Cellular strategies for retinal repair by photoreceptor replacement

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

Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

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

Multimodal analysis of ocular inflammation using the endotoxin-induced uveitis mouse model

Endotoxin-induced uveitis (EIU) in rodents is a model of acute Toll-like receptor 4 (TLR4)-mediated organ inflammation, and has been used to model human anterior uveitis, examine leukocyte trafficking and test novel anti-inflammatory therapeutics. Wider adoption has been limited by the requirement for manual, non-specific, cell-count scoring of histological sections from each eye as a measure of disease severity. Here, we describe a comprehensive and efficient technique that uses ocular dissection and multimodal tissue analysis. This allows matched disease scoring by multicolour flow cytometric analysis of the inflammatory infiltrate, protein analysis on ocular supernatants and qPCR on remnant tissues of the same eye. Dynamic changes in cell populations could be identified and mapped to chemokine and cytokine changes over the course of the model. To validate the technique, dose-responsive suppression of leukocyte infiltration by recombinant interleukin-10 was demonstrated, as well as selective suppression of the monocyte (CD11b+Ly6C+) infiltrate, in mice deficient for eitherCcl2orCcr2 Optical coherence tomography (OCT) was used for the first time in this model to allowin vivoimaging of infiltrating vitreous cells, and correlated with CD11b+Ly6G+ counts to provide another unique measure of cell populations in the ocular tissue. Multimodal tissue analysis of EIU is proposed as a new standard to improve and broaden the application of this model

Maternal genetic inheritance of red pericarp in the grain of maize

The diversity of colors in the grain of corn is wide, from whites to blacks and including a continuum of various shades of yellows, pinks, reds, purples and blues. The most abundant commercial colors are yellow and white, however other colors have become more important because of the presence of pigments to which are attributed favorable effects as a food. The pigments are also considered natural barriers of the grain against the invasion of pests and diseases in the production fields. The colors of the grain of corn occur in three different parts of the seed: the cover of the grain or pericarp, derived from the maternal tissue, with a diploid genetic content; the endo-sperm, including the aleurone layers that are cells in the grain immediately below the pericarp with a chromosome content of 3n; and the embryo, with a genetic content of 2n. The red color considered in this study is present in the pericarp ignoring possible effects in other tissues of grain and other organs of the plant. In this study, we used materials with colorless or red pericarp, and white or yellow endosperm; with the purpose of describing the type of inheritance of this character in the grain of corn. The results indicated a maternal genetic inheritance with classical complete dominance of the red color of pericarp over the clear or transparent phenotype, where the red color of the grains on ear is determined by the genotype of the mother grain but not by the seed embryo genotypes, which is characterized by uniformity of grain color of the ear. This type of inheritance could be useful in the development of pigmented varieties of higher food quality for humans

Development of an optimized AAV2/5 gene therapy vector for Leber congenital amaurosis owing to defects in RPE65

Leber congenital amaurosis is a group of inherited retinal dystrophies that cause severe sight impairment in childhood; RPE65-deficiency causes impaired rod photoreceptor function from birth and progressive impairment of cone photoreceptor function associated with retinal degeneration. In animal models of RPE65 deficiency, subretinal injection of recombinant adeno-associated virus (AAV) 2/2 vectors carrying RPE65 cDNA improves rod photoreceptor function, and intervention at an early stage of disease provides sustained benefit by protecting cone photoreceptors against retinal degeneration. In affected humans, administration of these vectors has resulted to date in relatively modest improvements in photoreceptor function, even when retinal degeneration is comparatively mild, and the duration of benefit is limited by progressive retinal degeneration. We conclude that the demand for RPE65 in humans is not fully met by current vectors, and predict that a more powerful vector will provide more durable benefit. With this aim we have modified the original AAV2/2 vector to generate AAV2/5-OPTIRPE65. The new configuration consists of an AAV vector serotype 5 carrying an optimized hRPE65 promoter and a codon-optimized hRPE65 gene. In mice, AAV2/5-OPTIRPE65 is at least 300-fold more potent than our original AAV2/2 vector

Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

Abstract Age-related macular degeneration (AMD) is a major cause of blindness and is associated with complement dysregulation. The disease is a potential target for stem cell therapy but success is likely to be limited by the inflammatory response. We investigated the innate immune properties of human induced-pluripotent stem cell (iPSC)-derived RPE cells, particularly with regard to the complement pathway. We focused on collectin-11 (CL-11), a pattern recognition molecule that can trigger complement activation in renal epithelial tissue. We found evidence of constitutive and hypoxia-induced expression of CL-11 in iPS-RPE cells, and in the extracellular fluid. Complement activation on the cell surface occurred in conjunction with CL-11 binding. CL-11 has been shown to activate inflammatory responses through recognition of L-fucose, which we confirmed by showing that fucosidase-treated cells, largely, failed to activate complement. The presence of CL-11 in healthy murine and human retinal tissues confirmed the biological relevance of CL-11. Our data describe a new trigger mechanism of complement activation that could be important in disease pathogenesis and therapeutic interventions

Fine-scale predictions of distributions of Chagas disease vectors in the state of Guanajuato, Mexico

One of the most daunting challenges for Chagas disease surveillance and control in Mexico is the lack of community level data on vector distributions. Although many states now have assembled representative domestic triatomine collections, only two triatomine specimens had been collected and reported previously from the state of Guanajuato. Field personnel from the stateÕs Secretarõ´a de Salud conducted health promotion activities in 43 of the 46 counties in the state and received donations of a total of 2,522 triatomine specimens between 1998 and 2002. All specimens were identiÞed, and live insects examined for Trypanosoma cruzi. In an effort to develop Þne-scale distributional data for Guanajuato, collection localities were georeferenced and ecological niches were modeled for each species by using evolutionary-computing approaches. Five species were collected: Triatoma mexicana (Herrich-Schaeffer), Triatoma longipennis (Usinger), Triatoma pallidipennis (Stål), Triatoma barberi (Usinger), and Triatoma dimidiata (Latreille) from 201 communities located at elevations of 870Ð2,200 m. Based on collection success, T. mexicana had the broadest dispersion, although niche mapping indicates that T. barberi represents the greatest risk for transmission of Chagas disease in the state. T. dimidiata was represented in collections by a single adult collected from one village outside the predicted area for all species. For humans, an estimated 3,755,380 individuals are at risk for vector transmission in the state, with an incidence of 3,500 new cases per year; overall seroprevalences of 2.6% indicate that 97,640 individuals are infected with T. cruzi at present, including 29,300 chronic cases

Multimodal analysis of ocular inflammation using the endotoxin-induced uveitis mouse model

Endotoxin-induced uveitis (EIU) in rodents is a model of acute Toll-like receptor 4 (TLR4)-mediated organ inflammation, and has been used to model human anterior uveitis, examine leukocyte trafficking and test novel anti-inflammatory therapeutics. Wider adoption has been limited by the requirement for manual, non-specific, cell-count scoring of histological sections from each eye as a measure of disease severity. Here, we describe a comprehensive and efficient technique that uses ocular dissection and multimodal tissue analysis. This allows matched disease scoring by multicolour flow cytometric analysis of the inflammatory infiltrate, protein analysis on ocular supernatants and qPCR on remnant tissues of the same eye. Dynamic changes in cell populations could be identified and mapped to chemokine and cytokine changes over the course of the model. To validate the technique, dose-responsive suppression of leukocyte infiltration by recombinant interleukin-10 was demonstrated, as well as selective suppression of the monocyte (CD11b+Ly6C+) infiltrate, in mice deficient for either Ccl2 or Ccr2. Optical coherence tomography (OCT) was used for the first time in this model to allow in vivo imaging of infiltrating vitreous cells, and correlated with CD11b+Ly6G+ counts to provide another unique measure of cell populations in the ocular tissue. Multimodal tissue analysis of EIU is proposed as a new standard to improve and broaden the application of this model

High-Throughput In Vitro, Ex Vivo, and In Vivo Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction

Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3′-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants

Recapitulation of Human Retinal Development from Human Pluripotent Stem Cells Generates Transplantable Populations of Cone Photoreceptors

Transplantation 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