A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

BACKGROUND: Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca(2+)-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. METHODOLOGY/PRINCIPAL FINDINGS: In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. CONCLUSIONS/SIGNIFICANCE: Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture

Stem Cells Have Different Needs for REST

REST is a well known repressor of neuronal gene expression. Genome-wide analysis of REST occupancy in different cell types now reveals new and cell-specific roles for REST in embryonic stem cells

Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells

AbstractThe Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, we have engineered a mouse embryonic stem (ES) cell line, in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells

Population-Specific Regulation of Chmp2b by Lbx1 during Onset of Synaptogenesis in Lateral Association Interneurons

Chmp2b is closely related to Vps2, a key component of the yeast protein complex that creates the intralumenal vesicles of multivesicular bodies. Dominant negative mutations in Chmp2b cause autophagosome accumulation and neurodegenerative disease. Loss of Chmp2b causes failure of dendritic spine maturation in cultured neurons. The homeobox gene Lbx1 plays an essential role in specifying postmitotic dorsal interneuron populations during late pattern formation in the neural tube. We have discovered that Chmp2b is one of the most highly regulated cell-autonomous targets of Lbx1 in the embryonic mouse neural tube. Chmp2b was expressed and depended on Lbx1 in only two of the five nascent, Lbx1-expressing, postmitotic, dorsal interneuron populations. It was also expressed in neural tube cell populations that lacked Lbx1 protein. The observed population-specific expression of Chmp2b indicated that only certain population-specific combinations of sequence specific transcription factors allow Chmp2b expression. The cell populations that expressed Chmp2b corresponded, in time and location, to neurons that make the first synapses of the spinal cord. Chmp2b protein was transported into neurites within the motor-and association-neuropils, where the first synapses are known to form between E11.5 and E12.5 in mouse neural tubes. Selective, developmentally-specified gene expression of Chmp2b may therefore be used to endow particular neuronal populations with the ability to mature dendritic spines. Such a mechanism could explain how mammalian embryos reproducibly establish the disynaptic cutaneous reflex only between particular cell populations

Processing unit resources in a distributed computing systems

The described program is designed for collecting, storing and processing data of distributed file system

Acute treatment with valproic acid and L-thyroxine ameliorates clinical signs of experimental autoimmune encephalomyelitis and prevents brain pathology in DA rats

This work was supported by grants from the Swedish Research Council (MJ (K2008-66X-20776-01-4 and K2012-99X-20776-05-3)), OH (2011-3457) and GCB (K2011-80P-21816-01-4 and K2011-80X- 21817-01-4)), Harald and Greta Jeanssons Foundation (MJ), Swedish Association for Persons with Neurological Disabilities (MJ), ÅkeWibergs Foundation (MJ), Åke Löwnertz Foundation (MJ), Swedish Brain Foundation (MJ and GCB), David and Astrid Hagélen Foundation (GCB), Swedish Society for Medical Research (GCB), Swedish Society of Medicine (GCB), Socialstyrelsen (MJ), Karolinska Institutet funds (MJ and GCB), Marie Curie Integration Grant, Seventh Framework Programme, European Union (GCB, PCIG12-GA-2012-333713)), Neuropromise LSHM-CT-2005-018637 (MZA, HL) and Theme Center for Regenerative Medicine at Karolinska Institutet (OH)

A Schematic Summary of Some Cell Type–Specific Roles for REST

<p>Recent reports show a novel role for REST in maintaining ESC phenotype by being a close part of the core transcriptional network of Oct4/Sox2/Nanog in ESCs (see text). REST has previously been suggested to repress neuronal differentiation in NSCs and medulloblastoma cells, in addition to its well-established role in repressing neuronal genes in non-neural cells. REST has also been shown to act as a tumor suppressor by repressing epithelial cell transformation, a role that could be linked to the regulation of cell adhesion genes (see text).</p

Recombinant Spider Silk Protein Matrices Facilitate Differentiation of Neural Stem Cells Into Mature and Functional Neurons

Neural stem cells (NSCs) show great promise in drug discovery and clinical application. Yet few efforts have been made to optimize biocompatible materials for such cells to be expanded and used in clinical conditions. We have previously demonstrated that NSCs are readily cultured on substrates of certain recombinant spider silk protein without addition of animal- or human-derived components. The question remains however whether this material allows differentiation into functional neurons, and whether such differentiation can take place also when the NSCs are cultured not only upon but also within the biodegradable material. Here we demonstrate that "foam"-like structures generated from recombinant spider silk protein (4RepCT) provided excellent matrices for the generation and multicellular analysis of functional excitatory neurons from NSCs without addition of animal- or human-derived components. NSCs isolated from the cerebral cortices of rat embryos were cultured at either 4RepCT matrices shaped as foam-like structures without coating, or on conventional polystyrene plates coated with poly-L-ornithine and fibronectin. Upon treatment with recombinant proteins including the extracellular signaling factor BMP4 or a combination of BMP4 and the signaling factor Wnt3a, the cortical NSCs cultured in 4RepCT foam-like structures differentiated efficiently into neurons that responded to glutamate receptor agonists, such as AMPA, to the same extent as control cultures. Matrices derived from recombinant spider silk proteins thus provide a functional microenvironment for neural stem cells with little or no animal- or human-derived components and can be employed in the development of new strategies in stem cell research and tissue engineering