61 research outputs found
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
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Pitx2 expression induces cell cycle exit and p21 expression in neural stem cells
Cortical development is a complex process that involves many events including proliferation, cell cycle exit, and differentiation that need to be appropriately synchronized.. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. The G1 phase of the cell cycle is mostly associated with cell cycle arrest and cell differentiation. Cell cycle progression and exit during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2, and found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≈20-fold) increase in expression of the cell cycle inhibitor p21Cip. In addition, Pitx2 bound directly to the p21Cip promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 directly regulates p21Cip expression and induces cell cycle exit in neural progenitors.Keywords: Neocortex, p27Kip2, Chromatin immunoprecipitation, Chromatin, p21Cip1, Transcription, Neural progenitors, p57Kip2, Pitx, Telencephalo
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
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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
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