Tracking STEMEZ hNP1 Progenitor Cell Fate

We would like to promote an important new discovery using our STEMEZ(TM) hNP1 Human Neural Progenitors Discovery Kit.

Human embryonic stem cell–derived neural progenitors (NP) present an important tool for understanding human development and disease. Optimal utilization of NP cells, however, requires an enhanced ability to monitor these cells in vitro and in vivo. Here we report production of the first genetically modified self-renewing human embryonic stem cell–derived NP cells that express fluorescent proteins under constitutive as well as lineage-specific promoters, enabling tracking and monitoring of cell fate. Nucleofection, transfection, and lentiviral transduction were compared for optimal gene delivery to NP cells. Transduction was most efficient in terms of transgene expression (37%), cell viability (39%), and long-term reporter expression (>3 months). Further, the constitutive gene promoters, cytomegalovirus, elongation factor 1α, and ubiquitin-C, exhibited comparable silencing (20–30%) in NP cells over a 2-month period, suggesting their suitability for long-term reporter expression studies. Transduced NP cells maintained their progenitor state and differentiation potential, as demonstrated by expression of endogenous NP markers and neuronal markers after differentiation. We also detected reporter expression in astrocytes generated from NP cells transduced with an astrocyte-specific gene promoter, glial fibrillary acidic protein, demonstrating the usefulness of this approach. The genetically manipulated NP cells described here offer great potential for live cell–tracking experiments, and a similar approach can as well be used for expression of proteins other than reporters.

Related Publication:
Sujoy K. Dhara, Brian A. Gerwe, Anirban Majumder, Mahesh C. Dodla, Nolan L. Boyd, David W. Machacek, Kowser Hasneen, Steven L. Stice. Genetic Manipulation of Neural Progenitors Derived from Human Embryonic Stem Cells. Tissue Engineering Part A. -Not available-, ahead of print. doi:10.1089/ten.tea.2009.0155

Neuromics' Stem Cell Markers Referenced:
Musashi-1, Rabbit, 1:100 , Catalog#:RA14128
Nestin, Mouse 1:500, Catalog#: MO15012
Tuj 1 (Neuron-specific class III beta-tubulin), Mouse, 1:500, Catalog#: MO15013
TH, Chicken, 1:80, Catalog#: CH23006

STEMEZ hNP1 Neural Progenitors Now Available

We are pleased to announce the introduction of our STEMEZ ^TMhNP1 Human Neural Progenitors Discovery Kit. The kit was developed by our partner, ArunA Biomedical.

Description/Data:
STEMEZ^TM hNP1 Human Progenitors are fully differentiated are derived as adherent cells from hESC WA09 line. The cells are shipped frozen in a vial with 1 x 10⁶ cells; once thawed they should be immediately plated on a Matrigel (or other suitable extracellular matrix protein)-coated dish and maintained in the accompanying serum-free medium. The neurons should be used within 14 days of thawing. These cells have the capabilities to:

• Display immunoreactive properties consistent with neural precursors and can be maintained in a proliferative state in monolayer cultures.
• Differentiate under serum-free conditions into neuronal phenotypes with functionally responsive transmitter receptors in vitro .
• Maintain multiple neural phenotypes in long term serum-free culture.
• Can be directed to alter phenotypic characteristics under different media conditions.

Applications: Gene Expression Analysis Western blotting, Flow Cytometry, Immunocytochemistry, FACS sorting, DNA Microarray, RT-PCR, Neurite Outgrowth assays, FLIPR calcium assays, cytotoxicity, and second messenger signaling.

Images: STEMEZ^TM hNP1 Human Neural Progenitor Cells are grown as monolayers (A), are karyotypically normal (B) and express NSC markers, Nestin Mouse and Sox-2 (C, D, E). Nuclei of the cells were visualized with DAPI (blue). The Sox-2 transcription factor is co-localized with the DAPI (blue) staining in the nucleus (F).

STEMEZ hNP1 FAQs

Note: STEMEZTM NP1 Progenitors and hN2 Human Neurons can be genetically manipulated with GFP reporters without altering the function and differentiation potential of these cells. In contrast to many stem cell sources, the reporter genes are not readily silence in the neural cultures. See: doi:10.1089/ten.tea.2009.0155.

DNA fingerprint cells: The loci match the DNA fingerprint pattern for the H9 (NIH designation, WA09) HESC line as published in http://stemcells.nih.gov/research/nihresearch/scunit/.

Viral tests cells: This lot was derived from the H9 hESC line that has been tested for Hepatitis B, Hepatitis C, HIV-1, HIV-2, HTLV-I/II, HSV1, HSV2, EBV, and CMV. The H9 cell line has been tested and shown to be negative. (Tests performed by GIVF Laboratories).

Related Reagents:

• STEMEZ(TM) hN2 Human Neurons Discovery Kit


• Neuron/Glial Marker Antibodies


• Neurotrophins and Growth Factor Antibodies


• Fibroblast Growth Factor Proteins


• Neurotrophins-Neuron/Glial Marker Proteins


• Stem Cell Reagents


• Transfection Reagents

Regenerating Axons Can Be Guided And Reform Connections After Spinal Cord Injury

This is an interesting update

Regenerating Axons Can Be Guided And Reform Connections After Spinal Cord Injury

We support this Spinal Cord Injury and Repair with:

• Axon Growth and Guidance Antibodies
• Neurotrophins-Neuron/Glial Marker
• Stem Cell Research Reagents
• STEMEZ (TM) Human Neuron Discovery Kits

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Link Between Inflammation and Tau Pathology in Alzheimer's Disease

Providing reagents for Alzheimer's Disease Researchers is a strategic focus area for us. This includes making a variety of Primary Neuronal Cultures available to these researchers.

We evolve the strategy based on Researcher input and how the cultures are being referenced in publications. This reference is especially satisfying because of the vangaurd findings:

Lisette T. Arnaud, Natura Myeku and Maria E. Figueiredo-Pereira. Proteasome–caspase–cathepsin sequence leading to tau pathology induced by prostaglandin J2 in neuronal cells. Journal of Neurochemistry. Volume 110 Issue 1, Pages 328 - 342.

"Our data suggest a potential sequence of events triggered by the neurotoxic product of inflammation PGJ2 leading to tau pathology. The accumulation of Ub proteins is an early response. If cells fail to overcome the toxic effects induced by PGJ2, including accumulation of Ub proteins, apoptosis kicks in triggering caspase activation and tau cleavage, the clearance of which by cathepsins could be compromised culminating in tau pathology. Our studies are the first to provide a mechanistic link between inflammation and tau pathology."

Featured Reagent:
E18 Primary Rat Cortical Neurons

Related Reagents:

STEMEZ(TM) hN2 Human Neurons Discovery Kit

Images: Neural phenotypes derived from hN2 cell lines. (A) Phase contrast image of differentiated culture. (B) Network including post-mitotic motoneurons (HB9). (C) Cholinergic neuron. (D) Tuj-1 positive cells that are DAT-positive (dopamine transporter; closed arrow) and DAT-negative (open arrow). (E) Gabaergic neurons, inset illustrates GABA in axon, but not the dendrites (arrow).

Tau (Tau 46) Mouse Antibody

Tau Chicken Antibody

Apoptosis Research Reagents

We will continue to post "whats new" in AD research and potential uses of our reagents.

survival of efferent synapses on mammalian outer hair cells.

Dr Douglas Vetter (Tufts University School of Medicine) and team recently published a study that amplifies the understanding of the development, function and maintenance of auditory system function.

Their data strongly suggest that hair cell responses induced and/or modulated by Olivocochlear (OC) activation are necessary for the survival of OC innervation and that these responses must involve SK2-mediated hyperpolarization.

It also includes an excellent image of Olivocochlear fibers degeneration in SK2−/− mice. One of the makers used was our Tuj 1 (Neuron-specific class III beta-tubulin).

Vidya Murthy, Stéphane F. Maison, Julián Taranda, Nadeem Haque, Chris T. Bond , A. Belén Elgoyhen, John P. Adelman, M. Charles Liberman, Douglas E. Vetter. SK2 channels are required for function and long-term survival of efferent synapses on mammalian outer hair cells. Molecular and Cellular Neuroscience 40 (2009) 39–49
...TuJ-1 (class III β-tubulin, Neuromics, Northfield, MN; cat. # MO15013) and processedwith Oregon Green labeled secondary antibodies (Molecular Probes/InVitrogen) for confocal microscopy...

Other Pubs Referencing Neuromics' Tuj 1 (Neuron-specific class III beta-tubulin):
Yoshifumi Saisho, Paul E. Harris, Alexandra E. Butler, Ryan Galasso, Tatyana Gurlo,1 Robert A. Rizza, and Peter C. Butler. Relationship between pancreatic vesicular monoamine transporter 2 (VMAT2) and insulin expression in human pancreas. J Mol Histol. 2008 October; 39(5): 543–551. Published online 2008 September 13. doi: 10.1007/s10735-008-9195-9.
Sujoy K. Dhara, Kowser Hasneen, David W. Machacek, Nolan L. Boyd, Raj R. Rao, Steven L. Stice (2008). Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures. Differentiation 76 (5) , 454–464 doi:10.1111/j.1432-0436.2007.00256.x
...NES (1:100, Neuromics, Edina, MN), MSI1 (1:100, Neuromics), Tuj1 (1:500, Neuromics)...
D. Conte, V. Lall, G. Dobson, S. Deuchars, J. Deuchars. Cerebrospinal fluid contacting neurones in the spinal cord of the mouse and rat: small cells with a big purpose? University of Leeds (2008) Proc Physiol Soc 10 PC45
...Preliminary immunohistochemical data supports the concept that the CSFcNs are immature neurones, since they express neuronal markers Tuj1 (Neuromics) ...

ENS Development Markers

This is an excellent study on the developing Enteric Nervous Stem (ENS). This system is often referred to as the "second brain".

Despite this, neurogenesis has been much less studied in the ENS than in the brain. Understanding how neurons are formed in the gut is the foundation for finding cures for ENS disorders. The key finding here is that the ability of 5-HT4 receptors to unmask a regulation of enteric neurogenesis in adult animals suggests that the mature ENS is capable of an unexpected degree of plasticity (potentially good new for discovering therapies for ENS related disorders).

Min-Tsai Liu, Yung-Hui Kuan, Jingwen Wang, René Hen, and Michael D. Gershon. 5-HT4 Receptor-Mediated Neuroprotection and Neurogenesis in the Enteric Nervous System of Adult Mice. The Journal of Neuroscience, August 5, 2009, 29(31):9683-9699; doi:10.1523/JNEUROSCI.1145-09.2009.

More details @ http://www.anxietyinsights.info/serotonin_the_gut_and_neurogenesis.htm

On a side note, this publication is rich with excellent ICC and Western Blot images of a variety of Neurogenesis Markers. This include referencing of 4 of our markers:

GFAP-CH22102-ICC Dilution 1:2000
Musashi-1-RA14128-ICC Dilution 1:100 WB Dilution 1:1000
Neurofilament NF-H-CH22104-ICC Dilution 1:2000
S100B-RA25022-ICC Dilution 1;1000

Spinal Cord Injury Repair

Neuromics has featured an overview of Dr. Matt Ramer's Research on Spinal Cord Injury and "Finding Fixes for Injured Nerves" on our Neuroscience News Blog.

We wanted to share a recent publication authored by Dr. Mark Tuszynski and his team of researchers at USCD. Here's the good news:

"NT-3 expression in the correct target led to reinnervation of the nucleus gracilis in a dose-related fashion, whereas NT-3 expression in the reticular formation led to mistargeting of regenerating axons. Axons regenerating into the nucleus gracilis formed axodendritic synapses containing rounded vesicles, reflective of pre-injury synaptic architecture. Thus, we report for the first time, to the best of our knowledge, the reinnervation of brainstem targets after SCI and an essential role for chemotropic axon guidance in target selection"

Laura Taylor Alto, Leif A Havton, James M Conner, Edmund R Hollis II, Armin Blesch & Mark H Tuszynski. Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury. Nature Neuroscience. Published online: 2 August 2009 doi:10.1038/nn.2365.

Featured Neuromics Reagent
NT-3
Related Reagents:
Neurotrophins and Growth Factor Antibodies
Neuron/Glial Marker Antibodies
Neurotrophins-Neuron/Glial Marker Proteins
Stem Cell Reagents

Here's to finding the cure!