RESUMEN
Stress and injury to the retinal pigment epithelium (RPE) often lead to dedifferentiation and epithelial-to-mesenchymal transition (EMT). These processes have been implicated in several retinal diseases, including proliferative vitreoretinopathy, diabetic retinopathy, and age-related macular degeneration. Despite the importance of RPE-EMT and the large body of data characterizing malignancy-related EMT, comprehensive proteomic studies to define the protein changes and pathways underlying RPE-EMT have not been reported. This study sought to investigate the temporal protein expression changes that occur in a human-induced pluripotent stem cell-based RPE-EMT model. We utilized multiplexed isobaric tandem mass tag labeling followed by high-resolution tandem MS for precise and in-depth quantification of the RPE-EMT proteome. We have identified and quantified 7937 protein groups in our tandem mass tag-based MS analysis. We observed a total of 532 proteins that are differentially regulated during RPE-EMT. Furthermore, we integrated our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into RPE-EMT mechanisms. To validate these results, we have performed a label-free single-shot data-independent acquisition MS study. Our integrated analysis indicates both the commonality and uniqueness of RPE-EMT compared with malignancy-associated EMT. Our comparative analysis also revealed that multiple age-related macular degeneration-associated risk factors are differentially regulated during RPE-EMT. Together, our integrated dataset provides a comprehensive RPE-EMT atlas and resource for understanding the molecular signaling events and associated biological pathways that underlie RPE-EMT onset. This resource has already facilitated the identification of chemical modulators that could inhibit RPE-EMT, and it will hopefully aid in ongoing efforts to develop EMT inhibition as an approach for the treatment of retinal disease.
Asunto(s)
Transición Epitelial-Mesenquimal , Epitelio Pigmentado de la Retina/metabolismo , Carcinogénesis , Células Cultivadas , Técnicas de Cocultivo , Células Madre Embrionarias , Humanos , Células Madre Pluripotentes Inducidas , ProteomaRESUMEN
In this study, we describe a simple and straightforward assay using induced pluripotent stem cell-derived melanocytes and high-throughput flow cytometry, to identify the effect induced by pigment regulating agents on melanin content. The assay is based on the correlation between forward light-scatter characteristics and melanin content, with pigmented cells displaying high light absorption/low forward light scatter, while the opposite is true for lowly pigmented melanocytes, as a result of genetic background or chemical treatments. Orthogonal validation is then performed by regular melanin quantification. Such approach was validated using a set of 80 small molecules and yielded a confirmed hit. The assay described in this study may prove a useful tool to identify modulators of melanogenesis in human melanocytes.
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Ensayos Analíticos de Alto Rendimiento/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Melanocitos/metabolismo , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Citometría de Flujo , Humanos , PigmentaciónRESUMEN
Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.
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Diferenciación Celular/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Epitelio Pigmentado de la Retina/citología , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Pluripotentes/citología , Reacción en Cadena de la PolimerasaRESUMEN
The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible regulation of visual cycle genes by common miRNAs.
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Proteínas del Ojo/genética , Regulación de la Expresión Génica , Epitelio Pigmentado de la Retina/metabolismo , Factor de Transcripción SOX9/fisiología , Animales , Sitios de Unión/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Proteínas del Ojo/metabolismo , Redes Reguladoras de Genes , Células HEK293 , Humanos , Inmunohistoquímica , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Noqueados , MicroARNs/genética , Modelos Genéticos , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , Epitelio Pigmentado de la Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/metabolismoRESUMEN
Retinal degenerative disease involving photoreceptor (PR) cell loss results in permanent vision loss and often blindness. Generation of induced pluripotent stem cell (iPSC)-derived retinal cells and tissues from individuals with retinal dystrophies is a relatively new and promising method for studying retinal degeneration mechanisms in vitro. Recent advancements in strategies to differentiate human iPSCs (hiPSCs) into 3D retinal eyecups with a strong resemblance to the mature retina raise the possibility that this system could offer a reliable model for translational drug studies. However, despite the potential benefits, there are challenges that remain to be overcome before stem-cell-derived retinal eyecups can be routinely used to model human retinal diseases. This chapter will discuss both the potential of these 3D eyecup approaches and the nature of some of the challenges that remain.
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Células Fotorreceptoras de Vertebrados/citología , Células Madre Pluripotentes/citología , Degeneración Retiniana/patología , Distrofias Retinianas/patología , Epitelio Pigmentado de la Retina/citología , Técnicas de Cultivo de Célula , Descubrimiento de Drogas , Humanos , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/genética , Distrofias Retinianas/tratamiento farmacológico , Distrofias Retinianas/genéticaRESUMEN
Driving efficient and pure skeletal muscle cell differentiation from pluripotent stem cells (PSCs) has been challenging. Here, we report an optimized protocol that generates skeletal muscle progenitor cells with high efficiency and purity in a short period of time. Human induced PSCs (hiPSCs) and murine embryonic stem cells (mESCs) were specified into the mesodermal myogenic fate using distinct and species-specific protocols. We used a specific maturation medium to promote the terminal differentiation of both human and mouse myoblast populations, and generated myotubes associated with a large pool of cell-cycle arrested PAX7+ cells. We also show that myotube maturation is modulated by dish-coating properties, cell density, and percentage of myogenic progenitor cells. Given the high efficiency in the generation of myogenic progenitors and differentiated myofibers, this protocol provides an attractive strategy for tissue engineering, modeling of muscle dystrophies, and evaluation of new therapeutic approaches in vitro.
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Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Humanos , Animales , Ratones , Células Cultivadas , Fibras Musculares Esqueléticas , Diferenciación Celular , Desarrollo de Músculos , Músculo EsqueléticoRESUMEN
Microorganisms can be equipped with synthetic genetic programs for the production of targeted therapeutic molecules. Cutibacterium acnes is the most abundant commensal of the human skin, making it an attractive chassis to create skin-delivered therapeutics. Here, we report the engineering of this bacterium to produce and secrete the therapeutic molecule neutrophil gelatinase-associated lipocalin, in vivo, for the modulation of cutaneous sebum production.
RESUMEN
Cutibacterium acnes (C. acnes) is one of the most prevalent bacteria that forms the human skin microbiota. Specific phylotypes of C. acnes have been associated with the development of acne vulgaris, while other phylotypes have been linked to healthy skin. In this scenario, bacterial extracellular vesicles (EVs) play a role in the interkingdom communication role with the human host. The purpose of this study was to examine the impact of EVs generated by various phylotypes of C. acnes on inflammation and sebum production using different in vitro skin cell types. The main findings of this study reveal that the proteomic profile of the cargo embodied in the EVs reflects distinct characteristics of the different C. acnes phylotypes in terms of life cycle, survival, and virulence. The in vitro skin cell types showed an extended pro-inflammatory modulation of SLST A1 EVs consistently triggering the activation of the inflammation-related factors IL-8, IL-6, TNFα and GM-CSF, in comparison to SLST H1 and SLST H2. Additionally, an acne-prone skin model utilizing PCi-SEB and arachidonic acid as a sebum inducer, was employed to investigate the impact of C. acnes EVs on sebum regulation. Our findings indicated that all three types of EVs significantly inhibited sebum production after a 24-h treatment period, with SLST H1 EVs exhibiting the most pronounced inhibitory effect when compared to the positive control. The results of this study highlight the protective nature of C. acnes SLST H1 EVs and their potential use as a natural treatment option for alleviating symptoms associated with inflammation and oily skin.
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Acné Vulgar , Vesículas Extracelulares , Enfermedades de la Piel , Humanos , Proteómica , Piel , Propionibacterium acnes , Factor VIII , InflamaciónRESUMEN
Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF-ß) and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), can induce RPE-EMT; however, small molecule inhibitors of RPE-EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKß) that selectively targets NF-κB signaling, can modulate TGF-ß/TNF-α-induced RPE-EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKß inhibition on RPE-EMT-associated factors using a second IKKß inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE-EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.
Asunto(s)
Epitelio Pigmentado de la Retina , Vitreorretinopatía Proliferativa , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Transición Epitelial-Mesenquimal , Quinasa I-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Vitreorretinopatía Proliferativa/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre/metabolismoRESUMEN
Pluripotency can be captured in vitro, providing that the culture environment meets the requirements that avoid differentiation while stimulating self-renewal. From studies in the mouse embryo, two kinds of pluripotent stem cells have been obtained from the early and late epiblast, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), representing the naive and primed states, respectively. All attempts to derive convincing ESCs in ungulates have been unsuccessful, although all attempts were based on the assumption that the conditions used to derive mouse ESCs or human ESC could be applied in other species. Pluripotent cells derived in primates, rabbit, and pig strongly indicate that the state of pluripotency of these cells is, in fact, closer to EpiSCs than to ESCs, and thus depend on fibroblast growth factor (FGF) and Activin signaling pathways. Based on this observation, we have tried to derive EpiSC from the epiblast of bovine elongated embryos as well as ESCs from Day-8 blastocysts. We here show that the core transcription factors Oct4/Sox2/Nanog can be used as markers of pluripotency in the bovine since their expression was restricted to the developing epiblast after Day 8, and disappeared following differentiation of both the ESC-like and EpiSC-like cultures. Although FGF and Activin pathways are indeed present and active in the bovine, it is not sufficient/enough to maintain a long-term pluripotency ex vivo, as was reported for mouse and pig EpiSCs.
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Diferenciación Celular , Células Madre Embrionarias/fisiología , Estratos Germinativos/metabolismo , Células Madre Pluripotentes/metabolismo , Activinas/metabolismo , Animales , Biomarcadores , Blastocisto/metabolismo , Bovinos , Células Cultivadas , Células Madre Embrionarias/citología , Factores de Crecimiento de Fibroblastos/metabolismo , Estratos Germinativos/citología , Proteínas de Homeodominio/biosíntesis , Ratones , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Células Madre Pluripotentes/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción SOXB1/biosíntesis , Transducción de SeñalRESUMEN
Mouse embryonic pluripotent stem cells can be obtained from the inner cell mass at the blastocyst stage (embryonic stem cells, ESCs) or from the late epiblast of postimplantation embryos (epiblast stem cells, EpiSCs). During normal development, the transition between these two stages is marked by major epigenetic and transcriptional changes including DNA de novo methylation. These modifications represent an epigenetic mark conserved in ESCs and EpiSCs. Pluripotent ESCs derived from blastocysts generated by nuclear transfer (NT) have been shown to be correctly reprogrammed. However, NT embryos frequently undergo abnormal development. In the present study, we have examined whether pluripotent cells could be derived from the epiblast of postimplantation NT embryos and whether the reprogramming process would affect the epigenetic changes occurring at this stage, which could explain abnormal development of NT embryos. We showed that EpiSCs could be derived with the same efficiency from NT embryos and from their fertilized counterparts. However, gene expression profile analyses showed divergence between fertilized- and nuclear transfer-EpiSCs with a surprising bias in the distribution of the differentially expressed genes, 30% of them being localized on chromosome 11. A majority of these genes were downregulated in NT-EpiSCs and imprinted genes represented a significant fraction of them. Notably, analysis of the epigenetic status of a downregulated imprinted gene in NT-EpiSCs revealed complete methylation of the two alleles. Therefore, EpiSCs derived from NT embryos appear to be incorrectly reprogrammed, indicating that abnormal epigenetic marks are imposed on cells in NT embryos during the transition from early to late epiblast.
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Estratos Germinativos/metabolismo , Células Madre/metabolismo , Animales , Biomarcadores , Línea Celular , Proliferación Celular , Forma de la Célula , Epigénesis Genética , Fertilización In Vitro , Perfilación de la Expresión Génica , Estratos Germinativos/citología , Ratones , Ratones Endogámicos C57BL , Técnicas de Transferencia Nuclear , Células Madre/citologíaRESUMEN
Purpose: RPE injury often induces epithelial to mesenchymal transition (EMT). Although RPE-EMT has been implicated in a variety of retinal diseases, including proliferative vitroretinopathy, neovascular and atrophic AMD, and diabetic retinopathy, it is not well-understood at the molecular level. To contribute to our understanding of EMT in human RPE, we performed a time-course transcriptomic analysis of human stem cell-derived RPE (hRPE) monolayers induced to undergo EMT using 2 independent, yet complementary, model systems. Methods: EMT of human stem cell-derived RPE monolayers was induced by either enzymatic dissociation or modulation of TGF-ß signaling. Transcriptomic analysis of cells at different stages of EMT was performed by RNA-sequencing, and select findings were confirmed by reverse transcription quantitative PCR and immunostaining. An ingenuity pathway analysis (IPA) was performed to identify signaling pathways and regulatory networks associated with EMT. Results: Proteocollagenolytic enzymatic dissociation and cotreatment with TGF-ß and TNF-α both induce EMT in human stem cell-derived RPE monolayers, leading to an increased expression of mesenchymal factors and a decreased expression of RPE differentiation-associated factors. Ingenuity pathway analysis identified the upstream regulators of the RPE-EMT regulatory networks and identified master switches and nodes during RPE-EMT. Of particular interest was the identification of widespread dysregulation of axon guidance molecules during RPE-EMT progression. Conclusions: The temporal transcriptome profiles described here provide a comprehensive resource of the dynamic signaling events and the associated biological pathways that underlie RPE-EMT onset. The pathways defined by these studies may help to identify targets for the development of novel therapeutic targets for the treatment of retinal disease.
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Transición Epitelial-Mesenquimal/fisiología , Células Madre Pluripotentes/citología , Epitelio Pigmentado de la Retina/metabolismo , Transcriptoma/fisiología , Diferenciación Celular , Células Cultivadas , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Epitelio Pigmentado de la Retina/citología , Transducción de Señal , Factores de TranscripciónRESUMEN
BACKGROUND: Genomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we apply allele-specific RNA-seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic tissues between E3.5 and E7.25 and in pluripotent cell lines to evaluate maintenance of imprinted gene expression. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos and from embryos obtained after nuclear transfer (NT) or parthenogenetic activation (PGA). RESULTS: As homozygous genomic regions of PGA-derived cells are not compatible with allele-specific RNA-seq, we developed an RNA-seq-based genotyping strategy allowing identification of informative heterozygous regions. Global analysis shows that proper imprinted gene expression as observed in embryonic tissues is largely lost in the ESC lines included in this study, which mainly consisted of female ESCs. Differentiation of ESC lines to embryoid bodies or NPCs does not restore monoallelic expression of imprinted genes, neither did reprogramming of the serum-cultured ESCs to the pluripotent ground state by the use of 2 kinase inhibitors. Fertilized EpiSC and EpiSC-NT lines largely maintain imprinted gene expression, as did EpiSC-PGA lines that show known paternally expressed genes being silent and known maternally expressed genes consistently showing doubled expression. Notably, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. With respect to female EpiSC, most of the lines displayed completely skewed X inactivation suggesting a (near) clonal origin. CONCLUSIONS: Altogether, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential.
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Alelos , Impresión Genómica , Células Madre Embrionarias de Ratones/metabolismo , ARN Mensajero/genética , Animales , Diferenciación Celular , Línea Celular , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Células Madre Embrionarias de Ratones/citologíaRESUMEN
Structural and biochemical cues of extracellular matrix can substantially influence the differentiation and maturation of cultured retinal pigment epithelial (RPE) cells. In this study, thin collagen vitrigels were engineered to create collagen nanofibrillar structures of different fibril densities in an effort to evaluate the maturation of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. The ultrastructure of the different collagen vitrigels was characterized by transmission electron microscopy, and the mechanical properties were evaluated by tensile testing. The pigmentation and polarization of cells, in addition to key RPE marker gene and protein expression levels, were analyzed to determine the differentiation of hESCs on the gels. The hESC-RPE differentiation was most significant in collagen vitrigels with low fibril density with mature collagen fibrils with diameter of around 60 nm and Young's modulus of 2.41 ± 0.13 MPa. This study provides insight into the influence of collagen nanofibrillar structures on hESC-RPE maturation and presents a potential bioengineered substratum for hESC-RPE for future preclinical and clinical applications.
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Diferenciación Celular , Colágeno/farmacología , Células Epiteliales/citología , Geles/farmacología , Epitelio Pigmentado de la Retina/citología , Vitrificación , Animales , Bovinos , Polaridad Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Reactivos de Enlaces Cruzados/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/ultraestructura , Humanos , Pigmentación/efectos de los fármacos , Poliésteres/farmacologíaRESUMEN
The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.
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Diferenciación Celular , Células Madre Pluripotentes/citología , Retina/citología , Retina/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Biomarcadores , Técnicas de Cultivo de Célula , Células Cultivadas , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestructura , Expresión Génica , Humanos , Hipoxia , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Factores de TiempoRESUMEN
The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.
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Proteínas Bacterianas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/genética , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Pollos , Biología Computacional , Células Madre Embrionarias/citología , Ingeniería Genética/métodos , Genoma Humano , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Nucleótidos/química , Plásmidos/metabolismo , Ratas , Streptococcus pyogenes , Pez CebraRESUMEN
Age-related macular degeneration (AMD), the leading cause of irreversible vision loss and blindness among the elderly in industrialized countries, is associated with the dysfunction and death of the retinal pigment epithelial (RPE) cells. As a result, there has been significant interest in developing RPE culture systems both to study AMD disease mechanisms and to provide substrate for possible cell-based therapies. Because of their indefinite self-renewal, human pluripotent stem cells (hPSCs) have the potential to provide an unlimited supply of RPE-like cells. However, most protocols developed to date for deriving RPE cells from hPSCs involve time- and labor-consuming manual steps, which hinder their use in biomedical applications requiring large amounts of differentiated cells. Here, we describe a simple and scalable protocol for the generation of RPE cells from hPSCs that is less labor-intensive. After amplification by clonal propagation using a myosin inhibitor, differentiation was induced in monolayers of hPSCs, and the resulting RPE cells were purified by two rounds of whole-dish single-cell passage. This approach yields highly pure populations of functional hPSC-derived RPE cells that display many characteristics of native RPE cells, including proper pigmentation and morphology, cell type-specific marker expression, polarized membrane and vascular endothelial growth factor secretion, and phagocytic activity. This work represents a step toward mass production of RPE cells from hPSCs.
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Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Pluripotentes/citología , Epitelio Pigmentado de la Retina/citología , Animales , Biomarcadores/metabolismo , Línea Celular , Polaridad Celular/genética , Separación Celular , Forma de la Célula , Supervivencia Celular , Células Clonales , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Cariotipificación , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fagocitosis , Células Madre Pluripotentes/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Trasplante de Células Madre , Factores de Tiempo , Transcripción Genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Nuclear reprogramming toward pluripotency has been now achieved either in vivo by somatic cell nuclear transfer into the ooplasm of an enucleated egg, or in vitro by three different approaches, namely cell fusion, treatment with cell extracts and more recently, forced expression of a reduced set of defined transcription factors. This last technique has expanded our view of genome plasticity with important applied perspectives in regenerative biomedicine. Because of their ease of generation, induced pluripotent stem cells represent a major hope in the field of regenerative medicine. However, the extent to which such an in vitro induced pluripotency can be considered to be equivalent to embryonic-derived pluripotency remains undetermined and also largely dependent on how pluripotency is assessed. Here, we provide an overwiew of the data published in the recent literature on the ability of each of the above techniques to reprogram somatic nuclei into pluripotent embryonic-like nuclei. These data support the view that even though nuclear transfer is technically demanding, it remains a fast and efficient means for a systematic derivation of bona fide embryonic stem cells from somatic donor cells. We conclude that nuclear transfer has still much to teach us about faithful nuclear reprogramming to pluripotency.