RESUMEN
Interactions between EphB4 receptor tyrosine kinases and their membrane-bound ephrin-B2 ligands on apposed cells play a regulatory role in neural stem cell differentiation. With both receptor and ligand constrained to move within the membranes of their respective cells, this signaling system inevitably experiences spatial confinement and mechanical forces in conjunction with receptor-ligand binding. In this study, we reconstitute the EphB4-ephrin-B2 juxtacrine signaling geometry using a supported-lipid-bilayer system presenting laterally mobile and monomeric ephrin-B2 ligands to live neural stem cells. This experimental platform successfully reconstitutes EphB4-ephrin-B2 binding, lateral clustering, downstream signaling activation, and neuronal differentiation, all in a configuration that preserves the spatiomechanical aspects of the natural juxtacrine signaling geometry. Additionally, the supported bilayer system allows control of lateral movement and clustering of the receptor-ligand complexes through patterns of physical barriers to lateral diffusion fabricated onto the underlying substrate. The results from this study reveal a distinct spatiomechanical effect on the ability of EphB4-ephrin-B2 signaling to induce neuronal differentiation. These observations parallel similar studies of the EphA2-ephrin-A1 system in a very different biological context, suggesting that such spatiomechanical regulation may be a common feature of Eph-ephrin signaling.
Asunto(s)
Diferenciación Celular , Efrina-B2/metabolismo , Fenómenos Mecánicos , Células-Madre Neurales/citología , Receptor EphB4/metabolismo , Transducción de Señal , Animales , Fenómenos Biomecánicos , Membrana Celular/metabolismo , RatonesRESUMEN
To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct â¼10(3) concurrent single-cell western blots (scWesterns) in â¼4 h. A microscope slide supporting a 30-µm-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (â¼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.
Asunto(s)
Western Blotting/métodos , Análisis de la Célula Individual/métodos , Animales , Diferenciación Celular , Proteínas Fluorescentes Verdes/biosíntesis , Células-Madre Neurales/fisiología , RatasRESUMEN
The activation of cellular signaling cascades, critical for regulating cell function and fate, often involves changes in the organization of receptors in the cell membrane. Using synthetic multivalent ligands to control the nanoscale organization of cellular receptors into clusters is an attractive approach to elicit desired downstream cellular responses, since multivalent ligands can be significantly more potent than their corresponding monovalent ligands. Synthetic multivalent ligands can serve as both versatile biological tools and potent nanoscale therapeutics, for example in applications to harness them to control stem cell fate in vitro and in vivo. Here we describe the use of recombinant protein expression and bioconjugate chemistry to synthesize multivalent ligands that have the potential to regulate cell signaling in a variety of cell types.
Asunto(s)
Ácido Hialurónico/metabolismo , Polímeros/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Animales , Pollos , Ligandos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology. Cellular signal transduction of growth factor and morphogen cues (which have critical roles in regulating cell function and fate) often begins with such multivalent binding of ligands, either secreted or cell-surface-tethered to target cell receptors, leading to receptor clustering. Cellular mechanisms that orchestrate ligand-receptor oligomerization are complex, however, so the capacity to control multivalent interactions and thereby modulate key signalling events within living systems is currently very limited. Here, we demonstrate the design of potent multivalent conjugates that can organize stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induce signalling in neural stem cells and promote their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organization of the receptor-ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhance human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates are therefore powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Efrina-B2/farmacología , Nanoconjugados/química , Células-Madre Neurales/citología , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Células Cultivadas , Humanos , Ligandos , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Receptores de la Familia Eph/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
One of the greatest challenges in cell therapy is to minimally invasively deliver a large quantity of viable cells to a tissue of interest with high engraftment efficiency. Low and inefficient homing of systemically delivered mesenchymal stem cells (MSCs), for example, is thought to be a major limitation of existing MSC-based therapeutic approaches, caused predominantly by inadequate expression of cell surface adhesion receptors. Using a platform approach that preserves the MSC phenotype and does not require genetic manipulation, we modified the surface of MSCs with a nanometer-scale polymer construct containing sialyl Lewis(x) (sLe(x)) that is found on the surface of leukocytes and mediates cell rolling within inflamed tissue. The sLe(x) engineered MSCs exhibited a robust rolling response on inflamed endothelium in vivo and homed to inflamed tissue with higher efficiency compared with native MSCs. The modular approach described herein offers a simple method to potentially target any cell type to specific tissues via the circulation.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Oligosacáridos/química , Animales , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Dinoprostona/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Células HL-60 , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Integrina beta1/metabolismo , Células Madre Mesenquimatosas/química , Ratones , Ratones Endogámicos BALB C , Selectinas/metabolismo , Antígeno Sialil Lewis X , Antígenos Thy-1/metabolismo , Trasplante HeterólogoRESUMEN
Embyroid body (EB) formation is a key step in many embryonic stem cell (ESC) differentiation protocols. The EB mimics the structure of the developing embryo, thereby providing a means of obtaining any cell lineage. Traditionally, the two methods of EB formation are suspension and hanging drop. The suspension method allows ESCs to self-aggregate into EBs in a nonadherent dish. The hanging drop method suspends ESCs on the lid of a dish and EBs form through aggregation at the bottom of the drops. Recently, alternative methods of EB formation have been developed that allow for highly accurate control of EB size and shape, resulting in reproducibly produced homogeneous EBs. This control is potentially useful for directed differentiation, as recent studies have shown that EB size may be a useful determinant of the resulting differentiated cell types. One particular approach to generate homogeneous EBs utilizes nonadhesive microwell structures. The methodology associated with this technique, along with the traditional approaches of suspension and hanging drop, is the focus of this chapter.