STEMEZ(TM) hNP1 Human Neural Progenitors and hN2 Primary Neurons Differentiation and Expression

I have received a growing number of requests regarding differentiation and expression patterns of our STEMEZ^TM cells. This is intended help you gain a clearer understanding. The focus of this document is G-protein Coupled Receptor Expression Patterns. This information was presented by Dr. Steve Stice and his team and the 2008 Neuroscience Conference.


INTRODUCTION

Human embryonic stem cells and their progeny can provide a novel Distribution of detectable transcripts for three cell populations tissue source for understanding developmental pathways, pharmaceutical screening and tissue replacement therapies. G-protein coupled receptors(GPCRs) comprise the largest cell-surface receptor superfamily and are the largest class of drug targets. The study of GPCR signaling in hES cells allows signaling mechanisms to be studied in endogenously expressed receptors in non-transformed cells. We characterized GPCR transcript expression in three cellular populations at different developmental stages: WAO9 human embryonic stem cells, Wa09 derived STEMEZ hNP1 and differentiated hN2 cells maintained 1 week in culture.

Goal: To characterize GPCR transcript expression in human hES cell derived neural tissue

CONCLUSIONS
• hES cells displayed the widest array of GPCR transcripts, while neural progenitors displayed the most restricted population.
• The Frizzled (FZD) family of receptors were among the most abundantly expressed transcripts across all populations.
• Neural progentitors up-regulated GPCR transcripts important to brain angiogenesis, cell proliferation, neurogenesis and cell adhesion.
• Further differentiated hN2 cells displayed up-regulation of a wider population of transcripts including GPCRs involved with neurotransmission.
• Functional assays demonstrated responses to sphingosine-1-phosphate in both hNP1 and hN2 populations of cells.
• hES cells and their derived tissue provide a unique model to study endogenous GPCR signaling in non-transformed cells for drug screening applications and to further our understanding of GPCRs role in developmental pathways.

G-protein Coupled Receptor Expression Patterns Are Altered as Human Embryonic Stem Details(pdf - 367Kb). From Poster Presented at Neuroscience 2008 by Dr. Steve Stice et al.

Dr. Steve Stice to Present the Power of StemEZ Neural Cells

Dr. Steve Stice to Present the Power of StemEZ Neural Cells

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I have profiled Steve Stice's research here. The focus has been the excellent research results he and his team at ArunA Biomedical have generated with STEMEZ(TM) hN2 Human Neurons and hNP1 Human Neural Progenitors.

The story continues. He will be presenting the latest at the 9th Annual World Pharmaceutical Congress in Philadelphia, June 14. Topics include: using these neural cell lines to study neurotoxicity in cell-based assays and disease modeling. Recent work conducted in outside laboratories demonstrates that these lines are more sensitive to environmental toxicants than traditional cellular models.

Sample high throughput assay applications:

• Cell morphology and neurite outgrowth


• Cell signaling and transcription factor expression


• Receptor and ion channel function


• Cytotoxicity
  
• Apoptosis, genotoxicity and DNA damage
  

These capabilities has been confirmed by our customers. I look for the use of the STEMEZ cell lines to continue to grow as researchers discover their value in Drug Discovery and Basic Neuroscience capabilities.

Amp up Your Results!

Labeling kits, tags, secondary antibodies and related reagents

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Labeling and tagging is an important step in your research process. This often drives the wow factor in published results. We offer some of the best and brightest including:

CHROMEO^TMsity-exhibit superior luminescence properties,
including a broad range of fluorescence excitation and emission, large
Stokes shifts, limited photobleaching and a broad pH tolerance.

ELISA Buffers and Diluents

Solulink™ Labeling Kits and Beads-The most efficient labeling kits delivering ready-to-use conjugates for the novice or the expert!

Cytoplasmic and Nuclear Staining

Strep-Tag®-One-STrEP-tag for protein complex purification.

Image: CHROMEOsity 488: HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 488 Goat anti-mouse IgG. The nuclei have been counterstained with DAPI.

Apoptosis Signaling-Visualization and Measurement.

Apoptosis-Oxidative Stress Research Reagents are widely used and frequently referenced in customer publications. We work hard to keep our fingers on the pulse of how they are utilized across the many research areas important to our customers and add new reagents based on evolving requirements.

I recently posted publications referencing our MitoPT^TM Kits for quantitating Tumor Apoptosis

New Pub referencing Polycaspase Assay Kit, green: L. Wei, D. Ding and R. Salvi. Salicylate-induced degeneration of cochlea spiral ganglion neurons-apoptosis signaling.
doi:10.1016/j.neuroscience.2010.03.015.

Images: Typical confocal photomicrographs of SGN stained with Polycaspase Assay Kit (green) and with an antibody against neuronal III ß-tubulin (red) to identify SGN. (A) In control cultures, most SGN have large, oval shaped soma and neurites extending from the soma; note absence of polycaspase labeling (green). (B) SGN treated for 3 h with 5 mM SS; polycaspase labeling was present on SGN with shrunken soma. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Related Reagents:

Polycaspase Assay Kit, green

Magic Red™ Real Time! Kits -Measure apoptosis in
whole living, intact cells - no lysis required

FLIVO™ Polycaspase Live!, in vivo Apoptosis Kits-New
-Designed for cancer and neurodegenerative disease applications.

FLICA™ in vitro Caspase Kits -Fast!-Use Caspase
kits to quantitate apoptosis via active caspases in whole, living
cells. These kits do not use ELISA or any antibodies for detection

FLISP™ Serine Protease Detection Kits -Measure
chymotrypsin-like protease activation in whole living cells.

MitoPT™ Kits -Quantitate mitochondrial
functionality and apoptosis

Angiogensis and CD antigens

We consider everything genesis as a core focus area. This includes embryogenesis, angiogenesis, neurogenesis and stem cell expansion and differentiation. This is important because there is an intersection between reagents and methods for studying embryo development and genesis pathways in "adult systems".

CD antigens serve as markers for growth and development. We are always on the look out for Customer Publications that reference use of these reagents in related applications. Here's one that just pinged our radar.

Karim Harhouri, Abdeldjalil Kebir, Benjamin Guillet, Alexandrine Foucault-Bertaud, Serge Voytenko, Marie-Dominique Piercecchi-Marti, Caroline Berenguer, Edouard Lamy, Frédéric Vely, Pascale Pisano, L'Houcine Ouafik, Florence Sabatier, José Sampol, Nathalie Bardin, Françoise Dignat-George, and Marcel Blot-Chabaud Soluble CD146 displays angiogenic properties and promotes neovascularization in experimental hind-limb ischemia. Blood, May 2010; 115: 3843 - 3851
Anti-rat antibodies used in this study are: anti-CD117 (Neuromics) and anti-CD146

Abstract: CD146, an endothelial molecule involved in permeability and monocyte transmigration, has recently been reported to promote vessel growth. As CD146 is also detectable as a soluble form (sCD146), we hypothesized that sCD146 could stimulate angiogenesis. Experiments of Matrigel plugs in vivo showed that sCD146 displayed chemotactic activity on endogenous endothelial cells, and exogenously injected late endothelial progenitor cells (EPCs). Recruited endothelial cells participated in formation of vascular-like structures. In vitro, sCD146 enhanced angiogenic properties of EPCs, with an increased cell migration, proliferation, and capacity to establish capillary-like structures. Effects were additive with those of vascular endothelial growth factor (VEGF), and sCD146 enhanced VEGFR2 expression and VEGF secretion. Consistent with a proangiogenic role, gene expression profiling of sCD146-stimulated EPCs revealed an up-regulation of endothelial nitric oxide synthase, urokinase plasminogen activator, matrix metalloproteinase 2, and VEGFR2. Silencing membrane-bound CD146 inhibited responses. The potential therapeutic interest of sCD146 was tested in a model of hind limb ischemia. Local injections of sCD146 significantly reduced auto-amputation, tissue necrosis, fibrosis, inflammation, and increased blood flow. Together, these findings establish that sCD146 displays chemotactic and angiogenic properties and promotes efficient neovascularization in vivo. Recombinant human sCD146 might thus support novel strategies for therapeutic angiogenesis in ischemic diseases

Image: c-KIT staining of rat skin (epidermis). c-KIT detection was done using anti-rabbit Cy3 conjugated antibodies (red color). DAPI was used to counterstain cell nuclei (blue color).Working dilution: 1:100-1:300.

Related Reagents:

• Immune Response Antibdodies


• Immune Response Proteins


• Cancer Research Antibodies


• Cancer Research Proteins


• Pain and Inflammation Research Antibodies


• Neurodegenerative Disease Antibodies


• Neurodegenerative Disease Proteins


• Apoptosis Research Reagents


• Stem Cell Research Reagents

TRPV1 Expression and Temporal Lobe Epilepsy

Dr Bret. Smith and his lab at Tulane have demontrated a link between Temporal Lobe Epilepsy (TLE) and VR1 N-Terminus (TRPV1) expression.

In this excellent study they showed an increase in expression in TLE mice vs controls.

Muthu D. Bhaskaran and Bret N. Smith. Effects of TRPV1 activation on synaptic excitation in the dentate gyrus of a mouse model of temporal lobe epilepsy. doi:10.1016/j.expneurol.2010.01.021
...with polyclonal VR1 N-terminus (1:2500) (Neuromics, Edina)..

Abstract: Temporal lobe epilepsy (TLE) is a condition characterized by an imbalance between excitation and inhibition in the temporal lobe. Hallmarks of this change are axon sprouting and accompanying synaptic reorganization in the temporal lobe. Synthetic and endogenous cannabinoids have variable therapeutic potential in treating intractable temporal lobe epilepsy, in part because cannabinoid ligands can bind multiple receptor types. This study utilized in vitro electrophysiological methods to examine the effect of transient receptor potential vanilloid type 1 (TRPV1) activation in dentate gyrus granule cells in a murine model of TLE. Capsaicin, a selective TRPV1 agonist had no measurable effect on overall synaptic input to granule cells in control animals, but significantly enhanced spontaneous and miniature EPSC frequency in mice with TLE. Exogenous application of anandamide, an endogenous cannabinoid that acts at both TRPV1 and cannabinoid type 1 receptors (CB1R), also enhanced glutamate release in the presence of a CB1R antagonist. Anandamide reduced the EPSC frequency when TRPV1 were blocked with capsazepine. Western blot analysis of TRPV1 receptor indicated protein expression was significantly greater in the dentate gyrus of mice with TLE compared with control mice. This study indicates that a prominent cannabinoid agonist can increase excitatory circuit activity in the synaptically reorganized dentate gyrus of mice with TLE by activating TRPV1 receptors, and suggests caution in designing anticonvulsant therapy based on modulating the endocannabinoid system.

Images: Western blot detection of TRPV1 receptor expression in the dentate gyrus. A. Diagram of dentate gyrus showing the microdissected area (box). B. Western blot showing TRPV1 receptor expression in two untreated mice and in two pilocarpine-treated mice that survived SE. Actin was used as the loading control which did not change significantly. C. Graph showing a significant (p less than 0.05; n=4) TRPV1 expression in epileptic mice.

Related Reagents:

TRPV (Vanilloid); TRPM; TRPA and TRPCs

Neurotransmission Research Antibodies-GPCRs, Ligand Gated Ion Channels, Biogenic Amines and more

Primary Neurons and Astrocytes- Primary human, rat and mouse neurons and astrocytes by Category.