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[This corrects the article DOI: 10.1371/journal.pone.0015730.].
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The generation of retinal organoids from human pluripotent stem cells (hPSC) is now a well-established process that in part recapitulates retinal development. However, hPSC-derived photoreceptors that exhibit well-organized outer segment structures have yet to be observed. To facilitate improved inherited retinal disease modeling, we determined conditions that would support outer segment development in maturing hPSC-derived photoreceptors. We established that the use of antioxidants and BSA-bound fatty acids promotes the formation of membranous outer segment-like structures. Using new protocols for hPSC-derived retinal organoid culture, we demonstrated improved outer segment formation for both rod and cone photoreceptors, including organized stacked discs. Using these enhanced conditions to generate iPSC-derived retinal organoids from patients with X-linked retinitis pigmentosa, we established robust cellular phenotypes that could be ameliorated following adeno-associated viral vector-mediated gene augmentation. These findings should aid both disease modeling and the development of therapeutic approaches for the treatment of photoreceptor disorders.
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Organoides , Células Madre Pluripotentes , Antioxidantes/farmacología , Suplementos Dietéticos , Humanos , Lípidos , Retina , Células Fotorreceptoras Retinianas ConosRESUMEN
Neuronal communication is typically mediated via synapses and gap junctions. New forms of intercellular communication, including nanotubes (NTs) and extracellular vesicles (EVs), have been described for non-neuronal cells, but their role in neuronal communication is not known. Recently, transfer of cytoplasmic material between donor and host neurons ("material transfer") was shown to occur after photoreceptor transplantation. The cellular mechanism(s) underlying this surprising finding are unknown. Here, using transplantation, primary neuronal cultures and the generation of chimeric retinae, we show for the first time that mammalian photoreceptor neurons can form open-end NT-like processes. These processes permit the transfer of cytoplasmic and membrane-bound molecules in culture and after transplantation and can mediate gain-of-function in the acceptor cells. Rarely, organelles were also observed to transfer. Strikingly, use of chimeric retinae revealed that material transfer can occur between photoreceptors in the intact adult retina. Conversely, while photoreceptors are capable of releasing EVs, at least in culture, these are taken up by glia and not by retinal neurons. Our findings provide the first evidence of functional NT-like processes forming between sensory neurons in culture and in vivo.
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Vesículas Extracelulares , Nanotubos , Animales , Comunicación Celular , Mamíferos , Neuronas , RetinaRESUMEN
Age-related macular degeneration and other macular diseases result in the loss of light-sensing cone photoreceptors, causing irreversible sight impairment. Photoreceptor replacement may restore vision by transplanting healthy cells, which must form new synaptic connections with the recipient retina. Despite recent advances, convincing evidence of functional connectivity arising from transplanted human cone photoreceptors in advanced retinal degeneration is lacking. Here, we show restoration of visual function after transplantation of purified human pluripotent stem cell-derived cones into a mouse model of advanced degeneration. Transplanted human cones elaborate nascent outer segments and make putative synapses with recipient murine bipolar cells (BCs), which themselves undergo significant remodeling. Electrophysiological and behavioral assessments demonstrate restoration of surprisingly complex light-evoked retinal ganglion cell responses and improved light-evoked behaviors in treated animals. Stringent controls exclude alternative explanations, including material transfer and neuroprotection. These data provide crucial validation for photoreceptor replacement therapy and for the potential to rescue cone-mediated vision.
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Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Macular/terapia , Organoides/trasplante , Recuperación de la Función/fisiología , Células Fotorreceptoras Retinianas Conos/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Masculino , Ratones , Ratones Transgénicos , Micotoxinas/genética , Micotoxinas/metabolismo , Organoides/citología , Organoides/metabolismo , Periferinas/genética , Periferinas/metabolismo , Estimulación Luminosa , Cultivo Primario de Células , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Células Bipolares de la Retina/citología , Células Bipolares de la Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/citología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Sinapsis/metabolismo , Trasplante Heterólogo , Visión Ocular/fisiologíaRESUMEN
Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGRex1-19 and RPGRORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGRORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.
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Proteínas del Ojo/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Retinitis Pigmentosa/genética , Empalme Alternativo/genética , Proteínas Portadoras/genética , Cilios/genética , Cilios/patología , Exones/genética , Femenino , Fibroblastos , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Humanos , Masculino , Mutación/genética , Isoformas de Proteínas/genética , Retina/metabolismo , Retina/patología , Retinitis Pigmentosa/patologíaRESUMEN
Degenerative retinal disease is the major cause of sight loss in the developed world and currently there is a lack of effective treatments. As the loss of vision is directly the result of the loss of retinal cells, effective cell replacement through stem-cell-based therapies may have the potential to treat a great number of retinal diseases whatever their underlying etiology. The eye is an ideal organ to develop cell therapies as it is immune privileged, and modern surgical techniques enable precise delivery of cells to the retina. Furthermore, a range of noninvasive diagnostic tests and high-resolution imaging techniques facilitate the evaluation of any therapeutic intervention. In this review, we evaluate the progress to date of current cell therapy strategies for retinal repair, focusing on transplantation of pluripotent stem-cell-derived retinal pigment epithelium (RPE) and photoreceptor cells.
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Degeneración Retiniana/terapia , Epitelio Pigmentado de la Retina/metabolismo , Trasplante de Células Madre/métodos , Trasplante de Células Madre/tendencias , Células Madre/citología , Animales , Investigación Biomédica/tendencias , Ensayos Clínicos como Asunto , Células Madre Embrionarias/citología , Femenino , Humanos , Masculino , Ratones , Células Madre Pluripotentes/citología , Regeneración , Retina/patología , Donantes de TejidosRESUMEN
BACKGROUND: The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required. METHODS: We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions. RESULTS: Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures. CONCLUSIONS: Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling, drug screening and cell transplantation studies.
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Reactores Biológicos/normas , Organoides/metabolismo , Células Fotorreceptoras/metabolismo , Células Madre Pluripotentes/metabolismo , Retina/metabolismo , HumanosRESUMEN
Adeno-associated viral vectors are showing great promise as gene therapy vectors for a wide range of retinal disorders. To date, evaluation of therapeutic approaches has depended almost exclusively on the use of animal models. With recent advances in human stem cell technology, stem cell-derived retina now offers the possibility to assess efficacy in human organoids in vitro. Here we test six adeno-associated virus (AAV) serotypes [AAV2/2, AAV2/9, AAV2/8, AAV2/8T(Y733F), AAV2/5, and ShH10] to determine their efficiency in transducing mouse and human pluripotent stem cell-derived retinal pigment epithelium (RPE) and photoreceptor cells in vitro. All the serotypes tested were capable of transducing RPE and photoreceptor cells in vitro. AAV ShH10 and AAV2/5 are the most efficient vectors at transducing both mouse and human RPE, while AAV2/8 and ShH10 achieved similarly robust transduction of human embryonic stem cell-derived cone photoreceptors. Furthermore, we show that human embryonic stem cell-derived photoreceptors can be used to establish promoter specificity in human cells in vitro. The results of this study will aid capsid selection and vector design for preclinical evaluation of gene therapy approaches, such as gene editing, that require the use of human cells and tissues.
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Dependovirus/fisiología , Vectores Genéticos/genética , Células Fotorreceptoras/citología , Células Fotorreceptoras/metabolismo , Células Madre Pluripotentes/citología , Epitelio Pigmentado de la Retina/citología , Tropismo Viral , Animales , Diferenciación Celular , Células Cultivadas , Dependovirus/clasificación , Técnica del Anticuerpo Fluorescente , Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros , Humanos , Ratones , Especificidad de Órganos/genética , Regiones Promotoras Genéticas , Transducción Genética , TransgenesRESUMEN
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.
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Células Madre Pluripotentes/citología , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/trasplante , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/ultraestructura , Humanos , Células Madre Pluripotentes/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/terapia , Factores de TiempoRESUMEN
The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor ß2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1-/- mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.
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Células Madre Embrionarias de Ratones/trasplante , Células Fotorreceptoras Retinianas Conos/citología , Degeneración Retiniana/terapia , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Factor Nuclear 6 del Hepatocito/metabolismo , Factor Inhibidor de Leucemia/farmacología , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/citología , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Opsinas/metabolismo , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Factores de Transcripción Otx/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Notch/antagonistas & inhibidores , Receptores Notch/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Degeneración Retiniana/patología , Transducción de Señal , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
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.
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Antígenos de Diferenciación/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Degeneración Retiniana/terapia , Células Fotorreceptoras Retinianas Bastones , Trasplante de Células Madre , Animales , Humanos , Ratones , Degeneración Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/trasplanteRESUMEN
Despite different aetiologies, most inherited retinal disorders culminate in photoreceptor loss, which induces concomitant changes in the neural retina, one of the most striking being reactive gliosis by Müller cells. It is typically assumed that photoreceptor loss leads to an upregulation of glial fibrilliary acidic protein (Gfap) and other intermediate filament proteins, together with other gliosis-related changes, including loss of integrity of the outer limiting membrane (OLM) and deposition of proteoglycans. However, this is based on a mix of both injury-induced and genetic causes of photoreceptor loss. There are very few longitudinal studies of gliosis in the retina and none comparing these changes across models over time. Here, we present a comprehensive spatiotemporal assessment of features of gliosis in the degenerating murine retina that involves Müller glia. Specifically, we assessed Gfap, vimentin and chondroitin sulphate proteoglycan (CSPG) levels and outer limiting membrane (OLM) integrity over time in four murine models of inherited photoreceptor degeneration that encompass a range of disease severities (Crb1rd8/rd8, Prph2+/Δ307, Rho-/-, Pde6brd1/rd1). These features underwent very different changes, depending upon the disease-causing mutation, and that these changes are not correlated with disease severity. Intermediate filament expression did indeed increase with disease progression in Crb1rd8/rd8 and Prph2+/Δ307, but decreased in the Prph2+/Δ307 and Pde6brd1/rd1 models. CSPG deposition usually, but not always, followed the trends in intermediate filament expression. The OLM adherens junctions underwent significant remodelling in all models, but with differences in the composition of the resulting junctions; in Rho-/- mice, the adherens junctions maintained the typical rod-Müller glia interactions, while in the Pde6brd1/rd1 model they formed predominantly between Müller cells in late stage of degeneration. Together, these results show that gliosis and its associated processes are variable and disease-dependent.
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Retina/patología , Retina/fisiopatología , Degeneración Retiniana/fisiopatología , Animales , Western Blotting , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteína Ácida Fibrilar de la Glía , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Proteínas del Tejido Nervioso/metabolismo , Periferinas/metabolismo , Proteoglicanos/metabolismo , Vimentina/metabolismoRESUMEN
Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. 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 ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas 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 compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.
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Ceguera/terapia , Técnicas de Cultivo de Célula , Células Madre Embrionarias/trasplante , Retina/citología , Animales , Ceguera/genética , Ceguera/patología , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Células Madre Embrionarias/citología , Proteínas Fluorescentes Verdes , Ratones , Células Fotorreceptoras , Células Fotorreceptoras de Vertebrados , Retina/metabolismo , Retina/patología , Rodopsina/genética , Rodopsina/metabolismo , Trasplante de Células MadreRESUMEN
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 (Crb1(rd8/rd8), Gnat1(-/-), Rho(-/-)), or remain constant (PDE6ß(rd1/rd1), Prph2(rd2/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.
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Ceguera Nocturna/cirugía , Atrofia Óptica Hereditaria de Leber/cirugía , Células Fotorreceptoras Retinianas Bastones/trasplante , Retinitis Pigmentosa/cirugía , Animales , Western Blotting , Recuento de Células , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Citometría de Flujo , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Transmisión , Proteínas del Tejido Nervioso/metabolismo , Ceguera Nocturna/genética , Atrofia Óptica Hereditaria de Leber/genética , Periferinas , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Retinitis Pigmentosa/genética , Transducina/genética , Transducina/metabolismo , Resultado del Tratamiento , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.
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Ojo/crecimiento & desarrollo , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Epitelio Pigmentado de la Retina/crecimiento & desarrollo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología , Animales , Aniridia/genética , Aniridia/patología , Apoptosis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/análisis , Proliferación Celular , Electrorretinografía , Eritropoyetina/metabolismo , Ojo/irrigación sanguínea , Ojo/citología , Eliminación de Gen , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microftalmía/genética , Microftalmía/patología , Epitelio Pigmentado de la Retina/citología , Factor A de Crecimiento Endotelial Vascular/análisis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
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.
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Células Madre Embrionarias/citología , Células Fotorreceptoras/citología , Animales , Células Cultivadas , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Inmunohistoquímica , Ratones , Células Fotorreceptoras/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante de Células MadreRESUMEN
Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions.
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Marcación de Gen , Genes Dominantes/genética , Proteínas Activadoras de la Guanilato-Ciclasa/genética , Retinitis Pigmentosa/genética , Animales , Enfermedades Asintomáticas , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Técnicas de Sustitución del Gen , Proteínas Activadoras de la Guanilato-Ciclasa/metabolismo , Humanos , Espacio Intracelular/metabolismo , Cinética , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/fisiopatologíaRESUMEN
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.
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Células Fotorreceptoras/citología , Retina/citología , Trasplante de Células Madre/métodos , Animales , Supervivencia Celular/inmunología , Células Cultivadas , Ciclosporina/uso terapéutico , Citometría de Flujo , Inmunohistoquímica , Inmunosupresores/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Células Fotorreceptoras/inmunología , Células Fotorreceptoras/metabolismo , Retina/efectos de los fármacos , Retina/inmunología , Retina/metabolismo , Linfocitos T/inmunología , Factores de TiempoRESUMEN
Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration.