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

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

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

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

I will keep you posted on these future Studies.

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.

Images: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.

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

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.

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

I did want to highlight yet another potential application for stem cells. For this, we send kudos to Dr. Yan Xu and his colleagues at University of Pittburgh for their findings on inflammatory response in Golbal Ischemia. Their work was recently published:

Aaron Hirko, Renee Dallasen, Sachiko Jomura, Yan Xu. Modulation of Inflammatory Responses after Global Ischemia by Transplanted Umbilical-Cord Matrix Stem Cells. Stem Cells First published online August 21, 2008; doi:doi:10.1634/stemcells.2008-0075

Secondary to Cardiac Arrest is Brain Damage do to lack of blood flow. This is marked by a delayed loss of Neurons in CA1 hippocampus region of the brain due to inflammatory response.

The story timeline of this response is good then bad with interesting twists. The delay in neuronal loss is linked to initial inflammation. It involves both reactive astrocytes (astrocytosis) and glia. Delaying the loss is, of course, good.

…But then, the reactive astrocytosis and related glial scarring cause a physical and biochemical barrier to regeneration of neurons…a bad thing. Protecting the microglia is a good thing, because they these cells serve as scavengers for clearing the cellular debris. They can also secrete a variety of cytotoxic and protective chemicals.

The wow factor in this research is that  implanted rat umbilical-cord matrix (RUCM) cells can provide partial protection against neuronal injury in rat brains. Rats treated with RUCM cells three days prior to an 8-min CA had only 25-32% neuronal loss in the hippocampal CA1 region compared to the typical 50-68% neuronal loss observed in the untreated or the vehicle-treated animals. This could be due to to the favaorable modulation of the “good-bad” inflammatory response.

The good news in the search for therapies for stroke and cardiac arrest victims is combined, stem-cell-like RUCM cells offer protection against neuronal injury after global cerebral ischemia by enhancing the survivability of the astroglia in the selectively vulnerable regions.

We are pleased that the research team used our GFAP antibody as an marker for astrotytic in their studies.

Dr Steve Stice and Human Stem Cells

I am pleased and honored for the privilege of profiling Dr. Steve Stice.  He has a history of working in areas that are Biotechnology Headliners…from cloning to stem cells. Here I will be focusing on his current work with Human Stem Cells and Neural Progenitors  at ArunA Biomedical and The University of Georgia. As with all the News Behind the Neuroscience News, I will highlight how it could impact Neuroscience Research and Drug Discovery.

The Back Story

Where it Starts

About Dr. Steve Stice

Dr. Steve Stice is CSO of Aruna Biomedical Inc and a Professor and Director of the Regenerative Bioscience Center and has a Georgia Research Alliance Eminent Scholar endowed chair.

Prior to joining the University of Georgia, Dr. Stice was a cofounder and Chief Scientific Officer at Advanced Cell Technology, a stem cell company.  Throughout his career he has published and lectured internationally on the topics of cloning and stem cells. 

In 2001, three of the human embryonic stem cell lines that Dr Stice’s lab derived were approved for federal funding by President Bush. In 2006, he was appointed by Gov. Perdue to the Post Natal Cord Blood Commission for the state of Georgia.

Dr. Stice founded Aruna Biomedical, Inc., and in cooperation with Millipore Inc. was first group to market a product derived from human embryonic stem cells (2007). The product is a neural stem cell used for research on neurological diseases and disorders, ranging from Parkinson’s disease to depression.

Contact Information:

sstice@arunabiomedical.com

Current Products

There is good news. Neuroscientists can now easily and inexpensively get human neural progenitor cells for Drug Discovery, Toxicity and Basic Research.

ENStem-A ™, Neural Progenitor Expansion Kit
hN2™, ArunA Human Neural Cell Kit 

So what was once difficult and frustrating, is now easy and convenient. Buy the kits and here’s an example of what you get.

What is Next

Knowing the needs and wants of the marketplace, ArunA’s products and capabilities excite me. Any tools that have they capabilities to bring researchers a steps closer to discovering cures for insidious Neuro-diseases need to be embraced. All of us have or will be touched by these diseases.

In my conversations with Steve, I am impressed with his clear understanding of how to evolve ArunA’s product to increase their value proposition. Available soon could be cultures developed to fit the niche needs of specific researrch areas like Parkinson’s, Pain’s and Alzheimer’s. I plan on communicating these evolutions here and at my company’s website @ www.neuromics.com.

My considerations are humble. They do not extend to the potential of manipulating pluripotent cells to grow transplantable human tissues and organs in the lab. I am more interested in the ability for scientists to manipulate progenitors to grow pure cell populations in vitro for basic research.  These cultures are useful for helping Scientists understand the molecular biology of diseases. This is but a baby step in the direction of actually discovering therapies for insidious human diseases. I would like to have these cultures available as research tools for my customers, but what are the ethical considerations even, say, if the cells were derived from government approved cell lines.

Ted Peters

In my journey of understanding, I happened upon a website that articulates  the roots of the debate and sheds light on the big questions that need to be answered by systematic theologians and public policy makers. These answers then could provide a moral and ethical framework for unleashing the promise of stem cells.

The Stem Cell Debate: Ethical Questions-About the author: Ted Peters is a professor of Systematic Theology at Pacific Lutheran Theological Seminary and the Graduate Theological Union (GTU) in Berkeley, California. He is author of GOD-The World’s Future (Fortress 2000) and Science, Theology, and Ethics (Ashgate 2003). He is editor-in-chief of Dialog, A Journal of Theology. He also serves as co-editor of Theology and Science published by the Center for Theology and the Natural Sciences in Berkeley.