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.

 

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

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.

“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.

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.

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.

In Search of Remyelination Therapies

Multiple Sclerosis (MS) is an inflammatory disease with no known cure. It affects over 400,000 people in the US and over 2.5 million people worldwide and is the leading cause of non-traumatic neurological disability in North America.

It is a chronic and brutal disease that attacks the brain and spinal cord. MS symptoms are due to the damage or loss of myelin sheath that surrounds, isolates and protects axons of brain and spinal cord. The results are often debilitating and afflict most sufferers in the prime of their lives. The annual costs to slow the disease and treat related
symptoms are in the billions of dollars and rising. There are currently no therapies to reverse damage of MS. At this point, there are only immune suppressive therapies that slow attack on the myelin sheath.

It is with hope and optimism that I present Dr. Satish Medicetty and his company, Renovo Neural Inc. (RNI) in this edition of the “News Behind the Neuroscience News”.

I became aware of Satish and his company in my search for Stem Cells that would broaden Neuromics ability to serve early phase Neuroscience Drug Discovery.

Satish Medicetty

Apr 2010 – Present: President and Board Director Renovo Neural Inc.

June 2008 – Mar 2010: Director of Stem Cell Research and Lab Operations
NeoStem Inc

July 2005 – June 2008: Senior Scientist Athersys

2006 – 2008: MBA, Case Western University

2002 – 2005: PhD, Kansas State University

After my first conversation with him, I was impressed with the capabilities RNI offered.

RNI

The company, founded in 2008 with a$3 million grant from the State of Ohio’s Third Frontier Commission, is leveraging cutting edge research from Dr. Bruce Trapp’s lab at the Cleveland Clinic.

At the core, RNI offers pioneering and propriety assays that give Drug Discovery Companies the ability to screen small molecules and compounds that could be lead therapy candidates for MS and other myelin-related diseases. These screens use a type of stem cell called adult oligodendrocyte precursor cells (OPCs).

The Power of OPCs

So what makes these OPCs an engine for finding cures for MS?  Inflammation associated with MS attacks destroys cells called oligodendrocytes that produce myelin. The only way to reverse this autoimmune related process is for the brain to produce healthy cells that can catalyze re-myelination. Enter OPCs.

OPCs are the raw material for processes the central nervous system uses to manufacture oligodendrocytes.  The brain’s inability to produce enough healthy cells to keep up with the destruction is a root cause of the disease. Understanding how to kick start and keep the oligodendrocyte factory running is a key to reversing this relentless destruction.

Delivering Value

RNI has the capabilities to the decrease time required and increase the odds for discovering potential MS therapies.  They have the raw material (OPCs) and the know how to encourage their transformation into myelinating cells. This expertise can be utilized can be used then to rapidly test new compounds both in vitro and in vivo.

In Vitro Assays Example

The features of their in vitro assays include:

• Stringent protocols to generate relatively homogeneous (>85% pure) and consistent population of OPCs as a reliable starting material for HCS assays
• Relatively high throughput primary screen to identify potential candidates that promote OPC proliferation and/or differentiation
• Secondary screen to confirm and qualify compounds for further pharmacological testing
• Positive and negative controls that demonstrate the utility of HCS assays to identify lead candidates that promote OPC proliferation and differentiation.

The features of their in vivo cuprizone assays include:

• Stringent protocols to generate highly reproducible demyelination/remyelination cuprizone model
• Cuprizone model recapitulates the in vivo process of demyelination and remyelination in the brain.
• Cuprizone model provides consistent and accurate results for key regions of the brain that are affected in MS patients including both white and gray matter regions – corpus callosum, hippocampus and cortex.
• Proof of concept studies demonstrate the utility of our in vivo remyelination assays to evaluate preclinical efficacy of potential remyelination therapies

The end goal is to discover therapies that repair neurons damaged by MS via remyelination and to get them in the hands of people that need them. I will keep you posted on their progress.

Obesity Energy, Thermogenesis and Appetite

Dr. Richard Rogers

In my routine follow up with researchers using our reagents, I started to get an appreciation on how these complex energy pathways are being unraveled and better understood. That appreciation forms the roots of this “News Behind the Neuroscience News” story. It is a story that has the hormones Leptin and TRH playing a starring role supported by hindbrain neuro/glial-circuitry and brown adipose tissue (BAT).

The Rogers Lab

I became aware of the Richard Rogers Lab in my follow up with Montina Van Meter checking on our  LepRb/OBRb antibody. She shared that it was giving them results better than most others they has used. She then gave me an overview of her research involved with  the control of feeding behavior and energy utilization including thermogenesis (“heat generation”) catalyzed in BAT.  Cool…this was a lab we wanted to make sure we served and served well.

Tina not only kept me informed on how our reagents were working, but also generously alerted to me publications referencing use of our reagents:
·        Maria J. Barnes, Richard C. Rogers, Montina J. Van Meter and Gerlinda E. Hermann. Co-localization of TRHR1 and LepRb
receptors on neurons in the hindbrain of the rat. doi:10.1016/j.brainres.2010.07.094…Included are excellent images of stained LepRb (OB-Rb)-(Dilution 1:500) and  GAD1-Dilution (2ug/ml) expressing neurons localized in loose clusters of cells in the DMN, NST, and the VLM…
·        Hung Hsuchou, Yi He, Abba J. Kastin, Hong Tu, Emily N. Markadakis, Richard C. Rogers, Paul B. Fossier, and Weihong Pan. Obesity induces functional astrocytic leptin receptors in hypothalamus. Brain, Mar 2009; doi:10.1093/brain/awp029…unique sequence of ObRb at its cytoplasmic tail (CH14104, Neuromics, Edina, MN, USA). This antibody was raised
against rat ObRb…

I found this research to be unique and intriguing. This led me to an interview with Dr. Rogers. Here is his backstory.

Beginnings

Dr. Rogers credits serendipity as a driving force in his interest in Neuroscience. It started with a bike ride and chance introduction with a ham radio operator when he was a youngster. This catalyzed his interest in electronics and circuitry.

This interest morphed to a passion for Neuroscience (circuits and signaling). He entered the first college program devoted to Neuroscience studies at UCLA.  He received his Ph. D. in 1979. His post-doc focused on digestive regulation. Here, he  investigated the neural circuitry involved in the normal control of gastric function.

Evolutions

In collaboration with his wife Dr. Gerlinda Hermann, his work evolved to solving the mystery of why we don’t eat (abnormal gastric function). What causes gastro-intestinal shutdown?  The breakthrough was their ability to show cross-talk between the immune system and nervous system. This research is a foundation for the discovery of therapies for sufferers of appetite shut down cause by cancer therapies and certain immune related pathologies.

The main culprit is TNF-α. The blood level of this peptide is elevated as a consequence of immune activation caused by infection, cancer, radiation exposure and chronic autoimmune disease. The breakthrough was showing that  TNF-α receptors are on neurons in the brainstem that control gastric functions, including emesis.  Neurons in the nucleus of the solitary tract (NST) respond to TNF-α greatly increasing the sensitivity of gastric vagal control circuitry.  This causes emptying of the gut to dramatically slow,l leading to nausea, vomiting and cessation of appetite.

Currently, they are delving into the complexities of  TNF-α signaling processes. This includes the role of astrocytes and glia.

See: Gerlinda E Hermann and Richard C Rogers.  TNF activates astrocytes and catecholaminergic neurons in the solitary nucleus: implications for autonomic control. doi: 10.1016/j.brainres.2009.03.059.

 Energy,Obesity and Thermogenesis-Active Astrocytes

Recently, the lab took another road less traveled. Dr. Gerlinda Hermann discovered interesting aspects of leptin and thyrotropin releasing hormone (TRH) signaling.  This research looks at signaling in thermogenesis and feeding behavior. A most interesting aspect includes conclusions concerning the role of astrocytes.  Their colleague Dr. Weihong Pan  showed that adult obese mice, (2 months after being placed on a high-fat diet) showed a striking increase of leptin receptor (+) astrocytes, most prominent in the dorsomedial hypothalamus and arcuate nucleus. Agouti viable yellow mice with their adult-onset obesity showed similar changes, but the increase of leptin receptor (+) astrocytes was barely seen in ob/ob or db/db mice with their early-onset obesity and defective leptin systems. The results indicate that metabolic changes in obese mice can rapidly alter leptin receptor expression and astrocytic activity, and that leptin receptor is responsible for leptin-induced calcium signalling in astrocytes. This novel and clinically relevant finding opens new avenues in astrocyte biology (doi: 10.1093/brain/awp029).

Non-shivering thermogenesis usually occurs in BAT. It uncouples the ATP energy producing process by generating heat rather than driving the conversion of ADP to ATP. This creates an ingenious way to untangle complex processes related to Leptin signaling. What happens when there is sufficient energy for the thermogenic process? Conversely, what happens when there is insufficient energy?

 Leptin and TRH act synergistically in the hindbrain to drive thermogenesis. However, in a starving condition there is a subsequent drop in Leptin and thermogenesis. Behind these simple facts are complex processes that occur in the hindbrain. The team is providing important insights including location of events and relevant signaling molecules. (doi:10.1016/j.brainres.2010.07.094).

The Future-Caged Compounds and Live Cell Signaling

Caged compounds are bioactive molecules attached to photolabile groups, that release the active component on contact with photons of the right energy level – the process of photolysis. Photolysis is now widely applied in biology to induce neurotransmitter and otherm ligand-receptor interactions in conditions that are otherwise subject to poor diffusional access and receptor desensitization, as well as for labile ligands.

This novel technology affords Dr. Rogers and his team the capability do live cell imaging of calcium signaling. From these they will help us gain a deeper understanding of what is happing and where. Specifically, we will more exactly learn the role that astrocytes and glia play in controlling the role of   Leptin and related signaling molecules in controlling energy, metabolism and feeding behavior. This could lead to important target for future therapies.

I also wanted to share a recent article on yet another intersection which focuses on thermogensis which occures in brown adipose tissue (BAT): Maria J. Barnes, Richard C. Rogers, Montina J. Van Meter and Gerlinda E. Hermann. Co-localization of TRHR1 and LepRb receptors on neurons in the hindbrain of the rat. doi:10.1016/j.brainres.2010.07.094.

Example images: Distribution of LepRb+ fibers in hindbrain. LepRb-ir (red) fibers and varicosities are seen among TRHR1-ir (green) cells and fibers. These red and green fibers are adjacent and co-mingle but do not show co-localization of receptors. This pattern is seen in (A) fascicles of the solitary tract (ST); (B) raphe pallidus (RP), and (C) raphe obscurrus (RO). (D) Border between the medial solitary nucleus (NST) and the area postrema (AP; white dashed line) showing an abundance of LepRb-ir (red) fibers and
 neurons (white arrows for selected neurons) in the NST but not the AP. (E) LepRb-ir staining is suppressed by pretreatment of tissue with LepRb epitope blocking peptide. (F) TRHR1-ir staining is suppressed by treatment with excess TRHR1. Scale bar A–D=100 microns; E, F=300 microns. cc=central canal. Note: this pus references use of our LepRb (OB-Rb) and GAD1 antibodies.

Drilling down further, I am pleased to present Electro-physiology and related data generated by Aruna and collaborators: hN2 Cells-Electro Phys Data Supplement

hN2-Whole Cell Voltage Clamp

Figure. hN2 cells can produce inward currents that generate action potentials. (A) Isolated hN2 with significant neurite growth 1 week  after plating . This cell was subjected to whole cell voltage clamp utilizing a potassium gluconate based intracellular solution. (B) Voltage gated inward and outward currents were elicited from this cell with depolarizing voltage steps. (C) Inward currents from another cell (potassium gluconate intracellular) were abolished by local application of 1 µM tetrodotoxin (red trace) while outward currents remained. Inward current recovered as TTX washed out of the region (green trace). (D) A different cell which exhibited voltage activated inward currents that inactivated in response to a 50 ms prepulse at different membrane potentials. The experiment was done 27 days after the removal of bFGF. A cesium gluconate based intracellular solution was used for this experiment to block outward potassium currents. The membrane potential for half maximal inactivation by standard Boltzman fitting (red line) was -40.1 mV with a slope of 4.7. (E) Recovery from fast inactivation utilizing a paired pulse protocol in the same cell as C. The single exponential time constant for recovery of inactivation was 1.7 ms (red line). (F) A different cell which elicited an overshooting action potential upon current injection under whole cell current clamp utilizing a potassium gluconate based intracellular solution. Inset: Response of the same cell under voltage clamp to a change in membrane potential from -80 mV to -10 mV elicited a peak current of 457 pA. Scale bars for inset: 5 ms, 0.2 nA.

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:

• gary@immunochemistry.com
• 952-888-8788  

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. Brain 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:

• FLIVO™ Polycaspase Live!, in vivo Apoptosis Kits
• 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
  • Measures chymotrypsin-like protease
    activation in whole living cells.
• Magic Red™ Real Time! Kits
  • Measures apoptosis in whole living, intact cells – no lysis required
• MitoPT™ Kits
• Quantitate mitochondrial functionality and apoptosis

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.

It was further confirmed  in Studies conducted by Dr. Philippe Serrat and his team at University of Sherbrooke.

Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); Jul;16(7):1331-9. Epub 2008 Jun 3 doi:10.1038/mt.2008.98.

Using ultra low dose of DsiRNAs complexed with Neuromics’  i-Fect , they were able to successfully reduce NTS2 gene expression by up to 86% in rat lumbar Dorsal Root Ganglia after only two intrathecal injections. This was confirmed by Western Blot and qPCR analysis.

We now have further confirmation of the capabilities of this delivery platform in a just released publication by Dr. Jeffrey Mogil and team:

Michael L. LaCroix-Fralish, Gary Mo, Shad B. Smith, Susana G. Sotocinal, Jennifer Ritchie, Jean-Sebastien Austin, Kara Melmed, Ara Schorscher-Petcu, Audrey C. Laferriere, Tae Hoon Lee, Dmitry Romanovsky, Guochun Liao, Mark A. Behlke, David J. Clark, Gary Peltz, Philippe Séguéla, Maxim Dobretsov and Jeffrey S. Mogil. The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test. doi:10.1016/j.pain.2009.04.028.

IT Delivery of siRNA in vivo supplement