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1.  Manipulation of the Recipient Retinal Environment by Ectopic Expression of Neurotrophic Growth Factors Can Improve Transplanted Photoreceptor Integration and Survival 
Cell transplantation  2012;21(5):871-887.
Degeneration of the neural retina is the leading cause of untreatable blindness in the developed world. Stem cell replacement therapy offers a novel strategy for retinal repair. Postmitotic photoreceptor precursors derived from the early postnatal (P) retina are able to migrate and integrate into the adult mouse retina following transplantation into the subretinal space, but it is likely that a large number of these cells would be required to restore vision. The adult recipient retina presents a very different environment to that from which photoreceptor precursor donor cells isolated from the developing postnatal retina are derived. Here we considered the possibility that modulation of the recipient environment by ectopic expression of developmentally regulated growth factors, normally present during photoreceptor development, might enhance the migration and integration of transplanted cells into the adult neural retina. Adeno-associated viral (AAV) vectors were used to introduce three growth factors previously reported to play a role in photoreceptor development, IGF1, FGF2, and CNTF, into the adult retina, prior to transplantation of P4 cells derived from the Nrl.GFP+ve neural retina. At 3 weeks posttransplantation the number of integrated, differentiated photoreceptor cells present in AAV-mediated neurotrophic factor-treated eyes was assessed and compared to control treated contralateral eyes. We show, firstly, that it is possible to manipulate the recipient retinal microenvironment via rAAV-mediated gene transfer with respect to these developmentally relevant growth factors. Moreover, when combined with cell transplantation, AAV-mediated expression of IGF1 led to significantly increased levels of cell integration, while overexpression of FGF2 had no significant effect on integrated cell number. Conversely, expression of CNTF led to a significant decrease in cell integration and an exacerbated glial response that led to glial scarring. Together, these findings demonstrate the importance of the extrinsic environment of the recipient retina for photoreceptor cell transplantation and show for the first time that it is possible to manipulate this environment using viral vectors to influence photoreceptor transplantation efficiency.
doi:10.3727/096368911X623871
PMCID: PMC3523316  PMID: 22325046
Photoreceptor; Retina; Transplantation; Neurotrophic factors; Gene therapy; Stem cell
2.  Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors 
Experimental Eye Research  2008;86(4):601-611.
Retinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3 weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72 h, and recovers by 2 weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72 h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor integration following transplantation into the subretinal space in the adult mouse. Reversible disruption of the OLM may provide a strategy for increasing cell integration in future therapeutic applications.
doi:10.1016/j.exer.2008.01.004
PMCID: PMC2394572  PMID: 18294631
retinal transplantation; Müller cell; outer limiting membrane; cell integration; photoreceptor; stem cells; mouse
3.  Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina 
Nature biotechnology  2013;31(8):10.1038/nbt.2643.
Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair1 and restoration of vision through transplantation of photoreceptor precursors obtained from post-natal retinas into visually impaired adult mice2,3. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages4-6. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs6. We show that Rhop.GFP-selected rod precursors derived by this protocol integrate within degenerate retinae of adult mice and mature into outer segment–bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently than cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.
doi:10.1038/nbt.2643
PMCID: PMC3826328  PMID: 23873086
4.  The Retinal Pigment Epithelium: a Convenient Source of New Photoreceptor cells? 
Recent success in restoring visual function through photoreceptor replacement in mouse models of photoreceptor degeneration intensifies the need to generate or regenerate photoreceptor cells for the ultimate goal of using cell replacement therapy for blindness caused by photoreceptor degeneration. Current research on deriving new photoreceptors for replacement, as regenerative medicine in general, focuses on the use of embryonic stem cells and induced pluripotent stem (iPS) cells to generate transplantable cells. Nonetheless, naturally occurring regeneration, such as wound healing, involves awakening cells at or near a wound site to produce new cells needed to heal the wound. Here we discuss the possibility of tweaking an ocular tissue, the retinal pigment epithelium (RPE), to produce photoreceptor cells in situ in the eye. Unlike the neural retina, the RPE in adult mammals maintains cell proliferation capability. Furthermore, progeny cells from RPE proliferation may differentiate into cells other than RPE. The combination of proliferation and plasticity opens a question of whether they could be channeled by a regulatory gene with pro-photoreceptor activity towards photoreceptor production. Studies using embryonic chick and transgenic mouse showed that indeed photoreceptor-like cells were produced in culture and in vivo in the eye using gene-directed reprogramming of RPE cells, supporting the feasibility of using the RPE as a convenient source of new photoreceptor cells for in situ retinal repair without involving cell transplantation.
PMCID: PMC4074479  PMID: 24982737
Photoreceptor; Regeneration; Replacement; Retina; Retinal Pigment Epithelium
5.  Transplantation of human embryonic stem cells derived photoreceptors restores some visual function in Crx deficient mice 
Cell stem cell  2009;4(1):73-79.
Summary
Some of the most common causes of blindness involve the degeneration of photoreceptors in the neural retina; photoreceptor replacement therapy might restore some vision in these individuals. Embryonic stem (ES) cells could in principle provide a source of photoreceptors to repair the retina. We have previously shown that retinal progenitors can be efficiently derived from human ES cells. We now show that retinal cells derived from human ES cells will migrate into mouse retinas following intra-ocular injection, settle into the appropriate layers and express markers for differentiated cells, including both rod and cone photoreceptor cells. After transplantation of the cells into the subretinal space of adult Crx -/- mice (a model of Leber's Congenital Amaurosis), the hES cell derived retinal cells differentiate into functional photoreceptors and restore light responses to the animals. These results demonstrate that hES cells can, in principle, be used for photoreceptor replacement therapies.
doi:10.1016/j.stem.2008.10.015
PMCID: PMC2713676  PMID: 19128794
6.  Transplantation of Photoreceptor and Total Neural Retina Preserves Cone Function in P23H Rhodopsin Transgenic Rat 
PLoS ONE  2010;5(10):e13469.
Background
Transplantation as a therapeutic strategy for inherited retinal degeneration has been historically viewed to restore vision as a method by replacing the lost retinal cells and attempting to reconstruct the neural circuitry with stem cells, progenitor cells and mature neural retinal cells.
Methods and Findings
We present evidence for an alternative strategy aimed at preventing the secondary loss of cones, the most crucial photoreceptors for vision, by transplanting normal photoreceptors cells into the eye of the P23H rat, a model of dominant retinitis pigmentosa. We carried out transplantation of photoreceptors or total neural retina in 3-month-old P23H rats and evaluated the function and cell counts 6 months after surgery. In both groups, cone loss was significantly reduced (10%) in the transplanted eyes where the cone outer segments were found to be considerably longer. This morphological effect correlated with maintenance of the visual function of cones as scored by photopic ERG recording, but more precisely with an increase in the photopic b-wave amplitudes by 100% and 78% for photoreceptor transplantation and whole retinal transplantation respectively.
Conclusions
We demonstrate here that the transplanted tissue prevents the loss of cone function, which is further translated into cone survival.
doi:10.1371/journal.pone.0013469
PMCID: PMC2957406  PMID: 20976047
7.  Defining the integration capacity of ES cell-derived photoreceptor precursors 
Stem cells (Dayton, Ohio)  2012;30(7):1424-1435.
Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human ES and iPS cells, potentially provide a renewable source of cells for retinal transplantation. Previously, we have shown both the functional integration of transplanted rod photoreceptor precursors, isolated from the postnatal retina, in the adult murine retina, and photoreceptor cell generation by stepwise treatment of ES cells with defined factors. In this study we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ES cell-derived retinal cells following transplantation using our established procedures. Optimized retinal differentiation via isolation of Rax.GFP retinal progenitors recreated a retinal niche and increased the yield of Crx+ and Rhodopsin+ photoreceptors. Rod birth peaked at day 20 of culture and expression of the early photoreceptor markers Crx and Nrl increased until day 28. Nrl levels were low in ES cell-derived populations compared with developing retinae. Transplantation of early stage retinal cultures produced large tumors, which were avoided by prolonged retinal differentiation (up to day 28) prior to transplantation. Integrated mature photoreceptors were not observed in the adult retina, even when more than 60% of transplanted ES cell-derived cells expressed Crx. We conclude that exclusion of proliferative cells from ES cell-derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ES cell-derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina.
doi:10.1002/stem.1123
PMCID: PMC3580313  PMID: 22570183
Embryonic stem cells; Retina; Cell transplantation; Photoreceptor cells; Fluorescent protein reporter genes; Stem cell transplantation
8.  Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells 
Cell Research  2013;23(6):788-802.
Various stem cell types have been tested for their potential application in treating photoreceptor degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Only embryonic stem cells (ESCs) have so far been shown to generate functional photoreceptor cells restoring light response of photoreceptor-deficient mice, but there is still some concern of tumor formation. In this study, we have successfully cultured Nestin+Sox2+Pax6+ multipotent retinal stem cells (RSCs) from the adult mouse retina, which are capable of producing functional photoreceptor cells that restore the light response of photoreceptor-deficient rd1 mutant mice following transplantation. After they have been expanded for over 35 passages in the presence of FGF and EGF, the cultured RSCs still maintain stable proliferation and differentiation potential. Under proper differentiation conditions, they can differentiate into all the major retinal cell types found in the adult retina. More importantly, they can efficiently differentiate into photoreceptor cells under optimized differentiation conditions. Following transplantation into the subretinal space of slowly degenerating rd7 mutant eyes, RSC-derived photoreceptor cells integrate into the retina, morphologically resembling endogenous photoreceptors and forming synapases with resident retinal neurons. When transplanted into eyes of photoreceptor-deficient rd1 mutant mice, a RP model, RSC-derived photoreceptors can partially restore light response, indicating that those RSC-derived photoreceptors are functional. Finally, there is no evidence for tumor formation in the photoreceptor-transplanted eyes. Therefore, this study has demonstrated that RSCs isolated from the adult retina have the potential of producing functional photoreceptor cells that can potentially restore lost vision caused by loss of photoreceptor cells in RP and AMD.
doi:10.1038/cr.2013.48
PMCID: PMC3674387  PMID: 23567557
retinal stem cells; photoreceptor cells
9.  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.
doi:10.1016/j.preteyeres.2012.06.003
PMCID: PMC3472113  PMID: 22771454
retinal degeneration; retinal transplantation; retinal progenitor sheets; electrophysiology; superior colliculus; trans-synaptic tracing; electron microscopy
10.  Adult Donor Rod Photoreceptors Integrate into the Mature Mouse Retina 
Cell based therapy offers hope to individuals that have lost their sight through retinal degenerations. Previous studies have found that photoreceptors from immature mice can be successfully transplanted to the mature mouse retina; however, rod photoreceptors from adult donors failed to integrate into the host retina following transplantation. The authors have found that rod photoreceptors from adult mice can be successfully transplanted to the mature retina and this provides a potentially new avenue for cell based therapy.
Purpose.
Previous studies indicate that early postnatal mouse photoreceptors have the ability to integrate into the mature host retina after transplantation, while progenitors and fully differentiated photoreceptors do not. The authors sought to determine whether the decline in the ability of photoreceptors to integrate after transplantation with increasing age is related to a loss of migratory ability in the adult neurons or by a decrease in their survival.
Methods.
Dissociated retinal cells were transferred from green fluorescent protein–positive (GFP+) donor mice of ages ranging from embryonic day (E)12.5 to adults (>28 days postnatal [P]). Immunofluorescence was used to assess marker expression and the morphology of integrated cells. In vitro cultures of dissociated Nrl-GFP mice were used to assay survival.
Results.
It was confirmed in previous reports that neonatal rods integrate into adult hosts. However, in contrast to previous reports, the age of the donor cell was not as critical as previously reported, because it was found that donor cells older than P11 effectively integrated into adult host retina. Although fully adult photoreceptors (P28 and older) show a higher transplant failure rate than immature ones (P5), successful transplants had very similar numbers of integrated cells for both mature and immature donors. Integrated cells from all ages were indistinguishable from those of the host in morphology and marker expression.
Conclusions.
Fully mature photoreceptors retain the ability to integrate into the mature retina. The authors propose that their potential for integration is limited primarily by their decreased survival after dissociation.
doi:10.1167/iovs.10-6329
PMCID: PMC3176050  PMID: 21436277
11.  Cone and rod photoreceptor transplantation in models of the childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive donor cells 
Human Molecular Genetics  2010;19(23):4545-4559.
Retinal degenerative disease causing loss of photoreceptor cells is the leading cause of untreatable blindness in the developed world, with inherited degeneration affecting 1 in 3000 people. Visual acuity deteriorates rapidly once the cone photoreceptors die, as these cells provide daylight and colour vision. Here, in proof-of-principle experiments, we demonstrate the feasibility of cone photoreceptor transplantation into the wild-type and degenerating retina of two genetic models of Leber congenital amaurosis, the Crb1rd8/rd8 and Gucy2e−/− mouse. Crx-expressing cells were flow-sorted from the developing retina of CrxGFP transgenic mice and transplanted into adult recipient retinae; CrxGFP is a marker of cone and rod photoreceptor commitment. Only the embryonic-stage Crx-positive donor cells integrated within the outer nuclear layer of the recipient and differentiated into new cones, whereas postnatal cells generated a 10-fold higher number of rods compared with embryonic-stage donors. New cone photoreceptors displayed unambiguous morphological cone features and expressed mature cone markers. Importantly, we found that the adult environment influences the number of integrating cones and favours rod integration. New cones and rods were observed in ratios similar to that of the host retina (1:35) even when the transplanted population consisted primarily of cone precursors. Cone integration efficiency was highest in the cone-deficient Gucy2e−/− retina suggesting that cone depletion creates a more optimal environment for cone transplantation. This is the first comprehensive study demonstrating the feasibility of cone transplantation into the adult retina. We conclude that flow-sorted embryonic-stage Crx-positive donor cells have the potential to replace lost cones, as well as rods, an important requirement for retinal disease therapy.
doi:10.1093/hmg/ddq378
PMCID: PMC2972691  PMID: 20858907
12.  Effective Transplantation of Photoreceptor Precursor Cells Selected Via Cell Surface Antigen Expression 
Stem cells (Dayton, Ohio)  2011;29(9):1391-1404.
Retinal degenerative diseases are a major cause of untreatable blindness. Stem cell therapy to replace lost photoreceptors represents a feasible future treatment. We previously demonstrated that postmitotic photoreceptor precursors expressing an NrlGFP transgene integrate into the diseased retina and restore some light sensitivity. As genetic modification of precursor cells derived from stem cell cultures is not desirable for therapy, we have tested cell selection strategies using fluorochrome-conjugated antibodies recognizing cell surface antigens to sort photoreceptor precursors. Microarray analysis of postnatal NrlGFP-expressing precursors identified four candidate genes encoding cell surface antigens (Nt5e, Prom1, Podxl, and Cd24a). To test the feasibility of using donor cells isolated using cell surface markers for retinal therapy, cells selected from developing retinae by fluorescence-activated cell sorting based on Cd24a expression (using CD24 antibody) and/or Nt5e expression (using CD73 antibody) were transplanted into the wild-type or Crb1rd8/rd8 or Prph2rd2/rd2 mouse eye. The CD73/CD24-sorted cells migrated into the outer nuclear layer, acquired the morphology of mature photoreceptors and expressed outer segment markers. They showed an 18-fold higher integration efficiency than that of unsorted cells and 2.3-fold higher than cells sorted based on a single genetic marker, NrlGFP, expression. These proof-of-principle studies show that transplantation competent photoreceptor precursor cells can be efficiently isolated from a heterogeneous mix of cells using cell surface antigens without loss of viability for the purpose of retinal stem cell therapy. Refinement of the selection of donor photoreceptor precursor cells can increase the number of integrated photoreceptor cells, which is a prerequisite for the restoration of sight.
doi:10.1002/stem.694
PMCID: PMC3303132  PMID: 21774040
Retina; Cell transplantation; Cell surface markers; fluorescence-activated cell sorting; Stem cell transplantation; Fluorescent protein reporter genes; Microarray; Embryonic stem cells
13.  Embryonic Stem Cell-Derived Neural Progenitors Incorporate into Degenerating Retina and Enhance Survival of Host Photoreceptors 
Stem Cells (Dayton, Ohio)  2005;24(2):274-283.
Embryonic stem (ES) cells differentiate into all cell types of the body during development, including those of the central nervous system (CNS). After transplantation, stem cells have the potential to replace host cells lost due to injury or disease or to supply host tissues with therapeutic factors and thus provide a functional benefit. In the current study, we assessed whether mouse neuralized ES cells can incorporate into retinal tissue and prevent retinal degeneration in mnd mice. These mice have an inherited lysosomal storage disease characterized by retinal and CNS degeneration. Sixteen weeks after intravitreal transplantation into adult mice, donor cells had incorporated into most layers of the retina, where they resembled retinal neurons in terms of morphology, location in the retina, and expression of cell type–specific marker proteins. Presence of these donor cells was correlated with a reduction in the sizes and numbers of lysosomal storage bodies in host retinal cells. The presence of transplanted donor cells was also accompanied by enhanced survival of host retinal neurons, particularly photoreceptors. These results demonstrate that neuralized ES cells protect host neurons from degeneration and appear to replace at least some types of lost neurons.
doi:10.1634/stemcells.2005-0059
PMCID: PMC3381839  PMID: 16123383
Embryonic stem cell; Retina; Transplantation; mnd mouse; Differentiation; Repair; Neuronal ceroid lipofuscinoses
14.  Photoreceptor Differentiation following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig 
Stem Cells International  2012;2012:939801.
Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the pig. Methods. Retinal progenitor cells were derived from the neural retina of GFP-transgenic pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival of allogeneic porcine RPCs without immune suppression in the setting of photoreceptor dystrophy. The expression of multiple photoreceptor markers by grafted cells included the rod outer segment-specific marker ROM-1. Further evidence of photoreceptor differentiation included the presence of numerous photoreceptor rosettes within GFP-positive grafts, indicative of the development of cellular polarity and self-assembly into rudiments of outer retinal tissue. Conclusion. Together, these data support the tolerance of RPCs as allografts and demonstrate the high level of rod photoreceptor development that can be obtained from cultured RPCs following transplantation. Strategies for further progress in this area, together with possible functional implications, are discussed.
doi:10.1155/2012/939801
PMCID: PMC3337587  PMID: 22567027
15.  RPE-secreted factors: influence differentiation in human retinal cell line in dose- and density-dependent manner 
Retinal pigment epithelial (RPE) cells play an important role in normal functioning of retina and photoreceptors, and some retinal degenerations arise due to malfunctioning RPE. Retinal pigment epithelium transplantation is being explored as a strategy to rescue degenerating photoreceptors in diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Additionally, RPE-secreted factors could rescue degenerating photoreceptors by prolonging survival or by their ability to differentiate and give rise to photoreceptors by transdifferentiation. In this study, we have explored what role cell density could play in differentiation induced in a human retinal progenitor cell line, in response to RPE-secreted growth factors. Retinal progenitors plated at low (1 × 104 cells/cm2), medium (2–4 × 104 cells/cm2), and high (1 × 105 cells/cm2) cell density were exposed to various dilutions of RPE-conditioned medium (secreted factors) under conditions of defined medium culture. Progenitor cell differentiation was monitored phenotypically (morphological, biochemical analysis, and immunophenotyping, and western blot analysis were performed). Our data show that differentiation in response to RPE-secreted factors is modulated by cell density and dilutions of conditioned medium. We conclude that before embarking on RPE transplantation as a modality for treatment of RP and AMD, one will have to determine the role that cell density and inhibitory and stimulatory neurotrophins secreted by RPE could play in the efficacy of survival of transplants. We report that RPE-conditioned medium enhances neuronal phenotype (photoreceptors, bipolars) at the lowest cell density in the absence of cell–cell contact. Eighty percent to 90% of progenitor cells differentiate into photoreceptors and bipolars at 50% concentration of conditioned medium, while exposure to 100% conditioned medium might increase multipolar neurons (ganglionic and amacrine phenotypes) to a small degree. However, no clear-cut pattern of differentiation in response to RPE-secreted factors is noted at higher cell densities.
doi:10.1007/s12177-011-9076-4
PMCID: PMC3289158  PMID: 23316262
Retinal progenitors; Retinal pigment epithelium; Secreted factors; Differentiation; Density
16.  Electrical Stimulation of Inner Retinal Neurons in Wild-Type and Retinally Degenerate (rd/rd) Mice 
PLoS ONE  2013;8(7):e68882.
Electrical stimulation of the retina following photoreceptor degeneration in diseases such as retinitis pigmentosa and age-related macular degeneration has become a promising therapeutic strategy for the restoration of vision. Many retinal neurons remain functional following photoreceptor degeneration; however, the responses of the different classes of cells to electrical stimuli have not been fully investigated. Using whole-cell patch clamp electrophysiology in retinal slices we investigated the response to electrical stimulation of cells of the inner nuclear layer (INL), pre-synaptic to retinal ganglion cells, in wild-type and retinally degenerate (rd/rd) mice. The responses of these cells to electrical stimulation were extremely varied, with both extrinsic and intrinsic evoked responses observed. Further examination of the intrinsically evoked responses revealed direct activation of both voltage-gated Na+ channels and K+ channels. The expression of these channels, which is particularly varied between INL cells, and the stimulus intensity, appears to dictate the polarity of the eventual response. Retinally degenerate animals showed similar responses to electrical stimulation of the retina to those of the wild-type, but the relative representation of each response type differed. The most striking difference between genotypes was the existence of a large amplitude oscillation in the majority of INL cells in rd/rd mice (as previously reported) that impacted on the signal to noise ratio following electrical stimulation. This confounding oscillation may significantly reduce the efficacy of electrical stimulation of the degenerate retina, and a greater understanding of its origin will potentially enable it to be dampened or eliminated.
doi:10.1371/journal.pone.0068882
PMCID: PMC3708954  PMID: 23874798
17.  Tissue Engineering the Retinal Ganglion Cell Nerve Fiber Layer 
Biomaterials  2013;34(17):4242-4250.
Retinal degenerative diseases, such as glaucoma and macular degeneration, affect millions of people worldwide and ultimately lead to retinal cell death and blindness. Cell transplantation therapies for photoreceptors demonstrate integration and restoration of function, but transplantation into the ganglion cell layer is more complex, requiring guidance of axons from transplanted cells to the optic nerve head in order to reach targets in the brain. Here we create a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal ganglion cell (RGC) axons radially, mimicking axon orientation in the retina. Using this scaffold we observed an increase in RGC survival and no significant change in their electrophysiological properties. When analyzed for alignment, 81% of RGCs were observed to project axons radially along the scaffold fibers, with no difference in alignment compared to the nerve fiber layer of retinal explants. When transplanted onto retinal explants, RGCs on ES scaffolds followed the radial pattern of the host retinal nerve fibers, whereas RGCs transplanted directly grew axons in a random pattern. Thus, the use of this scaffold as a cell delivery device represents a significant step towards the use of cell transplant therapies for the treatment of glaucoma and other retinal degenerative diseases.
doi:10.1016/j.biomaterials.2013.02.027
PMCID: PMC3608715  PMID: 23489919
18.  The lens controls cell survival in the retina: evidence from the blind cavefish Astyanax 
Developmental biology  2007;311(2):512-523.
The lens influences retinal growth and differentiation during vertebrate eye development but the mechanisms are not understood. The role of the lens in retinal growth and development was studied in the teleost Astyanax mexicanus, which has eyed surface-dwelling (surface fish) and blind cave-dwelling (cavefish) forms. A lens and laminated retina initially develop in cavefish embryos, but the lens dies by apoptosis. The cavefish retina is subsequently disorganized, apoptotic cells appear, the photoreceptor layer degenerates, and retinal growth is arrested. We show here by PCNA, BrdU and TUNEL labeling that cell proliferation continues in the adult cavefish retina but the newly born cells are removed by apoptosis. Surface fish to cavefish lens transplantation, which restores retinal growth and rod cell differentiation, abolished apoptosis in the retina but not in the RPE. Surface fish lens deletion did not cause apoptosis in the surface fish retina or affect RPE differentiation. Neither lens transplantation in cavefish nor lens deletion in surface fish affected retinal cell proliferation. We conclude that the lens acts in concert with another optic component, possibly the RPE, to promote retinal cell survival. Accordingly, deficiency in both optic structures may lead to eye degeneration in cavefish.
doi:10.1016/j.ydbio.2007.08.050
PMCID: PMC2151927  PMID: 17936264
retina; lens; retinal pigment epithelium; eye growth; eye degeneration; apoptosis; cavefish
19.  Recent Advances towards the Clinical Application of Stem Cells for Retinal Regeneration 
Cells  2012;1(4):851-873.
Retinal degenerative diseases constitute a major cause of irreversible blindness in the world. Stem cell-based therapies offer hope for these patients at risk of or suffering from blindness due to the deterioration of the neural retina. Various sources of stem cells are currently being investigated, ranging from human embryonic stem cells to adult-derived induced pluripotent stem cells as well as human Müller stem cells, with the first clinical trials to investigate the safety and tolerability of human embryonic stem cell-derived retinal pigment epithelium cells having recently commenced. This review aims to summarize the latest advances in the development of stem cell strategies for the replacement of retinal neurons and their supportive cells, the retinal pigment epithelium (RPE) affected by retinal degenerative conditions. Particular emphasis will be given to the advances in stem cell transplantation and the challenges associated with their translation into clinical practice.
doi:10.3390/cells1040851
PMCID: PMC3901131  PMID: 24710533
stem cells; retina; retinal pigment epithelium; photoreceptors; retinal ganglion cells; retinal degenerative diseases; transplantation; translational research
20.  Intraretinal Transplantation for Rod-Cell Replacement in Light-Damaged Retinas 
Blindness from retinal disease is often the consequence of extensive damage to the photoreceptor cell population, while other cell types which form the neural retina are relatively spared. In this setting, transplantation of photoreceptor cells could offer hope for the restoration of some degree of visual function. We testd the feasibility of this approach by transplanting immature retinal cells into the eyes of adult rats affected by late stage phototoxic retinopathy, which are almost totally devoid of photoreceptor cells.
Dissociated neuroretinal cells from newborn rats were injected into the hosts' retinas. These cells were labelled with the fluorescent tracer Fast-blue for identification within the host eye. Survival time ranged from 3 to 100 post-transplantation days.
Fundus examination of light-irradiated eyes showed pallor caused by a considerable reduction of the retino-choroidal vascular bed after light irradiation. Histologically the hosts exhibited decimation of the elements forming the outer layers.throughout the entire retina.
As visualized by light and electron microscopic procedures, we report the differentiation of clusters of transplanted photoreceptor cells, and the integration of these cells within the adjacent areas of the host retina.
Fluorescence microscopy showed these clusters to be formed by fluorescently labelled cells developing in intimate contact with the unlabelled host retina. Electron microscopically it was possible to determine that these photoreceptors had established synaptic contacts. These observations indicate that successful transplantation of immature retinal cells is feasible into adult eyes that have suffered extensive retino-choroidal damage. These findings also support the concept that retinal transplantation is a procedure which may open new avenues into the study of retinal repair.
doi:10.1155/NP.1989.1
PMCID: PMC2564997  PMID: 2519517
21.  Derivation of Traceable and Transplantable Photoreceptors from Mouse Embryonic Stem Cells 
Stem Cell Reports  2014;2(6):853-865.
Summary
Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC) line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas.
Highlights
•De novo isolation of Crx-GFP embryonic stem cell lines to trace photoreceptors•3D culture system fine-tuning to generate many integration-competent photoreceptors•Revealing in-vitro- and in-vivo-developing retina similarities•Characterization of the most appropriate stage to transplant photoreceptors
Photoreceptor replacement therapy is feasible because the transplantation of retina-derived photoreceptors can restore some visual functions of blind mice. To have an unlimited source of photoreceptors, Arsenijevic, Decembrini, and colleagues derived a specific ESC line to trace photoreceptors and established a 3D protocol to produce large amounts of integration-competent photoreceptors. Integrated cells develop active synapses, external segments, and make connections with the recipient retina.
doi:10.1016/j.stemcr.2014.04.010
PMCID: PMC4050344  PMID: 24936471
22.  Intraocular Retinal Transplantation: A Review 
This review covers intraocular transplantation of retinal tissue. This has importance both for theoretical understanding of retinal and neural development and for possible future clinical application. Transplantation sites have ranged from the anterior chamber through the retina to the subretinal space.
Transplanted tissue has ranged from whole retina to specific retinal layers or specific types of retinal cells. Both within-species and inter-species transplants have been performed, and donor age has ranged from embryonic to adult. The ability of transplanted tissue to be accepted and to differentiate in host eyes has been studied. The conditions under which successful transplants are obtained, host-graft interactions, and transplantation methodologies have been explored. Morphological, and to a small extent, also functional characteristics of the transplants have been studied.
doi:10.1155/NP.1991.81
PMCID: PMC2565094  PMID: 1747393
23.  Emerging options for the management of age-related macular degeneration with stem cells 
Age-related macular degeneration (AMD) is a devastating retinal disease that occurs in later life as the retinal pigment epithelium (RPE) cells die, with subsequent photoreceptor degeneration. In the past, RPE transplant surgeries gave evidence that AMD was potentially treatable, but it involved limited amounts of ocular tissue, and the complication rate was high. Then, stem cell transplants offered an unlimited supply of retinal precursors for endogenous repair and exogenous cell replacement. Debate continues as to which type of stem cell is most appropriate for treating AMD. The prospects include adult-derived progenitor stem cells (including progenitor cells from ocular tissues), hematopoietic stem cells, embryonic stem cells, and induced pluripotent stem cells. Now the therapy is expanding into phase I human trials. This review examines the collective research contributions toward a clinical model of AMD management with stem cells.
doi:10.2147/SCCAA.S7674
PMCID: PMC3781754  PMID: 24198525
age-related macular degeneration; pluripotent stem cells; stem cells; transplantation; retinal pigmented epithelium
24.  Long-Term Survival of Photoreceptors Transplanted into the Adult Murine Neural Retina Requires Immune Modulation 
Stem cells (Dayton, Ohio)  2010;28(11):1997-2007.
Stem cell therapy presents an opportunity to replace photoreceptors that are lost as a result of inherited and age-related degenerative disease. We have previously shown that murine postmitotic rod photoreceptor precursor cells, identified by expression of the rod-specific transcription factor Nrl, are able to migrate into and integrate within the adult murine neural retina. However, their long-term survival has yet to be determined. Here, we found that integrated Nrl.gfp+ve photoreceptors were present up to 12 months post-transplantation, albeit in significantly reduced numbers. Surviving cells had rod-like morphology, including inner/outer segments and spherule synapses. In a minority of eyes, we observed an early, marked reduction in integrated photoreceptors within 1 month post-transplantation, which correlated with increased numbers of amoeboid macrophages, indicating acute loss of transplanted cells due to an inflammatory response. In the majority of transplants, similar numbers of integrated cells were observed between 1 and 2 months post-transplantation. By 4 months, however, we observed a significant decrease in integrated cell survival. Macrophages and T cells were present around the transplantation site, indicating a chronic immune response. Immune suppression of recipients significantly increased transplanted photoreceptor survival, indicating that the loss observed in unsuppressed recipients resulted from T cell-mediated host immune responses. Thus, if immune responses are modulated, correctly integrated transplanted photoreceptors can survive for extended periods of time in hosts with partially mismatched H-2 haplotypes. These findings suggest that autologous donor cells are optimal for therapeutic approaches to repair the neural retina, though with immune suppression nonautologous donors may be effective.
doi:10.1002/stem.520
PMCID: PMC3272388  PMID: 20857496
Stem cell; Progenitor cell; Photoreceptor; Retina; Cell transplantation; Immune response
25.  XIAP Therapy Increases Survival of Transplanted Rod Precursors in a Degenerating Host Retina 
Survival of integrated cells decreases with time after transplantation but can be significantly increased with XIAP antiapoptotic therapy. Preventing programmed cell death through XIAP therapy may be an important component of future therapeutic retinal cell transplantation strategies.
Purpose.
To assess the survival of rod precursor cells transplanted into the Rd9 mouse, a model of X-linked retinal degeneration, and the effect of antiapoptotic therapy with X-linked inhibitor of apoptosis (XIAP) on preventing cell loss.
Methods.
Dissociated retinal cells from P4 Nrlp-GFP mice were transplanted into the subretinal space of 2-, 5-, and 8-month-old Rd9 mice. Histology, immunohistochemistry, and quantification of integrated cells were performed every month for up to 3 months after transplantation. XIAP delivery to donor cells was accomplished by transfection with adenoassociated virus (AAV-XIAP). Intraretinal activation of immune modulators was assessed using a quantitative real-time polymerase chain reaction-based immune response array.
Results.
GFP-positive rod precursors were able to integrate into the outer nuclear layer (ONL) of the Rd9 retina. Transplanted cells underwent morphologic differentiation with the formation of inner and outer segments and synaptic projections to bipolar cells. Integration of donor cells into the ONL increased as a function of host age at the time of transplantation. The number of integrated cells was maximal at 1 month after transplantation and then decreased with time. Survival of integrated cells was significantly increased when donor cells were pretreated with AAV-XIAP. We did not detect any donor cell-specific activation of inflammation within the host retina.
Conclusions.
Survival of integrated cells decreases with time after transplantation but can be significantly increased with XIAP antiapoptotic therapy. Preventing programmed cell death through XIAP therapy may be an important component of future therapeutic retinal cell transplantation strategies.
doi:10.1167/iovs.10-5998
PMCID: PMC3101692  PMID: 20926819

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