The Dance Between Immune System and Stem Cell Health

We named it the  immunoLinkTM 
We have been testing a growing number of Clients with our Quantibody Arrays. Many of of these clients have Autoimmune Disorder Diseases. These range from Rheumatoid Arthritis to Multiple Sclerosis.

These arrays are designed to precisely measure factors or markers(proteins) that are dysregulated by these diseases. We measure the levels of these biomarkers in our Clients’ Blood serum. The arrays have also been used to measure the levels of markers in plasma and cell culture supernatants.

Based on results, we are finding links between immune system and stem cell health. We call this the immunoLink. The link shows that when immune/inflammatory response markers are elevated, markers related to stem cell health are depleted.

Here we see the immune/inflammatory response markers IL-6, MCP-1 and TNF-alpha are elevated in our Clients with autoimmunity (A) vs Healthy Controls (HC). We also see lower levels of G-CSF and GM-CSF in these Clients.

G-CSF and GM-CSF are know to play a role in increasing circulating stem cells. GM-CSF is also know to be secreted by Mesenchymal Stem Cells (hMSCs) AND GM-CSF has anti-apoptotic functions on neurons, and is neuroprotective in animal stroke models while G-CSF has a prominent effect on the differentiation of adult neural stem cells (see: BMC Neuroscience 2007, doi:10.1186/1471-2202-8-88).

To us, the immunoLink means achieving a balance between immune system and stem cell health.

We provide immune system balancing and stem cell activating therapies for our Autoimmune Disease Clients and Children with Autism. We first do baseline and follow on testing (each 6 months) to determine how well the therapies are working. You can now benefit from one of our first Stem Cell Activating Product named Stem-Kine.

Dr. Joe Smarda, an applied immunologist, is user and promoter of Stem-Kine and oh, by the way. He is a European Porsche Cup Champion and pilots our Stem-Kine/Neuromics sponsored race car.

Our goal is to bring the many markers we test to healthier levels. As stem cell transplants become more common, moderating levels of immune/inflammatory response in patients could improve outcomes. If you would like to learn more, you can contact me at pshuster@neuromics.com or 612-801-1007.

Treating Autism and TBI

I am excited about this important development in the Neuromics and Vitro Biopharma partnership: We are in the planning stages of clinical trials to test mobilization of endogenous stem cells in the treatment of traumatic brain injury and Autism Spectrum Disorders. This is based on substantial pre-clinical research suggesting that activation of certain cellular pathways in combination with epigenetic modulation of select gene expression yields increased proliferation, migration and differentiation of adult stem cells including neural stem cells and MSCs.
See Press Release: http://finance.yahoo.com/news/vitro-biopharma-revenues-increase-early-143000008.html

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.

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.

    Dr. Jim Musick-Making MSCs Work

    Harnessing the Power of CellsTM

    Dr. Jim Musick

    Dr. Jim Musick

    Dr. Jim Musick and his company, Vitro Biopharma, give Basic and Drug Discovery Researchers the ability to harness the power of Human Mesenchymal Stem Cells (hMSCs). This power is essential for blazing new trails in the stem cell research and regenerative medicine frontier.

    I am pleased to welcome Jim as a partner in providing my company the expertise and knowledge highlighted in this profile. Together, we give our customers, colleagues and friends the ability to easily culture, grow, differentiate and maintain large stocks on hMSCs.

    Background

    Jim received his PhD from Northwestern University in 1975 and then joined the staff of University of Utah where he specialized in the study of Neuroscience and synaptic transmission. He joined UltraPure Laboratories in 1983.

    At UltraPure, he learned the art and science of commercializing biologicals. There he helped develop procedures for the commercial production of purified human pituitary hormones including, prolactin, growth hormone and TSH. This included developing QA/QC procedures to support commercial distribution of these products.

    He joined Vitro Diagnostics in 1988 and directed all operations involved in the establishment of a diagnostic product line that included about 30 different purified antigen products.  His direct responsibilities included research & development, manufacturing, intellectual property development and maintenance, marketing and sales.  He was also responsible for the development & initial commercialization of the fertility drug VITROPIN™ as well as the cell immortalization program of the Company.  He is an inventor or co-inventor of all issued and pending patents owned by the Company.

    In 1998 he also completed an Executive Program at JJ Kellogg Graduate School of Management, Northwestern University in Managing New Product Development.

    In 2000, he orchestrated the sale of the antigen manufacturing division to Aspen Biopharma (Nasdaq, APPY) while retaining IP related to use of FSH as a fertility drug and to cell line generation technology.

    He has the spirit of a polished scientist/entrepreneur with strong operational and process expertise.

    Harnessing the Power of hMSCs

    As President and CEO of Vito Biopharma, Jim leverages his expertise and experience to manufacture Cord Blood Derived hMSCs. The stem cell revolution demands large stocks of cells of the highest quality. Meeting the demand is the key to the development of stem cell related therapies. Vitro Biopharma has the capabilities to delivery.

    It is all about starting with vials of potent and pure hMSCs. From there, the customer can grow and differentiate large stocks and be confident in the quality because Jim’s company has the processes in place to insure this. The cell lines are well-characterized with regard to species authentication using sensitive PCR methods to quantify non-conserved genes including COX1, Cytochrome B and actin.  Vitro Biopharma also utilizes karyotyping to authenticate its  cell lines.  Adventitious agents are also tested negative by sensitive PCR methods including known viral contaminants and mycoplasma.  Performance is assured by rigorous testing of viability, growth rate and differentiation capacity for formation of chondrocytes, adipocytes and osteoblasts.  Finally these cells are characterized with regard to phenotypic cluster designation antigens.

    Current Products

    Native and fluorescent-labeled human MSCs including native and fluorescein/rhodamine-labeled MSC-derived chondrocytes and osteocytes along with MSC-GroTM  growth and differentiation media. MSC-Gro™ media is provided in low-serum, humanized and serum-free formulations for both growth and differentiation.  Humanized serum-free media may be supplemented with allogeneic or autologous serum for direct comparisons of growth and differentiation under these conditions.  Powdered MSC-Gro™ formulations are also provided.  Vitro Biopharma’s human MSCss have the capabilities to be expanded through at least 10 passages at rapid growth rates and can be further expanded to 16 passages (~50 population doublings) at slower growth rates.

    Human MSC-derived Osteoblasts stained with Alizarin red at 100 x.

    Image: Human MSC-derived Osteoblasts stained with Alizarin red at 100 x.

    Vitro Biopharma has recently launched a new and revised website complete with convenient online ordering and detailed product technical information (www.vitrobiopharma.com).

    Futures

    In our interview, Jim gave blinding glimpses of the future especially with regard to new products to extend Vitro Biopharma’s offering of clinical tools to fully explore the ever-expanding therapeutic applications of MSCs. I am excited about the potential. I will keep you posted as new products are commercialized.

    Lectin Binding Profiles among Human Embryonic Stem Cells

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

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

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

    hNP1_STEM_CELL_MARKERS_IF_IHC

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

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

    I will keep you posted on these future Studies.

    Differential healing properties of human ACL and MCL Stem Cells

    Autologous Stem Cell therapies for human injury and disease are gaining momentum. Understanding the properties of Stem Cell Colonies that have potential for these therapies is key to optimizing treatments. This study provides knowledge on the properties and their impact on future therapies for anterior cruciate ligament (hACL) and medial collateral ligament (hMCL) of the knee joint.

    Jianying Zhang, Tiffany Pan, Hee-Jeong Im, Freddie H Fu and James HC Wang. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. BMC Medicine 2011, 9:68doi:10.1186/1741-7015-9-68.

    Background: The human anterior cruciate ligament (hACL) and medial collateral ligament (hMCL) of the knee joint are frequently injured, especially in athletic settings. It has been known that, while injuries to the MCL typically heal with conservative treatment, ACL injuries usually do not heal. As adult stem cells repair injured tissues through proliferation and differentiation, we hypothesized that the hACL and hMCL contain stem cells exhibiting unique properties that could be responsible for the differential healing capacity of the two ligaments.

    Methods: To test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry.

    Self-renewal of hACL-SCs and hMCL-SCsImages:The expression of stem cell markers in hACL-SCs and hMCL-SCs. At passage 5, hACL-SCs had already become highly elongated in confluent culture, a typical fibroblast phenotype (A). In contrast, even at passage 13, confluent hMCL-SCs remained cobblestone-like (B). Moreover, hACL-SCs no longer expressed nucleostemin (C) or SSEA-4 (E) at passages > 5, whereas hMCL-SCs expressed both stem cell markers at passage 13 (D, F). Note, however, that hMCL-SCs at this high passage exhibited a lesser degree of nucleostemin expression compared to the cells at passage 1 (see Figure 3). The results shown here were obtained from a male donor of 27 years oldTo test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry.

     

    Results: We found that both hACL stem cells (hACL-SCs) and hMCL stem cells (hMCL-SCs) formed colonies in culture and expressed stem cell markers nucleostemin and stage-specific embryonic antigen-4 (SSEA-4). Moreover, both hACL-SCs and hMCL-SCs expressed CD surface markers for mesenchymal stem cells, including CD44 and CD90, but not those markers for vascular cells, CD31, CD34, CD45, and CD146. However, hACL-SCs differed from hMCL-SCs in that the size and number of hACL-SC colonies in culture were much smaller and grew more slowly than hMCL-SC colonies. Moreover, fewer hACL-SCs in cell colonies expressed stem cell markers STRO-1 and octamer-binding transcription factor-4 (Oct-4) than hMCL-SCs. Finally, hACL-SCs had less multi-differentiation potential than hMCL-SCs, evidenced by differing extents of adipogenesis, chondrogenesis, and osteogenesis in the respective induction media.

    Conclusions: This study shows for the first time that hACL-SCs are intrinsically different from hMCL-SCs. We suggest that the differences in their properties contribute to the known disparity in healing capabilities between the two ligaments.

    I will be posting more on autologous stem cell therapies research.

    STEMEZ hNeural Progenitors and Cell Migration

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

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

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

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

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

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

    Dr. Steve Stice to Present the Power of StemEZ Neural Cells

    STEMEZ hN2 Primary Human Neurons

    STEMEZ hN2 Primary Human Neurons

    I have profiled Steve Stice’s research here. The focus has been the excellent research results he and his team at ArunA Biomedical have generated with STEMEZ(TM) hN2 Human Neurons and hNP1 Human Neural Progenitors.

    The story continues. He will be presenting the latest at the 9th Annual World Pharmaceutical Congress in Philadelphia, June 14. Topics include: using these neural cell lines to study neurotoxicity in cell-based assays and disease modeling.  Recent work conducted in outside laboratories demonstrates that these lines are more sensitive to environmental toxicants than traditional cellular models.

    Sample high throughput assay applications:

    • Cell morphology and neurite outgrowth
    • Cell signaling and transcription factor expression
    • Receptor and ion channel function
    • Cytotoxicity
    • Apoptosis, genotoxicity and DNA damage

    These capabilities has been confirmed by our customers. I look for the use of the STEMEZ cell lines to continue to grow as researchers discover their value in Drug Discovery and Basic Neuroscience capabilities.

    Consistent Human Neurons

    We have featured Dr. Steve Stice here. He and his team at UGA and Aruna Biomedical are developing products that are highly desired by Neuroscience Researchers.

    We are in the process of finalizing details for distributing their human neuron cultures. Here is the related press release:

    ArunA Biomedical, Inc. announces alliance with Neuromics for distribution of normal human neural cells.

    Athens, Georgia – - March 23, 2009 – - ArunA Biomedical, Inc., announced today an agreement with Neuromics, Inc. of Edina, MN, giving Neuromics the right to non-exclusively market and sell the ArunA hN2™ Human Neural Cells and Neural Culture Medium to support applications in neurological research.ArunA has an exclusive worldwide license to develop and commercialize neural cells derived from human embryonic stem cells (hESC), and hN2 is a second generation product from this technology. These cells offer a consistent population of normal human neural cells that the neural research and pharmaceutical market highly desires.

     “ArunA has further developed its adherent monolayer technology by creating hN2™, a normal human neural cell ideal for drug screening, toxicology studies and basic neural research, and we are pleased to have Neuromics as a distribution partner,” said David Ray, Chief Executive Officer  of ArunA Biomedical

    “Neuromics growth is catalyzed by offering the unique products and expertise our customers require for research success through strategic alliances with companies like ArunA Biomedical. This relationship represents a growth opportunity for us. Their hN2™ cells fill a stated research need of the Neuroscience Community and we look forward to our customers having these cells and the related new discoveries they will help generate,” said Pete Shuster, CEO and Owner of Neuromics.

    Founded in 2003, ArunA Biomedical, Inc. is a privately held biotechnology corporation dedicated to the discovery, manufacturing and commercialization of emerging new technologies in human embryonic stem cell research for use in drug discovery and neuroscience research.

    Founded in 2003, Neuromics is a privately held Bio-regents Company focusing on providing research ready and proven products and methods expertise to Neuroscience, Diabetes/Obesity, Immunology and Researchers.
     
    This press release contains forward-looking statements regarding the company’s potential impact on scientific research and collaborations with third parties.  Certain conditions could alter the outcome or progress of these statements including but not limited to unexpected manufacturing issues, product performance and quality control/assurance issues.  Forward- looking statements are based on the opinions, beliefs and expectations of the company or individuals quoted in the press release and the company does not assume any obligation to update these forward-looking statements if circumstances change.