Donor and host photoreceptors engage in material transfer following transplantation of post-mitotic photoreceptor precursors.

Photoreceptor replacement by transplantation is proposed as a treatment for blindness. Transplantation of healthy photoreceptor precursor cells into diseased murine eyes leads to the presence of functional photoreceptors within host retinae that express an array of donor-specific proteins. The resulting improvement in visual function was understood to be due to donor cells integrating within host retinae. Here, however, we show that while integration occurs the majority of donor-reporter-labelled cells in the host arises as a result of material transfer between donor and host photoreceptors. Material transfer does not involve permanent donor-host nuclear or cell-cell fusion, or the uptake of free protein or nucleic acid from the extracellular environment. Instead, RNA and/or protein are exchanged between donor and host cells in vivo. These data require a re-evaluation of the mechanisms underlying rescue by photoreceptor transplantation and raise the possibility of material transfer as a strategy for the treatment of retinal disorders.

Restoration of vision after transplantation of photoreceptors.

Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1−/− mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1−/− mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.

Manipulation of the recipient retinal environment by ectopic expression of neurotrophic growth factors can improve transplanted photoreceptor integration and survival.

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.

Chromosome abnormalities and the genetics of congenital corneal opacification.

Congenital corneal opacification (CCO) encompasses a broad spectrum of disorders that have different etiologies, including genetic and environmental. Terminology used in clinical phenotyping is commonly not specific enough to describe separate entities, for example both the terms Peters anomaly and sclerocornea have been ascribed to a clinical picture of total CCO, without investigating the presence or absence of iridocorneal adhesions. This is not only confusing but also unhelpful in determining valid genotype-phenotype correlations, and thereby revealing clues for pathogenesis. We undertook a systematic review of the literature focusing on CCO as part of anterior segment developmental anomalies (ASDA), and analyzed its association specifically with chromosomal abnormalities. Genes previously identified as being associated with CCO are also summarized. All reports were critically appraised to classify phenotypes according to described features, rather than the given diagnosis. Some interesting associations were found, and are discussed.

Effective transplantation of photoreceptor precursor cells selected via cell surface antigen expression.

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 Crb1(rd8/rd8) or Prph2(rd2/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 donorphotoreceptor precursor cells can increase the number of integrated photoreceptor cells,which is a prerequisite for the restoration of sight.

Cone and rod photoreceptor transplantation in models of the childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive donor cells.

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 Crb1(rd8/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.

Targeted disruption of outer limiting membrane junctional proteins (Crb1 and ZO-1) increases integration of transplanted photoreceptor precursors into the adult wild-type and degenerating retina.

Diseases culminating in photoreceptor loss are a major cause of untreatable blindness. Transplantation of rod photoreceptors is feasible, provided donor cells are at an appropriate stage of development when transplanted. Nevertheless, the proportion of cells that integrate into the recipient outer nuclear layer (ONL) is low. The outer limiting membrane (OLM), formed by adherens junctions between Müller glia and photoreceptors, may impede transplanted cells from migrating into the recipient ONL. Adaptor proteins such as Crumbs homologue 1 (Crb1) and zona occludins (ZO-1) are essential for localization of the OLM adherens junctions. We investigated whether targeted disruption of these proteins enhances donor cell integration. Transplantation of rod precursors in wild-type mice achieved 949 +/- 141 integrated cells. By contrast, integration is significantly higher when rod precursors are transplanted into Crb1(rd8/rd8) mice, a model of retinitis pigmentosa and Lebers congenital amaurosis that lacks functional CRB1 protein and displays disruption of the OLM (7,819 +/- 1,297; maximum 15,721 cells). We next used small interfering (si)RNA to transiently reduce the expression of ZO-1 and generate a reversible disruption of the OLM. ZO-1 knockdown resulted in similar, significantly improved, integration of transplanted cells in wild-type mice (7,037 +/- 1,293; maximum 11,965 cells). Finally, as the OLM remains largely intact in many retinal disorders, we tested whether transient ZO-1 knockdown increased integration in a model of retinitis pigmentosa, the rho(-/-) mouse; donor cell integration was significantly increased from 313 +/- 58 cells without treatment to 919 +/- 198 cells after ZO-1 knockdown. This study shows that targeted disruption of OLM junctional proteins enhances integration in the wild-type and degenerating retina and may be a useful approach for developing photoreceptor transplantation strategies.

Cell transplantation strategies for retinal repair.

Cell transplantation is a novel therapeutic strategy to restore visual responses to the degenerate adult neural retina and represents an exciting area of regenerative neurotherapy. So far, it has been shown that transplanted postmitotic photoreceptor precursors are able to functionally integrate into the adult mouse neural retina. In this review, we discuss the differentiation of photoreceptor cells from both adult and embryonic-derived stem cells and their potential for retinal cell transplantation. We also discuss the strategies used to overcome barriers present in the degenerate neural retina and improve retinal cell integration. Finally, we consider the future translation of retinal cell therapy as a therapeutic strategy to treat retinal degeneration.

Isolation and characterisation of neural progenitor cells from the adult Chx10(orJ/orJ) central neural retina.

Retinal stem cells have been isolated from the ciliary epithelium (CE) of the mammalian retina. However, the central neural retina (CNR) lacks the capability to regenerate, a phenomenon retained by lower vertebrates. Mutations in the Chx10 homeobox gene cause reduced proliferation of retinal progenitor cells during development, leading to microphthalmia. Recently, we showed that in Chx10(orJ/orJ) mice, dividing cells persist in the adult CNR, suggesting the existence of a dormant progenitor population. Here, we show that these cells are proliferative and give rise to neurospheres in vitro, a characteristic of neural stem cells. However, these adult-derived CNR progenitors differ from those of the wildtype CE, leading to de-pigmented, larger and more numerous neurospheres expressing Müller glial cell markers. Our results suggest that lack of Chx10 leads to maintenance of a dormant neural progenitor population in the adult CNR. Furthermore, Chx10 is not required for in vitro proliferation of these progenitors.

Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors.

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.

Molecular and developmental mechanisms of anterior segment dysgenesis.

Anterior segment dysgenesis (ASD) is a failure of the normal development of the tissues of the anterior segment of the eye. It leads to anomalies in the structure of the mature anterior segment, associated with an increased risk of glaucoma and corneal opacity. Several different gene mutations have been identified underlying these anomalies with the majority of ASD genes encoding transcriptional regulators. In this review, the role of the ASD genes, PITX2 and FOXC1, is considered in relation to the embryology of the anterior segment, the biochemical function of these proteins, and their role in development and disease aetiology. The emerging view is that these genes act in concert to specify a population of mesenchymal progenitor cells, mainly of neural crest origin, as they migrate anteriorly around the embryonic optic cup. These same genes then regulate mesenchymal cell differentiation to give rise to distinct anterior segment tissues. Development appears critically sensitive to gene dosage, and variation in the normal level of transcription factor activity causes a range of anterior segment anomalies. Interplay between PITX2 and FOXC1 in the development of different anterior segment tissues may partly explain the phenotypic variability and the genetic heterogeneity characteristic of ASD.

Retinal repair by transplantation of photoreceptor precursors.

Photoreceptor loss causes irreversible blindness in many retinal diseases. Repair of such damage by cell transplantation is one of the most feasible types of central nervous system repair; photoreceptor degeneration initially leaves the inner retinal circuitry intact and new photoreceptors need only make single, short synaptic connections to contribute to the retinotopic map. So far, brain- and retina-derived stem cells transplanted into adult retina have shown little evidence of being able to integrate into the outer nuclear layer and differentiate into new photoreceptors. Furthermore, there has been no demonstration that transplanted cells form functional synaptic connections with other neurons in the recipient retina or restore visual function. This might be because the mature mammalian retina lacks the ability to accept and incorporate stem cells or to promote photoreceptor differentiation. We hypothesized that committed progenitor or precursor cells at later ontogenetic stages might have a higher probability of success upon transplantation. Here we show that donor cells can integrate into the adult or degenerating retina if they are taken from the developing retina at a time coincident with the peak of rod genesis. These transplanted cells integrate, differentiate into rod photoreceptors, form synaptic connections and improve visual function. Furthermore, we use genetically tagged post-mitotic rod precursors expressing the transcription factor Nrl (ref. 6) (neural retina leucine zipper) to show that successfully integrated rod photoreceptors are derived only from immature post-mitotic rod precursors and not from proliferating progenitor or stem cells. These findings define the ontogenetic stage of donor cells for successful rod photoreceptor transplantation.

Expression of Drosophila omb-related T-box genes in the developing human and mouse neural retina.

PURPOSE: To examine the role of Drosophila optomotor blind (omb)-related T-box genes in development of human and mouse retina. METHODS: Mouse Tbx2, Tbx3, and Tbx5 and human TBX2 cDNAs were isolated from retinal cDNA libraries by hybridization to the Drosophila omb gene. Gene expression patterns in developing retina were analyzed by in situ hybridization. RESULTS: TBX2/Tbx2, TBX3/Tbx3, and TBX5/Tbx5 were expressed asymmetrically across the embryonic neural retina with highest levels of mRNA within dorsal and peripheral retina. The dorsoventral gradient of TBX2 expression disappeared before the ganglion cell layer (GCL) formed. Its expression then became restricted to the inner neuroblastic retina and later to the GCL and inner nuclear layer (INL). The dorsal expression domains of TBX5/Tbx5 and TBX3/Tbx3 were maintained during formation of the GCL. As the retina matured, TBX3/Tbx3 expression was restricted to the INL, and TBX5/Tbx5 was expressed within the GCL. CONCLUSIONS: The expression pattern of TBX2, TBX3, and TBX5 within the developing retina supports the idea that the encoded transcription factors play a role in providing positional information important for topographic mapping and in differentiation of distinct cell types across the laminar axis of the retina.

Foxn4--a new member of the forkhead gene family is expressed in the retina.

We report the cloning and expression of a novel murine forkhead/winged helix family member--Foxn4--that is expressed during neural development in the retina, the ventral hindbrain and spinal cord and dorsal midbrain. Retinal Foxn4 expression is associated with the zone of proliferating progenitor cells. In the mouse mutant ocular retardation (or(J)), Foxn4 expression in the retina is significantly reduced and terminates prematurely.

Temporal and spatial expression patterns of the CRX transcription factor and its downstream targets. Critical differences during human and mouse eye development.

Cone--rod homeobox (CRX), a paired-like homeobox transcription factor, plays a major role in photoreceptor development and maintenance of the retina. Fifteen different mutations in the CRX gene have been identified as a cause of blinding retinal dystrophy. As a step towards characterizing the underlying pathophysiology of disease, temporal and spatial gene expression patterns during human and mouse eye development were investigated for CRX and for downstream retinally expressed genes, postulated to be transactivated by CRX. We found that human CRX was expressed at 10.5 weeks post-conception (p.c.). This was significantly later than observed in mouse development. Immunocytochemistry in human retina showed that CRX protein was not detected until >4 weeks later at 15 weeks p.c., implying that it would be unable to transactivate PDEB, IRBP and arrestin, which were all expressed before 15 weeks. These data therefore eliminate CRX as the major transcriptional activator of these three genes from a wide group of retinal genes that can be transactivated by CRX in vitro. Additionally, PDEB was expressed 2 weeks before CRX whereas murine Pdeb was expressed after Crx, highlighting a potential difference for the role of PDEB in human eye development. Previous data had shown CRX expression in the adult human retina to be photoreceptor-specific; however, we demonstrate that this gene is also expressed in the inner nuclear layer (INL) of the human and mouse retina by in situ hybridization and immunocytochemistry. INL localization of murine Crx was confirmed in rd/rd,cl mice, as in this mouse model the photoreceptors are absent. We have found important differences in the temporal expression of this gene in human and mouse retina, although spatial expression of the CRX gene appears to be conserved. In addition, downstream targets of CRX in vitro might not represent in vivo function during development. These data support concerns about the extent to which we can extrapolate from rodent models regarding embryonic development and disease pathophysiology.

Human microphthalmia associated with mutations in the retinal homeobox gene CHX10.

Isolated human microphthalmia/anophthalmia, a cause of congenital blindness, is a clinically and genetically heterogeneous developmental disorder characterized by a small eye and other ocular abnormalities. Three microphthalmia/anophthalmia loci have been identified, and two others have been inferred by the co-segregation of translocations with the phenotype. We previously found that mice with ocular retardation (the or-J allele), a microphthalmia phenotype, have a null mutation in the retinal homeobox gene Chx10 (refs 7,8). We report here the mapping of a human microphthalmia locus on chromosome 14q24.3, the cloning of CHX10 at this locus and the identification of recessive CHX10 mutations in two families with non-syndromic microphthalmia (MIM 251600), cataracts and severe abnormalities of the iris. In affected individuals, a highly conserved arginine residue in the DNA-recognition helix of the homeodomain is replaced by glutamine or proline (R200Q and R200P, respectively). Identification of the CHX10 consensus DNA-binding sequence (TAATTAGC) allowed us to demonstrate that both mutations severely disrupt CHX10 function. Human CHX10 is expressed in progenitor cells of the developing neuroretina and in the inner nuclear layer of the mature retina. The strong conservation in vertebrates of the CHX10 sequence, pattern of expression and loss-of-function phenotypes demonstrates the evolutionary importance of the genetic network through which this gene regulates eye development.

Retinoschisin, the X-linked retinoschisis protein, is a secreted photoreceptor protein, and is expressed and released by Weri-Rb1 cells.

X-linked retinoschisis is characterized by microcystic-like changes of the macular region and schisis within the inner retinal layers, leading to visual deterioration in males. Many missense and protein-truncating mutations of the causative gene RS1 have now been identified and are thought to be inactivating. RS1 encodes a 224 amino acid protein, retinoschisin, which contains a discoidin domain but is of unknown function. We have generated a polyclonal antibody against a peptide from a unique region within retinoschisin, which detects a protein of approximately 28 kDa in retinal samples reduced with dithiothreitol, but multimers sized >40 kDa under non-reducing conditions. A screen of human tissues with this antibody reveals retinoschisin to be retina specific and the antibody detects a protein of similar size in bovine and murine retinae. We investigated the expression pattern in the retina of both RS1 mRNA (using in situ hybridization with riboprobes) and retinoschisin (using immunohistochemistry). The antisense riboprobe detected RS1 mRNA only in the photoreceptor layer but the protein product of the gene was present both in the photoreceptors and within the inner portions of the retina. Furthermore, differentiated retinoblastoma cells (Weri-Rb1 cells) were found to express RS1 mRNA and to release retinoschisin. These results suggest that retinoschisin is released by photo-receptors and has functions within the inner retinal layers. Thus, X-linked retinoschisis is caused by abnormalities in a putative secreted photoreceptor protein and is the first example of a secreted photo-receptor protein associated with a retinal dystrophy.

Characterization of the human TBX20 gene, a new member of the T-Box gene family closely related to the Drosophila H15 gene.

T-box transcription factors contain a novel type of DNA-binding domain, the T-box domain, and are encoded by an ancient gene family. Four T-box genes, omb, Trg, org-1, and H15, have been identified in Drosophila, whereas in mammals the T-box gene family has expanded, and 12 human T-box genes have been isolated. We have identified a new human T-box gene, TBX20, and its mouse homologue Tbx20, which are more closely related to the Drosophila H15 gene than to any known vertebrate gene. H15 expression in leg imaginal discs correlates with commitment to a ventral fate, implicating this gene in early patterning events. We find that TBX20 is expressed in the fetal heart, eye, and limb, and during embryogenesis in the mouse, Tbx20 is expressed in the developing heart, eye, ventral neural tube, and limbs, indicating a possible role in regulating development of these tissues. The TBX20 gene maps to chromosome 7p14-p15. An association between TBX20 and loci for retinitis pigmentosa, RP9, and blepharophimosis syndrome, BPES, have been excluded.