Musculoskeletal Disorders-Stem Cell Based Drug Discovery

A common Neuromics’ theme is harnessing the power of cellsTM. The raw cost of the cells are often the biggest consideration. We encourage our customers to focus on true costs. These include the # of cells (how many times can they be passaged?), culture viability (how long do the cells live) and bioactivity (how closely do cultures mimic in vivo behavior?). I would like to present a presentation and publication confirming our competitive advantage when analyzing true costs.

Setting a higher bar for Neuron-Glial Based Assays!

Dr. Randen Patterson and his team at UC Davis have developed new culturing techniques using our e18 Rat Primary Hippocampal Neurons. They have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons. This is good news indeed: Todd GK, Boosalis CA, Burzycki AA, Steinman MQ, Hester LD, et al. (2013) Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons. PLoS ONE 8(4): e58996. doi:10.1371/journal.pone.0058996.

We will continue to raise the bar. Better cultures=lower costs and better outcomes!

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

Claudia Zylberberg-Cell Culturing Innovator

Taking You Cultures to New Dimensions

I am pleased to feature Dr. Claudia Zylberberg, President and CEO of Akron Biotech, in this edition of “News Behind the News”.  She is an expert and innovator in providing tools and methods for the discovery and development of cell based therapies.  This starts with potent cell based assays and culminates with the ability to provide GMP produced reagents to support animal testing and other pre-clinical trial drug discovery processes.

Scientist and Entrepreneur a Synopsis

Claudia has a background that uniquely positions her to understand and address the growing needs and requirements of the basic and drug discovery research community. This includes researchers using stem cells as for discovery and potential therapies. Here’s an overview.

With a PhD in Biotechnology from the University of British Columbia and University of Buenos Aires, Claudia has over 25 years of experience in the international biopharmaceutical industry.  At NABI Biopharmaceuticals, she and her team developed and scaled plasma-derived products and recombinant vaccines. This included harmonizing products between EMEA and FDA. She has authored and co-authored many scientific articles and developed several commercial products for use in the field of cellular biology. She has also authored a children’s book on genetics and has several patent pending products in the area of cryopreservation and QC of stem cells.

She is as an advisor for the US Pharmacopoeia in standards setting for biologics and ancillary materials critical for the production of cellular therapies.  She is a member of the BioFlorida Board of Directors, Board member of Business Development Board of Palm Beach County, Scientific Advisor for ISCT (International Society for Cell Therapy), Executive Committee Member for the Alliance of Regenerative Medicine and the Chair of the Business Advisory Board for the Banner Center of the State of Florida and is part of the organizing committee for the World Stem Cell Summit coming up in Palm Beach December 2012.

Excellence in Cell Based Assays

Excellent in cell based assays means lower research and development costs. There are two sides of the “cost coin”. On one side, if any of the raw materials (plates, cells, media, growth factors, markers, probes or detection, etc. are a weak link), the whole chain is destroyed and all time and material cost are wasted. On the other side, if culture conditions do not promote an environment enables in vivo like conditions, the data may prove to be unsupported in pre-clinical testing. This results in big costs in both opportunity and related expenses.

This is why Akron Biotech’s product and expertise are so important.  This is also why they could become important partners for Neuromics.  They have the ability to deliver a large cross section of the capabilities required for excellence in cell based assays. 

These capabilities include:

  • Best manufacturing practices (GMP) guarantee products will work as expected.
  • Delivery of tools and methods that support research from the bench to pre-clinical studies.
  • Product strategies that insure current and future fill known gaps in driving cell based assay excellence.

These includes:  media, growth factors, 2D/3-D culturing ECMS and Polyfibers, recombinant proteins and cryopreservatives.  Many fit hand in glove with my strategic offerings. I plan on continue to publishing update on new developments from Akron Biotech.

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.

Nanofiber 3-D Cell Based Assays

This “News behind the News” is a historic event.  It demonstrates how nanofiber scaffolds can be used to engineer organs for human transplants. Good news for researchers looking solutions are in vivo like environments for cell based assays.

Nanofibers Solutions work in transplants-imagine how well they will work in your 3-D based cell based assays.

3-D Cell Based Assays for Drug Discovery are the future. Like any new model, adoption rates are a function of how well the new solutions works. “The proof is in the pudding”.

Here’re highlights of a historic event based on transplants using nanofiber engineered laryngotrachea : Collaboration between Nanofiber Solutions and the Karolinska Institutet produces first synthetic laryngotracheal implants seeded with the patient’s stem cells to be successfully transplanted into human patients in Russia.

COLUMBUS, Ohio, June 26, 2012 – Nanofiber Solutions, LLC, an Ohio-based developer, manufacturer and marketer of 3-D synthetic scaffolds to advance basic research, tissue engineering and regenerative medicine announced today the first and second successful transplants of its tissue engineered laryngotracheal implants seeded with cells from the patients’ bone marrow.

The surgeries were performed June 19th and 21st at the Krasnodar Regional Hospital (Russia) by Dr. Paolo Macchiarini, Professor of Regenerative Surgery at the Karolinska Institutet (Stockholm, Sweden), and colleagues. Dr. Macchiarini led an international team that included Dr. Vladimir Porhanov, head of Oncological and Thoracic Surgery at Kuban State Medical University (Russia), Dr. Jed Johnson, Nanofiber Solution’s Chief Technology Officer who created the synthetic organs, Harvard Bioscience (Boston, USA) who produced the bioreactor, and Dr. Alessandra Bianco at University of Rome, Tor Vergata, who performed mechanical testing during scaffold development.

Both patients, a 33 year-old mother from St. Petersburg and a 28 year-old man from Rostov-on-Don, were in au to accidents and suffered from a narrowing of the laryngotracheal junction for which they already had failed previous surgeries. Transplantation was the last option for the patients to have normal quality of life. Immediately following transplantation, both patients were able to speak and breathe normally.

Nanofiber Solutions, lead by Dr. Johnson, designed and built the nanofiber laryngotracheal scaffolds specifically to match the dimensions of each patient’s natural larynx and trachea, while Harvard Bioscience provided a bioreactor used to seed the scaffold with the patients’ own stem cells.  Although this procedure represents the world’s first and second successful use of synthetic synthetic laryngotracheal implants, it is Nanofiber Solution’s second and third successful organ implants using their synthetic scaffolds within the last year.

Nanofiber Solutions’ scaffolds mimic the body’s physical structure and allow for a more successful seeding, growth and differentiation of stem cells. Because the cells used to regenerate the larynx and trachea were the patients’ own, doctors report there has been no rejection of the transplants and the patients are not taking immunosuppressive drugs. (more).

Capabilities of 3-D nanofiber scaffolds for cell based assays:

Human brain tumor biopsy showing migrating tumor cells along the alligned nanofiber.
  • Nanofibers are optically transparent to allow for live-cell imaging and real time quantification of cell mobility using an inverted microscope
  • Nanofibers mimic the 3D topography found in vivo which produces a more realistic cellular response to therapeutics.
  • More realistic cellular behavior means you can use fewer animals and decrease time-to-market for drug discovery and development.
  • Nanofibers can easily be coated with ECM proteins using existing protocols for standard lab ware.
  • Cells can be easily removed for protein or gene analysis using trypsin, EDTA, etc.
  • We will continue posting relevant press releases, pubs and data that prove the capabilities of these important solutions.

    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.

    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.