Short-wave infrared fluorescence imaging of near-infrared dyes with robust end-tail emission using a small-animal imaging device.

Commercially available near-infrared (NIR) dyes, including indocyanine green (ICG), display an end-tail of the fluorescence emission spectrum detectable in the short-wave infrared (SWIR) window. Imaging methods based on the second NIR spectral region (1,000-1,700 nm) are gaining interest within the biomedical imaging community due to minimal autofluorescence and scattering, allowing higher spatial resolution and depth sensitivity. Using a SWIR fluorescence imaging device, the properties of ICG vs. heptamethine cyanine dyes with emission >800 nm were evaluated using tissue-simulating phantoms and animal experiments. In this study, we tested the hypothesis that an increased rigidity of the heptamethine chain may increase the SWIR imaging performance due to the bathochromic shift of the emission spectrum. Fluorescence SWIR imaging of capillary plastic tubes filled with dyes was followed by experiments on healthy animals in which a time series of fluorescence hindlimb images were analyzed. Our findings suggest that higher spatial resolution can be achieved even at greater depths (>5 mm) or longer wavelengths (>1,100 nm), in both tissue phantoms and animals, opening the possibility to translate the SWIR prototype toward clinical application.

Biotin oligonucleotide labeling reactions: A method to assess their effectiveness and reproducibility.

The strong molecular interaction between biotin and streptavidin is widely used in the growing field of nucleic acid nanotechnology. Several biotin labeled oligonucleotide tools have been developed for the detection of biological molecules as well as for protein purification. For these reasons, biotinylation can be considered one of the main chemical reactions for nucleic acid labeling. However, despite its widespread application and the presence on the market of many reagents for the conjugation of biotin to oligonucleotides, it is not yet available a cheap, easy and sensitive system able to assess the effectiveness and reproducibility of this reaction. Here, we present an accurate and reliable method to achieve a qualitative and quantitative analysis of oligonucleotide biotinylation. The protocol employs basic laboratory instruments and standard software for molecular biology applications and does not require advanced expertise for its execution. Most importantly, our method is independent from complex kinetic equilibrium parameters and shows a limit of detection more than one order of magnitude lower than the current fluorometric gold standard assay. Therefore, this method could become a standard, inexpensive and routinely used quality test for post-synthesis evaluation of biotin conjugation reactions.

Neural stem cells engrafted in the adult brain fuse with endogenous neurons.

Neural stem cells (NSCs) have become promising tools for basic research and regenerative medicine. Intracerebral transplantation studies have suggested that these cells may be able to adopt neuronal phenotypes typical of their engraftment site and to establish appropriate connections in the recipient circuitries. Here, we examined the in vivo neurogenic competence of well-characterized NSC lines subjected to in vitro priming and subsequent implantation into the adult intact mouse brain. Upon implantation into the hippocampus and, less frequently, in the striatum and in the cerebral cortex, numerous green fluorescent protein (GFP)-tagged cells acquired differentiated features indistinguishable from resident neurons. Upon closer examination, however, we found that this outcome resulted from fusion of donor cells with local neuronal elements generating long-term persistent GFP(+) neuronal hybrids. This fusogenic behavior of NSCs was unexpected and also observed in coculture with E18 hippocampal immature neural cells, but not with microglia or astrocytes. Similar findings were consistently obtained with different NSC lines, mouse recipients, and donor cell-labeling methods. The frequent and cell type-specific fusion of donor NSCs with host neurons highlights a previously underestimated biological property of the nervous tissue that might prove profitable for basic and therapeutically oriented studies.

Neural stem cell systems: diversities and properties after transplantation in animal models of diseases.

Currently available effective treatments of the diseased or damaged central nervous system (CNS) are restricted to a limited pharmacological relief of symptoms or those given to avoid further damage. Therefore the search is on for treatments that can restore function in the CNS. During recent years replacement of damaged neurons by cell transplantation is being enthusiastically explored as a potential treatment for many neurodegenerative diseases, stroke and traumatic brain injury. Several references in both scientific journals and popular newspapers concerning different types of cultured stem cells, potentially exploitable to treat pathological conditions of the brain, raise important questions pertinent to the fundamental and realistic differences between grafts of primary neural cells and the transplantation of in vitro expanded neural stem cells (NSCs). Our aim is to review the available information on the grafting of different NSC types into the adult rodent brain, focusing on critical aspects for the development of clinical therapies to replace damaged neurons.

Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.

The function of the neuronal proteins Shc and huntingtin in stem cells and neurons: pharmacologic exploitation for human brain diseases.

The identification of intracellular molecules and soluble factors that are important for neuronal differentiation and survival are of critical importance for development of therapeutic strategies for brain diseases. First, the activity of these factors/molecules may be enhanced in vivo in the attempt to induce proper neuronal differentiation and integration of the resident stem cells. Second, these factors may be applied ex vivo to increase the recovery of neurons from stem cells. Third, for those intracellular molecules that play crucial roles in neuronal survival, identification of their downstream targets may give us the chance to develop drug screening assays that use these targets for therapeutic purposes. In recent years, it has become evident that intracellular signaling processes are critical mediators of the responses of neural stem cells and neurons to growth factors. Analysis of the mechanisms of signal transduction has led to the striking finding that a handful of conserved signaling pathways appear to be used in different combinations to specify a wide variety of tissues or cells. This review will focus on the mechanisms by which specific molecules control the transition from proliferation to differentiation of neural progenitor cells and the subsequent survival of postmitotic neurons; it also discusses how this knowledge may be exploited to increase the potential efficacy of stem cell replacement in the damaged brain.