Network vs Individual Bursting Neurons

Motor Neurons and MEA
Dysregulated bursting is at the root of many motor neuron/neuromuscular junction disease. ArunA Biomedical teaming with Axion Biosystems have generated relevant bursting data from our Mouse Motor Neurons cultured on Axion-Bioystem’s Maestro MEA.

Figure: Mouse Motor Neuron Network Modulation by Bicuculline-ckeck out the entire presentation to learn more: GFP+ Motor Neurons: Development and in-vitro Functional Assessment on Microelectrode Arrays

Protocol User’s Guide for Culturing Motor Neuron on MEA(pdf – 679Kb)

Name Catalog # Type Species Applications Size Price
Motor Neurons-GFP+ Quick Start Kit mMN7205.QS Primary Neurons M Cell Assays 750,000 $349
Motor Neurons-GFP+ HTS Kit mMN7205-HTS Primary Neurons M Cell Assays 4 X 750,000 $989
GDNF (Human, Mouse) PR27022-2 Protein H; M 2 ug 10 ug $108 $205
AB2™ Basal Neural Medium AB27011.3 Cell Growth Media H; M Cell Assays 500 ml $69

We will continue providing you content we believe important. Should you have questions, do not hesitate to contact us. Thank you and we stand ready to serve you and your team.

Pete Shuster-CEO and Owner, Neuromics, 612-801-1007, pshuster@neuromics.com

Neuromics’ hNP1 Neural Progenitors, DJ-1 and Stroke

Dr. Cesar V. Borlongan, University of South Florida and a team of researchers have successfully identified DJ-1 as a potential therapeutic target for treating stroke. They used our hNP1TM Human Neural Progenitors to confirm the neuroprotective properties of the DJ-1 protein: Yuji Kaneko, Hideki Shojo, Jack Burns, Meaghan Staples, Naoki Tajiri, Cesar V. Borlongan, DJ-1 ameliorates ischemic cell death in vitro possibly via mitochondrial pathway, Neurobiology of Disease, Available online 21 September 2013, ISSN 0969-9961, http://dx.doi.org/10.1016/j.nbd.2013.09.007.

hNP1 Human Neural Progenitors in Culture

hNP1 Human Neural Progenitors in Culture

Highlights

•DJ-1 translocation was assayed in oxygen–glucose deprived human neural progenitor cells.
•Immunofluorescent microscopy and ELISA were used to measure DJ-1 translocation.
•DJ-1 translocated preferentially into polarized mitochondria.
•DJ-1 translocation is associated with the preservation of functional mitochondria.
•DJ-1 exhibits antioxidative stress effects following ischemic stroke.

I will continue to post updates on Research success with our Cell Based Assay Solutions.

Inflammatory Macrophages in ALS Spinal Cord

In my many conversation with Neuro-disease researchers, I often learn of discoveries that beg to be shared. I have been collaborating with Dr. Milan Fiala to explore how our hN2 Primary Human Neurons could be best used to study the role of inflammatory cytokines in amyotrophic lateral sclerosis (ALS). This would build on the excellent research he and his team are conducting at UCLA.

He shared with me that these inflammatory cytokines could be the bad actors in ALS. Specifically, in vitro, superoxide dismutase-1 (SOD-1) stimulates expression of inflammatory cytokines, including IL-1β, IL-6, and TNF-α, through activation of cyclooxygenase-2 (COX-2) and caspase-1. Further, they have discovered The lipid mediator resolvin D1 (RvD1) inhibited IL-6 and TNF-α production in ALS macrophages with 1,100 times greater potency than its parent molecule docosahexaenoic acid. ALS peripheral blood mononuclear cells (PBMCs) showed increased transcription of inflammatory cytokines and chemokines at baseline and after stimulation by aggregated wild-type SOD-1, and these cytokines were down regulated by RvD1. Thus the neurons are impacted by macrophages expressing inflammatory cytokines. RvD1 strongly inhibits in ALS macrophages and PBMCs cytokine transcription and production. Resolvins offer a new approach to suppression of inflammatory activation in ALS. To learn more see: Guanghao Liu, Milan Fiala, Mathew T. Mizwicki, James Sayre, Larry Magpantay, Avi Siani, Michelle Mahanian, Madhuri Chattopadhyay, Antonio La Cava, and Martina Wiedau-Pazos. Neuronal phagocytosis by inflammatory macrophages in ALS spinal cord: inhibition of inflammation by resolvin D1.
Am J Neurodegener Dis. 2012;1(1):60-74.

Images: Co localization of TNF-a- and IL-6- expressing macrophages with caspase-3-and the chemokine RANTES (CCL5) – stained neurons in ALS and control spinal cords. Frozen sections of ALS and control lumbar spinal cord were stained with anti-NeuN (red), anti-CD68 (green), anti-caspase-3 (magenta) or anti-RANTES (magenta), and DAPI (blue) (Immunofluorescence microscopy (20X)). The experiment was repeated with 2 other ALS spinal cords and 2 other control spinal cords and yielded comparable results.
Photomicrographs are shown in 2 patients (A, B, C, D) and 2 controls (E, F). (A) Co
localization (yellow) of TNF-a-positive (magenta) and (CD68-positive, green) macrophages with NeuN–positive (red) neurons; (B) Co localization (yellow) of IL-6-positive (magenta) and CD68-positive (green) macrophages with NeuN–positive (red) neurons; (C) Co localization of macrophages (CD68-positive, green) with apoptotic, caspase-3-positive (magenta) and non-apoptotic (caspase-3-negative (red)) neurons. Eight neurons are impacted by macrophages; 3 neurons are caspase-3-positive (arrows) and 5 neurons are caspase-3- negative (asterisk); (D) Co localization of macrophages (yellow) with RANTES-positive (magenta) and CD 68-positive (green) macrophages with NeuN-positive (red) neurons. (E&F) No macrophages (green) are detected in 3 control spinal cords. (G&H) The table shows that in three ALS spinal cords 19.2 +/−4.8% NeuN-positive (red) neurons co localize with TNF-a -positive (magenta) macrophages (green) and 18.5 +/− 4.9 % NeuN-positive (red) neurons co localize with IL-6-positive (magenta) macrophages (green), whereas in control spinal cords 0% neurons (red) co localize with macrophages (green).

I will keep you posted on progress.

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.

Studying Apoptosis In Tumors

I have featured Gary Johnson here.

I value my partnership with his compant, ICT. They provide our customers with potent and research proven Apoptosis Kits and Methods. Here we feature publications referencing our MitoPT™ Kits. These Kits easily assess changes in mitochondrial membrane potential. Changes in mitochondrial membrane potential can correlate with cytochrome c release and the initiation of apoptosis.
A431 cells, treated with predetermined IC50
concentration of novel anticancer agents, fluoresce green and orange-red with MitoPT JC-1. Data courtesy of Zayas/ Carro, Universidad Metropolitana.
Anticancer Effects of Alpinia pricei Hayata Roots.
CL Hsu, YS Yu, GC Yen. J. Agric. Food Chem., Jan 2010, 58 (4), pp 2201–2208.

Anticancer Effects of Flavonoid Derivatives Isolated from Millettia
reticulata Benth in SK-Hep-1 Human Hepatocellular Carcinoma Cells.

SC Fang, CL Hsu, HT Lin, GC Yen. J. Agric. Food Chem., Jan 2010, 58
(2), pp 814–820.

Mechanisms of Apoptotic Effects Induced by Resveratrol,
Dibenzoylmethane, and Their Analogues on Human Lung Carcinoma Cells.

CJ Weng, YT Yang, CT Ho, GC Yen. J. Agric. Food Chem., Jun 2009; 57
(12), pp 5235–5243.

Gary Johnson-Apoptosis Ace

 Gary Johnson

1994-present-President, ICT

1993-1996-Conjugation Chemist, R&D Systems

19989-1993-Supervisor Protein Conjugation & ELISA Development Group, Solvay Animal Health

1986-1989-Immunologists, Biosciences Lab, 3M

1976-1986-Various Lab, U of MN

Gary’s Conatct Info:

Inventing Better Ways to Measure Apoptosis 

This profile features another Scientist Entrepreneur. Dr Gary Johnson is the Founder and President of Immunochemistry Technologies LLC (ICT). His company manufactures kits that have the capabilities to quantitatively measure apoptosis effects. This is important to Neuromics, because these are core to many diseases of research interest to our customers. These range from Cancer where apoptosis detection can be used to to visualize the efficacy of tumor killing therapies to Neuroscience where apoptosis could be a root cause of many cognitive and neuro-muscular diseases.

I am excited about featuring Gary. I have been working with him and his team over the past 5 years. They have actively supported my company in providing Apoptosis Research Kits. The strength in our relationship is built on his company supplying best of breed reagents. The feedback I receive from users is overwhelmingly positive. In addition to these kits, ICT is also recoginized for their rock solid ELISA Buffers and Diluents.

It takes a unique blend of business and scientific acumen to build a company like ICT. So let’s start with Gary’s background and experience and then on to the specifics on his company and products and what sets ICT apart from competitors.

Gary’s Background

Gary’s began his career at the University of Minnesota in 1978 where he worked in a variety of labs. There he gained a wealth of experience and expertise in research techniqes. These included chromatography, immunoelectrophoresis, radiolabeling, mass spectrometry,  proton NMR spectroscopy and western blotting.

He leveraged his abilities and became more deeply involved in immunobiology. He  joined Dr. Harry Orr’s lab in 1981. There he used recombinant DNA techniques to study the class I genes of the major histocompatibility complex and he also supervised the tissue culture work. This provided the stepping stone to Dr. David Klein’s lab in 1984. There he studied the difference between diabetic and non-diabetic glomerular basement membrane proteoglycans in kidney disease. In order to do this research Gary developed in vivo or in vivo labeling techniques.

Gary then moved from University to commercial labs. We will see how his growing expertise morphed into the founding of ICT and why his broad knowledge and experise enabled a successful launch of the company.

From 1986 until founding ICT Gary worked at 3M, Solvay Animal Health and R&D Systems. Over his tenure, he worked as an Immunologist, Supervised an ELISA and Protein Purification and was a Conjugation Chemist. Having mastered a unique range of basic and commercial bio-research techniques, the evolution to Scientist-Entreprenuer was a natural next step.

In 1994, Dr. Brian Lee and Gary launched ICT. The company’s early success was in contract assay development. The revenue generated from these programs, has enabled ICT to manufacture and release a growing catalog of Apoptosis Detection Kits.

ICT’s Products and Capabilties

ICT’s provides proprietary probes for measuring apoptosis in vitro and in vivo. These probes are used by researchers  to detect caspases, cathepsins, serine proteases, cholinesterase enzymes, and assess mitochondrial health.Applications include: assessing the efficacy of chemotherapy, to quantifying  neurodegeneration, and early detectionof eye disease, to name a few.

Specific Products Include:

keratconus1

Images: Normal (left) and keratoconus (right) corneal fibroblasts were labeled with Caspase 3 & 7 Assay Kit, green.

Pacing the Field

ICT is setting the pace in Apoptosis Detection by  recognizing and resolving issues inherent in competitive offerings. These include:

  1. Difficulty permeating cells.
  2. High background problems.
  3. Does not bind to early stage apoptotic cells.
  4. Not as sensitive as a cell permeant inhibitor probe.
  5. Does not bind to all apoptotic tumor cells (Dicker, Cancer Biol. Ther., 2005. 9:1014-1017).
  6. Binds positively to normal and healthy bone marrow derived cells (Dillon, J. of Immunol., 2001. 166:58-71).
  7. Many in vitro protocols involve lysing the red blood cells before running flow cytometry, this method results in the binding of Annexin V to all of the cells in the sample (Tait, Blood, Cells, Molecules, and Diseases., 1999. 25:271-278).  The inversion of PS and cells containing large amounts of PS may not be related to apoptosis and this adds to the background issues.
  8. Does not measure a process of apoptosis, but rather an effect of apoptosis.

Capabilities that will enable them strengthen their leadership position include:

  1.  Uses a cell permeant probe that can easily penetrate tissues and cells.
  2. Very sensitive.
  3. Specific, no reported false positives.
  4. It is a direct measurement of an intracellular process of apoptosis, detects only active caspases and caspase active cells are always apoptotic.
  5. Passage through the blood-brain barrier has been demonstrated.
  6. Passage through the blood-retinal barrier has been demonstrated.
  7. No background problems when injected intravenously.
  8. Detects very early through late stage apoptosis.

ICT is continuing to invest heavily in developing new capabilties. Gary highlighlighted some of the breakthroughs that are on the horizon. I plan on announcing these as they become public.Stay tuned.

Advancing the Study of Apoptosis

gary-johnson1Featuring Gary Johnson and his team at Immunochemistry Technologies LLC.

The ability to accurately measure apoptosis processes is a core  research component for many of our customers and colleagues.  Neuromics has leveraged our growing partnership with Gary and ICT to meet the exacting requirements of  Researchers studying apoptosis.

I am excited to be featuring Gary and his company in our June News Behind the News. Gary and his team have

Polycaspase Apoptsis

Polycaspase Apoptsis

proven to me time  and again the ability to deliver methods and kits that meet our customers’ needs.  I can count on the feedback to be positive and use to expand in user labs.

In the feature, I will provide details of Gary’s unique background. The path that lead him to founding ICT and the development of current capabilities. Most importantly, I will provide a glimpse of coming new methods and products. These could significantly improve the development of therapies for diseases that involve aptotosis.

Image: Jurkat cells dually stained with Hoechst and Polycaspase Assay Kit, green-FAM-VAD-FMK. Caspase activity is revealed by green fluorescence in cell #2, indicating that only this cell is apoptotic. Cell #1 is also dying (scattered blue), but is not apoptotic because it is not green. Cell #3 is healthy (concentrated blue nucleus).