“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.

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.

When differentiated, these  neural progenitors express subunits of glutamatergic,  GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium  and calcium channel subunits were also expressed. Functionally, virtually all the NP cells exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited  tetrodotoxin sensitive, voltage-dependent sodium channel current under whole-cell voltage clamp and action potentials could be elicited by current injection under whole-cell current clamp.  These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and also results in the capability to produce excitable cells that can generate action potentials. This is the first data demonstrating capabilitiesof these cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.

Images: Glutamate receptor expression in hNP cells and differentiated hNP cells The expression of ionotropic glutamate receptors might also be an indicator of neuronal maturation. These receptors are composed of three distinct families: NMDA, kainate and AMPA receptors. The hNP cells and differentiated hNP cells cultured in the absence of bFGF for 2 weeks were analyzed for mRNA expression of subunits of each glutamate receptor subtype relative to hESCs. Significant increases (p<0.05) in Grin2b were seen in hNP cells (20 fold) and differentiated hNP cells (25 fold) relative to hESCs (Figure 3A). Additionally, Grin1 and Grin2d were significantly increased (p<0.05) only in differentiated hNP cells relative to hESCs, but not in undifferentiated hNP cells (Figure 3A). Of the kainate receptors, Grik4 and Grik5 were significantly (p<0.05) increased only in undifferentiated hNP cells relative to hESCs (Figure 3B); whereas, Grik2 was significantly (p<0.05) increased only in hNP cells where bFGF had been removed (Figure 3B). AMPA receptor subunits were also examined. Gria1 and Gria4 were up regulated in hNP cells relative to hESCs (Figure 3C). Two week differentiated hNP cells showed significant (p<0.05) up regulation of Gria2 and Gira4 relative to hESCs (Figure 3C). To determine if functional glutamate channels exist in differentiated hNP cells, calcium influx in response to AMPA, kainic acid or NMDA application was measured on hNP cells, 14 days after the removal of bFGF. Figure 3G indicates that NMDA could not depolarize differentiated or undifferentiated hNP cells enough to cause significant calcium influx above background. In contrast, AMPA and kainic acid can cause calcium influx which can be potentiated by AMPA receptor specific modulator, cyclothiazide (50 μM, Figure 3G).Calcium influx was detected in the presence of cyclothiazide in calcium activity as measured (Figure 3H).

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.

Drilling down further, I am pleased to present Electro-physiology and related data generated by Aruna and collaborators: hN2 Cells-Electro Phys Data Supplement

hN2-Whole Cell Voltage Clamp

Figure. hN2 cells can produce inward currents that generate action potentials. (A) Isolated hN2 with significant neurite growth 1 week  after plating . This cell was subjected to whole cell voltage clamp utilizing a potassium gluconate based intracellular solution. (B) Voltage gated inward and outward currents were elicited from this cell with depolarizing voltage steps. (C) Inward currents from another cell (potassium gluconate intracellular) were abolished by local application of 1 µM tetrodotoxin (red trace) while outward currents remained. Inward current recovered as TTX washed out of the region (green trace). (D) A different cell which exhibited voltage activated inward currents that inactivated in response to a 50 ms prepulse at different membrane potentials. The experiment was done 27 days after the removal of bFGF. A cesium gluconate based intracellular solution was used for this experiment to block outward potassium currents. The membrane potential for half maximal inactivation by standard Boltzman fitting (red line) was -40.1 mV with a slope of 4.7. (E) Recovery from fast inactivation utilizing a paired pulse protocol in the same cell as C. The single exponential time constant for recovery of inactivation was 1.7 ms (red line). (F) A different cell which elicited an overshooting action potential upon current injection under whole cell current clamp utilizing a potassium gluconate based intracellular solution. Inset: Response of the same cell under voltage clamp to a change in membrane potential from -80 mV to -10 mV elicited a peak current of 457 pA. Scale bars for inset: 5 ms, 0.2 nA.

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

Here they isolate, and maintain in culture, neural progenitors demonstrating properties of these neural epithelial cells from WA09 human embryonic stem cells (hESCs):

D.W. Machacek, S. K. Dhara, C. Sturkie, K. Hasneen, D. Carter, L. Murrah Hanson, P.R. MacLeish, M. Benveniste, S.L. Stice. DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELL DERIVED NEURAL PROGENITORS INTO FUNCTIONALLY RESPONSIVE POPULATIONS IN THE ABSENCE OF EXOGENOUS EGF.

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.

Podcast by ClearCast

This Podcast gives is a good primer on Embryonic Stem Cells (ESCs)…where they come from, how they are used and the promise they have for helping researchers cure human diseases.

The listener will gain insight how ECSs when manipulated into mature neuronal cell lines can accelerate the pace of neurological research for scientists working on treatments for spinal cord injuries and neurological diseases such as Parkinson’s, Huntington’s, Alzheimer’s, ALS and possibly even depression.