The study of neuronal morphology and neurite outgrowth has been enhanced by the combination of imaging informatics and high content screening, in which thousands of images are acquired using robotic fluorescent microscopy. To understand the process of neurite outgrowth in the context of neuroregeneration, we used mouse neuroblastoma N1E115 as our model neuronal cell. Six-thousand cellular images of four different culture conditions were acquired with two-channel widefield fluorescent microscopy. We developed a software package called NeuronCyto. It is a fully automatic solution for neurite length measurement and complexity analysis. A novel approach based on topological analysis is presented to segment cells. The detected nuclei were used as references to initialize the level set function. Merging and splitting of cells segments were prevented using dynamic watershed lines based on the constraint of topological dependence. A tracing algorithm was developed to automatically trace neurites and measure their lengths quantitatively on a cell-by-cell basis. NeuronCyto analyzes three important biologically relevant features, which are the length, branching complexity, and number of neurites. The application of NeuronCyto on the experiments of Toca-1 and serum starvation show that the transfection of Toca-1 cDNA induces longer neurites with more complexities than serum starvation. ' 2008 International Society for Advancement of Cytometry Key terms image cytometry; cell segmentation; neurite outgrowth; fluorescent microscopy CELLULAR morphogenesis and its relationship with cell functions are significant and critical in many biological studies. Advances in digital microscopy and robotic techniques in cell cultures have enabled thousands of cell images to be acquired through high-content screenings. Manual processing of those images is subjective, labor intensive, and inaccurate. Automatic analysis and extracting quantitative information are challenging, but vital for inferring new biological insights.Neurites, including axons and dendrites, are a unique feature of neurons that allow formation and maintenance of the nervous system. Understanding the process of neurite outgrowth is important for neuroregeneration strategies in the treatment of diseases, such as Alzheimer's disease and Parkinson's. In this article, N1E115 cells are used as our model to follow neurite outgrowth. N1E115 mouse neuroblastoma cells have been used as a model neuronal cell for a number of years. N1E115 cells grow as round cells and upon serum starvation (or cDNA transfection) produce neurites and a neuronal morphology.Transducer of cdc42-dependent actin-1 assembly (Toca-1) is a protein that has three distinct domains; F-BAR, Cdc42 binding site, and SH3 domain. The F-BAR domain can deform membranes while the SH3 domain binds N-WASP, linking to actin polymerization. The domain structure of Toca-1 is very similar to the Insulin Receptor Substrate 53 kDa (IRSp53) and both proteins induce the formation of filopodia (complexity) and neurites in ...
J. Neurochem. (2011) 117, 565–578. Abstract Neural stem cells (NSCs) are self‐renewing multipotent cells that undergo symmetric and asymmetric cell division during development of the nervous system. The behavior of NSCs is tightly regulated by intrinsic processes such as transcriptional and post‐transcriptional control, as well as the stem cell niche factors that activate ligand‐receptor‐mediated signaling pathways. However, the role of these niche factors that regulate NSC behavior is not clearly understood. We identified chondroitin sulfate proteoglycan, apolipoprotein E (ApoE) and cystatin C as factors derived from the mouse neurosphere conditioned medium. Here, we show that ApoE is an autocrine/paracrine factor that regulates NSC survival. Stimulation of NSC survival is mediated by ApoE receptor interaction and the downstream extracellular signal‐regulated kinase/mitogen‐activated protein kinase signaling pathway. In addition, ApoE also enhanced neurosphere formation of mouse embryonic stem cell‐derived NSCs. Finally, in vitro differentiation studies with ApoE knock‐out NSCs suggest a role for ApoE in oligodendrogenesis.
Analyzing cellular morphologies on a cell-by-cell basis is vital for drug discovery, cell biology, and many other biological studies. Interactions between cells in their culture environments cause cells to touch each other in acquired microscopy images. Because of this phenomenon, cell segmentation is a challenging task, especially when the cells are of similar brightness and of highly variable shapes. The concept of topological dependence and the maximum common boundary (MCB) algorithm are presented in our previous work (Yu et al., Cytometry Part A 2009;75A:289-297). However, the MCB algorithm suffers a few shortcomings, such as low computational efficiency and difficulties in generalizing to higher dimensions. To overcome these limitations, we present the evolving generalized Voronoi diagram (EGVD) algorithm. Utilizing image intensity and geometric information, EGVD preserves topological dependence easily in both 2D and 3D images, such that touching cells can be segmented satisfactorily. A systematic comparison with other methods demonstrates that EGVD is accurate and much more efficient. ' 2010 International Society for Advancement of Cytometry Key terms image cytometry; cell segmentation; fluorescence microscopy; generalized Voronoi diagram ANALYZING cellular morphology is crucial in drug discovery, cell and developmental biology. Automated high-content image-based approaches are preeminent tools, which enable thousands of images to be acquired. However, acquiring high quality images is only the first step towards biological discoveries. Image processingcomputer-based interrogation is essential to extract useful data from the images acquired. To extract the quantitative information on a cell-by-cell basis, a critical but challenging task is to segment individual cells. Once cells have been segmented successfully, subsequent analysis including cell counting, morphology, and migration becomes possible.In the images acquired by high-content screening experiments, the cells can be classified into three groups; (i) isolated, (ii) touching, and (iii) overlapping. In monolayer cell cultures, as shown in Figure 1, isolated cells are the cells that are well separated from other cells, touching cells are the cells that adhere to other cells and share some common boundaries, and overlapping cells are the cells that lie on top of each other with no clear boundary. Touching cells form the majority of cells in normal culture conditions. Segmentation of the isolated cells is straightforward as simple thresholding can successfully segment them, while segmentation of the touching cells is much more challenging.Among many existing cell segmentation approaches, active contours represented through the level set is a successful way to segment cells of irregular shapes. The Mumford-Shah model (1) is proposed to segment two-phase piecewise constant images. This model is enhanced by Chan-Vese (2) using the level set concept introduced in (3). However, the level set formulation lacks the ability to constrain
Lineage commitment of human mesenchymal stem cells (hMSCs) could be directed through micro/nanopatterning of the extracellular matrix (ECM) between cells and substrate. Integrin receptors, integrator of the ECM and cell cytoskeleton, function as molecular bridges linking cells to different biophysical cues translated from patterned ECM. Here we report the distinct recruitment of active integrin β1 (ITG-β1) in hMSCs when they were committed toward the cardiomyogenic lineage on a micropatterned surface. In addition, a systematic study of the distribution of ITG-β1 was performed on focal adhesions (FAs) using a direct stochastic optical reconstruction microscopy (dSTORM) technique, a super-resolution imaging technique to establish the relationship between types of integrin expression and its distribution pattern that are associated with cardiomyogenic differentiation of hMSCs. We ascertained that elongated FAs of ITG-β1 expressed in patterned hMSCs were more prominent than FAs expressed in unpatterned hMSCs. However, there was no significant difference observed between the widths of FAs from both experimental groups. It was found in patterned hMSCs that the direction of FA elongation coincides with cell orientation. This phenomenon was however not observed in unpatterned hMSCs. These results showed that the biophysical induction methods like FAs patterning could selectively induce hMSCs lineage commitment via integrin-material interaction.
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