Activin signaling limits the competence for retinal regeneration from the pigmented epithelium

Regeneration of the retina in amphibians is initiated by the transdifferentiation of the retinal pigmented epithelium (RPE) into neural progenitors. A similar process occurs in the early embryonic chick, but the RPE soon loses this ability. The factors that limit the competence of RPE cells to regenerate neural retina are not understood; however, factors normally involved in the development of the eye (i.e. FGF and Pax6) have also been implicated in transdifferentiation. Therefore, we tested whether activin, a TGFbeta family signaling protein shown to be important in RPE development, contributes to the loss in competence of the RPE to regenerate retina. We have found that addition of activin blocks regeneration from the RPE, even during stages when the cells are competent. Conversely, a small molecule inhibitor of the activin/TGFbeta/nodal receptors can delay, and even reverse, the developmental restriction in FGF-stimulated neural retinal regeneration.     

Mutant gene expression in mouse aggregation chimeras. 6. An analysis of the effects of the white gene using a histochemical marker

The Miwh expression was studied using a genetical marker, acid beta-galactosidase, in 20 day old chimeric mice Miwh/Miwh Bglb/Bglb C/C in equilibrium +/+ Bgld/Bgld C/Ca and Miwh/Miwh Bglb/Bglb C/C in equilibrium +/+ Bgld/Bgld c/c. Three phenotypically new types of the retinal pigment epithelium (RPE) regions were found in the chimeras, which were absent in the RPE of the parental strains. The presence in the chimeras of the RPE regions consisting of pigmented and normally differentiated cells with a high activity of the enzyme suggests the normalization of differentiation of the Miwh/Miwh RPE cells, due, apparently, to the inducing influence of the normal (+/+ C/C) mesenchyme cells. In addition, the presence of nonpigmented, both hyperplasied and nonhyperplasied, RPE regions (+/+ C/C) suggests an insufficient growth-inhibiting influence and the absence of melanogenesis-stimulating activity of defective Miwh/Miwh ectomesenchyme. The histochemical and cytological analysis of the RPE in the obtained chimeras has, thus, shown that the Miwh gene primarily affects ectomesenchyme, which does not exert melanogenesis-stimulating and growth-inhibiting influence on the RPE ensuring its hyperplasia and the absence of pigmentation.     

Lipofuscin-formation in cultured retinal pigment epithelial cells is related to their melanin content

Age-related macular degeneration (AMD), the leading cause of blindness in the developed world, is accompanied by degeneration of the retinal pigment epithelial (RPE) cells. There is an inverse correlation between the melanin content of the eye and the incidence of AMD. Lipofuscin (LF)-accumulation in RPE cells accompanies the process of aging, and may also be related to AMD. This study was designed to evaluate the effect of melanin/melanosomes on the rate of LF formation in cultured rabbit and bovine RPE cells subjected to oxidative stress (40% normobaric O(2)) and daily supplementation with photoreceptor outer segments for 4 weeks. The LF content was measured at 0, 2, and 4 weeks in RPE cells from pigmented and albino rabbits, as well as in pigment-rich and pigment-poor bovine cells. Albino rabbit and pigment-poor bovine cells accumulated significantly higher amounts of LF than pigmented rabbit cells and pigment-rich bovine RPE cells after both 2 and 4 weeks of exposure. Autometallography of melanin-containing cells, without previous exposure to ammonium sulfide, showed a positive outcome, indicating either the occurrence of pre-existing iron-sulphur clusters or an extremely high intrinsic reducing capacity. These results suggest that melanin acts as an efficient antioxidant, perhaps by interacting with transition metals.     

Contact relationships between chick embryo cells growing in monolayer culture after infection with Rous sarcoma virus

An electron microscopic examination was made of cell contacts and associated microfilament arrays in subconfluent cultures of chick embryo fibroblasts (CEF) and chick embryo retinal pigmented epithelium cells (RPE) transformed by strains of Rous sarcoma virus (RSV) imparting a rounded (Morph r) or fusiform (Morph f) transformed morphology. A few cell substrate contact specializations were found in Morph r-transformed CEF and RPE cells. These resembled cell/substrate plaques of uninfected fibroblasts, but lacked associated microfilament tracts. In contrast Morph f-transformed CEF and RPE resembled untransformed fibroblasts having well developed cell/substrate and cell/cell contact specializations with extensive associated microfilament arrays. Morph r- and Morph f-transformed RPE cells had lost the junctional complex typical of untransformed RPE cultures and additionally no melanosomes were found. SEM and TEM demonstrated differences in adhesive properties of CEF and RPE cell surfaces, few virions adhering to the free cell surface of RPE cells but being found in clumps and singly on CEF cells.     

多能干细胞分化来源视网膜色素上皮细胞移植治疗视网膜变性研究进展

视网膜色素上皮（RPE）对视觉功能的维持起着至关重要的作用。视网膜变性是全球不可治愈性致盲疾病的重要原因,它由视网膜色素上皮功能失常所引起。因此,视网膜色素上皮移植是视网膜变性患者恢复视力的一种最有前景的手段之一。随着干细胞技术的快速发展,从多能干细胞（PSC）到有功能的视网膜色素上皮细胞的体外分化诱导技术已经成熟,其中包括胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）等。此外,从患者特异性iPSCs分化而来的RPE更能用于阐明发病机理并有针对性地个体治疗。更值得一提的是,经诱导得到RPE的移植不论在动物模型中,还是在临床试验里都已经得到了可喜的治疗效果。本文回顾PSC来源RPE干预治疗视网膜变性的最新研究进展。     

The myc oncogene and transdifferentiation of the retinal pigment epithelium]

The retinal pigment epithelium (RPE) develops from the same sheet of neuroepithelium as the neuroretina. When infected with MC29, a v-myc expressing virus, the RPE cells can be induced to transdifferentiate and to take a neuroretinal epithelium fate. After a PCR-based differential screening from these cells we have identified three genes of interest. Qath5, a quail basic helix-loop-helix (bHLH) gene that is closely related to the Drosophila atonal, and whose expression is found in the developing neuroretina. A Chx10-related homeobox gene also expressed in the developing neuroretina and HuD, a RNA-binding protein not expressed in the RPE but expressed during neurogenesis. Beside these genes whose function is involved in regulating neuronal differentiation myc also induced a transient Mitf expression. Mitf is expressed in the entire optic cup, later restricted to the pigmented retina. Mitf is involved in the regulation of the pigmented differentiation. We conclude that v-myc can reverse the RPE to the bipotential retinal primordia.     

A comparative study of the effect of pigment on drug toxicity in human choroidal melanocytes and retinal pigment epithelial cells

The aim of this study was to investigate whether the presence of pigment affects the sensitivity of pigmented cells of the eye, retinal pigment epithelium (RPE) and choroidal melanocytes (CMs) to the cytotoxic effects of xenobiotic drugs. Two approaches were used to compare pigmented versus unpigmented cells: RPE cells were repigmented by phagocytosis of synthetic melanin; UVB irradiation was used to induce an increase in pigment in both RPE and CMs. Three drugs known to induce toxicity in the eye, tamoxifen, chloroquine and thioridazine, were used to assess the sensitivity of cells to xenobiotic drugs. RPE cells were more resistant than CMs to the cytotoxic effects of all three drugs by a factor of 5-fold for tamoxifen, 7-fold for thioridazine and 30-fold for chloroquine. When RPE cells were repigmented using synthetic melanin, their sensitivity to tamoxifen was unchanged, they showed a slightly improved response to thioridazine (after 3 days of incubation with this drug), but they showed greatly increased toxicity to chloroquine (after 1 and 3 days of exposure to the drug), suggesting accumulation of this latter drug on the synthetic melanin. UVB irradiation was used to achieve an increase in the pigment content of both RPE and CMs. CMs were much more sensitive to UVB than RPE cells. CMs appeared to synthesise pigment via DOPA oxidase activity; RPE cells showed an increase in fluorescent material independent of any detectable DOPA oxidase activity. Irrespective of the nature of the pigment that UVB induced in melanocytes and RPE cells, their subsequent response to thioridazine and chloroquine was unchanged by the presence of this pigment.     

In vitro differentiation of retinal pigment epithelium from adult retinal stem cells

One of the limitations in molecular and functional studies of the retinal pigment epithelium (RPE) has been the lack of an in vitro system retaining all the features of in vivo RPE cells. Retinal pigment epithelium cell lines do not show characteristics typical of a functional RPE, such as pigmentation and expression of specific markers. The present study was aimed at the development of culture conditions to differentiate, in vitro, retinal stem cells (RSC), derived from the adult ciliary body, into a functional RPE. Retinal stem cells were purified from murine eyes, grown as pigmented neurospheres and induced to differentiate into RPE on an extracellular matrix substrate using specific culture conditions. After 7-15 days of culture, pigmented cells with an epithelial morphology showed a polarized organization and a capacity for phagocytosis. We detected different stages of melanogenesis in cells at 7 days of differentiation, whereas RPE at 15 days contained only mature melanosomes. These data suggest that our protocol to differentiate RPE in vitro can provide a useful model for molecular and functional studies.     

Influence of Donor Age on Establishing Well-Differentiated Clonal Cultures from Embryonic Chicken Retinal Pigmented Epithelium

Retinal pigmented epithelia (RPE) isolated from chicken embryos of various developmental stages were dissociated into single cells, and their ability to re-express defferentiated characteristics in clonal culture was investigated. The lighty pigmented, columnar cells isolated from stage 25 to 29 embryos dissociated more easily than the heavily pigmeted, cuboidal cells from embryos of stages 30 to 34. The yield of RPE cells per embryo increased with donor age, paralleling the growth of the epithelium in vivo . However, the potential these cells to attach, to proliferate, and to form typical, welldifferentiated RPE colonies declined with donor age. Cells from stage 25 embryos developed exclusively into large, typical epithelial colonies which expressed all stages of differentiation from flat, unpigmented cells at the margin to cuboidal, pigmented cells in the centre. At the other end of the spectrum, cells from stage 34 embryos frequently formed small, atypical colonies of unpigmented cells, in addition to typical but relatively small colonies. The plating efficiency (calculated on the basis of pigmented colonies formed within 3 weeks) dropped from more than 2% at stage 25 to 0.01% at stage 34.     

Evaluation of different procedures for the dissociation of retinal pigmented epithelium into single viable cells

Retinal pigmented epithelium (RPE) from 7-day-old chicken embryos (stages 29 to 31) was isolated and dissociated into single cells using different procedures. The results were assessed in two ways. (1) The yield of single RPE cells per embryo was determined, and their ability to form pigmented colonies in clonal culture was tested. The most efficient and gentle procedure included isolation of the RPE in EDTA solution, trypsinization at low temperature and low enzyme concentration in the presence of EDTA, followed by incubation in culture medium for up to 4 hr. The completely dissociated cells thus obtained had a much higher plating efficiency and more uniform pattern of colony growth and differentiation than those obtained under any other conditions tested. (2) The effects of different treatments on cell junctions and morphological integrity of the cells were determined by transmission electron microscopy. EDTA solution yielded excellent separation of the epithelial sheet from the mesenchyme by dissociating it from Bruch's membrane, but had little effect on the junctions between adjacent RPE cells. Trypsinization of the epithelium under various conditions separated the basal lateral cell borders and caused loss of gap junctions, but left many cells still joined by apical tight junctions. Final disruption of the tight junctions occurred during recovery of the trypsinized cells in culture medium and was accompanied by dedifferentiation of the RPE cells.     

Evidence of decreased adhesion between the neural retina and retinal pigmented epithelium of the Mitf vit (vitiligo) mutant mouse

In order for the retina to function properly, photoreceptor cell outer segments must be in contact with the adjacent retinal pigmented epithelium (RPE). A mouse model homozygous for the vitiligo mutation of the microphthalmia (Mitf) gene manifests disruption of the outer segment/RPE interdigitation and demonstrates progressive loss of the photoreceptor cells. The mouse nevertheless has near normal levels of rhodopsin for many weeks and it is not known whether there is an in vivo loss of adhesion or whether the disruption is visible following tissue processing for histology. To assess this, a mechanical separation experiment was performed in which neural retinas were peeled free from the RPE and examined for the amount of pigment adherent to them. The peeling experiment indicated that control neural retinas retained significant amounts of adherent pigment at all ages examined. Neural retinas of mutant mice at age 2 weeks demonstrated adherent pigment, but older animals retained minimal pigment. Scanning electron microscopy indicated that the RPE cells of control mice were markedly damaged upon peeling and displayed different planes of cleavage, whereas those of mutants showed minimal cellular damage upon peeling, suggestive of decreased adhesion. A recombination experiment revealed that the mutant RPE/eyecup could reappose mutant and control retinas under in vitro conditions, suggesting that RPE fluid transport abilities were intact. The data provide the first direct experimental evidence that the Mitf^vit mutant mouse has a naturally occurring retinal detachment and hence support its value as a model for studies of retina/RPE adhesion.     

Amyloid‐β up‐regulates complement factor B in retinal pigment epithelial cells through cytokines released from recruited macrophages/microglia: Another mechanism of complement activation in age‐related macular degeneration

One of the earliest signs of age‐related macular degeneration (AMD) is the formation of drusen which are extracellular deposits beneath the retinal pigmented epithelium (RPE). To investigate the relationship between drusen and AMD, we focused on amyloid β (Aβ), a major component of drusen and also of senile plaques in the brain of Alzheimer's patients. We previously reported that Aβ was accumulated in drusen‐like structure in senescent neprilysin gene‐disrupted mice. The purpose of this study was to investigate the influence of Aβ on factor B, the main activator of the complement alternative pathway. The results showed that Aβ did not directly modulate factor B expression in RPE cells, but increased the production of monocyte chemoattractant protein‐1 (MCP‐1). Aβ also increased the production of IL‐1β and TNF‐α in macrophages/microglia, and exposure of RPE cells to IL‐1β and TNF‐α significantly up‐regulated factor B. Co‐cultures of RPE cells and macrophages/microglia in the presence of Aβ significantly increased the expression of factor B in RPE. These findings indicate that cytokines produced by macrophages/microglia that were recruited by MCP‐1 produced in RPE cells stimulated by Aβ up‐regulate factor B in RPE cells. Thus, a combined mechanism exists for Aβ‐induced for the activation of the complement alternative pathway in the subretinal space; cytokine‐induced up‐regulation of activator factor B and dysfunction of the inhibitor factor I by direct binding to Aβ as suggested in our earlier study. J. Cell. Physiol. 220: 119–128, 2009. © 2009 Wiley‐Liss, Inc.     

Demonstration of Transdifferentiation of Neural Retina from Pigmented Retina in Culture1

The formation of neural retina (NR) from retinal pigmented epithelium (RPE) of chick embryos in culture was investigated. In cultures of explants of PRE, depigmented, preretinal foci, consisting of 50 to 100 cells appeared in the pigmented central portion of the explant within three days. Then these depigmented cells increased rapidly in number and by about day 14 they formed characteristic spherical bodies, which were identified as a neural retinal-like structure (NR structure) by electron microscopic observations. Culture of explants of RPE from embryos of different stages showed that the capacity of embryonic RPE to form an NR structure decreased steadily with embryonic age from st. 24 to 27. At and after stage 27, no foci leading to the neural retinal differentiation were formed in the explants. Medium conditioned by cell cultures of chicken embryonic NR, RPE or chondrocytes had no effect on the formation of NR structures by explants of RPE.     

Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses

Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.     

L-Dopa and the Albino Riddle: Content of L-Dopa in the Developing Retina of Pigmented and Albino Mice

Background

The absence or deficiency of melanin as in albinos, has detrimental effects on retinal development that include aberrant axonal projections from eye to brain and impaired vision. In pigmented retinal pigment epithelium (RPE), dihydroxyphenalanine (L-Dopa), an intermediate in the synthetic path for melanin, has been hypothesized to regulate the tempo of neurogenesis. The time course of expression of retinal L-Dopa, whether it is harbored exclusively in the RPE, the extent of deficiency in albinos compared to isogenic controls, and whether L-Dopa can be restored if exogenously delivered to the albino have been unknown.

Methodology/Principal Findings

L-Dopa and catecholamines including dopamine extracted from retinas of pigmented (C57BL/6J) and congenic albino (C57BL/6J-tyr^c2j) mice, were measured throughout development beginning at E10.5 and at maturity. L-Dopa, but not dopamine nor any other catecholamine, appears in pigmented retina as soon as tyrosinase is expressed in RPE at E10.5. In pigmented retina, L-Dopa content increases throughout pre- and postnatal development until the end of the first postnatal month after which it declines sharply. This time course reflects the onset and completion of retinal development. L-Dopa is absent from embryonic albino retina and is greatly reduced in postnatal albino retina compared to pigmented retina. Dopamine is undetectable in both albino and pigmented retinas until after the postnatal expression of the neuronal enzyme tyrosine hydroxylase. If provided to pregnant albino mothers, L-Dopa accumulates in the RPE of the fetuses.

Conclusions

L-Dopa in pigmented RPE is most abundant during development after which content declines. This L-Dopa is not converted to dopamine. L-Dopa is absent or at low levels in albino retina and can be restored to the RPE by administration in utero. These findings further implicate L-Dopa as a factor in the RPE that could influence development, and demonstrate that administration of L-Dopa could be a means to rescue developmental abnormalities characteristic of albinos.     

The many different cellular functions of MYO7A in the retina

Mutations in MYO7A (myosin VIIa) cause Usher syndrome type?1B, a disorder involving profound congenital deafness and progressive blindness. In the retina, most MYO7A is localized in the apical region of the RPE (retinal pigmented epithelial) cells, and a small amount is associated with the ciliary and periciliary membranes of the photoreceptor cells. Its roles appear to be quite varied. Studies with MYO7A-null mice indicate that MYO7A participates in the apical localization of RPE melanosomes and in the removal of phagosomes from the apical RPE for their delivery to lysosomes in the basal RPE. In the first role, MYO7A competes with microtubule motors, but in the second one, it may function co-operatively. An additional role of MYO7A in the RPE is indicated by the requirement for it in the light-dependent translocation of the ER (endoplasmic reticulum)-associated visual cycle enzyme RPE65 and normal functioning of the visual retinoid cycle. In photoreceptor cells lacking MYO7A, opsin accumulates abnormally in the transition zone of the cilium, suggesting that MYO7A functions as a selective barrier for membrane proteins at the distal end of the transition zone. It is likely that the progressive retinal degeneration that occurs in Usher syndrome 1B patients results from a combination of cellular defects in the RPE and photoreceptor cells.     

Electron microscopic observations on the retinal pigment epithelium and choroid in the domestic pig (Sus scrofa)

Summary The morphology of the retinal pigment epithelium (RPE) and adjacent choroid has been investigated by electron microscopy in the domestic pig. The RPE consists of a single layer of cells which are columnar posteriorly but become cuboidal and even squamous moving peripherally in the fundus. The cells of the RPE layer regardless of location display basal (scleral) infoldings and apical (vitreal) processes and are joined laterally by junctional complexes. Throughout the retina the epithelial cells are rich in smooth endoplasmic reticulum and mitochondria but less so in rough endoplasmic reticulum and polysomes. The epithelial nucleus is vesicular and basally located. In the superior fundus an area of the RPE is very lightly pigmented and richer in lysosomes than is this layer in the inferior and peripheral fundus. The choroid overlying this area is also lightly pigmented and contains much collagen in a lamellar arrangement. This region may represent a vestigial tapetum fibrosum. Bruch's membrane is slightly thicker posteriorly but is everywhere seen to have a pentalaminate substructure. The choriocapillaris is a single layer of large capillaries which show numerous fenestrations facing the RPE. In the superior fundus the choriocapillaris is also highly fenestrated facing the choroid.