RESUMEN
Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.
Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular , Células Madre Pluripotentes Inducidas , Organoides , Epitelio Pigmentado de la Retina , Humanos , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Organoides/citología , Organoides/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Técnicas de Cultivo de Célula/métodos , Células CultivadasRESUMEN
Purpose: Metabolic defects in the retinal pigment epithelium (RPE) underlie many retinal degenerative diseases. This study aims to identify the nutrient requirements of healthy and diseased human RPE cells. Methods: We profiled nutrient use of various human RPE cells, including differentiated and dedifferentiated fetal RPE (fRPE), induced pluripotent stem cell-derived RPE (iPSC RPE), Sorsby fundus dystrophy (SFD) patient-derived iPSC RPE, CRISPR-corrected isogenic SFD (cSFD) iPSC RPE, and ARPE-19 cell lines using Biolog Phenotype MicroArray Assays. Results: Differentiated fRPE cells and healthy iPSC RPE cells can use 51 and 48 nutrients respectively, including sugars, intermediates from glycolysis and tricarboxylic acid (TCA) cycle, fatty acids, ketone bodies, amino acids, and dipeptides. However, when fRPE cells lose their epithelial phenotype through dedifferentiation, nutrient use becomes restricted to 17 nutrients, primarily sugar and glutamine-related amino acids. SFD RPE cells can use 37 nutrients; however, compared to cSFD RPE and healthy iPSC RPE, they are unable to use lactate, some TCA cycle intermediates, and short-chain fatty acids. Nonetheless, they show increased use of branch-chain amino acids (BCAAs) and BCAA-containing dipeptides. Dedifferentiated ARPE-19 cells grown in traditional culture media cannot use lactate and ketone bodies. In contrast, nicotinamide supplementation promotes differentiation toward an epithelial phenotype, restoring the ability to use these nutrients. Conclusions: Epithelial phenotype confers metabolic flexibility to healthy RPE for using various nutrients. SFD RPE cells have reduced metabolic flexibility, relying on the oxidation of BCAAs. Our findings highlight the potentially important roles of nutrient availability and use in RPE differentiation and diseases.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas , Fenotipo , Epitelio Pigmentado de la Retina , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Diferenciación Celular/fisiología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Cultivadas , Línea CelularRESUMEN
The human retinal pigment epithelium (RPE) cell line ARPE-19 is widely used as an alternative to primary RPE despite losing many features of primary RPE. We aimed to determine whether a combination of RPE-specific laminin (LN) and nicotinamide (NAM) could improve ARPE-19 redifferentiation to resemble mature RPE and improve the assessment of RPE-specific gene therapy strategies. ARPE-19 cells were propagated on tissue culture plastic supplemented with NAM and human recombinant LN521-coating. RPE maturation was performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno-associated virus (AAV)1 or AAV2, carrying a VMD2-driven GFP, were assessed at 2- and 4-weeks post-plating in the different culturing conditions with a low multiplicity of infection. The combination of LN521 coating with NAM supplementation promoted cytoskeletal and tight junction protein reorganization. The expression of maturation markers bestrophin-1 and RPE 65 was promoted concomitantly with a reduction of several epithelial-mesenchymal transition markers, such as TNF-α, TGF-ß, CDH2, and vimentin. Redifferentiated ARPE-19 transduced at low multiplicity of infection of both AAV1- and AAV2-VMD2-GFP. Expression of GFP was detected at 2 weeks and increased at 4 weeks post-plating. AAV1 exhibited a greater expression efficacy compared to AAV2 in maturated ARPE-19 cells already after 2 weeks with increased efficiency after 4 weeks. Our study demonstrates an improved maturation protocol for ARPE-19 cells in vitro, mimicking an in vivo phenotype with the expression of signature genes and improved morphology. Viral-mediated RPE-specific gene expression demonstrates that the combination cultures mimic in vivo AAV tropism essential to test new gene therapies for RPE-centered diseases.
Asunto(s)
Dependovirus , Terapia Genética , Epitelio Pigmentado de la Retina , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Terapia Genética/métodos , Línea Celular , Dependovirus/genética , Diferenciación Celular , Laminina/metabolismo , Laminina/genética , Transición Epitelial-Mesenquimal , Bestrofinas/genética , Bestrofinas/metabolismoRESUMEN
Purpose: Progressive choroid and retinal pigment epithelial (RPE) degeneration causing vision loss is a unique characteristic of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), a fatty acid oxidation disorder caused by a common c.1528G>C pathogenic variant in HADHA, the α subunit of the mitochondrial trifunctional protein (TFP). We established and characterized an induced pluripotent stem cell (iPSC)-derived RPE cell model from cultured skin fibroblasts of patients with LCHADD and tested whether addition of wildtype (WT) HAHDA could rescue the phenotypes identified in LCHADD-RPE. Methods: We constructed an rAAV expression vector containing 3' 3xFLAG-tagged human HADHA cDNA under the transcriptional control of the cytomegalovirus (CMV) enhancer-chicken beta actin (CAG) promoter (CAG-HADHA-3XFLAG). LCHADD-RPE were cultured, matured, and transduced with either AAV-GFP (control) or AAV-HADHA-3XFLAG. Results: LCHADD-RPE express TFP subunits and accumulate 3-hydroxy-acylcarnitines, cannot oxidize palmitate, and release fewer ketones than WT-RPE. When LCHADD-RPE are exposed to docosahexaenoic acid (DHA), they have increased oxidative stress, lipid peroxidation, decreased viability, and are rescued by antioxidant agents potentially explaining the pathologic mechanism of RPE loss in LCHADD. Transduced LCHADD-RPE expressing a WT copy of TFPα incorporated TFPα-FLAG into the TFP complex in the mitochondria and accumulated significantly less 3-hydroxy-acylcarnitines, released more ketones in response to palmitate, and were more resistant to oxidative stress following DHA exposure than control. Conclusions: iPSC-derived LCHADD-RPE are susceptible to lipid peroxidation mediated cell death and are rescued by exogenous HADHA delivered with rAAV. These results are promising for AAV-HADHA gene addition therapy as a possible treatment for chorioretinopathy in patients with LCHADD.
Asunto(s)
Dependovirus , Vectores Genéticos , Células Madre Pluripotentes Inducidas , Peroxidación de Lípido , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga , Epitelio Pigmentado de la Retina , Transfección , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Células Madre Pluripotentes Inducidas/metabolismo , Dependovirus/genética , Células Cultivadas , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/genética , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Errores Innatos del Metabolismo Lipídico/genética , Errores Innatos del Metabolismo Lipídico/metabolismo , Errores Innatos del Metabolismo Lipídico/terapia , Proteína Trifuncional Mitocondrial/genética , Proteína Trifuncional Mitocondrial/deficiencia , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/metabolismo , Terapia Genética/métodos , Cardiomiopatías , Enfermedades del Sistema Nervioso , RabdomiólisisRESUMEN
Galectins have the potential to interact with transmembrane glycoproteins to modulate their functions. Since galectin-1 interacts with PDGF-Rß, we analyzed the effect of galectin-1 on PDGF-BB-mediated AKT signaling in primary human retinal pigment epithelial (RPE) cells and galectin-1-deficient immortalized human RPE cells (LGALS1-/-/ARPE-19) following incubation with PDGF-BB and galectin-1. Expression and localization of galectin-1, PDGF-Rß and pAKT were investigated using western blot analysis and immunohistochemical staining. Cell proliferation of RPE cells was analyzed using BrdU ELISA. Following treatment of human RPE cells with human recombinant (hr)-galectin-1 and PDGF-BB, an intense clustering of PDGF-Rß and colocalization with galectin-1 were detected. By Western blot analysis and immunocytochemistry of human RPE cells, an enhanced PDGF-BB-mediated expression of pAKT was observed, which was substantially reduced by additional incubation with hr-galectin-1. Vice versa, in LGALS1-/-/ARPE-19 cells, the PDGF-BB-induced pAKT signal was enhanced compared to wild-type cells. Furthermore, a decreased expression of PDGF-Rß in human RPE cells was observed after treatment with PDGF-BB and hr-galectin-1, while in untreated LGALS1-/-/ARPE-19 cells, its constitutive expression was increased. In addition, after treatment of RPE cells with hr-galectin-1, the PDGF-BB-induced proliferation was markedly reduced. In summary, galectin-1 has the distinct potential to reduce PDGF-mediated pAKT signaling and proliferation in human RPE cells-an effect that is most likely facilitated via a decreased expression of PDGF-Rß.
Asunto(s)
Becaplermina , Proliferación Celular , Galectina 1 , Proteínas Proto-Oncogénicas c-akt , Epitelio Pigmentado de la Retina , Transducción de Señal , Humanos , Galectina 1/metabolismo , Galectina 1/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Becaplermina/metabolismo , Becaplermina/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Línea Celular , Células Epiteliales/metabolismoRESUMEN
To elucidate the possible biological roles of fatty acid-binding protein 5 (FABP5) in the intraocular environment, the cells from which FABP5 originates were determined by using four different intraocular tissue-derived cell types including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cell lines, and the effects of FABP ligand 6, a specific inhibitor for FABP5 and FABP7 were analyzed by RNA sequencing and seahorse cellular metabolic measurements. Among these four different cell types, qPCR analysis showed that FABP5 was most prominently expressed in HNPCE cells, in which no mRNA expression of FABP7 was detected. In RNA sequencing analysis, 166 markedly up-regulated and 198 markedly down-regulated differentially expressed genes (DEGs) were detected between non-treated cells and cells treated with FABP ligand 6. IPA analysis of these DEGs suggested that FABP5 may be involved in essential roles required for cell development, cell survival and cell homeostasis. In support of this possibility, both mitochondrial and glycolytic functions of HNPCE cells, in which mRNA expression of FABP5, but not that of FABP7, was detected, were shown by using a Seahorse XFe96 Bioanalyzer to be dramatically suppressed by FABP ligand 6-induced inhibition of the activity of FABP5. Furthermore, in IPA upstream analysis, various unfolded protein response (UPR)-related factors were identified as upstream and causal network master regulators. Analysis by qPCR analysis showed significant upregulation of the mRNA expression of most of UPR-related factors and aquaporin1 (AQP1). The findings in this study suggest that HNPCE is one of intraocular cells producing FABP5 and may be involved in the maintenance of UPR and AQP1-related functions of HNPCE.
Asunto(s)
Proteínas de Unión a Ácidos Grasos , Humanos , Proteínas de Unión a Ácidos Grasos/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Línea Celular , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Células Epiteliales/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , Regulación de la Expresión Génica , Cuerpo Ciliar/metabolismo , Cuerpo Ciliar/citología , GlucólisisRESUMEN
Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.
Asunto(s)
Consumo de Oxígeno , Epitelio Pigmentado de la Retina , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Humanos , Consumo de Oxígeno/fisiología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiologíaRESUMEN
Glaucine is an aporphine alkaloid with anti-inflammatory, bronchodilator and anti-cancer activities. However, the effects of glaucine in the regulation of age-related macular degeneration (AMD) remain unclear. Herein, we aimed to investigate the anti-angiogenetic and anti-inflammatory effects of glaucine in ARPE-19 cells. ARPE-19 cells were treated with N-(methoxyoxoacetyl)-glycine, methyl ester (DMOG) and cobalt chloride (CoCl2) for induction of hypoxia, while lipopolysaccharide (LPS) treatment was used for elicitation of inflammatory response. Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The expression of hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) were measured by Western blot. The secretion of VEGF, interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) was detected using enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were used for tube formation analysis. Expression of HIF-1α and secretion of VEGF were significantly increased under DMOG and CoCl2 induction, whereas glaucine significantly attenuated both HIF-1α expression and VEGF secretion by DMOG- and CoCl2-induced ARPE-19 cells. In addition, glaucine suppressed the tube formation by DMOG- and CoCl2-induced HUVEC cells. Moreover, glaucine also attenuated the production of IL-6 and MCP-1 by LPS-induced ARPE-19 cells. This study indicated that glaucine exhibited anti-angiogenic and anti-inflammatory effects, suggesting that glaucine might have benefits for the treatment of AMD.
Asunto(s)
Aporfinas , Supervivencia Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia , Lipopolisacáridos , Epitelio Pigmentado de la Retina , Factor A de Crecimiento Endotelial Vascular , Humanos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Aporfinas/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Hipoxia de la Célula/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Antiinflamatorios/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Inhibidores de la Angiogénesis/farmacología , Cobalto/toxicidad , Cobalto/farmacología , Quimiocina CCL2/metabolismo , AngiogénesisRESUMEN
During tissue regeneration, proliferation, dedifferentiation and reprogramming are necessary to restore lost structures. However, it is not fully understood how metabolism intersects with these processes. Chicken embryos can regenerate their retina through retinal pigment epithelium (RPE) reprogramming when treated with fibroblast factor 2 (FGF2). Using transcriptome profiling, we uncovered extensive regulation of gene sets pertaining to proliferation, neurogenesis and glycolysis throughout RPE-to-neural retina reprogramming. By manipulating cell media composition, we determined that glucose, glutamine or pyruvate are individually sufficient to support RPE reprogramming, identifying glycolysis as a requisite. Conversely, the activation of pyruvate dehydrogenase by inhibition of pyruvate dehydrogenase kinases, induces epithelial-to-mesenchymal transition, while simultaneously blocking the activation of neural retina fate. We also identified that epithelial-to-mesenchymal transition fate is partially driven by an oxidative environment. Our findings provide evidence that metabolism controls RPE cell fate decisions and provide insights into the metabolic state of RPE cells, which are prone to fate changes in regeneration and pathologies, such as proliferative vitreoretinopathy.
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Glucólisis , Epitelio Pigmentado de la Retina , Animales , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Embrión de Pollo , Transición Epitelial-Mesenquimal , Diferenciación Celular , Reprogramación Celular , Proliferación Celular , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glucosa/metabolismo , Pollos , Neurogénesis/fisiología , Glutamina/metabolismoRESUMEN
Retinal pigment epithelial (RPE) cells play a crucial role in the health of the retina, and their dysfunction is associated with various ocular diseases. The transplantation of RPE cells has been proposed as a potential treatment for numerous degenerative diseases, including geographic atrophy from macular degeneration. However, current models to induce RPE damage in animal models prior to transplantation involve mechanical scraping, chemical administration, or laser photocoagulation techniques, which can damage the overlying neurosensory retina. This study aims to investigate the feasibility and efficacy of nanosecond duration laser treatment to safely remove large areas of RPE cells without causing damage to the adjacent tissue or affecting the retinal architecture. Twelve pigmented rabbits were treated with a nanosecond laser on each eye at a laser energy ranging from 200 to 800 nJ with a treated area of 5 × 5 mm2. Human induced pluripotent stem cells-differentiated to RPE (hiPSC-RPE) cells labeled with indocyanine green (ICG), an FDA approved dye, were transplanted subretinally into the damaged RPE areas at day 14 post-laser treatment. The RPE atrophy and hiPSC-RPE cell survival was evaluated and monitored over a period of 14 days using color photography, fluorescein angiography (FA), photoacoustic microscopy (PAM), and optical coherence tomography (OCT) imaging. All treated eyes demonstrated focal RPE loss with a success rate of 100%. The injured RPE layers and the transplanted hiPSC-RPE cells were visualized in three dimensions using PAM and OCT. By performing PAM at an optical wavelength of 700 nm, the location of hiPSC-RPE cells were identified and distinguished from the surrounding RPE cells, and the induced PA signal increased up to 18 times. Immunohistochemistry results confirmed the grafted hiPSC-RPE replaced regions of RPE damage. This novel technique has the potential to serve as an animal model of RPE degeneration, to improve models of RPE transplantation, and may help accelerate translation of this therapeutic strategy for clinical use.
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Epitelio Pigmentado de la Retina , Epitelio Pigmentado de la Retina/citología , Animales , Conejos , Humanos , Células Madre Pluripotentes Inducidas/citología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Tomografía de Coherencia Óptica , Terapia por Láser/métodos , Rayos Láser , Diferenciación Celular , Angiografía con FluoresceínaRESUMEN
The aim of the present study was to elucidate unknown effects of intraocular fatty acids (ioFAs) including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), arachidonic acid (C20:4), eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6) on the outer blood-retinal barrier (oBRB). For this purpose, human retinal pigment epithelium cell line ARPE19 was subjected to analyses for evaluating the following biological phenotypes: (1) cell viability, (2) cellular metabolic functions, (3) barrier functions by trans-epithelial electrical resistance (TEER), and (4) expression of tight junction (TJ) molecules. In the presence of 100 nM ioFAs, no significant effects on cell viability of ARPE19 cells was observed. While treatment with EPA or DHA tended to reduce non-mitochondrial oxygen consumption, most indices in mitochondrial functions were not markedly affected by treatment with ioFAs in ARPE19 cells. On the other hand, ioFAs except for palmitic acid and stearic acid significantly increased basal extracellular acidification rates, suggesting activated glycolysis or increased lactate production. Interestingly, TEER values of planar ARPE19 monolayer were significantly increased by treatment any ioFAs. Consistently, gene expression levels of TJ proteins were increased by treatment with ioFAs. Collectively, the findings presented herein suggest that ioFAs may contribute to reinforcement of barrier functions of the oBRB albeit there are some differences in biological effects depending on the type of ioFAs.
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Barrera Hematorretinal , Epitelio Pigmentado de la Retina , Humanos , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/citología , Línea Celular , Ácidos Grasos/metabolismo , Ácidos Grasos/farmacología , Supervivencia Celular/efectos de los fármacos , Ácido Palmítico/farmacología , Ácidos Docosahexaenoicos/farmacología , Ácidos Esteáricos/farmacología , Ácido Linoleico/farmacología , Ácido Eicosapentaenoico/farmacología , Ácido Oléico/farmacología , Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Ácido Araquidónico/farmacología , Ácido Araquidónico/metabolismoRESUMEN
Physiologically relevant in vitro models of the human outer retina are required to better elucidate the complex interplay of retinal tissue layers and investigate their role in retinal degenerative disorders. Materials currently used to mimic the function of Bruch's membrane fail to replicate a range of important structural, mechanical, and biochemical properties. Here, we detail the fabrication of a surface-functionalized, fibrous collagen I membrane. We demonstrate its ability to better replicate a range of important material properties akin to the function of human Bruch's membrane when compared with a commonly utilized synthetic polyethylene terephthalate alternative. We further reveal the ability of this membrane to support the culture of the ARPE-19 cell line, as well as human pluripotent stem cell-derived RPE-like cells and human umbilical vein endothelial cells. This material could provide greater physiological relevance to the native Bruch's membrane than current synthetic materials and further improve the outcomes of in vitro outer retinal models.
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Lámina Basal de la Coroides , Colágeno Tipo I , Retina , Humanos , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Lámina Basal de la Coroides/metabolismo , Lámina Basal de la Coroides/química , Línea Celular , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Tereftalatos Polietilenos/química , Retina/citología , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacosRESUMEN
PURPOSE: The abnormal growth factors-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells was known as a vital pathogenesis of proliferative vitreoretinopathy (PVR). This study aims to explore how survivin inhibition affects EMT induced by epidermal growth factor (EGF) in RPE cells. METHODS: Human primary RPE cells were identified in vitro. EMT in RPE cells was induced by EGF. Inhibition of survivin in RPE cells was accomplished through the use of a survivin inhibitor (YM155) and survivin siRNA. The viability, proliferation and migration of RPE cells was detected by methylthiazol tetrazolium assay, bromodeoxyuridine labeling assay, and wound healing assay, respectively. The EGF receptor /mitogen-activated protein kinase (EGFR/MAPK) proteins and EMT-related proteins were measured by western blot and immunofluorescence assay. RESULTS: EGF induced significant EMT in RPE cells, activated the phosphorylation of EGFR/MAPK signaling proteins, and caused changes to EMT-related proteins. YM155 suppressed RPE cells' viability, proliferation, and migration; induced the phosphorylation of EGFR, JNK, and P38MAPK; and down regulated EGFR and phosphorylated ERK. YM155 also increased expression of E-cadherin and ZO-1 proteins and reduced expression of N-cadherin, Vimentin, and α-SMA proteins. The EGF-induced increase of RPE cell proliferation and migration was constrained by survivin inhibition. Moreover, survivin inhibition in RPE cells suppressed the EGF-caused phosphorylation of EGFR/MAPK proteins and attenuated the EGF-induced reduction of E-cadherin and ZO-1 proteins and increase of N-cadherin, Vimentin, and α-SMA proteins. CONCLUSIONS: Survivin inhibition attenuates EGF-induced EMT of RPE cells by affecting the EGFR/MAPK signaling pathway. Survivin might be a promising target for preventing PVR.
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Cadherinas , Movimiento Celular , Proliferación Celular , Factor de Crecimiento Epidérmico , Transición Epitelial-Mesenquimal , Receptores ErbB , Imidazoles , Sistema de Señalización de MAP Quinasas , Naftoquinonas , Epitelio Pigmentado de la Retina , Survivin , Humanos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Survivin/metabolismo , Survivin/antagonistas & inhibidores , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inhibidores , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Naftoquinonas/farmacología , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Cadherinas/metabolismo , Supervivencia Celular/efectos de los fármacos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Fosforilación/efectos de los fármacos , Vimentina/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Células Cultivadas , Actinas/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Cell adhesion to the extracellular matrix and its natural outcome of cell spreading, along with the maintenance of barrier activity, are essential behaviors of epithelial cells, including retinal pigment epithelium (RPE). Disruptions in these characteristics can result in severe vision-threatening diseases such as diabetic macular edema and age-related macular degeneration. However, the precise mechanisms underlying how RPE cells regulate their barrier integrity and cell spreading are not fully understood. This study aims to elucidate the relative importance of upper glycolytic components in governing these cellular behaviors of RPE cells. Electric Cell-Substrate Impedance Sensing (ECIS) technology was utilized to assess in real-time the effects of targeting various upper glycolytic enzymes on RPE barrier function and cell spreading by measuring cell resistance and capacitance, respectively. Specific inhibitors used included WZB117 for Glut1 inhibition, Lonidamine for Hexokinase inhibition, PFK158 for PFKFB3/PFK axis inhibition, and TDZD-8 for Aldolase inhibition. Additionally, the viability of RPE cells was evaluated using a lactate dehydrogenase (LDH) cytotoxicity assay. The most significant decrease in electrical resistance and increase in capacitance of RPE cells were observed due to dose-dependent inhibition of Glut1 using WZB117, as well as Aldolase inhibition with TDZD-8. LDH level analysis at 24-72 h post-treatment with WZB117 (1 and 10 µM) or TDZD-8 (1 µM) showed no significant difference compared to the control, indicating that the disruption of RPE functionality was not attributed to cell death. Lastly, inhibition of other upper glycolytic components, including PFKFB3/PFK with PFK158 or Hexokinase with Lonidamine, did not significantly affect RPE cell behavior. This study provides insights into the varied roles of upper glycolytic components in regulating the functionality of RPE cells. Specifically, it highlights the critical roles of Glut1 and Aldolase in preserving barrier integrity and promoting RPE cell adhesion and spreading. Such understanding will guide the development of safe interventions to treat RPE cell dysfunction in various retinal disorders.
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Glucólisis , Epitelio Pigmentado de la Retina , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/citología , Glucólisis/efectos de los fármacos , Humanos , Transportador de Glucosa de Tipo 1/metabolismo , Hexoquinasa/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Impedancia Eléctrica , Fosfofructoquinasa-2/metabolismo , Fosfofructoquinasa-2/antagonistas & inhibidoresRESUMEN
Inspired by natural metallopeptides, our work focuses on engineering self-assembling nanostructures of C2-symmetric metallopeptide conjugates (MPC) from a pyridine-bis-tripeptide bioprobe that uniquely detects lead (Pb2+) ions by emitting a fluorescence signal at 450 nm, which is further intensified in the presence of DAPI (λem = 458 nm), enhancing the bioimaging quality. This study enables precise lead quantification by modulating the ionic conformation and morphology. Experimental and theoretical insights elucidate the nanostructure formation mechanism, laying the groundwork for materials encapsulation and advancing lead detoxification. Our proof-of-principle experiment, demonstrating actin filament recovery in lead-treated cells, signifies therapeutic potential for intracellular lead aggregation and introduces novel avenues in biotechnological applications within biomaterials science.
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Plomo , Humanos , Plomo/química , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Nanoestructuras/química , Línea Celular , Péptidos/química , Péptidos/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Piridinas/químicaRESUMEN
Age-related macular degeneration (AMD) is characterized by visual impairment observed in elderly population. Two forms of the disease are generally described, the atrophic (AMDa) and exudative forms (AMDe). Up until now, no curative treatment is available for this disease. The retinal pigment epithelium (RPE) plays a central role in the pathogenesis of age-related macular degeneration. Here, involvement of RPE dysfunction in AMD onset and progression was analyzed by a comparison of transcriptome profiles of hiPSC-RPE derived from healthy individuals or individuals affected by AMDa or AMDe. The analysis highlighted almost 1000 genes differentially expressed between the three comparison groups. Among these genes, 33 genes were already known to be involved in AMD pathogenesis. To establish an AMD genetic signature, we focused on genes differentially expressed in both AMDa/e cell lines compared to control cells and focused on the three genes (ABCA1, RPN2, RB1CC1) that were related to lipidic homeostasis. Differences in level expression of these three genes are found not only in control and AMDa/e cell lines, but also between AMDa and AMDe populations.
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Homeostasis , Células Madre Pluripotentes Inducidas , Metabolismo de los Lípidos , Degeneración Macular , Epitelio Pigmentado de la Retina , Línea Celular , Homeostasis/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Metabolismo de los Lípidos/genética , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , RNA-Seq , Humanos , Persona de Mediana Edad , Anciano , Anciano de 80 o más AñosRESUMEN
Cell therapeutic applications based on induced pluripotent stem cells (iPSCs) appear highly promising and challenging at the same time. Good manufacturing practice (GMP) regulations impose necessary yet demanding requirements for quality and consistency when manufacturing iPSCs and their differentiated progeny. Given the scarcity of accessible GMP iPSC lines, we have established a corresponding production workflow to generate the first set of compliant cell banks. Hence, these lines met a comprehensive set of release specifications and, for instance, displayed a low overall mutation load reflecting their neonatal origin, cord blood. Based on these iPSC lines, we have furthermore developed a set of GMP-compatible workflows enabling improved gene targeting at strongly enhanced efficiencies and directed differentiation into critical cell types: A new protocol for the generation of retinal pigment epithelium (RPE) features a high degree of simplicity and efficiency. Mesenchymal stromal cells (MSCs) derived from iPSCs displayed outstanding expansion capacity. A fully optimized cardiomyocyte differentiation protocol was characterized by a particularly high batch-to-batch consistency at purities above 95%. Finally, we introduce a universal immune cell induction platform that converts iPSCs into multipotent precursor cells. These hematopoietic precursors could selectively be stimulated to become macrophages, T cells, or natural killer (NK) cells. A switch in culture conditions upon NK-cell differentiation induced a several thousand-fold expansion, which opens up perspectives for upscaling this key cell type in a feeder cell-independent approach. Taken together, these cell lines and improved manipulation platforms will have broad utility in cell therapy as well as in basic research.
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Diferenciación Celular , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Inmunoterapia/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Línea Celular , Neoplasias/terapia , Neoplasias/inmunología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Flujo de Trabajo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismoRESUMEN
Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.
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Oftalmoscopía , Epitelio Pigmentado de la Retina , Humanos , Oftalmoscopía/métodos , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/química , Adulto , Masculino , Femenino , Envejecimiento/fisiología , Persona de Mediana Edad , Anciano , Adulto Joven , Imagen Óptica/métodos , Lipofuscina/metabolismo , Lipofuscina/análisis , Lipofuscina/química , Estudios de FactibilidadRESUMEN
The choroid, a vascularized tissue situated between the retina and the sclera, plays a crucial role in maintaining ocular homeostasis. Despite its significance, research on choroidal abnormalities and the establishment of effective in vitro models have been limited. In this study, we developed an in vitro choroid model through the co-culture of human induced pluripotent stem cells (hiPSC)-derived endothelial cells (ECs) and mouse choroidal fibroblasts (msCFs) with hiPSC-derived retinal pigment epithelial (RPE) cells via a permeable membrane. This model, inclusive of ECs, CFs, and RPE cells, exhibited similarities with in vivo choroidal vessels, as confirmed through immunohistochemistry of extracellular matrix markers and vascular-related markers, as well as choroid angiogenesis sprouting assay analysis. The effectiveness of our in vitro model was demonstrated in assessing vascular changes induced by drugs targeting vasoregulation. Our model offers a valuable tool for gaining insights into the pathological mechanisms underlying choroid development and the progression of choroidal vascular diseases.
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Coroides , Técnicas de Cocultivo , Células Endoteliales , Células Madre Pluripotentes Inducidas , Epitelio Pigmentado de la Retina , Coroides/irrigación sanguínea , Coroides/metabolismo , Animales , Humanos , Ratones , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Endoteliales/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Fibroblastos/metabolismo , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Células CultivadasRESUMEN
The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer. In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored. Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization. In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation. Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures. CUT&RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites. These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.