RESUMEN
Welding fumes induce lung toxicity and are carcinogenic to humans but the molecular mechanisms have yet to be clarified. The aim of this study was to evaluate the toxicity of stainless and mild steel particles generated via gas-metal arc welding using primary human small airway epithelial cells (hSAEC) and ToxTracker reporter murine stem cells, which track activation of six cancer-related pathways. Metal content (Fe, Mn, Ni, Cr) of the particles was relatively homogenous across particle size. The particles were not cytotoxic in reporter stem cells but stainless steel particles activated the Nrf2-dependent oxidative stress pathway. In hSAEC, both particle types induced time- and dose-dependent cytotoxicity, and stainless steel particles also increased generation of reactive oxygen species. The cellular metal content was higher for hSAEC compared to the reporter stem cells exposed to the same nominal dose. This was, in part, related to differences in particle agglomeration/sedimentation in the different cell media. Overall, our study showed differences in cytotoxicity and activation of cancer-related pathways between stainless and mild steel welding particles. Moreover, our data emphasizes the need for careful assessment of the cellular dose when comparing studies using different in vitro models.
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Contaminantes Ocupacionales del Aire/toxicidad , Acero Inoxidable/toxicidad , Acero/toxicidad , Soldadura , Contaminantes Ocupacionales del Aire/química , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Humanos , Exposición por Inhalación/efectos adversos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/ultraestructura , Tamaño de la Partícula , Especies Reactivas de Oxígeno/metabolismo , Acero Inoxidable/química , Acero/química , Soldadura/métodosRESUMEN
While embryonic stem cells and cancer cells are known to have many similarities in signalling pathways, healthy somatic cells are known to be different in many ways. Characterization of embryonic stem cell is crucial for cancer development and cancer recurrence due to the shared signalling pathways and life course with cancer initiator and cancer stem cells. Since embryonic stem cells are the sources of the somatic and cancer cells, it is necessary to reveal the relevance between them. The past decade has seen the importance of interdisciplinary studies and it is obvious that the reflection of the physical/chemical phenomena occurring on the cell biology has attracted much more attention. For this reason, the aim of this study is to elementally and topologically characterize the mouse embryonic stem cells, mouse lung squamous cancer cells, and mouse skin fibroblast cells by using Atomic Force Microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM) supported with Electron Dispersive Spectroscopy (EDS) techniques in a complementary way. Our AFM findings revealed that roughness data of the mouse embryonic stem cells and cancer cells were similar and somatic cells were found to be statistically different from these two cell types. However, based on both XPS and SEM-EDS results, surface elemental ratios vary in mouse embryonic stem cells, cancer cells and somatic cells. Our results showed that these complementary spectroscopic and microscopic techniques used in this work are very effective in cancer and stem cell characterization and have the potential to gather more detailed information on relevant biological samples.
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Fibroblastos , Neoplasias Pulmonares , Células Madre Embrionarias de Ratones , Neoplasias de Células Escamosas , Piel , Animales , Línea Celular Tumoral , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/ultraestructura , Ratones , Microscopía de Fuerza Atómica , Microscopía Electroquímica de Rastreo , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/ultraestructura , Neoplasias de Células Escamosas/metabolismo , Neoplasias de Células Escamosas/ultraestructura , Piel/metabolismo , Piel/ultraestructuraRESUMEN
Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.
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Microscopía Fluorescente/métodos , Microtúbulos/ultraestructura , Células Madre Embrionarias de Ratones/ultraestructura , Osteoblastos/ultraestructura , Coloración y Etiquetado/métodos , Animales , Anticuerpos/química , Biotina/química , Línea Celular Tumoral , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Microtúbulos/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Osteoblastos/metabolismo , Estreptavidina/química , Succinimidas/químicaRESUMEN
INTRODUCTION: Embryonic stem cells (ESCs) possess great application prospects in biological research and regenerative medicine, so it is important to obtain ESCs with excellent and stable cellular states during in vitro expansion. The feeder layer culture system with the addition of leukemia inhibitory factor (LIF) is currently applied in ESC cultures, but it has a series of disadvantages that could influence the culture efficiency and quality of the ESCs. With the development of nanotechnology, many studies have applied nanomaterials to optimize the stem cell culture system and regulate the fate of stem cells. In this study, we investigated the layer-number-dependent biofunction of graphene oxide (GO) on the pluripotency of ESCs from mice (mESCs). METHODS: Single-layer GO (SGO) and multi-layer GO (MGO) were characterized and their effects on the cytotoxicity and self-renewal of mESCs were detected in vitro. The differentiation potentials of mESCs were identified through the formation of embryoid bodies and teratomas. The regulatory mechanism of GO was verified by blocking the target receptors on the surface of mESCs using antibodies. RESULTS: Both SGO and MGO were biocompatible with mESCs, but only MGO effectively sustained their self-renewal and differentiation potential. In addition, GO influenced the cellular activities of mESCs by regulating the interactions between extracellular matrix proteins and integrins. CONCLUSION: This work demonstrates the layer-number-dependent effects of GO on regulating the cell behavior of mESCs and reveals the extracellular regulatory mechanism of this process.
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Matriz Extracelular/metabolismo , Grafito/farmacología , Integrinas/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/ultraestructura , Estrés Oxidativo/efectos de los fármacosRESUMEN
Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.
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Reparación del ADN/efectos de los fármacos , Flavonoides/toxicidad , Genisteína/toxicidad , Quempferoles/toxicidad , Luteolina/toxicidad , Quercetina/toxicidad , Animales , Línea Celular , Puntos de Rotura del Cromosoma/efectos de los fármacos , Cromosomas de los Mamíferos/efectos de los fármacos , ADN/química , Roturas del ADN de Doble Cadena/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Dexrazoxano/farmacología , Etopósido/toxicidad , Histonas/genética , Histonas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/ultraestructura , Inhibidores de Topoisomerasa II/farmacología , Translocación Genética/efectos de los fármacosRESUMEN
Embryonic stem cells (ESCs) exhibit high levels of ribosomal RNA (rRNA) transcription and ribosome biogenesis. Here, we reveal an unexpected role for an essential DEAD-box helicase, DDX18, in antagonizing the polycomb repressive complex 2 (PRC2) to prevent deposition of the repressive H3K27me3 mark onto rDNA in pluripotent cells. DDX18 binds and sequesters PRC2 in the outer layer of the nucleolus and counteracts PRC2 complex formation in vivo and in vitro. DDX18 knockdown leads to increased occupancy of PRC2 and H3K27me3 at rDNA loci, accompanied by drastically decreased rRNA transcription and reduced ribosomal protein expression and translation. Auxin-induced rapid degradation of DDX18 enhances PRC2 binding at rDNA. The inhibition of PRC2 partially rescues the effects of DDX18 depletion on rRNA transcription and ESC self-renewal. These results demonstrate a critical role for DDX18 in safeguarding the chromatin and transcriptional integrity of rDNA by counteracting the epigenetic silencing machinery to promote pluripotency.
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ARN Helicasas DEAD-box/metabolismo , ADN Ribosómico/metabolismo , Células Madre Pluripotentes/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Animales , Nucléolo Celular/metabolismo , Cromatina/metabolismo , ADN Ribosómico/genética , Desarrollo Embrionario/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Metilación , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/ultraestructura , Células Madre Pluripotentes/citología , Unión Proteica , Proteolisis , ARN Ribosómico/metabolismo , Transcripción GenéticaRESUMEN
The genome of pluripotent stem cells adopts a unique three-dimensional architecture featuring weakly condensed heterochromatin and large nucleosome-free regions. Yet, it is unknown whether structural loops and contact domains display characteristics that distinguish embryonic stem cells (ESCs) from differentiated cell types. We used genome-wide chromosome conformation capture and super-resolution imaging to determine nuclear organization in mouse ESC and neural stem cell (NSC) derivatives. We found that loss of pluripotency is accompanied by widespread gain of structural loops. This general architectural change correlates with enhanced binding of CTCF and cohesins and more pronounced insulation of contacts across chromatin boundaries in lineage-committed cells. Reprogramming NSCs to pluripotency restores the unique features of ESC domain topology. Domains defined by the anchors of loops established upon differentiation are enriched for developmental genes. Chromatin loop formation is a pervasive structural alteration to the genome that accompanies exit from pluripotency and delineates the spatial segregation of developmentally regulated genes.
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Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Células-Madre Neurales/metabolismo , Animales , Diferenciación Celular , Cromatina/ultraestructura , Ratones , Células Madre Embrionarias de Ratones/fisiología , Células Madre Embrionarias de Ratones/ultraestructura , Células-Madre Neurales/fisiología , Células-Madre Neurales/ultraestructura , Unión Proteica , CohesinasRESUMEN
A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.
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Cromatina/química , Microscopía Fluorescente/métodos , Células Madre Embrionarias de Ratones/metabolismo , Imagen Individual de Molécula/métodos , Animales , Línea Celular , Cromatina/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Células Madre Embrionarias de Ratones/ultraestructura , FotoblanqueoRESUMEN
Mouse embryonic stem (ES) cells are widely used in developmental biology and transgenic research. Despite numerous studies, ultrastructural reorganization of inner cell mass (ICM) cells during in vitro culture has not yet been described in detail. Here, we for the first time performed comparative morphological and morphometric analyses of three ES cell lines during their derivation in vitro. We compared morphological characteristics of blastocyst ICM cells at 3.5 and 4.5 days post coitum on feeder cells (day 6, passage 0) with those of ES cells at different passages (day 19, passage 2; day 25, passage 4; and passage 15). At passage 0, there were 23-36% of ES-like cells with various values of the medium cross-sectional area and nucleocytoplasmic parameters, 55% of fibroblast-like (probably trophoblast derivatives), and ~ 19% of dying cells. ES-like cells at passage 0 contained autolysosomes and enlarged mitochondria with reduced numerical density per cell. There were three types of mitochondria that differed in matrix density and cristae width. For the first time, we revealed cells that had two and sometimes three morphologically distinct mitochondria types in the cytoplasm. At passage 2, there were mostly ES cells with a high nucleocytoplasmic ratio and a cytoplasm depleted of organelles. At passage 4, ES cell morphology and morphometric parameters were mostly stable with little heterogeneity. According to our data, cellular structures of ICM cells undergo destabilization during derivation of an ES cell line with subsequent reorganization into the structures typical for ES cells. On the basis of ultrastructural analysis of mitochondria, we believe that the functional activity of these organelles changes during early stages of ES cell formation from the ICM.
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Mitocondrias/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Línea Celular , Citoplasma/metabolismo , Citoplasma/ultraestructura , Ratones , Mitocondrias/ultraestructura , Células Madre Embrionarias de Ratones/ultraestructura , Orgánulos/metabolismo , Orgánulos/ultraestructuraRESUMEN
Extracellular vesicles (EVs) released by mouse embryonic stem cells (mESCs) are considered a source of bioactive molecules that modulate their microenvironment by acting on intercellular communication. Either intracellular endosomal machinery or their derived EVs have been considered a relevant system of signal circuits processing. Herein, we show that these features are found in mESCs. Ultrastructural analysis revealed structures and organelles of the endosomal system such as coated pits and endocytosis-related vesicles, prominent rough endoplasmic reticulum and Golgi apparatus, and multivesicular bodies (MVBs) containing either few or many intraluminal vesicles (ILVs) that could be released as exosomes to extracellular milieu. Besides, budding vesicles shed from the plasma membrane to the extracellular space is suggestive of microvesicle biogenesis in mESCs. mESCs and mouse blastocyst express specific markers of the Endosomal Sorting Complex Required for Transport (ESCRT) system. Ultrastructural analysis and Nanoparticle Tracking Analysis (NTA) of isolated EVs revealed a heterogeneous population of exosomes and microvesicles released by mESCs. These vesicles contain Wnt10b and the Notch ligand Delta-like 4 (DLL4) and also the co-chaperone stress inducible protein 1 (STI1) and its partner Hsp90. Wnt10b and Dll4 colocalize with EVs biogenesis markers in mESCs. Overall, the present study supports the function of the mESCs endocytic network and their EVs as players in stem cell biology.
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Vesículas Extracelulares/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Línea Celular , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Vesículas Extracelulares/ultraestructura , Aparato de Golgi/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Células Madre Embrionarias de Ratones/ultraestructura , Cuerpos Multivesiculares/metabolismoRESUMEN
The production of erythropoietin (EPO) by the kidneys, a principal hormone for the hematopoietic system, is reduced in patients with chronic kidney disease (CKD), eventually resulting in severe anemia. Although recombinant human EPO treatment improves anemia in patients with CKD, returning to full red blood cell production without fluctuations does not always occur. We established a method to generate EPO-producing cells from human induced pluripotent stem cells (hiPSCs) by modifying previously reported hepatic differentiation protocols. These cells showed increased EPO expression and secretion in response to low oxygen conditions, prolyl hydroxylase domain-containing enzyme inhibitors, and insulin-like growth factor 1. The EPO protein secreted from hiPSC-derived EPO-producing (hiPSC-EPO) cells induced the erythropoietic differentiation of human umbilical cord blood progenitor cells in vitro. Furthermore, transplantation of hiPSC-EPO cells into mice with CKD induced by adenine treatment improved renal anemia. Thus, hiPSC-EPO cells may be a useful tool for clarifying the mechanisms of EPO production and may be useful as a therapeutic strategy for treating renal anemia.
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Anemia/terapia , Eritropoyetina/biosíntesis , Riñón/patología , Células Madre Pluripotentes/citología , Trasplante de Células Madre , Anemia/patología , Animales , Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Eritropoyesis/efectos de los fármacos , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/ultraestructura , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/ultraestructura , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/ultraestructuraRESUMEN
Poly-É-caprolactone (PCL) based microspheres have received much attention as drug or growth factor delivery carriers and tissue engineering scaffolds due to their biocompatibility, biodegradability, and tunable biophysical properties. In addition, PCL and polydimethylsiloxane (PDMS) can be fabricated into thermoresponsive shape memory polymers for various biomedical applications (e.g., smart sutures and vascular stents). However, the influence of biophysical properties of PCL-PDMS based microspheres on stem cell lineage commitment has not been well understood. In this study, PDMS was used as soft segments of varying length to tailor the elastic modulus of PCL-based copolymers. It was found that lower elastic modulus (<10 kPa) of the tri-block copolymer PCL-PDMS-PCL promoted vascular differentiation of embryonic stem cells, but the range of 60-100 MPa PCL-PDMS-PCL had little influence on cardiovascular differentiation. Then different sizes (30-140 µm) of PCL-PDMS-PCL microspheres were fabricated and incorporated with embryoid bodies (EBs). Differential expression of KDR, CD31, and VE-cadherin was observed for the EBs containing microspheres of different sizes. Higher expression of KDR was observed for the condition with small size of microspheres (32 µm), while higher CD31 and VE-cadherin expression was observed for the group of medium size of microspheres (94 µm). Little difference in cardiac marker α-actinin was observed for different microspheres. This study indicates that the biophysical properties of PCL-PDMS-PCL microspheres impact vascular lineage commitment and have implications for drug delivery and tissue engineering.
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Diferenciación Celular , Dimetilpolisiloxanos/química , Microesferas , Células Madre Embrionarias de Ratones/citología , Miocitos Cardíacos/citología , Poliésteres/química , Animales , Materiales Biocompatibles/farmacología , Línea Celular , Dimetilpolisiloxanos/síntesis química , Módulo de Elasticidad , Ratones , Peso Molecular , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/ultraestructura , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/ultraestructura , Poliésteres/síntesis química , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
BACKGROUND: Nuclear size and shape are specific to a cell type, function, and location, and can serve as indicators of disease and development. We previously found that lamin A/C and associated nuclear envelope structural proteins were upregulated when murine embryonic stem (ES) cells differentiated to primitive endoderm cells. Here we further investigated the morphological changes of nuclei that accompany this differentiation. RESULTS: The nuclei of undifferentiated wild type cells were found shaped as flattened, irregular ovals, whereas nuclei of Gata4-positive endoderm cells were more spherical, less flattened, and with a slightly reduced volume. The morphological change was confirmed in the trophectoderm and primitive endoderm lineages of E4.5 blastocysts, compared to larger and more irregularly shaped of the nuclei of the inner cell mass. We established ES cells genetically null for the nuclear lamina proteins lamin A/C or the inner nuclear envelope protein emerin, or compound mutant for both lamin A/C and emerin. ES cells deficient in lamin A/C differentiated to endoderm but less efficiently, and the nuclei remained flattened and failed to condense. The size and shape of emerin-deficient nuclei also remained uncondensed after treatment with RA. The emerin/lamin A/C double knockout ES cells failed to differentiate to endoderm cells, though the nuclei condensed but retained a generally flattened ellipsoid shape. Additionally, ES cells deficient for lamin A/C and/or emerin had compromised ability to undergo endoderm differentiation, where the differentiating cells often exhibited coexpression of pluripotent and differentiation markers, such as Oct3/4 and Gata4, respectively, indicating an infidelity of gene regulation. CONCLUSIONS: The results suggest that changes in nuclear size and shape, which are mediated by nuclear envelope structural proteins lamin A/C and/or emerin, also impact gene regulation and lineage differentiation in early embryos. Nevertheless, mice lacking both lamin A/C and emerin were born at the expected frequency, indicating their embryonic development is completed despite the observed protein deficiency.
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Diferenciación Celular/genética , Forma del Núcleo Celular/genética , Regulación del Desarrollo de la Expresión Génica , Lamina Tipo A/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Animales , Blastocisto/citología , Linaje de la Célula/genética , Células Cultivadas , Cruzamientos Genéticos , Cuerpos Embrioides/citología , Cuerpos Embrioides/ultraestructura , Desarrollo Embrionario/genética , Endodermo/citología , Femenino , Genotipo , Masculino , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/ultraestructura , Mutación/genéticaRESUMEN
Chromosome conformation capture (3C)-based techniques such as chromosome conformation capture carbon copy (5C) and Hi-C revealed that the folding of mammalian chromosomes is highly hierarchical. A fundamental structural unit in the hierarchy is represented by topologically associating domains (TADs), sub-megabase regions of the genome within which the chromatin fibre preferentially interacts. 3C-based methods provide the mean contact probabilities between chromosomal loci, averaged over a large number of cells, and do not give immediate access to the single-cell conformations of the chromatin fibre. However, coarse-grained polymer models based on 5C data can be used to extract the single-cell conformations of single TADs. Here, we extend this approach to analyse around 2500 TADs in murine embryonic stem cells based on high-resolution Hi-C data. This allowed to predict the cell-to-cell variability in single contacts within genome-wide TADs and correlations between them. Based on these results, we predict that TADs are more similar to ideal chains than to globules in terms of their physical size and three-dimensional shape distribution. Furthermore, we show that their physical size and the degree of structural anisotropy of single TADs are correlated with the level of transcriptional activity of the genes that it harbours. Finally, we show that a large number of multiplets of genomic loci co-localize more often than expected by random, and these loci are particularly enriched in promoters, enhancers and CTCF-bound sites. These results provide the first genome-wide structural reconstruction of TADs using polymeric models obeying the laws of thermodynamics and reveal important universal trends in the correlation between chromosome structure and transcription.
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Cromosomas/ultraestructura , Genoma , Células Madre Embrionarias de Ratones , Animales , Ensamble y Desensamble de Cromatina , Cromosomas/química , Ratones , Modelos Biológicos , Células Madre Embrionarias de Ratones/ultraestructura , Transcripción GenéticaRESUMEN
OBJECTIVE: To investigate the effect of T-2 toxin on murine embryonic stem cells (ESCs) cardiac differentiation and mitochondrial biogenesis in vitro. METHODS: Cardiac differentiation of the mouse ESCs was initiated by embryoid bodies (EBs) formation in hanging drops. EBs were exposed to 0.5ng/ml T-2 toxin for 24, 72 and 120h. Cultures were observed daily for the appearance of contracting clusters, and cardiac-specific protein (α-actiniin) were measured by Western blot and immunocytochemistry. Mitochondrial ultrastructure was observed by confocal laser scanning microscopy and transmission EM photography. Reactive oxygen species (ROS) was monitored by H2-dichlorofluorescein-diacetate (H2DCF-DA). The phosphorylation of the p38 (p-p38) and p38 mitogen-activated protein kinase (MAPK) and the expression of mitochondrial biogenesis proteins, including peroxisome proliferator activated receptor coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (mtTFA), and mitochondrial respiratory chain complex IV (COXIV) were analyzed using Western blot. In some experiments, mESCs were pre-treated with the antioxidant Trolox (200µM) for 30min, then exposed to Trolox (200µM) and T-2 toxin (0.5ng/ml) for 72h. RESULTS: Contracting clusters were observed under the microscope light and cardiac-specific protein (α-actinin) expressed positively indicated mESCs directly differentiated in cardiomyocytes. However, the cardiac differentiation was inhibited by T-2 toxin treatment 72 and 120h. ROS accumulated in murine ES cells in a time-dependent manner. The expression of p-p38 significantly increased in 24h group and decrease in 72 and 120h groups. The decrease of mitochondrial number and the mitochondrial biogenesis-related proteins expression, including PGC-1α, NRF-1, mtTFA, and COXIV decreased in a time-dependent manner with T-2 toxin treatment. However, the inhibition of mitochondrial biogenesis by T-2 toxin in differentiated mESCs was recovered significantly in the presence of the antioxidant Trolox. CONCLUSION: Taken together, T-2 toxin decreased the expression of PGC-1α, NRF-1, and mtTFA, inhibited mitochondrial biogenesis, and then inhibited the cardiac differentiation of murine ES cells, and the effect was partly responsible for the p38 MAPK mediated by ROS.
Asunto(s)
Dinámicas Mitocondriales/efectos de los fármacos , Células Madre Embrionarias de Ratones/efectos de los fármacos , Mioblastos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Toxina T-2/toxicidad , Teratógenos/toxicidad , Animales , Antioxidantes/farmacología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Técnicas de Cocultivo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/ultraestructura , Mioblastos Cardíacos/citología , Mioblastos Cardíacos/metabolismo , Mioblastos Cardíacos/ultraestructura , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Biogénesis de Organelos , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Toxina T-2/antagonistas & inhibidores , Teratógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Culture conditions have been established to maintain the pluripotency of mouse naïve and primed embryonic stem cells (ESCs) using human amnion epithelial cells (hAECs) as the feeder layer. In this study, the ultrastructures of mouse primed ESCs grown on hAECs were analyzed by transmission electron microscopy. Consistent with mouse naïve ESCs, the undifferentiated mouse primed ESC line ESD-EpiSC [ESC-derived epiblast stem cells (EpiSCs)] revealed typical characteristics, including large nuclei, reticulated nucleoli, scanty cytoplasm, and low cytoplasm-to-nuclear ratios. Cells had prominent Golgi apparatus and well-developed endoplasmic reticulum. Adjacent cells were tightly in contact with dense junction desmosomes. However, in EpiSC colonies, cell contact was no longer close like naïve ESCs, and differentiated cells existed. The differentiated cells had small nucleoli with large cytoplasm, which represented primitive mesenchyme. Phagosomes or apoptotic cells were also common in the cytoplasm of differentiated cells, which suggests a differentiation potential. When exposed to leukemia inhibitory factor (LIF), ESD-EpiSCs could convert to naïve-like cells. We further analyzed the ultrastructure of converted EpiSCs (cESCs). As compared to ESD-EpiSCs, cESCs showed similar ultrastructural characteristics as naïve ESCs. These findings suggest that ultrastructure could be used to evaluate the pluripotency of ESCs.
Asunto(s)
Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/ultraestructura , Amnios/citología , Animales , Diferenciación Celular , Células Cultivadas , Células Nutrientes , Estratos Germinativos/citología , Estratos Germinativos/ultraestructura , Humanos , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/ultraestructuraRESUMEN
Functional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase) footprinting to probe the functions of two chromatin-remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs), we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene-regulatory factors.