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.

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 donorspecific 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.

SummaryTransplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium cones survive and become incorporated within the adult mouse retina, supporting the potential of photoreceptor transplantation for treating retinal degeneration.

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 donor photoreceptor precursor cells can increase the number of integrated photoreceptor cells, which is a prerequisite for the restoration of sight. STEM

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 1 and Rhodopsin 1 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 ESCderived 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 CELLS

SummaryLoss of cone photoreceptors, crucial for daylight vision, has the greatest impact on sight in retinal degeneration. Transplantation of stem cell-derived L/M-opsin cones, which form 90% of the human cone population, could provide a feasible therapy to restore vision. However, transcriptomic similarities between fetal and stem cell-derived cones remain to be defined, in addition to development of cone cell purification strategies. Here, we report an analysis of the human L/M-opsin cone photoreceptor transcriptome using an AAV2/9.pR2.1:GFP reporter. This led to the identification of a cone-enriched gene signature, which we used to demonstrate similar gene expression between fetal and stem cell-derived cones. We then defined a cluster of differentiation marker combination that, when used for cell sorting, significantly enriches for cone photoreceptors from the fetal retina and stem cell-derived retinal organoids, respectively. These data may facilitate more efficient isolation of human stem cell-derived cones for use in clinical transplantation studies.

Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell‐derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post‐mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(−) facilitates the isolation of photoreceptor precursors from three‐dimensional self‐forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell‐derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation‐competent cells for therapeutic application. Stem Cells 2015;33:2469—2482

Loss of photoreceptors due to retinal degeneration is a major cause of blindness in the developed world. While no effective treatment is currently available, cell replacement therapy, using pluripotent stem cell-derived photoreceptor precursor cells, may be a feasible future treatment. Recent reports have demonstrated rescue of visual function following the transplantation of immature photoreceptors and we have seen major advances in our ability to generate transplantation-competent donor cells from stem cell sources. Moreover, we are beginning to realise the possibilities of using endogenous populations of cells from within the retina itself to mediate retinal repair. Here, we present a review of our current understanding of endogenous repair mechanisms together with recent progress in the use of both ocular and pluripotent stem cells for the treatment of photoreceptor loss. We consider how our understanding of retinal development has underpinned many of the recent major advances in translation and moved us closer to the goal of restoring vision by cellular means.