Biopharma musculoskeletal disorders_4-30-2013 from Pete Shuster

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!

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 37^OC 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

They are designed for:

• Osteogenesis/Bone Formation Studies
• Compound/Small Molecule Testing
• Gene Expression Analysis

Images: (A) Human cord-blood MSCs were expanded in low-serum MSC-Gro^TM to confluence as shown here. (B) were differentiated in osteogenic MSC-Gro^TM. Early stage osteoblasts are shown here; the arrow shows early formation of mineralized matrix. (C)&(D) Mature osteoblasts stained positive for Alizarin red. Phase contrast image at 200 x, scale bar is 50 mmeters.

I will continue to update you on new Cell Based Assay Solutions.

Phase contrast image (200x) of chondrocyte and associated cells in day five of culture as described (SC00A7 Protocol). Scale bar = 50 m. Chondrocyte is darker multicellular structure overlying attached cells, ~ 70 m in diameter.

Steve Stice (right) and Franklin West, UGA College of Agricultural and Environmental Sciences animal scientists, have developed a new method to measure the effects of environmental toxicants on cells, photographed in their lab on Monday, Dec. 3, 2012. (R

I have multiple posting on Dr. Steve Stice and his work at ArunA Biomedical and UGA. He and his team developed our hNP1™ Human Neural Progenitor & hN2™ Neuron Discovery Kits and hMPro™ Human Mesenchymal Progenitors (hMPC) Kits. These kits are increasingly becoming important for basic human disease research and drug discovery.

I am pleased to report here that he and his colleagues at UGA have developed a better way to study the effects of environmental toxins on fetal development.  Franklin West and Steve Stice, animal scientists in the UGA College of Agricultural and Environmental Sciences, and Mary Alice Smith, a toxicologist in environmental health science in the UGA College of Public Health, began testing the efficiency of a technology West and Stice developed to measure the effects of environmental toxicants. They use cells that come from stem cells and represent the early cells that eventually will form sperm cells in adults. The cells are highly vulnerable to toxicants. Their findings were published recently in Toxicological Sciences: Franklin D. West, W. Matthew Henderson, Ping Yu, Jeong-Yeh Yang, Steven L. Stice, and Mary Alice Smith. Metabolomic Response of Human Embryonic Stem Cell–Derived Germ-like Cells After Exposure to Steroid Hormones. Toxicol. Sci. (2012) 129 (1): 9-20. doi: 10.1093/toxsci/kfs185.

Highlights: “There is no human system to study toxicant effects on early sperm development,” West said. “But, we can expose these human cells to different toxicants and predict how changes will impact birth defects and fertility later in life.”

Currently, most developmental toxicological studies are done using mice or rats. ”We need to move away from rodent tests because they often won’t tell us how detrimental or safe chemicals are in humans,” West said. “For example, the potential effects of BPA in humans, especially in the unborn child, is still hotly contested. This cell culture system moves us away from animal testing, which most agree is preferable as long as the studies are equally or more reliable.”

The new testing model fills a void left by current methods and provides human-specific results. ”Using animal studies, you are looking at more than a year to test a chemical in rodents,” Stice said. “Using this test, we get results in two to three weeks at most and possibly shorter.” Considering the vast number of untested chemicals humans are exposed to, with new ones coming out every day, animal testing will never be practical to prioritize which chemicals need further testing, Stice said. There are more than 80,000 chemicals in the environment that haven’t been tested for human impact mainly because of the cost and time required using current procedures.

The reliability of those results also must be considered.

“Mice don’t have three or four (of the) critically important genes that are leading causes of infertility,” West explained. “Sperm and egg cells are the only cells that go through meiosis, a special type of cell division necessary for sexual reproduction. You can’t use another type of cell to find the impact a chemical has on these cells and, in turn, fetal development.”

West first developed the germ-like cells during his doctoral work in Stice’s UGA laboratory.

“Our next step with the cells is to test more chemicals and see what happens with more compounds,” he said.

Stice also plans to use neural and bone forming cells he developed to test chemicals for developmental neurological and skeletal toxicology. Scientists at the Environmental Protection Agency already have demonstrated in peer-reviewed studies that these types of cells are very sensitive to known toxins such as lead and mercury. The team is now testing more chemicals on these cells.

“If the assay is predictive and repeatable, the chemical industry will have better, faster and less expensive test systems and, as a result, will reduce the use of animal testing and experiments,” Stice said. ”Testing more known chemicals for developmental toxicology will validate these assays for later use by the chemical industry. Our shared long-term goal is to ensure the health and well being of our children,” he said.

I will keep you posted on progress on these assays.  I anticipate commercially available assays will be an outcome of this important research.

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.

Vitro Biopharma Launches Products for Drug Development & Advancement of Joint Regeneration Technology-GOLDEN, Colo., Oct. 9, 2012 (GLOBE NEWSWIRE) — Vitro Diagnostics, Inc. (OTCQB: VODG), dba Vitro Biopharma, announced the launch of a series of products for use in the development of advanced treatment of injury and diseases of joints. The new products are chondrocytes derived from human adult stem cells, mesenchymal stem cells (MSCs). These cells produce collagen and are critical to proper joint function. Joint disease and injury often involve defects in collagen production and/or damage to chondrocytes due to inflammation, disease or injury. The new products include native and fluorescent labeled MSC-derived human chondrocytes together with SPIO-labeled chondrocytes. The later cells are labeled with super paramagnetic iron oxide (SPIO) and may be used for in-vivo imaging of chondrocytes using MRI (magnetic resonance imaging) a common clinical imaging procedure. The availability of native and multiply-labeled chondrocytes provides customers with numerous options to conduct in-vitro high throughput screening cell assays and to coordinate these studies with in-vivo studies.

Images: Chondrocyte Cultures

Our plans call for the addition of more types of primary cells as well as cells that can be tracked in vivo by MRI. I will keep you posted on these developments. 

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.

Vitro Biopharma Strengthens and Expands Its Distribution Agreement With Neuromics
Published: July 24, 2012
GOLDEN, Colo., July 24, 2012 (GLOBE NEWSWIRE) — Vitro Diagnostics, Inc. (OTCQB:VODG), dba Vitro Biopharma, announced an expanded relationship with a key distributor of its stem cell-based products, privately-held Neuromics, Inc (www.neuromics.com). Vitro Biopharma officials recently met Neuromics principals at their corporate headquarters in Minneapolis, MN. Neuromics was formed in 2005 by Pete Shuster, a marketing and sales professional with extensive experience in the computer industry who is now focused on providing integral products to the life science industry. Neuromics competitive advantage is based on providing products that lower costs and raise the probability of successful outcomes and include bio-markers, growth factors, transfection reagents, apoptosis detection kits, primary cells and related media. They are designed to serve basic and drug discovery researchers focusing on finding root causes and therapies for neuro-degenerative, autoimmune, inflammatory response diseases and certain forms of cancer.

Vitro Biopharma and Neuromics recently entered a distribution agreement whereby Vitro provides its stem cell-based products to Neuromics’ customers. Stem cell research continues to grow, based on positive results from several studies showing potential clinical benefit of stem cell-based cellular therapy. There is a growing demand for adult stem cells known as mesenchymal stem cells (MSCs). Vitro Biopharma manufactures and sells several fundamental tools needed to advance adult stem cell research and clinical studies, including human MSCs and derivatives, the MSC-Gro™ Brand of media optimized for growth and differentiation of MSCs as well as assay kits for determination of MSC quality, potency and response to toxic agents.

Based on the growing demand for MSC products provided by Vitro Biopharma for both research and clinical studies, Neuromics officials have committed increased resources to promote these products. Furthermore, both Vitro Biopharma and Neuromics are evaluating additional products that are perceived to enhance the competitive advantage of Vitro Biopharma’s existing MSC product line through strategic combination. A strategic goal moving forward, is an additional partner to supply devices needed for FDA-compliant manufacture of clinical materials for use in pre-clinical and clinical studies. These devices are known as bio-reactors that allow for highly controlled and reproducible production of large numbers of stem cells needed to provide therapeutic benefit. Several characteristics of these bioreactors are essential in reaching the demanding goals needed to achieve regulatory compliant manufacturing to support stem cell therapy that maintains rigorous standards of safety and efficacy. We are presently evaluating suitable devices for this application that appear to be well-suited to the required needs, specifications and characteristics.

Dr. Jim Musick, Vitro’s President & CEO, said, “We are very pleased to expand our relationship with Neuromics, Inc and look forward to a long-term mutually beneficial business relationship. While we focus on product development, manufacturing and advancement of our product pipeline, our sales partnerships are important alliances in the expansion of revenue generation. Neuromics is an ideally suited sales partner due to their broad experience and success in distribution of critical life science products. We also see the importance of a complete manufacturing system to support stem cell-based cellular therapy. It is through the highly complex interactions of cells, cell culture media, extracellular matrices and other environmental conditions that together determine the characteristics of the stem cells intended for clinical use. We also anticipate additional expansion of our relationship with Neuromics as Vitro Biopharma’s product pipeline advances through the commercialization process.”

“Neuromics’ strategic direction aligns tightly with Vitro’s goal of ‘Harnessing the Power of Cells™’”, said Shuster, “expanding capabilities enable us to serve an increasing segment of the Biotech and Pharma research community. This is especially true of the stem cell and regenerative medicine research community. Our relationship with Vitro Biopharma adds horsepower to these capabilities. Mesenchymal Stem Cells and Media are raw materials for high throughput and high content cell-based assays. Better materials help insure only the best targets, biologicals and compounds are passed on to downstream processes.”

About Vitro Diagnostics, Inc.

Vitro Diagnostics, Inc. dba Vitro Biopharma (OTCQB:VODG);(http://www.vitrobiopharma.com), owns U.S. patents for production of FSH, immortalization of pituitary cells, and a cell line that produces beta islets for use in treatment of diabetes. Vitro also owns a pending international patent for generation of pluripotent stem cells. Vitro’s mission is “Harnessing the Power of Cells™” for the advancement of regenerative medicine to its full potential. Vitro operates within a modern biotechnology manufacturing, R&D and corporate facility in Golden, Colorado. Vitro manufactures and sells “Tools for Stem Cell and Drug Development™”, including human mesenchymal stem cells and derivatives, MSC-Gro™ optimized media for stem cell self-renewal and lineage-specific differentiation. Vitro recently formed a strategic alliance with HemoGenix®, Inc. (http://www.hemogenix.com/) to jointly manufacture and distribute LUMENESC™ and LumiSTEM™ quantitative assays for determination of stem cell quality, potency and response to toxic agents.

About Neuromics, Inc.

Neuromics (http://www.neuromics.com), located in Minneapolis, MN, is a profitable and growing bio-regents company. The company was initially built by supplying bio-markers to Neuroscience Researchers. Today, Neuromics provides a range of solutions that include markers, growth factors, gene expression analysis tools, apoptosis detection kits, primary cells and related media. These solutions are increasingly being used in combinations by customers to help accelerate or improve the process of drug discovery.

Safe Harbor Statement

Certain statements contained herein and subsequent statements made by and on behalf of the Company, whether oral or written may contain “forward-looking statements” within the meaning of the Private Securities Litigation Reform Act of 1995. Such forward looking statements are identified by words such as “intends,” “anticipates,” “believes,” “expects” and “hopes” and include, without limitation, statements regarding the Company’s plan of business operations, product research and development activities, potential contractual arrangements, receipt of working capital, anticipated revenues and related expenditures. Factors that could cause actual results to differ materially include, among others, acceptability of the Company’s products in the market place, general economic conditions, receipt of additional working capital, the overall state of the biotechnology industry and other factors set forth in the Company’s filings with the Securities and Exchange Commission. Most of these factors are outside the control of the Company.

Investors are cautioned not to put undue reliance on forward-looking statements. Except as otherwise required by applicable securities statutes or regulations, the Company disclaims any intent or obligation to update publicly these forward looking statements, whether as a result of new information, future events or otherwise.

CONTACT:
Dr. James Musick
Chief Executive Officer
Vitro Biopharma
(303) 999-2130 Ext. 3
E-mail: jim@vitrobiopharma.com

Source: Vitro Diagnostics, Inc.