Emma L. West

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.

Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina

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.

Repair of the degenerate retina by photoreceptor transplantation

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2+/Δ307), decrease (Crb1rd8/rd8, Gnat1−/−, Rho−/−), or remain constant (PDE6βrd1/rd1, Prph2rd2/rd2) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho−/−), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.

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.

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 Crb1rd8/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.

Defining the Integration Capacity of Embryonic Stem Cell-Derived Photoreceptor Precursors

Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), 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 ESCs with defined factors. In this study, we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ESC‐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 ESC‐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 ESC‐derived cells expressed Crx. We conclude that exclusion of proliferative cells from ESC‐derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ESC‐derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina. Stem Cells2012;30:1424–1435

Long-term survival of photoreceptors transplanted into the adult murine neural retina requires immune modulation.

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. STEM CELLS 2010;28:1997–2007

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.

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 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 donorphotoreceptor precursor cells can increase the number of integrated photoreceptor cells,which is a prerequisite for the restoration of sight. STEM CELLS 2011;29:1391–1404

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.