Characterization of Tunneling Nanotubes in Wharton's jelly Mesenchymal Stem Cells. An Intercellular Exchange of Components between Neighboring Cells.

Intercellular communication is one of the most important events in cell population behavior. In the last decade, tunneling nanotubes (TNTs) have been recognized as a new form of long distance intercellular connection. TNT function is to allow molecular and subcellular structure exchange between neighboring cells via the transfer of molecules and organelles such as calcium ions, prions, viral and bacterial pathogens, small lysosomes and mitochondria. New findings support the concept that mesenchymal stem cells (MSCs) can affect cell microenvironment by the release of soluble factors or the transfer of cellular components to neighboring cells, in a way which significantly contributes to cell regulation and tissue repair, although the underlying mechanisms remain poorly understood. MSCs have many advantages for their implementation in regenerative medicine. The TNTs in these cell types are heterogeneous in both structure and function, probably due to their highly dynamic behavior. In this work we report an extensive and detailed description of types, structure, components, dynamics and functionality of the TNTs bridging neighboring human umbilical cord MSCs obtained from Wharton"s jelly. Characterization studies were carried out through phase contrast, fluorescence, electron microscopy and time lapse images with the aim of describing cells suitable for an eventual regenerative medicine.

uPA-uPAR molecular complex is involved in cell signaling during neuronal migration and neuritogenesis.

BACKGROUND: In the development of the central nervous system (CNS), neuronal migration and neuritogenesis are crucial processes for establishing functional neural circuits. This relies on the regulation exerted by several signaling molecules, which play important roles in axonal growth and guidance. The urokinase-type plasminogen activator (uPA)-in association with its receptor-triggers extracellular matrix proteolysis and other cellular processes through the activation of intracellular signaling pathways. Even though the uPA-uPAR complex is well characterized in nonneuronal systems, little is known about its signaling role during CNS development. RESULTS: In response to uPA, neuronal migration and neuritogenesis are promoted in a dose-dependent manner. After stimulation, uPAR interacts with α5- and ß1-integrin subunits, which may constitute an αß-heterodimer that acts as a uPA-uPAR coreceptor favoring the activation of multiple kinases. This interaction may be responsible for the uPA-promoted phosphorylation of focal adhesion kinase (FAK) and its relocation toward growth cones, triggering cytoskeletal reorganization which, in turn, induces morphological changes related to neuronal migration and neuritogenesis. CONCLUSIONS: uPA has a key role during CNS development. In association with its receptor, it orchestrates both proteolytic and nonproteolytic events that govern the proper formation of neural networks.

Anaglyph of retinal stem cells and developing axons: selective volume enhancement in microscopy images.

Retinal stem cell culture has become a powerful research tool, but it requires reliable methods to obtain high-quality images of living and fixed cells. This study describes a procedure for using phase contrast microscopy to obtain three-dimensional (3-D) images for the study of living cells by photographing a living cell in a culture dish from bottom to top, as well as a procedure to increase the quality of scanning electron micrographs and laser confocal images. The procedure may also be used to photograph clusters of neural stem cells, and retinal explants with vigorous axonal growth. In the case of scanning electron microscopy and laser confocal images, a Gaussian procedure is applied to the original images. The methodology allows for the creation of anaglyphs and video reconstructions, and provides high-quality images for characterizing living cells or tissues, fixed cells or tissues, or organs observed with scanning electron and laser confocal microscopy. Its greatest advantage is that it is easy to obtain good results without expensive equipment. The procedure is fast, precise, simple, and offers a strategic tool for obtaining 3-D reconstructions of cells and axons suitable for easily determining the orientation and polarity of a specimen. It also enables video reconstructions to be created, even of specimens parallel to the plastic base of a tissue culture dish, It is also helpful for studying the distribution and organization of living cells in a culture, as it provides the same powerful information as optical tomography, which most confocal microscopes cannot do on sterile living cells.

EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

Optic tectum morphogenesis: a step-by-step model based on the temporal-spatial organization of the cell proliferation. Significance of deterministic and stochastic components subsumed in the spatial organization.

BACKGROUND: Cell proliferation plays an important morphogenetic role. This work analyzes the temporal-spatial organization of cell proliferation as an attempt to understand its contribution to the chick optic tectum (OT) morphogenesis. RESULTS: A morphogenetic model based on space-dependent differences in cell proliferation is presented. Step1: a medial zone of high mitotic density (mZHMD) appears at the caudal zone. Step2: the mZHMD expands cephalically forming the dorsal curvature and then duplicates into two bilateral ZHMDs (bZHMD). Step3: the bZHMDs move toward the central region of each hemitectum. Step4: the planar expansion of both bZHMD and a relative decrement in the dorsal midline growth produces a dorsal medial groove separating the tectal hemispheres. Step5: a relative caudal displacement of the bZHMDs produces the OT caudal curvature. Numerical sequences derived from records of mitotic cells spatial coordinates, analyzed as stochastic point processes, show that they correspond to 1/f((ß)) processes. The spatial organization subsumes deterministic and stochastic components. CONCLUSIONS: The deterministic component describes the presence of a long-range influence that installs an asymmetric distribution of cell proliferation, i.e., an asymmetrically located ZHMD that print space-dependent differences onto the tectal corticogenesis. The stochastic component reveals short-range anti-correlations reflecting spatial clusterization and synchronization between neighboring cells.