hN2 Human Neurons & hNP1 Neural Progenitors in Action

I have been promoting Dr. Steve Stice and his team. They are the brains behind our  hN2TM Human Neuron and hNP1TM Human Neural Progenitor Discovery Kits. I would like to share 2 recent publication referencing use of these discovery kits. These validate the postings on capabilities. They are the best solutions available for researchers searching for Neuron or Neural Progenitor Based Assays for basic research, toxicology studies or drug discovery.

Xiugong Gao, Hsiuling Lin, Radharaman Ray, Prabhati Ray. Toxicogenomic Studies of Human Neural Cells Following Exposure to Organophosphorus Chemical Warfare Nerve Agent VX. Neurochemical Research. February 2013…Human hN2 neurons were obtained from Neuromics…

Abstract: Organophosphorus (OP) compounds represent an important group of chemical warfare nerve agents that remains a significant and constant military and civilian threat. OP compounds are considered acting primarily via cholinergic pathways by binding irreversibly to acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Many studies over the past years have suggested that other mechanisms of OP toxicity exist, which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis. Here we performed a microarray study in which cultured human neural cells were exposed to 0.1 or 10 μM of VX for 1 h. Global gene expression changes were analyzed 6, 24, and 72 h post exposure. Functional annotation and pathway analysis of the differentially expressed genes has revealed many genes, networks and canonical pathways that are related to nervous system development and function, or to neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In particular, the neuregulin pathway impacted by VX exposure has important implications in many nervous system diseases including schizophrenia. These results provide useful information valuable in developing suitable antidotes for more effective prevention and treatment of, as well as in developing biomarkers for, VX-induced chronic neurotoxicity.

Protocol: Human hN2 neural cellswere obtained from Neuromics (Edina, MN). The hN2 cells were fully differentiated

normal human neural cells derived as adherent cells from human embryonic stem cell (hESC) WA09 line [34] and thus are considered as ‘‘matured’’ neuronal cells. It should
be noted that the universal neural cell marker Tuj (beta tubulin III) indicates that [80 % of the hN2 cells are neural. The other cell types which constitute\20 % of the cell population are mostly astrocytes and microglia, which are common glial cells found in the brain and spinal cord. The inclusion of the small amount of glial cells in the cell population better mimics real life situation in the central nervous system. The hN2 cells were seeded in 12-well plates at *500,000 cells/well in the AB2 Basal Medium complemented with ANS Supplement (cholinesterase free) provided by Neuromics and cultured at 37OC under
humidified 5 % CO2 for 48 h (without changing media) before VX exposure.

Xiufang Guo, Severo Spradling, Maria Stancescu, Stephen Lambert, James J. Hickman. Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials, In Press, Corrected Proof,Mar 2013.doi:10.1016/j.biomaterials.2013.02.061 ...hNP1, were obtained from Neuromics (Edina, Minnesota)…

Abstract: Although sensory neurons constitute a critical component for the proper function of the nervous system, the in vitro differentiation of functional sensory neurons from human stem cells has not yet been reported. This study presents the differentiation of sensory neurons (SNs) from a human neural progenitor cell line, hNP1, and their functional maturation in a defined, in vitro culture system without murine cell feeder layers. The SNs were characterized by immunocytochemistry and their functional maturation was evaluated by electrophysiology. Neural crest (NC) precursors, as one of the cellular derivatives in the differentiation culture, were isolated, propagated, and tested for their ability to generate sensory neurons. The hSC-derived SNs, as well as the NC precursors provide valuable tools for developing in vitro functional systems that model sensory neuron-related neural circuits and for designing therapeutic models for related diseases.

Images: Generation of Schwann cells from the differentiated culture. Immunostaining of a day 38 culture with the Schwann cell marker S100 demonstrating a significant number of Schwann cells in the culture. Schwann cells were located either within the neuronal clusters (A) or along the axonal bundles (B). The neuronal clusters and axonal bundles were marked by Peripherin immunostaining. doi.org/10.1016/j.biomaterials.2013.02.061

I will continue to post more proof regarding the capabilties and value of our human neurons & neural progenitors as pubs/data/images becomes available

More on STEMEZ hN2 Primary Human Neurons

My company’s STEMEZTM hN2 Primary Human Neuron Discovery Kits have been a frequent topic on “News Behind the Neuroscience News”. My friends at Aruna Biomedical continue to broaden the capabilities of these Kits based on customer feedback.

I am seeing increasing demand for these cells as these capabilities are published. Here’s the latest:

A. Young, D.W. Machacek, S.K. Dhara, P.R. MacLeish, M. Benveniste, M.C. Dodla, C.D. Sturkie and S.L. Stice. Ion channels and ionotrophic receptors in a human embryonic stem cell derived neural progenitors. doi:10.1016/j.neuroscience.2011.04.039. Markers used:…mouse nonoclonal anti nestin (neuromics), mouse monoclonal anti tuj-1 (neuromics)…

Abstract: Human neural progenitor cells differentiated from human embryonic stem cells offer a potential cell source for studying neurodegenerative diseases and for drug screening assays. Previously, we demonstrated that human neural progenitors could be maintained in a proliferative state with the addition of leukemia inhibitory factor and basic fibroblast growth factor. Here we demonstrate that 96 h after removal of basic fibroblast growth factor the neural progenitor cell culture was significantly altered and cell replication halted. Fourteen days after the removal of basic fibroblast growth factor, most cells expressed microtubule-associated protein 2 and TUJ1, markers characterizing a post-mitotic neuronal phenotype as well as neural developmental markers Cdh2 and Gbx2. Real-time PCR was performed to determine the ionotrophic receptor subunit expression profile. Differentiated neural progenitors express subunits of glutamatergic, GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium and calcium channel subunits were also expressed. Functionally, virtually all the hNP cells tested under whole-cell voltage clamp exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited tetrodotoxin-sensitive, voltage-dependent sodium channel current. Action potentials could also be elicited by current injection under whole-cell current clamp in a minority of cells. These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and has the capability to produce excitable cells that can generate action potentials, a landmark characteristic of a neuronal phenotype. This is the first report of an efficient and simple means of generating human neuronal cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.

STEMEZ hN2 Cells-Electrophysiology Data

STEMEZ hN2 Cells-Electrophysiology Data

 

 

 

 

 

I will continue to post updates here.

25 Best Blogs for Following Stem Cell Research

Providing research proven and reasonably priced Stem Cell Research Reagents is core to our business growth.  Part of my business strategy includes keeping the Stem Cell research community up to date on latest news, methods and publications. This helps oil the engines of basic research and drug discovery.

hN2 Cell-Differentiation

Images Courtesy of Paula M. Keeney, Laboratory and Research Manager, VCU Parkinson's Disease Center of Excellence.

This listing comes to me from my friend Roxanne McAnn at Nursingdegree.net.

Stem cell research has been a contentious issue in both the scientific and political spheres for quite some years. Despite the ongoing battle between those who support and those who oppose the research and treatments, new discoveries and advances in the field are being made all the time. Whether you’re pursuing a career in medicine or science, if you’d like to keep up with these advances, then blogs on the issue are one of the best tools out there. Here, you’ll find a collection of blogs that provide all the information you’ll need to stay on top of the latest in stem cell discoveries.

News-These blogs will let you stay on the cutting edge of new developments in the stem cell research community.

  1. The Stem Cell Blog: Through this blog, you’ll be able to get updates on the latest and greatest in stem cell research.
  2. Stem Cell News Blog: This blog collects a wide range of articles related to stem cell treatments, research and policy.
  3. Ben’s Stem Cell News: Ben Kaplan is a stem cell activist, blogger and a biotech professional who shares his thoughts and the latest information on stem cells here.
  4. Stem Cell Directory: No matter what kind of stem cell information you’re looking for, you’ll find it here through articles, news and videos.
  5. All Things Stem Cell: From treating baldness to cancer, learn about the myriad of ways stem cells may be able to help patients on this blog.
  6. Cell News: This blog will make it simple to be in-the-know when it comes to everything related to stem cells.
  7. The Stem Cell Trekker: Use this blog to learn more about stem cell innovations around the globe.
  8. StemSave: You might not think dental care when you think of stem cells, but this blog will show you that stem cells may be able to be taken from the teeth, giving you a whole new appreciation for those chompers.
  9. Joescamp’s Stem Cell Blog: This blog offers up news, information and insights into adult stem cell research.

Businesses and Organizations-Check out these blogs to see what research corporations and organizations
invested in stem cells are doing.

  1. International Stem Cell Corporation: Visit this blog to learn more about stem cell research that’s being done overseas, as many countries don’t have the same restrictions on research as the U.S.
  2. ViaCord Blog: This company, invested in cord blood baking and research, shares advances in the field of stem cells and cord blood treatments through this blog.
  3. Stem Cell Network Blog: Based out of Canada, this organization’s blog will help readers stay on top of new studies being done in the field, as well as some political issues that will affect researchers in Canada and around the world.
  4. Stem Cell Aware: Here you’ll find articles and information that can help you learn more about individuals who are receiving treatment with adult stem cells around the world.
  5. Umbilical Cord Blood Blog: Learn more about donating umbilical blood and the stem cell research being done with it through this organization’s blog.

Commentary Here, you’ll get not only news, but commentary on stem cell issues as well.

  1. David Granovsky’s Stem Cell Blog: Ranked as one of the top health bloggers by Wellsphere, David Granovsky’s blog on stem cells is sure to provide you more  information on the subject than you’ll have time to read.
  2. California Stem Cell Report: See how stem cell politics are affecting research and development in California through this blog written by journalist David Jensen.
  3. Advance Stem Cell Research: Follow the latest news and commentary on stem cells with this blog.

Research-These blogs, many from labs and experts in the field, focus on providing news and information on the best research being done with stem cells in the world.

  1. Knoepfler Lab Stem Cell Blog: The UC Davis School of Medicine maintains this blog, providing readers with information on everything stem cell as well as other science-related issues.
  2. CIRM Research Results: The California Institute for Regenerative Medicine shares their latest discoveries and political battles here.
  3. Robert Lanza, MD: Dr. Robert Lanza is a scientist and professor working on issues related to cell technology and engineering; his blog will provide readers with some insights into the field and his research.
  4. Stem Cell Gateway: Whether you live in the U.S. or abroad, this blog is the place to visit for information geared towards the stem cell research community.
  5. Tissue and Cellular Innovation Center Blog: Focused on tissue engineering and stem cell biology, this center is at the forefront of much of the research they share via this blog.
  6. Stem Cell Breaking Research: Need to know the absolute latest on stem cell research? This blog may be one of your best bets, with updates posted every day.
  7. Stem Cell Digest.net: On this blog, you’ll find information about stem cell research, progress, new applications and companies who are doing the work.
  8. Stem Cell Methods: Researchers, scientists and medical professionals can learn more about the protocols and methods being used in stem cell research and treatment through this blog.

Author’s not (6/1/2011). This excellent site was brought to my attention by Dr. Anthony G. Payne- www.stemcelltherapies.org: This site is run by Steenblock Research Institute (San Clemente, California) which is a 501(c)(3) non-profit organization devoted to stem cell related education and research (SRI has a massive library facility and  stem cell R & D laboratory).

Ion Channels and Neuromics’ STEMEZ Cells

In my conversation with neuro-drug discover researchers, I am frequently being asked about the potential of using our STEMEZ(TM) hNP1 Human Neural Progenitors Expansion Kits for studying ion channels. How effective are these cells as a source for studying neurodegenerative diseases and for drug screening assays?  There is good news from Dr. Steve Stice and my friends from ArunA and UGA.

When differentiated, these  neural progenitors express subunits of glutamatergic,  GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium  and calcium channel subunits were also expressed. Functionally, virtually all the NP cells exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited  tetrodotoxin sensitive, voltage-dependent sodium channel current under whole-cell voltage clamp and action potentials could be elicited by current injection under whole-cell current clamp.  These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and also results in the capability to produce excitable cells that can generate action potentials. This is the first data demonstrating capabilitiesof these cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.
hNP1_Gene_Expression

Images: Glutamate receptor expression in hNP cells and differentiated hNP cells The expression of ionotropic glutamate receptors might also be an indicator of neuronal maturation. These receptors are composed of three distinct families: NMDA, kainate and AMPA receptors. The hNP cells and differentiated hNP cells cultured in the absence of bFGF for 2 weeks were analyzed for mRNA expression of subunits of each glutamate receptor subtype relative to hESCs. Significant increases (p<0.05) in Grin2b were seen in hNP cells (20 fold) and differentiated hNP cells (25 fold) relative to hESCs (Figure 3A). Additionally, Grin1 and Grin2d were significantly increased (p<0.05) only in differentiated hNP cells relative to hESCs, but not in undifferentiated hNP cells (Figure 3A). Of the kainate receptors, Grik4 and Grik5 were significantly (p<0.05) increased only in undifferentiated hNP cells relative to hESCs (Figure 3B); whereas, Grik2 was significantly (p<0.05) increased only in hNP cells where bFGF had been removed (Figure 3B). AMPA receptor subunits were also examined. Gria1 and Gria4 were up regulated in hNP cells relative to hESCs (Figure 3C). Two week differentiated hNP cells showed significant (p<0.05) up regulation of Gria2 and Gira4 relative to hESCs (Figure 3C). To determine if functional glutamate channels exist in differentiated hNP cells, calcium influx in response to AMPA, kainic acid or NMDA application was measured on hNP cells, 14 days after the removal of bFGF. Figure 3G indicates that NMDA could not depolarize differentiated or undifferentiated hNP cells enough to cause significant calcium influx above background. In contrast, AMPA and kainic acid can cause calcium influx which can be potentiated by AMPA receptor specific modulator, cyclothiazide (50 μM, Figure 3G).Calcium influx was detected in the presence of cyclothiazide in calcium activity as measured (Figure 3H).

hNP1_Electrophysiology

Images: Sodium channel activity in differentiated hNP cells was measured using whole cell voltage clamp. 81 total hNP cells cultured in the absence of bFGF from 4 to 27 days were analyzed. Of these, 34 exhibited no fast inward currents in response to a step depolarization indicating the 348 absence of functional voltage gated sodium channels (Figure 4G). The remaining cells yielded between 0.04 – 1.5 nA of inward current in response to the step depolarization (Figures 4B and 4G). These currents inactivated rapidly in all cases (Figures 4B and 4C) and could be abolished with the addition of 1 μM TTX (n = 3 cells; Figure 4C). Voltage-dependent steady state inactivation (n = 11 cells; Figure 4D) and recovery from fast inactivation (n = 5 cells; Figure 4E) were also observed on several positive cells. A subset of these cells was subjected to current clamp and action potentials were elicited by current injection (n = 8 cells, Figure 4F). In support of this, increasing concentrations of a sodium channel activator veratridine in a FLIPR assay on differentiated hNP cells show an increasing calcium response (Figure 4H). This probably resulted from voltage-gated sodium channel depolarization of cells that subsequently allowed calcium influx through calcium channels. These data indicate that differentiation of hNP cells by removal of bFGF can lead to a neuronal cell that can generate action potentials and depolarize the cell. The 58% hit rate for voltage-gated sodium channel function (Figure 4G), does not reflect the true proportion of sodium channel positive cells in our differentiated hNP cells, but rather our ability to morphologically distinguish these cells from negative cells by eye. An example of the morphology of a sodium channel positive cell is shown in Figure 4A. The positive cells were phase bright with a few long processes.

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

STEMEZ hN2 Primary Human Neurons

STEMEZ hN2 Primary Human Neurons

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.