The Importance of in vivo Like Astroglial-Neuron Co-Cultures

The Power of Neuromics’-Aruna Biomedical’s hAstroPro™-NeuroNet™ Co-Cultures. We are pleased to be the first to market with our in vivo like co-cultures. Here are key points of what make these cultures unique:

  • Pure, Potent and Proven

  • Proof That These areTrue Co-Cultures

  •  Co-Cultures vs Neuron Only Cultures

What have we learned? The mix of Astroglia vs Neurons can have a dramatic impact on your data end points. This can lead to wrong conclusion being drawn from your tox and compound/small molecule neurodegenerative disease assays. We stand ready to serve you and your team. Questions? Don not hesitate to call 612-801-1007 or e-mail me pshuster@neuromics.com. Pete Shuster, CEO and Owner, Neuromics

Gerry Shaw-Master of World Class Neuronal/Glial Markers

Build it and They will Come

Gerry and One of His Triumph's MCs
Gerry and One of His Triumph’s MCs

I am pleased to profile Dr. Gerry Shaw, a Professor at the University of Florida and also the Head of EnCor Biotechnology Inc.  His story is a guide for incubating and spinning out a successful biotech company (EnCor Biotechnology, Inc.) from a university research laboratory. It should provide an inspiration for fledgling entrepreneurs as the model required little capital investment and has enjoyed profitable growth.

The Backstory

Gerry’s major area of research interest can be summarized as the study of cellular changes resulting from central nervous system damage and disease states. These changes help neuroscience researchers understand the progression and hopefully discover root causes of diseases like Alzheimer’s, Parkinson’s and ALS. Understanding which proteins are involved in particular disease states also has the potential of identifying targets for therapies.

The story starts with Gerry’s Post Doctoral research at the Max Planck Institute for Biophysical Chemistry in Goettingen, in what was at the time West Germany. Here he joined the world renowned laboratory of Klaus Weber and Mary Osborn. This lab had pioneering several important techniques, notably SDS-PAGE for protein analysis and the use of antibodies in immunocytochemistry. Later, after Gerry left the same lab made key contributions leading to the routine use of RNAi in “knock down” of normal cellular proteins. The lab had developed antibodies to tag the subunit proteins of microtubules, microfilaments, intermediate filaments and other cellular proteins, and then used these antibodies to visualize the proteins in immunofluorescence microscopy and on western blots. This enabled researchers to look at changes in the cellular expression of these proteins in powerful new way. These methods have become vital tools for understanding normal cellular function and what happens when cells transition from healthy to diseased states. This lab was an ideal location for Gerry to learn how to make quality monoclonal and polyclonal antibodies. Good antibody reagents are vital for the correct interpretation of immunofluorescence microscopy and western blots, and he was soon supplying his reagents to friends, collaborators and other researchers all around the world. Success is value as antibodies that do not as work as expected waste research time and resources, while quality reagents soon become appreciated and may get to be standard lab reagents.

University of Florida

The University of Florida, in Gainesville imported his expertise when Gerry joined the institute in 1986. Here he continued to make antibodies to Neurofilaments or NFs and other Neuronal-Glial Markers. It’s hard to keep a good thing a secret and Gerry faced growing demand from all over for these reagents. This proved a drain both financially and in terms of time commitment, as well as a significant conflict of interest with his basic biomedical research program.

MAP2_Doering IHC Image: Co-culture of embryonic mouse hippocampal neurons and astrocytes. Primary embryonic hippocampal neurons at 7 days in vitro, were stained with Microtubule Associated Protein-2 (MAP, green) to enable the visualization of the dendritic arbors. These neurons were cultured on top of a monolayer of primary cortical astrocytes, stained with an antibody directed against

Glial Fibrillary Acidic Protein (GFAP, red). The cell nuclei were visualized by staining with 4′,6-diamidino-2-phenylindole (DAPI, blue). BMC Image of the Month October 2010

As a result Gerry took his first entrepreneurial step by selling his most popular reagents in bulk initially to Chemicon (now Millipore-Merck). Like any new business venture, he did not really know what to expect. It should come as no surprise that the reagents sold like hot cakes and the check started rolling in. Other immunoreagent companies approached Gerry and soon he was supplying antibodies to pretty much every major biotechnology vendor.

ABC Biologicals to EnCor Biotechnology Inc.

Success breeds success and as sales increased over the 1990s, it was time to form an independent business and so ABC Biologicals Inc. was incorporated in 1999 initially to buy equipment and develop licensing agreements. Since Gerry had income from sales, he was in the unusual and enviable position of not needing grants, investors, loans or cash from any other source, and so could proceed with almost total independence. The company was renamed EnCor Biotechnology Inc. in 2002, and at the same time moved into the Sid Martin Biotechnology Incubator, a lab dedicated to commercialization of intellectual property generated by the faculty of the University of Florida. The University of Florida is unusually experienced at this and is well known for launching Gatorade, Trusopt and many other products. After 4 years EnCor “graduated” from the Incubator and now occupies a facility in Gainesville. The company now has almost 100 products with many more under development. This is good news for the Neuroscience community.

The EnCor-Neuromics Connection

Neuromics provides EnCor Biotechnology reagents to researchers studying neuro-degeneration, neuro-regeneration, neuro-development, neural stem cells, mood disorders, brain injury and spinal cord injury. My customers have found EnCor’s reagents to be rock solid and versatile.

In addition, Gerry and his team have proved adept at culturing our E18 hippocampal neurons and ESC derived hN2TM primary neurons. This is a big plus as we can actually see how the cells and markers could resonate together for use in cell based assays.

Hippo_MAPT_DC1 Image: E18 hippocampal neurons stained with Tau (red) and Doublecortin (green). The two proteins overlap in the proximal dendrites (yellow) Axons (low doublecortin content) are red. Blue staining is the nuclear DNA.

Futures

I am excited by the glimpse of the future that Gerry shared. We can expect many new, novel and important markers in the coming months and years. In addition, he will be manufacturing various Enzyme-linked immunosorbent assays (ELISA). These kits have the potential to help clinicians diagnose the early onset of diseases like ALS, Parkinson’s and Alzheimer’s.

For example, his company currently sells an ELISA kit for sensitive detection of Phosphorylated Neurofilament-H (pNF-H). Expression of this protein is up regulated in a variety of damage and disease states, and can be used to accurately quantify this up regulation. The kit can also detect pNF-H in the sera and spinal cord fluid (CSF) of animals with spinal cord and brain lesions. This protein is not normally found in sera or CSF, so its presence indicates recent axonal injury as a result of either damage or disease. This suggests pNF-H is a useful biomarker of neuronal and more specifically axonal injury or degeneration, a suggestion supported by a growing list of basic science publications on various animal models and patient types from Gerry’s research lab (e.g. Shaw et al. 2005, Lewis et al. 2008, Boylan et al. 2009, Lewis et al. 2010).

Given the capabilities of EnCor’s markers, the development of more kits is coming. There could be a day in the not distant future where they give clinicians tools to better diagnose and monitor serious neurodegenerative diseases, leading to better disease treatment and management.

I will keep you informed on Gerry’s and EnCor’s future developments.

Coming Soon-Dr. Gerry Shaw

Zen and the Art of Bio-marker Production

Up next will be Dr. Gerry Shaw.  Gerry is the founder and head of EnCor Biotechnology, Inc. His company is recognized for creating markers that are engines of Neuroscience and Stem Cell Research.

Dr. Gerry Shaw with Triumph MC

Dr. Gerry Shaw with Triumph MC

I am pleased to represent his company’s reagents. They are well designed, thoroughly tested and proven to work in my customers’ many application.

They have proven especially effective in working in cell based assays using our eSC derived hNP1 human neurons and e18 primary rat hippocampal neurons.

Applications include the study of TBI, SCI, ALS, AD, MS and PD.

Image:  hN2 cells stained with our chicken polyclonal antibody to Vimentin, in red. Islands of Hn2 cells form after 4 days in culture forming beautiful flower like structures. Vimentin is a well established marker of early differentiating neuronal lineage cells. Taken with a 10X objective lens. Blue staining is the nuclear DNA.

hN2 Cells stained with Vimentin

hN2 Cells stained with Vimentin

Dr. Ivan Rich and HemoGenix

Stem Cells Testing Tools that enlighten Drug Discovery and Cell Therapy Researchers
I am pleased to profile Dr. Ivan Rich. He is the founder, chairman and CEO of HemoGenix and an internationally recognized leader in hematology.  I am timing this profile to coincide with Neuromics launch of HemoGenix’s first to market fully standardized, proven and cost effective  ATP-based, in vitro bioluminescence and high-throughput screening (HTS) cell based assay systems.

These assays represent best in class solutions for detecting and measuring cell viability, functionality, growth, proliferation and cytotoxicity of stem and progenitor cells for stem cell and basic research, cellular therapy, in vitro toxicity testing and veterinary applications.

Hemogenix_Pic

 ivan-rich

2000-Present- Hemogenix-CEO
and Chairman

1996-2000-Palmetto Richland Memorial Hospital

 1995-Second Thesis in Experimental Hematology, University of Ulm

1981-1983-Post Doc University of Chicago

1973-1978-Ph.D. University of Ulm, Biology

 

Ivan’s journey leading to founding of HemoGenix provided him a unique blend of scientific, entrepreneurial and operational expertise.  These traits are the drivers that enable him to invent, successfully commercialize and continuously improve cell based assay systems. These systems meet a wide range of demanding requirements. These include, for example, meeting the requirement by Standards Organizations and Regulatory Agencies for “appropriate” and “validated” assays that can be used by cord blood banks and stem cell transplantation centers to determine whether a stem cell product has the necessary potency characteristics and can be released for transplantation into a patient…high standards indeed!

The Back Story-Hematology and Hemopoietic Stem Cells

Ivan received his PhD from the University of Ulm, in Germany in 1973 in Human Biology. He then completed a second thesis in 1995 in experimental hematology.  Our story starts here.  As a background we need to understand:  the hemopoietic stem cell compartment consists of cells which are responsible for maintaining the steady-state production of some two million red blood cells and two hundred thousand white blood cells every second of a person’s life!

Beginning in 1973, he worked extensively with “classic” colony-forming cell (CFC) assay.  At the same time, He also gained experience in culturing erythropoietic progenitor cells (BFU-E and CFU-E) under low oxygen tension. His group was the first to demonstrate that macrophages grown in vitro could respond to low oxygen tension by regulating erythropoietin production at a local level. His group also demonstrated the role of HOXB6 in erythropoietic development as well as the role of the Na/H exchanger in hematopoiesis. “Necessity being the mother of invention”, Ivan began developing these assays into miniaturized format.  Assays necessary for fully understanding the potential and associated risks of using of these cells for human therapies.

This opened the door for him to do a post doc with the late Dr. Eugene Goldwasser at the University of Chicago. Dr. Goldwasser was renowned for discovering the first partial amino acid sequence of erythropoietin (EPO). This discovery eventually led to the production of human recombinant EPO by Amgen and the development of first EPO related therapeutic (Epogen). It is used to treat anemia from kidney disease and certain cancers.

We now move to Palmetto Richland Memorial Hospital in South Carolina where Ivan served as Director of Basic Research for Transplantation Medicine. From this research,  we learn that the most primitive stem cells have the greatest potential for proliferation and long-term reconstitution of the hemopoietic system, while the most mature stem cells have only short-term reconstitution potential. These primitive cells then become the most excellent candidates for future therapies. BUT how do we know the population of cells derived from cord blood or bone marrow contain the required population of potent and safe (phenotypically stable) primitive stem cells for effective therapies? We can ask the same questions for other stem cell populations that are candidates for therapies. These include mesenchymal stem cells, neural stem cells and others.

Introducing Quantitative, Accurate and Proven High Throughput (HTS) Stem Cell Assays

Ivan and HemoGenix began answering these questions in 2002 with help from National Cancer Insitute (NCI) SBIR grants. This led to the successful launch of the HALO® family of kits. These kits are based on Bioluminomics™ which is the science of using the cell’s energy source in the form of ATP (adenosine triphosphate) to provide us with a wealth of information. The production of ATP is an indicator of the cell’s cellular and mitochondrial integrity, which, in turn, is an indicator of its viability and cellular functionality. ATP also changes in proportion to cell number, proliferation status and potential, its cytotoxicity and even its apoptotic status.

HemoGenix continues to develop and evolve kits key to developing effective and safe stem cell related drugs and cell based therapies.

Practical Applications

Here are examples of the kits in action.

  • HemoGenix and Vitro Diagnostic-Via this partnership, LUMENESC kits for mesenchymal stem cells include high performance growth media for research, quality control or potency or cytotoxicity to the mesenchymal stem cell system
  • LumiSTEM™ for testing  hNP1™ Human Neural Progenitors Expansion Kit-enables  fast, accurate and multiplex detection system for hastening advances in drug safety and discovery as well as environmental toxicology. . LumiSTEM™[now LumiCYTE-HT]  kits are used for in vitro detection of liver toxicity, with an overall reduction in drug development cost for drug candidates
  • High Throughput (HTS) Screening of Multiple Compounds using HALO®-(to learn more see: TOXICOLOGICAL SCIENCES 87(2), 427–441 (2005) doi:10.1093/toxsci/kfi25). Eleven reference compounds from the Registry of Cytotoxicity (RC) and eight other compounds, including anticancer drugs, were studied over an 8- to 9-log dose range for their effects on seven cell populations from both human and mouse bone marrow simultaneously. The cell populations studied included a primitive (HPP-SP) and mature (CFC-GEMM) stem cell, three hematopoietic (BFU-E, GM-CFC, Mk-CFC) and two lymphopoietic (T-CFC, B-CFC) populations. The results reveal a five-point prediction paradigm for lympho-hematotoxicity.
HSC Toxicity Data

HSC Toxicity Data

Futures

The dawn is breaking for stem cells therapies. These cells are the reparative engines for damaged cells in our bodies. These therapies have the potential to alleviate the world’s most insidious, chronic and costly diseases. Tools that enable us to understand the true properties and potency of these cells lower the cost of discovering drugs and cell based therapies.

I look for more tools to spring from the vision of Dr. Ivan Rich that will play an ever increasing and important role in the world of basic stem cell research, stem cell based therapies and regenerative medicine. I plan to keep you updated on the evolution and capabilities of these inventions.

Harnessing the Power of Neural Stem Cells

I wanted to share an important presentation by Dr. Steve Stice. He is a featured researcher in “News Behind the Neuroscience News”.

“Does amplification of neural progenitor cells derived from embryonic stem cells solve problems of cell production and FDA safety standards?”
Steven L. Stice, PhD
Professor, GRA Eminent Scholar
Director of the Regenerative Bioscience Center at University of Georgia
CSO, Aruna Biomedical Inc.

Lectin Binding Profiles among Human Embryonic Stem Cells

I have featured  numerous posting of innovations by Dr. Steve Stice and our friends at Aruna Biomedical. Here I would like to share a publication by Steve and his team featuring a new slant on isolating eSC Derived hNP Neural Progenitors. This study also includes methods for sorting hESCs, hNP cells and hMP cells.

Mahesh C. Dodla, Amber Young, Alison Venable, Kowser Hasneen1, Raj R. Rao, David W. Machacek, Steven L. Stice. Differing Lectin Binding Profiles among Human Embryonic Stem Cells and Derivatives Aid in the Isolation of Neural Progenitor Cells. PLoS ONE 6(8): e23266. doi:10.1371/journal.pone.0023266.

Abstract: Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells.

hNP1_STEM_CELL_MARKERS_IF_IHC

Figure 1. Defining the stem cell phenotype using immunocytochemistry and flow cytometry.Phase contrast image of hESCs (A), hNPs (B), and hMPs (C). hESCs express pluripotency markers: Oct 4 (D,GG, JJ), SSEA-4 (G), and Sox 2 (J,GG); lack expression of Nestin (M, JJ), CD 166 (P,DD), CD73 (DD), and CD105 (AA). hNPs have low expression of pluripotency markers: Oct 4 (E,KK), SSEA-4 (H); and mesenchymal markers CD 166 (Q,EE), CD73 (EE), and CD105 (BB). hNPs express neural markers: Sox 2 (J,HH) and Nestin (N,HH,KK). hMPs lack expression of pluripotency markers: Oct 4 (F,LL), SSEA-4 (I), and Sox 2 (L,II); however, hMPs express Nestin (O,II,LL), CD 166 (R,FF), CD73 (FF), CD90 (CC) and CD105 (CC). All the cells have been stained with the nuclear marker DAPI (blue) in panels D- P. Scale bar: 10 µm. In the dot plots, red dots indicate isotype control or secondary antibody only; black dots indicate the antigen staining. doi:10.1371/journal.pone.0023266.g001

 By comparing hESCs, hNP cells and hMP cells, we have identified glycan structures that are unique to hNP cells: GalNac end groups (VVA), α-linked N-acetylgalactosamine (DBA), and fucose moieties α-linked to GlcNAc (LTL). Future studies help in identifying the roles of these glycans in cell maintenance, proliferation and differentiation fate.

I will keep you posted on these future Studies.

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.

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.

STEMEZ hNeural Progenitors and Cell Migration

I first featured Dr. Steve Stice in August 2008. I have since done follow up posts based on the excellent studies they have been conducting using our  STEMEZ (TM) Human Neural Progenitor & Neuron Discovery Kits.

I would like to highlight a poster based on research Steve and his Team conducted with Platypus Technologies.

Allan C. Powe, Jr., Kathryn L. Hodges, Jamie M. Chilton, Scott Gehler, Renee L. Herber, Keren I. Hulkower, Steven L. Stice. Identification of stimulators and inhibitors of cell migration in human embryonic stem cell derived neural progenitors using a novel, high throughput amenable assay platform.

Investigates the migratory behavior of an adherent monolayer neural progenitor cell line derived from human embryonic stem cells (hNP1 ™; ArunA Biomedical)using a novel 96‐well based cell migration assay platform (Oris™ Cell Migration Assay; Platypus Technologies) amenable for high throughput screening. The assay platform uses stoppers to create central exclusion zones within the wells; cells are plated outside the zone and migrate inward once the stopper is removed.

Data suggest this is a tool for understanding proper nervous system development, development of therapies for cell migration defects, and identifying novel environmental neurotoxicants.

Conclusions:
—The hNP1™ Oris™ Cell Migration Assay can quantitatively detect both stimulators and inhibitors of cell migration.
—Method development to date indicates that the assay has the potential for adaptation as a homogenous HTS‐suitable cell‐based assay.
—Preliminary results suggest that bFGF alone has a potent chemokinetic effect while LIF and GDNF act synergistically to drive migratory behavior during dopaminergic differentiation.