The neurogenic competence of progenitors from the postnatal rat retina in vitro

The mammalian retina develops from stem or progenitor cells that are of neuroectodermal origin and derive from bilateral invaginations of the neuroepithelium, the optic vesicles. Shortly after birth, around 12 days postnatal in rats, the retina is fully developed in its cellular parts. Even though different cell types in the adult might be potential sources for retinal stem cells or progenitor cells, the retina is a non-neurogenic region and the diseased retina is devoid of any spontaneous regeneration. In an attempt to link late developmental processes to the adult situation, we analyzed the presence and the neurogenic potential of retinal progenitors during the postnatal period and compared it to adult ciliary body (CB) derived retinal progenitors and subventricular zone (SVZ) derived neural stem cells. Retinal progenitor properties were identified by the capacity to proliferate and by the expression of the progenitor markers Nestin, Flk-1, Chx10, Pax6 and the radial glia marker BLBP. The neurogenic potential was assayed by the expression of the neuronal markers doublecortin, betaIII Tubulin, Map2 and NSE, the glial makers A2B5, NG2, GalC and GFAP, and by incorporation of BrdU. The number of Flk-1 positive cells and concomitantly the number of newly born betaIII Tubulin-positive cells decreased within the first postnatal week in retinal progenitor cultures and no newly generated betaIII Tubulin, but GFAP positive cells were detected thereafter. In contrast to neural stem cells derived from the adult SVZ, postnatal and adult CB derived progenitors had a lower and a restricted proliferation potential and did not generate oligodendrocytes. The work demonstrates, however, that the existence of retinal progenitor cells is not restricted to embryonic development. In the sensory retina the differentiation potential of late retinal progenitors becomes restricted to the glial lineage, whereas neurogenic progenitor cells are still present in the CB. In addition, major differences in growth and differentiation potential of adult neural stem cells and postnatal and adult retinal progenitors are presented.     

Retinal regional differences in photoreceptor cell death and regeneration in light-lesioned albino zebrafish

Teleost fish regenerate retinal cells from a population of inner nuclear layer (INL) stem cells. To characterize photoreceptor regeneration in zebrafish (Danio rerio), adult albino fish were subjected to constant intense light to cause photoreceptor cell death. Retinal morphometry was performed on histological sections of control and light-lesioned albino retinas to compare the extent of light damage in the ventral, central and dorsal retinal regions. In addition, opsin immunohistochemistry and TUNEL were used to compare photoreceptor cell death in these different retinal areas, while PCNA immunolabeling quantified the cell proliferation that precedes the photoreceptor regeneration. Transgenic albino; Tg(alpha1-tubulin:egfp) zebrafish were also exposed to the intense light in order to examine regeneration-related gene expression changes. The light-lesioned retinas are characterized by extensive rod and cone photoreceptor cell death in the central and dorsal regions. In contrast, many of the rods and cones survive in the ventral retina. The highest levels of INL cell proliferation, which occurs subsequent to photoreceptor death, correspond to the retinal regions that suffer the greatest levels of photoreceptor damage. In the ventral retina, where photoreceptor cell death is minimal, cell proliferation is confined to the ONL. In addition, EGFP expression from the alpha1-tubulin promoter is increased in Müller glial cells in the light-damaged central and dorsal retina, while transgene expression in the ventral retina is restricted to small, round INL cells. Furthermore, expression of the HuC/D neuronal antigen is detected in a subpopulation of the Müller cells in the light-damaged superior retinal region. These data demonstrate that adult albino zebrafish display retinal regional differences in photoreceptor cell death and in the regeneration-related INL cell proliferation response. The high levels of INL cell proliferation and alpha1-tubulin:egfp transgene expression in the Müller cells may be graded in response to the degree of photoreceptor cell death. This suggests that the levels of photoreceptor damage may directly influence cell responses in the underlying retinal layers.     

Cell therapy for retinitis pigmentosa: From rats to pigs

Retinitis pigmentosa (RP) is a leading cause of blindness worldwide and lacks effective clinical treatment. Stem cell-based therapy offers a novel experimental therapeutic approach, based on the strategy that transplanted progenitor cells can replace or rescue damaged photoreceptor cells. However, many factors remain to be determined, for example, what is the optimal time to choose for targeting the host tissue during the progression of the degeneration, what the characteristics and potential capacities in different stem cells, do stem cells differentiate into functional daughter cells, and to what degree can host retinal function be restored? We have used Royal College of Surgeons rats and light-induced retinal degeneration minipigs as animal models of retinitis pigmentosa to study the effectiveness of cell transplant therapies and the functional capacity of the host retina. Stem cells from rat retina and bone marrow, neonatal pig, and human fetal retina have been investigated to find the proper donor cells. The dedifferentiation and then redifferentiation of Müller cells following retinal stem cell transplantation may contribute to host visual function and presents a promising line of research.     

Transplantation of ocular stem cells: the role of injury in incorporation and differentiation of grafted cells in the retina

The incorporation of transplanted cells into the host retina is one of the prerequisites for successful cell replacement therapy to treat retinal degeneration. To test the hypothesis that injury promotes cell incorporation, stem cells/progenitors were isolated from the retina, ciliary epithelium or limbal epithelium and transplanted into the eyes of rats with retinal injury. Different stem cell/progenitor populations incorporated into traumatized or diseased retina but not into the normal retina. The proportion of cells incorporated into the inner retina was consistently higher than in the outer retina. The transplanted cells expressed markers specific to cells of the lamina into which they were incorporated suggesting that cues for specific differentiation are localized within the inner and outer retina. These findings demonstrate that injury-induced cues play a significant role in promoting the incorporation of ocular stem cells/progenitors regardless of their origin or their differentiation along specific retinal sublineage.     

Stem cell therapy and the retina

Retinal degeneration culminating in photoreceptor loss is the leading cause of untreatable blindness in the developed world. In this review, we consider how photoreceptors might be replaced by transplantation and how stem cells might be optimised for use as donor cells in future clinical strategies for retinal repair. We discuss the current advances in human and animal models of retinal cell transplantation, focussing on stem cell and reproductive cloning biology, in relation to the practical issues of retinal transplantation surgery. Stem and progenitor cells can be isolated from a number of sources including embryonic tissue, adult brain and even the retina, prompting many researchers to investigate the potential for using these cells to generate photoreceptors for transplantation. Nevertheless, several obstacles need to be overcome before these techniques can be applied in a clinical setting. Embryonic or stem cells have so far shown little ability to differentiate into retinal phenotypes when transplanted into the adult retina. We have recently noted, however, that donor cells harvested much later, at the photoreceptor precursor developmental stage, can be transplanted successfully and restore visual function. The current challenge is to understand the developmental processes that guide embryonic or adult stem cells towards photoreceptor differentiation, so that large numbers of these cells might be transplanted at the optimal stage. Future advances in reproductive cloning technology could lead to the successful generation of stem cells from adult somatic cells, thereby facilitating auto-transplantation of genetically identical cells in patients requiring photoreceptor replacement.     

Persistent and injury-induced neurogenesis in the vertebrate retina

The brains of all vertebrates are persistently neurogenic. However, this is not true for the neural retinas. Only three extant classes of vertebrates show significant posthatch/postnatal retinal neurogenesis: amphibians, birds and fish. The retinas of these animals contain an annulus of progenitors at the margin, from which differentiated neurons emerge. In posthatch amphibians and fish the vast majority of the adult retina is added from the margin and neurogenesis is lifelong, whereas in posthatch birds neurogenesis is limited. Unique to fish, rod photoreceptors are added in situ from stem cells within the mature retina. Strikingly, for each class of animal retinal lesions stimulate neuronal regeneration, however the cellular source differs for each: the retinal pigmented epithelium in amphibians and embryonic birds, Müller glia in posthatch birds and intrinsic stem cells in fish. The molecular events surrounding injury-induced neuronal regeneration are beginning to be identified.     

Neural regeneration in the chick retina

In warm-blooded vertebrates, possibilities for retinal regeneration have recently become reality with the discovery of neural stem cells in the mature eye. A number of different cellular sources of neural stem cells have been identified. These sources include stem cells at the retinal margin, pigmented cells in the ciliary body and iris, non-pigmented cells in the ciliary body and Müller glia within the retina. This review focuses on recent reports of neural stem cells and regeneration in the postnatal chicken retina. In the chicken eye sources of neurogensis and regeneration include: (1) retinal stem cells at the peripheral edge of the retina; (2) Müller glia in central regions of the retina; (3) non-pigmented epithelial cells in the posterior portion of the ciliary body; and (4) possibly pigmented cells in the pars plana of the ciliary body. This review discusses the similarities between the retinal progenitor cells in the postnatal eye and those found in the embryo. In addition, I discuss combinations of growth factors, (insulin, IGF-I, EGF and FGF2) that are capable of stimulating the proliferation and production of neurons from neural progenitors, non-neural epithelial cells, and postmitotic support cells in the avian eye. In summary, the mechanisms that regulate the proliferation and differentiation of cells with neurogenic potential are beginning to be understood and the postnatal chicken eye has proven to be a useful model system to study retinal regeneration.     

Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay

PURPOSE: The present study describes a method for isolating neural stem cells/progenitors directly from the freshly dissociated embryonic retina (prospective identification) and compares their characteristics with those enriched from mitogen-exposed embryonic retinal cell culture. METHODS: Cell dissociates from embryonic rat retina and mitogen-exposed embryonic retinal cultures were stained with Hoechst 33342 fluorescent dye. The emission patterns of cells were analyzed in both blue and red wavelength using flow cytometry to enrich cells that retained or excluded the dye. The phenotype characteristics and differentiation potential of enriched cells were analyzed by immunocytochemical, RT-PCR, and electrophysiological analyses. RESULTS: The Hoechst dye efflux assay identified a minor population of cells, called side population (SP) cells, in fresh retinal dissociates. These cells that preferentially excluded the Hoechst 33342 fluorescent dye were proliferative and expressed both neural progenitor and retinal progenitor markers. The retinal SP cells generated functional neurons and glia and possessed the ability to differentiate along lineages of different late-born retinal cell types. Cells of similar phenotypes and potential were observed in the SP obtained from mitogen-exposed retinal culture. CONCLUSIONS: The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.     

Stem cell therapy for retinal degeneration: retinal neurons from heterologous sources

Over the past few years a great deal of interest has been generated in using stem cells/progenitors to treat degenerative diseases that afflict different tissues, including retina. This interest is due to the defining properties of stem cells/progenitors, the ability of these cells to self-renew and generate all the basic cell types of the particular tissue to which they belong. In addition, the recent reports of plasticity of the adult tissue-specific stem cells/progenitors and directed differentiation of the embryonic cells (ES) has fueled the hope for cell and gene therapy using stem cells from heterologous sources. Will this approach work for treating retinal degeneration? Here, we review the current state of knowledge about obtaining retinal cells from heterologous sources, including ES cells.     

Cones regenerate from retinal stem cells sequestered in the inner nuclear layer of adult goldfish retina

PURPOSE: To determine whether retinal progenitor cells in the inner nuclear layer give rise to regenerated cones after laser ablation of photoreceptors in adult goldfish retina. METHODS: Using a technique developed previously in this laboratory, photoreceptors in the retina of adult goldfish were ablated with an argon laser. The mitotic marker, bromodeoxyuridine, was used to label proliferating and regenerated cells, which were identified with cell-specific markers. RESULTS: Cells proliferating locally within lesion included microglia, Müller glia, and retinal progenitors in the inner nuclear layer (INL). The nuclei of both Müller glia and associated retinal progenitors migrated from the inner to the outer nuclear layer. The proliferating retinal progenitors, which express Notch-3 and N-cadherin, regenerated cone photoreceptors and then rod photoreceptors. CONCLUSIONS: Previous work has demonstrated that photoreceptors in the goldfish retina regenerate selectively after laser ablation, but the source of regenerated cones has not been identified. The results reported here provide support for the existence of retinal stem cells within the adult fish retina that are capable of regenerating cone photoreceptors. The data also support the involvement of Müller glia in the production of regenerated cones.     

Cell replacement and visual restoration by retinal sheet transplants

Retinal diseases such as age-related macular degeneration (ARMD) and retinitis pigmentosa (RP) affect millions of people. Replacing lost cells with new cells that connect with the still functional part of the host retina might repair a degenerating retina and restore eyesight to an unknown extent. A unique model, subretinal transplantation of freshly dissected sheets of fetal-derived retinal progenitor cells, combined with its retinal pigment epithelium (RPE), has demonstrated successful results in both animals and humans. Most other approaches are restricted to rescue endogenous retinal cells of the recipient in earlier disease stages by a ‘nursing’ role of the implanted cells and are not aimed at neural retinal cell replacement. Sheet transplants restore lost visual responses in several retinal degeneration models in the superior colliculus (SC) corresponding to the location of the transplant in the retina. They do not simply preserve visual performance – they increase visual responsiveness to light. Restoration of visual responses in the SC can be directly traced to neural cells in the transplant, demonstrating that synaptic connections between transplant and host contribute to the visual improvement. Transplant processes invade the inner plexiform layer of the host retina and form synapses with presumable host cells. In a Phase II trial of RP and ARMD patients, transplants of retina together with its RPE improved visual acuity.In summary, retinal progenitor sheet transplantation provides an excellent model to answer questions about how to repair and restore function of a degenerating retina. Supply of fetal donor tissue will always be limited but the model can set a standard and provide an informative base for optimal cell replacement therapies such as embryonic stem cell (ESC)-derived therapy.     

Function of glial cell network as a modulator of neural cell death during retinal degeneration

PURPOSE: Recent studies have demonstrated the mechanism of neural cell death, neuroprotection, and regeneration. However, the functional importance of glial cells during retinal degeneration is not well understood. In this review, we summarize our recent progress regarding the function of glial cells in neurotrophic factor production and neural cell death during retinal degeneration. METHODS: We made a rat model of photoreceptor degeneration by continuous light exposure, and examined the distribution and expression levels of neurotrophins and their receptors. In addition, we carried out quantitative analysis of neurotrophic factor production in cultured Müller glial cells and microglia. RESULTS: In the light-degenerated retina, microglia invade the photoreceptor layer from the inner part of the retina and increase the production of nerve growth factor (NGF). NGF decreases the production of basic fibroblast growth, factor, which prevents photoreceptor cell death, in Müller glial cells through low-affinity neurotrophin receptor p 75. Blockade of p 75 decreased photoreceptor cell death during light-induced retinal degeneration. CONCLUSIONS: These results suggest that a gliaglia network plays a critical role in neural cell death during retinal degeneration. Thus, a glia-glia network as well as a glia-neuron network could be a possible therapeutic target for inhibition of retinal degeneration.     

Proapoptotic bcl-2 family members,Bax and Bak,are essential for developmental photoreceptor apoptosis

PURPOSE: Apoptosis has been implicated in retinal development and degeneration, but the specific apoptotic pathways used are incompletely understood. The purpose of this study was to characterize the roles in retinal development of the proapoptotic Bcl-2 family members Bax and Bak. METHODS: Eyes from mice at postnatal day (P)7, during the peak of developmental apoptosis in the retina, were processed for TdT-dUTP terminal nick-end labeling (TUNEL) to determine whether Bax knockout or double Bax/Bak knockout causes a defect in developmental apoptosis. Adult (>2-month-old) eyes from wild-type, Bak(-/-), Bax(-/-), and Bax(-/-)Bak(-/-) mice were analyzed by histology and immunocytochemistry to identify persistent retinal cells. RESULTS: Adult Bax(-/-)Bak(-/-) eyes showed significant increases in the number of inner retinal cells, with an almost complete absence of TUNEL-positive cell death at P7. Some of these persistent cells in the inner retina notably included rod photoreceptors that normally undergo apoptosis after failure to migrate to the outer retina. These inner nuclear layer (INL) rods contained markers of early rod differentiation: rod opsin, arrestin, and recoverin. However, they did not form ectopic outer segments or contain the associated markers ROM-1, peripherin-2, and RP1. CONCLUSIONS: Bax and Bak are important for retinal development and are the first apoptotic factors identified as essential for developmental photoreceptor apoptosis. Future studies will investigate the potential role of Bax and Bak in mediating pathologic photoreceptor death.     

Retinal oxygenation and oxygen metabolism in Abyssinian cats with a hereditary retinal degeneration

PURPOSE: To investigate the effects of a hereditary retinal degeneration on retinal oxygenation and determine whether it is responsible for the severe attenuation of retinal circulation in hereditary photoreceptor degenerations. METHODS: Seven adult Abyssinian cats affected by hereditary retinal degeneration were studied. Oxygen microelectrodes were used to collect spatial profiles of retinal oxygenation in anesthetized animals. A one-dimensional model of oxygen diffusion was fitted to the data to quantify photoreceptor oxygen utilization (Qo(2)). RESULTS: Photoreceptor Qo(2) progressively decreased until it reached zero in the end stage of the disease. Average inner retinal oxygen tension remained within normal limits at all disease stages, despite the observed progressive retinal vessel attenuation. Light affected photoreceptors normally, decreasing Qo(2) by approximately 50% at all stages of the disease. CONCLUSIONS: Loss of photoreceptor metabolism allows choroidal oxygen to reach the inner retina, attenuating the retinal circulation in this animal model of retinitis pigmentosa (RP) and probably also in human RP. As the degeneration progresses, there is a strong relationship between changes in the a-wave of the ERG and changes in rod oxidative metabolism, indicating that these two functional measures change together.     

Current studies on induced pluripotent stem cells in retinal degenerative diseases

Retinal degeneration diseases are a group of severe eye diseases that can lead to blindness. They are characterized by degeneration and apoptosis of photoreceptor cells and still lacking effective therapeutic procedures. Pluripotent stem cells (induced pluripotent stem cells, iPS cells) obtained from somatic cell reprogramming are similar to the embryonic stem cells (embryonic stem cells, ES cells), which have unlimited proliferation, differentiation and memory characteristics. Retinal cells from iPS cells have been used in cell transplantation for the treatment of retinal diseases, for the study of pathogenesis and drug toxicity evaluation in retinal degenerative diseases. This may provide new ideas and novel procedures for the treatment of retinal degenerative diseases in the future.     

CNTF induces dose-dependent alterations in retinal morphology in normal and rcd-1 canine retina

Ciliary neurotrophic factor (CNTF) provides morphologic preservation of rods in several animal models of retinitis pigmentosa (RP). However, CNTF may alter photoreceptor morphology and rod photoreceptor differentiation in vitro, as well as affecting normal retinal electrophysiology. In addition, the capacity of CNTF to support other cell types affected secondarily in RP (cones and ganglion cells) is unclear. The purposes of this study were to examine the effects of CNTF upon a canine model of RP, the rod-cone degeneration (rcd-1) dog. Archival tissue from a previous study assessing the capacity of CNTF to rescue photoreceptors in rcd-1 dogs was used. One eye was treated for 7 weeks before being explanted. The contralateral eye was untreated. A total of 23 rcd-1 dogs and seven control dogs (four untreated and three CNTF-treated) were used. Morphometric data describing outer and inner nuclear layer thickness, inner retinal thickness, cones and ganglion cells were collected at nine evenly spaced points along each retina and analysed using a mixed effects model. Immunohistochemistry was performed on a subset of 11 dogs for expression of rhodopsin, human cone arrestin (hCAR) and recoverin. CNTF protected the outer nuclear layer and increased inner retinal thickness in a dose-dependent manner (both were maximal at CNTF doses of 1-6 ng day-1). Significant cone loss or reduction of inner nuclear layer width in rcd-1 did not occur in this model, therefore we were unable to assess the protective effect of CNTF upon these parameters. CNTF did not afford significant ganglion cell protection. CNTF induced morphologic changes in rods and ganglion cells, as well as reducing expression of hCAR and rhodopsin, but not recoverin. The dose of CNTF which provided optimal outer nuclear layer protection also resulted in several other effects, including altered ganglion cell morphology, increased thickness of the entire retina, and reduced expression of some phototransduction proteins. These changes were more marked in rcd-1 retinas than in wild-type retinas. This implies that the consequences of CNTF treatment may be substantially influenced by the cellular context into which it is introduced.     

Identification and subcellular localization of the RP1 protein in human and mouse photoreceptors.

PURPOSE: Mutations in the RP1 gene account for 6% to 10% of autosomal dominant retinitis pigmentosa (adRP). Previous studies have shown that the RP1 gene is expressed specifically in photoreceptor cells. So far, little is known about the RP1 protein or how mutations in RP1 lead to photoreceptor cell death. The goal of this study was to identify the RP1 protein and investigate its location in photoreceptor cells. METHODS: A combination of RT-PCR and rapid amplification of cDNA ends (RACE) was used to isolate the full-length mouse Rp1 cDNA. Antibodies against different regions of the predicted mouse Rp1 protein were generated. Western blot analyses were used to identify the RP1/Rp1 proteins. The subcellular location of RP1 in human and mouse retinas was determined by immunostaining retinal sections. RESULTS: The full-length mouse Rp1 cDNA is 6944 bp, encoding a predicted protein of 2095 amino acids. Rp1 was found to be a soluble protein of approximately 240 kDa, consistent with predictions based on the cDNA sequence. Immunofluorescence analyses revealed that both the human RP1 and mouse Rp1 proteins are specifically localized in the connecting cilia of rod and cone photoreceptors. CONCLUSIONS: The presence of RP1/Rp1 in connecting cilia suggests that it may participate in transport of proteins between the inner and outer segments of photoreceptors or in maintenance of cilial structure. This study forms the basis for further investigation of the function of RP1 in retina and the mechanism by which mutations in RP1 lead to photoreceptor cell death.     

The rod photoreceptor lineage of teleost fish

The retinas of postembryonic teleost fish continue to grow for the lifetime of the fish. New retinal cells are added continuously at the retinal margin, by stem cells residing at the circumferential germinal zone. Some of these retinal cells differentiate as Müller glia with cell bodies that reside within the inner nuclear layer. These glia retain some stem cell properties in that they carry out asymmetric cell divisions and continuously generate a population of transit-amplifying cells--the rod photoreceptor lineage--that are committed to rod photoreceptor neurogenesis. These rod progenitors progress through a stereotyped sequence of changes in gene expression as they continue to divide and migrate to the outer nuclear layer. Now referred to as rod precursors, they undergo terminal mitoses and then differentiate as rods, which are inserted into the existing array of rod and cone photoreceptors. The rod lineage displays developmental plasticity, as rod precursors can respond to the loss of rods through increased proliferation, resulting in rod replacement. The stem cells of the rod lineage, Müller glia, respond to acute damage of other retinal cell types by increasing their rate of proliferation. In addition, the Müller glia in an acutely damaged retina dedifferentiate and become multipotent, generating new, functional neurons. This review focuses on the cells of the rod lineage and includes discussions of experiments over the last 30 years that led to their identification and characterization, and the discovery of the stem cells residing at the apex of the lineage. The plasticity of cells of the rod lineage, their relationships to cone progenitors, and the applications of this information for developing future treatments for human retinal disorders will also be discussed.