RESUMEN
Mesenchymal stromal cells (MSCs) show immunosuppressive effects both via cell-to-cell contact (direct) with immune cells and by producing paracrine factors and extracellular vesicles (indirect). A key challenge in delivering this therapeutic effect in vivo is retaining the MSCs at the site of injection. One way to address this is by encapsulating the MSCs within suitable biomaterial scaffolds. Here, we assess the immunosuppressive effect of alginate-encapsulated murine MSCs on proliferating murine splenocytes. Our results show that MSCs are able to significantly suppress splenocyte proliferation by â¼50% via the indirect mechanism and almost completely (â¼98%) via the direct mechanism. We also show for the first time that MSCs as monolayers on tissue culture plastic or encapsulated within alginate, when physically isolated from the splenocytes via transwells, are able to sustain immunosuppressive activity with repeated exposure to fresh splenocytes, for as long as 9 days. These results indicate the need to identify design strategies to simultaneously deliver both modes of MSC immunosuppression. By designing cell-biomaterial constructs with tailored degradation profiles, we can achieve a more sustained (avoiding MSCs migration and apoptosis) and controlled release of both the paracrine signals and eventually the cells themselves enabling efficient MSC-based immunosuppressive therapies for wound healing.
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Alginatos , Células Madre Mesenquimatosas , Alginatos/farmacología , Animales , Materiales Biocompatibles , Inmunosupresores , Ratones , Bazo , Cicatrización de HeridasRESUMEN
BACKGROUND: Airway smooth muscle (ASM) cells are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyperresponsiveness and airway remodelling. The extracellular matrix (ECM) can influence tissue remodelling pathways; however, to date no study has investigated the effect of ASM ECM stiffness and cross-linking on the development of asthmatic airway remodelling. We hypothesised that transforming growth factor-ß (TGF-ß) activation by ASM cells is influenced by ECM in asthma and sought to investigate the mechanisms involved. METHODS: This study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGF-ß activation and expression of ECM cross-linking enzymes. Human bronchial biopsies from asthmatic and nonasthmatic donors were used to confirm lysyl oxidase like 2 (LOXL2) expression in ASM. A chronic ovalbumin (OVA) model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling. RESULTS: We found that asthmatic ASM cells activated more TGF-ß basally than nonasthmatic controls and that diseased cell-derived ECM influences levels of TGF-ß activated. Our data demonstrate that the ECM cross-linking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGF-ß activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an OVA mouse model of asthma. CONCLUSION: These data are the first to highlight a role for LOXL2 in the development of asthmatic airway remodelling and suggest that LOXL2 inhibition warrants further investigation as a potential therapy to reduce remodelling of the airways in severe asthma.
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Remodelación de las Vías Aéreas (Respiratorias) , Aminoácido Oxidorreductasas/metabolismo , Asma , Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Animales , Asma/metabolismo , Ratones , Músculo Liso/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Proteína-Lisina 6-Oxidasa/farmacología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses. Graphical abstract.
RESUMEN
Hydrogels have been used extensively in bioengineering as artificial cell culture supports. Investigation of the interrelationship between cellular response to the hydrogel and its chemistry ideally requires methods that allow characterization without labels and can map species in three-dimensional to follow biomolecules adsorbed to, and absorbed into, the open structure before and during culture. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has the potential to be utilized for through thickness characterization of hydrogels. The authors have established a simple sample preparation procedure to successfully achieve analysis of frozen hydrated hydrogels using ToF-SIMS without the need for dry glove box entry equipment. They demonstrate this on a poly(2-hydroxyethyl methacrylate) (pHEMA) film where a model protein (lysozyme) is incorporated using two methods to demonstrate how protein distribution can be determined. A comparison of lysozyme incorporation is made between the situation where the protein is present in a polymer dip coating solution and where lysozyme is in an aqueous medium in which the film is incubated. It is shown that protonated water clusters H(H2O)n (+) where n = 5-11 that are indicative of ice are detected through the entire thickness of the pHEMA. The lysozyme distribution through the pHEMA hydrogel films can be determined using the intensity of a characteristic amino acid secondary ion fragment.
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Congelación , Hidrogeles/química , Proteínas/análisis , Espectrometría de Masa de Ion Secundario/métodos , Argón , Rayos Láser , Muramidasa/análisisRESUMEN
The accurate study of cellular microenvironments is limited by the lack of technologies that can manipulate cells in 3D at a sufficiently small length scale. The ability to build and manipulate multicellular microscopic structures will facilitate a more detailed understanding of cellular function in fields such as developmental and stem cell biology. We present a holographic optical tweezers based technology to accurately generate bespoke cellular micro-architectures. Using embryonic stem cells, 3D structures of varying geometries were created and stabilized using hydrogels and cell-cell adhesion methods. Control of chemical microenvironments was achieved by the temporal release of specific factors from polymer microparticles positioned within these constructs. Complex co-culture micro-environmental analogues were also generated to reproduce structures found within adult stem cell niches. The application of holographic optical tweezers-based micromanipulation will enable novel insights into biological microenvironments by allowing researchers to form complex architectures with sub-micron precision of cells, matrices and molecules.
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Pinzas Ópticas , Células Madre Adultas/fisiología , Animales , Apoptosis , Agregación Celular , Microambiente Celular , Técnicas de Cocultivo , Medios de Cultivo/química , Células Madre Embrionarias/fisiología , Holografía , Humanos , Hidrogeles/química , Células Madre Mesenquimatosas/fisiología , Ratones , Micromanipulación/métodos , PolímerosRESUMEN
Cell-secreted matrices (CSMs), where extracellular matrix (ECM) deposited by monolayer cell cultures is decellularized, have been increasingly used to produce surfaces that may be reseeded with cells. Such surfaces are useful to help us understand cell-ECM interactions in a microenvironment closer to the in vivo situation than synthetic substrates with adsorbed proteins. We describe the production of CSMs from mouse primary osteoblasts (mPObs) exposed to cytokine challenge during matrix secretion, mimicking in vivo inflammatory environments. Time-of-flight secondary ion mass spectrometry data revealed that CSMs with cytokine challenge at day 7 or 12 of culture can be chemically distinguished from one another and from untreated CSM using multivariate analysis. Comparison of the differences with reference spectra from adsorbed protein mixtures points towards cytokine challenge resulting in a decrease in collagen content. This is supported by immunocytochemical and histological staining, demonstrating a 44% loss of collagen mass and a 32% loss in collagen I coverage. CSM surfaces demonstrate greater cell adhesion than adsorbed ECM proteins. When mPObs were reseeded onto cytokine-challenged CSMs they exhibited reduced adhesion and elongated morphology compared to untreated CSMs. Such changes may direct subsequent cell fate and function, and provide insights into pathological responses at sites of inflammation.
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Citocinas/farmacología , Matriz Extracelular/metabolismo , Espectrometría de Masa de Ion Secundario/métodos , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Matriz Extracelular/efectos de los fármacos , Humanos , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Análisis de Componente Principal , RatasRESUMEN
Nonunion fractures and large bone defects are significant targets for osteochondral tissue engineering strategies. A major hurdle in the use of these therapies is the foreign body response of the host. Herein, we report the development of a bone tissue engineering scaffold with the ability to release anti-inflammatory drugs, in the hope of evading this response. Porous, sintered scaffolds composed of poly(D,L-lactic acid-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) were prepared with and without the anti-inflammatory drug diclofenac sodium. Analysis of drug release over time demonstrated a profile suitable for the treatment of acute inflammation with â¼80% of drug released over the first 4 days and a subsequent release of around 0.2% per day. Effect of drug release was monitored using an in vitro osteoblast inflammation model, comprised of mouse primary calvarial osteoblasts stimulated with proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Levels of inflammation were monitored by cell viability and cellular production of nitric oxide (NO) and prostaglandin E2 (PGE2). The osteoblast inflammation model revealed that proinflammatory cytokine addition to the medium reduced cell viability to 33%, but the release of diclofenac sodium from scaffolds inhibited this effect with a final cell viability of â¼70%. However, releasing diclofenac sodium at high concentrations had a toxic effect on the cells. Proinflammatory cytokine addition led to increased NO and PGE2 production; diclofenac-sodium-releasing scaffolds inhibited NO release by â¼64% and PGE2 production by â¼52%, when the scaffold was loaded with the optimal concentration of drug. These observations demonstrate the potential use of PLGA/PEG scaffolds for localized delivery of anti-inflammatory drugs in bone tissue engineering applications.
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Diclofenaco/uso terapéutico , Sistemas de Liberación de Medicamentos , Inflamación/tratamiento farmacológico , Osteoblastos/patología , Polietilenglicoles/química , Poliglactina 910/química , Andamios del Tejido/química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diclofenaco/administración & dosificación , Diclofenaco/farmacología , Dinoprostona/biosíntesis , Humanos , Inflamación/patología , Interferón gamma/farmacología , Interleucina-1beta , Ratones , Modelos Biológicos , Óxido Nítrico/biosíntesis , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Cráneo/patología , Factor de Necrosis Tumoral alfaRESUMEN
There are well-established approaches for osteogenic differentiation of embryonic stem cells (ESCs), but few show direct comparison with primary osteoblasts or demonstrate differences in response to external factors. Here, we show comparative analysis of in vitro osteogenic differentiation of mouse ESC (osteo-mESC) and mouse primary osteoblasts. Both cell types formed mineralized bone nodules and produced osteogenic extracellular matrix, based on immunostaining for osteopontin and osteocalcin. However, there were marked differences in the morphology of osteo-mESCs and levels of mRNA expression for osteogenic genes. In response to the addition of proinflammatory cytokines interleukin-1ß, tumor necrosis factor-α, and interferon-γ to the culture medium, primary osteoblasts showed increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) at early time points and decreases in cell viability. In contrast, osteo-mESCs maintained viability and did not produce NO and PGE2 until day 21. The formation of bone nodules by primary osteoblasts was reduced markedly after cytokine stimulation but was unaffected in osteo-mESCs. Cell sorting of osteo-mESCs by cadherin-11 (cad-11) showed clear osteogenesis of cad-11(+) cells compared to unsorted osteo-mESCs and cad-11(-) cells. Moreover, the cad-11(+) cells showed a significant response to cytokines, similar to primary osteoblasts. Overall, these results show that while osteo-mESC cultures, without specific cell sorting, show characteristics of osteoblasts, there are also marked differences, notably in their responses to cytokine stimuli. These findings are relevant to understanding the differentiation of stem cells and especially developing in vitro models of disease, testing new drugs, and developing cell therapies.
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Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Interferón gamma/metabolismo , Interleucina-1beta/metabolismo , Osteogénesis/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/citología , Ratones , Osteoblastos/citologíaRESUMEN
Embryoid bodies (EBs) generated from embryonic stem cells are used to study processes of differentiation within a three dimensional (3D) cell environment. In many instances however, EBs are dispersed to single cell suspensions with a subsequent monolayer culture. Moreover, where the 3D integrity of an EB is maintained, cytokines or drugs of interest to stimulate differentiation are often added directly to the culture medium at fixed concentrations and effects are usually limited to the outer layers of the EB. The aim of this study was to create an EB model with localised drug and or growth factor delivery directly within the EB. Using poly(DL-lactic acid-co-glycolic acid) microparticles (MPs) with an average diameter of 13µm, we have demonstrated controllable incorporation of defined numbers of MPs within human ES cell derived EBs, down to 1 MP per EB. This was achieved by coating MPs with human ES cell lysate and centrifugation of specific ratios of ES cells and MPs to form 3D aggregates. Using MPs loaded with simvastatin (pro or active drug) or BMP-2, we have demonstrated osteogenic differentiation within the 3D aggregates, maintained in culture for up to 21days, and quantified by real time QPCR for osteocalcin. Immunostaining for RUNX2 and osteocalcin, and also histochemical staining with picrosirius red to demonstrate collage type 1 and Alizarin red to demonstrate calcium/mineralisation further demonstrated osteogenic differentiation and revealed regional staining associated with the locations of MPs within the aggregates. We also demonstrated endothelial differentiation within human ES cell-derived aggregates using VEGF loaded MPs. In conclusion, we demonstrate an effective and reliable approach for engineering stem aggregates with definable number of MPs within the 3D cellular structure. We also achieved localised osteogenic and endothelial differentiation associated with MPs releasing encapsulated drug molecules or cytokines directly within the cell aggregate. This provides a powerful tool for controlling and investigating differentiation within 3D cell cultures and has applications to drug delivery, drug discovery, stem cell biology, tissue engineering and regenerative medicine.
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Proteína Morfogenética Ósea 2/administración & dosificación , Portadores de Fármacos/química , Células Madre Embrionarias/metabolismo , Ácido Láctico/química , Ácido Poliglicólico/química , Simvastatina/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Proteína Morfogenética Ósea 2/química , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Portadores de Fármacos/administración & dosificación , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Humanos , Osteocalcina/genética , Osteocalcina/metabolismo , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Simvastatina/química , Factor A de Crecimiento Endotelial Vascular/químicaRESUMEN
The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2 in mouse embryonic stem cells were monitored over a 48 hour period after exposure to the growth factors BMP2 and TGFß1. Candidate GRNs of early osteogenesis were constructed based on published experimental findings and simulation results of Boolean and ordinary differential equation models were compared with our experimental data in order to test the validity of these models. Three gene regulatory networks were found to be consistent with the data, one of these networks exhibited sustained oscillation, a behaviour which is consistent with the general view of embryonic stem cell plasticity. The work cycle presented in this paper illustrates how mathematical modelling can be used to elucidate from gene expression profiles GRNs that are consistent with experimental data.
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Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Osteogénesis/genética , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 2/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Células Madre Embrionarias/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Homeodominio/genética , Ratones , Modelos Biológicos , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , ARN/genética , ARN/metabolismo , Biología de Sistemas , Transcriptoma , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
OBJECTIVES: The purpose of this study was to identify a cell source, scaffold substrate and culture environment suitable for use in engineering an in-vitro model of rodent cartilage. METHODS: The chondrogenic activity and stability of cells isolated at Day 18 of gestation was assessed under normoxia and hypoxia using a cytokine stimulation assay and gene expression analysis. The ability of the selected cells seeded in fibrous electrospun scaffolds to form cartilaginous tissue during longterm static and dynamic culture was assessed using immunocytochemistry and biochemical analysis. KEY FINDINGS: Rodent fetal chondrocytes appear to have enhanced phenotypic stability compared with other cell sources. Following 16 weeks under static culture, the engineered constructs were found to have greater cellularity and collagen content that native rodent cartilage. CONCLUSIONS: A cell source, scaffold and culture environment have been identified that support the generation of in-vitro rodent cartilage. In future work, cytokine treatment of the engineered tissues will take place to generate in-vitro osteoarthritis models.
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Cartílago/citología , Condrocitos/citología , Colágeno/metabolismo , Ratas/embriología , Ingeniería de Tejidos/métodos , Animales , Cartílago/metabolismo , Bovinos , Células Cultivadas , Condrocitos/metabolismo , Citocinas/farmacología , Investigación Fetal , Expresión Génica , Edad Gestacional , Hipoxia , Modelos Biológicos , Oxígeno/farmacología , Fenotipo , Ratas Wistar , Valores de ReferenciaRESUMEN
The development of an anterior-posterior (AP) polarity is a crucial process that in the mouse has been very difficult to analyse, because it takes place as the embryo implants within the mother. To overcome this obstacle, we have established an in-vitro culture system that allows us to follow the step-wise development of anterior visceral endoderm (AVE), critical for establishing AP polarity. Here we use this system to show that the AVE originates in the implanting blastocyst, but that additional cells subsequently acquire AVE characteristics. These 'older' and 'younger' AVE domains coalesce as the egg cylinder emerges from the blastocyst structure. Importantly, we show that AVE migration is led by cells expressing the highest levels of AVE marker, highlighting that asymmetry within the AVE domain dictates the direction of its migration. Ablation of such leading cells prevents AVE migration, suggesting that these cells are important for correct establishment of the AP axis.
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Tipificación del Cuerpo , Biología Evolutiva/métodos , Endodermo/metabolismo , Animales , Blastocisto/citología , Movimiento Celular , Técnicas de Cultivo de Embriones , Femenino , Marcadores Genéticos/genética , Proteínas Fluorescentes Verdes/metabolismo , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Modelos Genéticos , Factores de TiempoRESUMEN
BACKGROUND: To investigate how patterns of cell differentiation are related to underlying intra- and inter-cellular signalling pathways, we use a stochastic individual-based model to simulate pattern formation when stem cells and their progeny are cultured as a monolayer. We assume that the fate of an individual cell is regulated by the signals it receives from neighbouring cells via either diffusive or juxtacrine signalling. We analyse simulated patterns using two different spatial statistical measures that are suited to planar multicellular systems: pair correlation functions (PCFs) and quadrat histograms (QHs). RESULTS: With a diffusive signalling mechanism, pattern size (revealed by PCFs) is determined by both morphogen decay rate and a sensitivity parameter that determines the degree to which morphogen biases differentiation; high sensitivity and slow decay give rise to large-scale patterns. In contrast, with juxtacrine signalling, high sensitivity produces well-defined patterns over shorter lengthscales. QHs are simpler to compute than PCFs and allow us to distinguish between random differentiation at low sensitivities and patterned states generated at higher sensitivities. CONCLUSIONS: PCFs and QHs together provide an effective means of characterising emergent patterns of differentiation in planar multicellular aggregates.
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Diferenciación Celular , Células Madre/citología , Animales , Técnicas de Cultivo de Célula , Linaje de la Célula , Ratones , Modelos Biológicos , Transducción de Señal , Células Madre/metabolismoRESUMEN
Hydrogen silsesquioxane (HSQ) is a material with the potential for studying the effect of surface stiffness on stem cell differentiation. Here, the effects of electron beam dose on the topography and the mechanical properties of HSQ obtained with or without trimethylamine (TMA) development are characterised by atomic force microscopy imaging and indentation. A correlation between the surface stiffness (uniform across the sample) and electron beam exposure is observed. Surface roughness of HSQ samples developed in TMA decreases exponentially with increasing electron beam exposure. Surface coating with plasma polymerised allylamine (ppAAm) leads to an overall decrease in stiffness values. However, the increase in surface stiffness with increasing electron beam exposure is still evident. The ppAAm coating is shown to facilitate human mesenchymal stem cell adhesion.
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Raman microspectroscopy (RMS) was used to detect and image molecular markers specific to cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs). This technique is noninvasive and thus can be used to discriminate individual live CMs within highly heterogeneous cell populations. Principal component analysis (PCA) of the Raman spectra was used to build a classification model for identification of individual CMs. Retrospective immunostaining imaging was used as the gold standard for phenotypic identification of each cell. We were able to discriminate CMs from other phenotypes with >97% specificity and >96% sensitivity, as calculated with the use of cross-validation algorithms (target 100% specificity). A comparison between Raman spectral images corresponding to selected Raman bands identified by the PCA model and immunostaining of the same cells allowed assignment of the Raman spectral markers. We conclude that glycogen is responsible for the discrimination of CMs, whereas myofibril proteins have a lesser contribution. This study demonstrates the potential of RMS for allowing the noninvasive phenotypic identification of hESC progeny. With further development, such label-free optical techniques may enable the separation of high-purity cell populations with mature phenotypes, and provide repeated measurements to monitor time-dependent molecular changes in live hESCs during differentiation in vitro.
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Células Madre Embrionarias/citología , Imagen Molecular/métodos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Animales , Biomarcadores/metabolismo , Supervivencia Celular , Humanos , Rayos Láser , Ratones , Fenotipo , Análisis de Componente Principal , Espectrometría RamanRESUMEN
Osteoblasts are the cells that contribute to the formation and function of bone tissue. Knowledge of their biology is important to understanding of the normal processes of bone repair, the development of diseases affecting bone tissue, and to the investigation of approaches to improve bone repair and to treat or prevent bone diseases. Osteoblasts can be readily isolated from bone tissues and grown in culture, and under relatively simple culture conditions, they will recapitulate many aspects of their normal biology. These culture conditions can be also applied to adult stem cells, such as mesenchymal/bone marrow stromal stem cells. More recently, these studies have been extended to include embryonic stem cells. This chapter provides detailed step-by-step protocols to investigate the differentiation of embryonic stem cells into osteoblasts. Several 2D and 3D culture methods are presented and enable comparisons to be made on the efficiency and mechanisms of osteogenic differentiation. Emphasis is also placed on methods to analyse and confirm osteogenic differentiation.
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Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Embrionarias/citología , Osteogénesis , Alginatos/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Bioensayo , Cadherinas/metabolismo , Agregación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Cuerpos Embrioides/citología , Cuerpos Embrioides/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/enzimología , Células Madre Embrionarias/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/farmacología , Humanos , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Minerales/metabolismo , Osteogénesis/efectos de los fármacos , Poliésteres/farmacología , Coloración y Etiquetado , Andamios del Tejido/química , Microtomografía por Rayos XRESUMEN
The unlimited proliferative ability and plasticity to generate other cell types ensures that stem cells represent a dynamic system apposite for the identification of new molecular targets and the production and development of novel drugs. These cell lines derived from embryos could be used as a model for the study of basic and applied aspects in medical therapeutics, environmental mutagenesis and disease management. As a consequence, these can be tested for safety or to predict or anticipate potential toxicity in humans. Human ES cell lines may, therefore, prove clinically relevant to the development of safer and more effective drugs for patients presenting with diabetes mellitus.
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Diabetes Mellitus/terapia , Células Madre Embrionarias/citología , Células Madre Adultas/citología , Animales , Diferenciación Celular , Línea Celular , Humanos , RatonesRESUMEN
Pluripotent embryonic stem (ES) cells hold great promise for the field of tissue engineering, with numerous studies investigating differentiation into various cell types including cardiomyocytes, chondrocytes, and osteoblasts. Previous studies have detailed osteogenic differentiation via dissociated embryoid body (EB) culture in osteoinductive media comprising of ascorbic acid, beta-glycerophosphate, and dexamethasone. It is hoped that these osteogenic cultures will have clinical application in bone tissue repair and regeneration and pharmacological testing. However, differentiation remains highly inefficient and generates heterogeneous populations. We have previously reported an engineered three-dimensional culture system for controlled ES cell-ES cell interaction via the avidin-biotin binding complex. Here we investigate the effect of such engineering on ES cell differentiation. Engineered EBs exhibit enhanced osteogenic differentiation assessed by cadherin-11, Runx2, and osteopontin expression, alkaline phosphatase activity, and bone nodule formation. Results show that cultures produced from intact EBs aggregated for 3 days generated the greatest levels of osteogenic differentiation when cultured in osteoinductive media. However, when cultured in control media, only engineered samples appeared to exhibit bone nodule formation. In addition, polymerase chain reaction analysis revealed a decrease in endoderm and ectoderm expression within engineered samples. This suggests that engineered ES cell aggregation has increased mesoderm homogeneity, contributing to enhanced osteogenic differentiation.
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Diferenciación Celular , Células Madre Embrionarias/citología , Osteogénesis , Ingeniería de Tejidos/métodos , Animales , Agregación Celular , Línea Celular , Linaje de la Célula , Forma de la Célula , Embrión de Mamíferos/citología , Embrión de Mamíferos/ultraestructura , Células Madre Embrionarias/ultraestructura , Estratos Germinativos/citología , Ratones , Factores de TiempoRESUMEN
Realizing the potential clinical and industrial applications of human embryonic stem cells (hESCs) is limited by the need for costly, labile, or undefined growth substrates. Here we demonstrate that trypsin passaging of the hESC lines, HUES7 and NOTT1, on oxygen plasma etched tissue culture polystyrene (PE-TCPS) in conditioned medium is compatible with pluripotency. This synthetic culture surface is stable at room temperature for at least a year and is readily prepared by placing polystyrene substrates in a radio frequency oxygen plasma generator for 5 min. Modification of the polystyrene surface chemistry by plasma etching was confirmed by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), which identified elemental and molecular changes as a result of the treatment. Pluripotency of hESCs cultured on PE-TCPS was gauged by consistent proliferation during serial passage, expression of stem cell markers (OCT4, TRA1-60, and SSEA-4), stable karyotype and multi-germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Generation of cost-effective, easy-to-handle synthetic, defined, stable surfaces for hESC culture will expedite stem cell use in biomedical applications.
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Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , Medios de Cultivo Condicionados/metabolismo , Células Madre Embrionarias/química , Células Madre Embrionarias/metabolismo , Humanos , Células Madre Pluripotentes/química , Células Madre Pluripotentes/metabolismo , Propiedades de SuperficieRESUMEN
This work investigates an application of the alginate encapsulation technology to the differentiation of embryonic stem (ES) cells into insulin-producing cells. It shows that the ES cells can efficiently be encapsulated within the alginate beads, retaining a high level of cell viability. The alginate encapsulation achieves approximately 10-fold increase in the cell density in the culture, in comparison to the two-dimensional conditions, opening a potential benefit of the technology in large-scale cell culture applications. Manipulations of encapsulation conditions, particularly of the initial alginate concentration, allow the control over both the diffusion of molecules into the alginate matrix (e.g. differentiation factors) as well as control over the matrix porosity/flexibility to permit the proliferation and growth of encapsulated ES aggregates within the bead. Post-differentiation analysis confirms the presence of insulin-positive cells, as judged from immunostaining, insulin ELISA and RT-PCR analysis. The functionality of the encapsulated and differentiated cells was confirmed by their insulin production capability, whereby on glucose challenge the insulin production by the cells differentiated within alginate beads was found to be statistically significantly higher than for the cells from conventional two-dimensional differentiation system.