Mesenchymal Differentiation Pathways

I will soon be profiling Dr. Jim Musick of Vitro Biopharma.  He manufacturers and provides us a wealth of expertise on our Human Mesenchymal Stem Cells (hMSCs) and MSCGro™ Mesenchymal Stem Cell Media.  As the demand for these grow, we are receiving a variety of questions on differentiation. Specifically, researchers desire to drive these cells to specific progenitor and cell phenotypes like Osteocytes, Adipocytes and Chondroytes.

I would like to share a pathway map that gives a snapshot of these pathways:

Regenerative Biology of the Spine and Spinal Cord. Edited by: Rahul Jandial, Mike Y. Chen, Bihong T. Chen and Joseph Ciacci. ISBN: 978-1-4614-4089-5. Publication date: May 25, 2012. Series: Special Books

I will continue to post information that will enable the researchers to harness the power of Mesenchymal Stem Cells.

Jim Musick and Vitro Biopharma

Coming Soon

Neuromics recently added key products from Dr. Jim Musick and our friends @ Vitro Biopharma. These include potent umbilical cord derived human mesenchymal stem cells and MSCGroTM (best of breed growth and differentiation media). These cells are capable of many passages enabling researchers to build large stocks.

MSCs_MSCGro

We will be posting Jim’s profile in several weeks.

Stem Cell and Cell Based Assays Groups on Linkedin

I wanted to share some links to groups on Linkedin that have proven a useful resource for me. They are also additive to the stories and data posted here:

Stem Cell Clinical TrialsStem Cell Clinical Trials

Note: I am the moderator the the Stem Cell Clinical Trail group and welcome all new members. We are currently 400+ strong and growing.

3D cell biology : tools & techniques3D cell biology : tools & techniques

Stem Cell Research Stem Cell Research

California Institute for Regenerative Medicine (CIRM)California Institute for Regenerative Medicine (CIRM)

neuropathy and neuropathic painneuropathy and neuropathic pain

The Gene Silencing Collaboration (RNAi, siRNA, miRNA, Dicer, etc.)The Gene Silencing Collaboration (RNAi, siRNA, miRNA, Dicer, etc.)

Enjoy.

Scripps Florida Scientists Awarded $3 Million to Develop New, More Effective Pain Treatments

We profiled Dr. Laura Bohn research in one of our news stories. We are excited to share the news.Dr. Laura Bohn

JUPITER, FL, February 29, 2012 – Scripps Florida scientists have been awarded $3.1 million by the National Institute on Drug Abuse, part of the National Institutes of Health, to study and develop several new compounds that could prove to be effective in controlling pain without the unwanted side effects common with opiate drugs, such as morphine, Oxycontin®, and Vicoden®.

Laura Bohn, an associate professor in the Department of Molecular Therapeutics and Neuroscience at Scripps Research, and Thomas Bannister, an assistant professor in the Department of Chemistry and associate scientific director in the Translational Research Institute at Scripps Research, will serve as joint principal investigators for the new five-year study.

Their study will focus on four new classes of compounds that appear to differ fundamentally from opiates inthe side effects that they can produce.

“Once we more fully understand how these compounds work, we expect to optimize and develop them as novel drugs,”said Bohn. “We hope to produce potent pain relievers without the problems associated with current treatments.” Full article: http://www.scripps.edu/news/press/20120229bohn-bannister.html

We wish her great success in her research aimed at discovering improved solution for managing pain.

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.

Harnessing the Power of Neural Stem Cells

I wanted to share an important presentation by Dr. Steve Stice. He is a featured researcher in “News Behind the Neuroscience News”.

“Does amplification of neural progenitor cells derived from embryonic stem cells solve problems of cell production and FDA safety standards?”
Steven L. Stice, PhD
Professor, GRA Eminent Scholar
Director of the Regenerative Bioscience Center at University of Georgia
CSO, Aruna Biomedical Inc.

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.