CECs) have been seeded. RPE cells have been stimulated by an inflammatory cytokine, interleukin-1 (IL-1beta), and the ability of the underlying choroidal endothelium to form vascular tubes has been tested. IL-1beta stimulation of the RPE insert increased the number of tubes formed by CECs in the gel as early as 3 d. By 7 d, tubes began to regress. Both IL-8 and monocyte chemotactic protein-1 (MCP-1) were found to be secreted in greater amounts in stimulated RPE. Because MCP-1 is also a chemokine for monocytes, which in turn secrete angiogenic factors, monocytes were added to the upper surface of the choroidal gel sandwich and then incubated with the stimulated RPE insert as above. By day 7, more tubes formed and there was no regression over the experimental time period. The versatility of this model has been illustrated in that both RPE and CECs can be cultured in a more natural construct and their molecular interactions tested by physiologically altering one cell type and not the other.     

Prenatal human ocular degeneration occurs in Leber's congenital amaurosis (LCA2)

Background

Leber's congenital amaurosis (LCA) encompasses the most precocious and severe forms of inherited retinal dystrophy, displaying very significant visual handicap at or soon after birth 1 . Among the currently identified mutations, alterations in the gene coding for retinal pigment epithelium 65‐kDa protein (RPE65) lead to LCA2 2 . Existing animal models for LCA2 (RPE65^‐/‐ null mice 3 and naturally occurring RPE65^‐/‐ Briard dogs 4 ) exhibit near normal retinal histology at birth, although no recordable photofunction can be detected. Structural degeneration in both cases occurs with delayed onset, cone death generally preceding that of rods.

Methods

We obtained retinal tissue from a voluntarily aborted embryo of an LCA2 carrier in order to compare histopathology and immunohistochemistry with age‐matched normal foetal retina.

Results

Compared to normal retinas, affected retina displayed cell loss and thinning of the outer nuclear (photoreceptor) layer, decreased immunoreactivity for key phototransduction proteins, and aberrant synaptic and inner retinal organisation. The gene mutation abolished detectable expression of RPE65 within the retinal pigment epithelium (RPE) of affected eyes, and ultrastructural examination revealed the presence of lipid and vesicular inclusions not seen in normal RPE. In addition, mutant eyes demonstrated thickening, detachment and collagen fibril disorganisation in the underlying Bruch's membrane, and the choroid was distended and abnormally vascularised, in comparison with controls.

Conclusions

Such data contrast with the late‐onset ocular changes observed in animal models, indicating caution should be exercised when inferring human retinal pathophysiology from information based on other species. Copyright © 2002 John Wiley & Sons, Ltd.

     

Human adult retinal pigment epithelial cells as potential cell source for retina recovery

The retinal pigment epithelium (RPE), as well as the neural retina, develops from the neuroectoderm and plays a key role in photoreceptor functions. Several degenerative eye diseases, e.g., macular degeneration or retinitis pigmentosa, associated with an impaired RPE function cause the loss of the photoreceptor and partial or complete blindness. Cultured RPE cells obtained from human cadaver eyes could be a valuable source for transplantation to cure retinal degenerative diseases. The paper describes RPE cell isolation, maintenance in culture, and immunohistochemical characteristics of dedifferentiated cells. It was found that RPE cells from human adults exhibit neural cell properties in vitro.     

Cultivation of the retinal pigment epithelium in the cavity of the lentectomized eye of newts

A study was made of proliferative activity and transdifferentiation of the cells of retinal pigment epithelium (RPE) cultivated in the cavity of the lensectomized eye of adult newt. Implantation of the newt RPE together with vascular membrane and scleral coat resulted in the regeneration of retina. In this process the character of changes in the proliferative activity of RPE and differentiation of retinal cells were the same as in the regeneration of retina in situ. RPE implanted with the vascular membrane alone, despite a high level of proliferation during the first ten days of cultivation, no differentiated retina was formed. Possible causes of these differences are discussed, and the comparison is made of the data obtained with those on RPE cultivation in vitro. After lens removal, with RPE implants present in the eye cavity, in addition to the regenerated lens, 2-3 extra lenses and retina were formed from the cells of the inner layer of the recipient's dorsal iris. Also some cases were revealed of lens formation from the cells of ventral iris. With a complete detachment of the recipient's retina (an after-effect of transplantation) a second differentiated retina regenerated in situ from the recipient's RPE cells.     

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.     

Directional protein secretion by the retinal pigment epithelium: roles in retinal health and the development of age‐related macular degeneration

The structural and functional integrity of the retinal pigment epithelium (RPE) is fundamental for maintaining the function of the neuroretina. These specialized cells form a polarized monolayer that acts as the retinal–blood barrier, separating two distinct environments with highly specialized functions: photoreceptors of the neuroretina at the apical side and Bruch's membrane/highly vascularized choriocapillaris at the basal side. The polarized nature of the RPE is essential for the health of these two regions, not only in nutrient and waste transport but also in the synthesis and directional secretion of proteins required in maintaining retinal homoeostasis and function. Although multiple malfunctions within the RPE cells have been associated with development of age‐related macular degeneration (AMD), the leading cause of legal blindness, clear causative processes have not yet been conclusively characterized at the molecular and cellular level. This article focuses on the involvement of directionally secreted RPE proteins in normal functioning of the retina and on the potential association of incorrect RPE protein secretion with development of AMD. Understanding the importance of RPE polarity and the correct secretion of essential structural and regulatory components emerge as critical factors for the development of novel therapeutic strategies targeting AMD.     

Basic fibroblast growth factor induces retinal regeneration in vivo

In the present study, we have investigated the effect of basic fibroblast growth factor (bFGF) on retinal regeneration in the stage 22-24 chick embryo. The neural retina was surgically removed in ovo leaving the retinal pigment epithelium (RPE) intact and then slow-release, plastic implants containing bFGF were inserted into the eye. Light microscopic examination of eyes 7 days later revealed that bFGF induced retinal regeneration in a dose-dependent manner. The absence of the RPE in these eyes and the reversed polarity of the regenerated neural retina is consistent with the hypothesis that this process occurs by transdifferentiation of the RPE. This represents the first time that a known molecule has been shown to induce retinal regeneration in vivo.     

β-Catenin Inactivation Is a Pre-Requisite for Chick Retina Regeneration

In the present study we explored the role of β-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear β-catenin in the CM and RPE after injury (retinectomy). β-catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear β-catenin was maintained as long as FGF2 was present. However, nuclear β-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear β-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear β-catenin. Moreover, retinectomy followed by disruption of active β-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that β-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of β-catenin is a pre-requisite for chick retina regeneration.     

Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in the silver mutation of the Japanese quail

The neural retina and retinal pigment epithelium (RPE) diverge from the optic vesicle during early embryonic development. They originate from different portions of the optic vesicle, the more distal part developing as the neural retina and the proximal part as RPE. As the distal part appears to make contact with the epidermis and the proximal part faces mesenchymal tissues, these two portions would encounter different environmental signals. In the present study, an attempt has been made to investigate the significance of interactions between the RPE and mesenchymal tissues that derive from neural crest cells, using a unique quail mutant silver (B/B) as the experimental model. The silver mutation is considered to affect neural crest-derived tissues, including the epidermal melanocytes. The homozygotes of the silver mutation have abnormal eyes, with double neural retinal layers, as a result of aberrant differentation of RPE to form a new neural retina. Retinal pigment epithelium was removed from early embryonic eyes (before the process began) and cultured to see whether it expressed any phenotype characteristic of neural retinal cells. When RPE of the B/B mutant was cultured with surrounding mesenchymal tissue, neural retinal cells were differentiated that expressed markers of amacrine, cone or rod cells. When isolated RPE of the B/B mutant was cultured alone, it acquired pigmentation and did not show any property characteristic of neural retinal cells. The RPE of wild type quail always differentiated to pigment epithelial cells. In the presence of either acidic fibroblast growth factor (aFGF) or basic FGF (bFGF), the RPE of the B/B mutant differentiated to neural retinal cells in the absence of mesenchymal tissue, but the RPE of wild type embryos only did so in the presence of 10–40 times as much aFGF or bFGF. These observations indicate that genes responsible for the B/B mutation are expressed in the RPE as well as in those cells that have a role in the differentiation of neural crest cells. They further suggest that development of the neural retina and RPE is regulated by some soluble factor(s) that is derived from or localized in the surrounding embryonic mesenchyme and other ocular tissues, and that FGF may be among possible candidates.     

A complex choreography of cell movements shapes the vertebrate eye

Optic cup morphogenesis (OCM) generates the basic structure of the vertebrate eye. Although it is commonly depicted as a series of epithelial sheet folding events, this does not represent an empirically supported model. Here, we combine four-dimensional imaging with custom cell tracking software and photoactivatable fluorophore labeling to determine the cellular dynamics underlying OCM in zebrafish. Although cell division contributes to growth, we find it dispensable for eye formation. OCM depends instead on a complex set of cell movements coordinated between the prospective neural retina, retinal pigmented epithelium (RPE) and lens. Optic vesicle evagination persists for longer than expected; cells move in a pinwheel pattern during optic vesicle elongation and retinal precursors involute around the rim of the invaginating optic cup. We identify unanticipated movements, particularly of central and peripheral retina, RPE and lens. From cell tracking data, we generate retina, RPE and lens subdomain fate maps, which reveal novel adjacencies that might determine corresponding developmental signaling events. Finally, we find that similar movements also occur during chick eye morphogenesis, suggesting that the underlying choreography is conserved among vertebrates.