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3.
Nat Commun ; 11(1): 1609, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32231223

RESUMO

In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and -521 without the need for manual isolation.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Neurônios/metabolismo , Pigmentos da Retina/metabolismo , Animais , Antígeno CD56 , Células-Tronco Embrionárias , Humanos , Laminina/genética , Degeneração Macular/metabolismo , Coelhos , Epitélio Pigmentado da Retina/metabolismo
4.
J Vis Exp ; (131)2018 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-29443034

RESUMO

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.


Assuntos
Atrofia Geográfica/diagnóstico , Células-Tronco Embrionárias Humanas/transplante , Degeneração Macular/terapia , Retina/transplante , Epitélio Pigmentado da Retina/transplante , Animais , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Atrofia Geográfica/patologia , Humanos , Coelhos , Retina/citologia , Epitélio Pigmentado da Retina/citologia , Transplante Heterólogo
5.
Invest Ophthalmol Vis Sci ; 58(2): 1314-1322, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28241319

RESUMO

Purpose: Subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the capacity to form functional monolayers in naive eyes. We explore hESC-RPE integration when transplanted in suspension to a large-eyed model of geographic atrophy (GA). Methods: Derivation of hESC-RPE was performed in a xeno-free and defined manner. Subretinal bleb injection of PBS or sodium iodate (NaIO3) was used to induce a GA-like phenotype. Suspensions of hESC-RPE were transplanted to the subretinal space of naive or PBS-/NaIO3-treated rabbits using a transvitreal pars plana technique. Integration of hESC-RPE was monitored by multimodal real-time imaging and by immunohistochemistry. Results: Subretinal blebs of PBS or NaIO3 caused different degrees of outer neuroretinal degeneration, RPE hyperautofluorescence, focal RPE loss, and choroidal atrophy; that is, hallmark characteristics of GA. In nonpretreated naive eyes, hESC-RPE integrated as subretinal monolayers with preserved overlying photoreceptors, yet not in areas with outer neuroretinal degeneration and native RPE loss. When transplanted to eyes with PBS-/NaIO3-induced degeneration, hESC-RPE failed to integrate. Conclusions: In a large-eyed preclinical model, subretinal suspension transplants of hESC-RPE did not integrate in areas with GA-like degeneration.


Assuntos
Células Epiteliais/transplante , Atrofia Geográfica/terapia , Células-Tronco Embrionárias Humanas/citologia , Epitélio Pigmentado da Retina/citologia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Humanos , Injeções Intraoculares , Coelhos
6.
Invest Ophthalmol Vis Sci ; 56(4): 2423-30, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25788649

RESUMO

PURPOSE: To analyze the morphologic effects of subretinal blebs in rabbits using real-time imaging by spectral-domain optical coherence tomography (SD-OCT), infrared-confocal scanning laser ophthalmoscopy (IR-cSLO), and blue-light fundus autofluorescence (BAF). METHODS: Subretinal blebs of PBS or balanced salt solution (BSS) were induced in albino or pigmented rabbits using a transvitreal pars plana technique. Spectral-domain optical coherence tomography, IR-cSLO, and BAF were done at multiple intervals for up to 12 weeks after subretinal bleb injection. The morphologic effects were compared with histologic analysis on hematoxylin-eosin-stained sections of the neurosensory retina and on flat-mounts of phalloidin-labeled RPE. RESULTS: Scans of SD-OCT of the normal rabbit posterior segment revealed 11 bands including six layers of the photoreceptors. Subretinal blebs of PBS or BSS caused acute swelling of the neurosensory retina followed by gradual atrophy. Outer retinal thickness was significantly reduced with pronounced degeneration of all the photoreceptor OCT layers. En face IR-cSLO showed a hyperreflective area corresponding to the progressive photoreceptor degeneration, whereas BAF revealed both hyper- and hypofluorescent changes in the RPE layer. The in vivo results were confirmed by histology and on subretinal flatmounts demonstrating extensive photoreceptor loss and disruption of the RPE mosaic. CONCLUSIONS: Subretinal blebs induce pronounced photoreceptor degeneration and RPE changes in the rabbit as demonstrated by in vivo imaging using SD-OCT, IR-cSLO, and BAF.


Assuntos
Diagnóstico por Imagem , Angiofluoresceinografia/métodos , Oftalmoscopia/métodos , Células Fotorreceptoras de Vertebrados/patologia , Segmento Posterior do Olho/patologia , Degeneração Retiniana/diagnóstico , Tomografia de Coerência Óptica/métodos , Animais , Modelos Animais de Doenças , Fundo de Olho , Coelhos , Reprodutibilidade dos Testes , Degeneração Retiniana/etiologia
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