eSC Derived hNP1 Neural Progenitors Astrocytic Differentiation

Protocol for Driving hNP1TM Human Neural Progenitors to Astrocytes

There is a great demand for an easy way to generate human astrocytes in culture. I am pleased to present a protocol for differentiating our hNP1 Cells to Astrocytes. This comes from my friend Dr. Steve Stice and his team at ArunA Biomedical and University of Georgia: Majumder A, Dhara SK, Swetenburg R, Mithani M, Cao K, Medrzycki M, Fan Y, Stice SL. Inhibition of DNA methyltransferases and histone deacetylases induces astrocytic differentiation of neural progenitors. Stem Cell Res. 2013 Jul;11(1):574-86. doi: 10.1016/j.scr.2013.03.003. Epub 2013 Apr 2.

These enriched non-transformed human astrocyte progenitors will provide a critical cell source to further our understanding of how astrocytes play a pivotal role in neural function and development. Human neural progenitors derived from pluripotent embryonic stem cells and propagated in adherent serum-free cultures provide a fate restricted renewable source for quick production of neural cells; however, such cells are highly refractive to astrocytogenesis and show a strong neurogenic bias, similar to neural progenitors from the early embryonic central nervous system (CNS). We found that several astrocytic genes are hypermethylated in such progenitors potentially preventing generation of astrocytes and leading to the proneuronal fate of these progenitors. However, epigenetic modification by Azacytidine (Aza-C) and Trichostatin A (TSA), with concomitant signaling from BMP2 and LIF in neural progenitor cultures shifts this bias, leading to expression of astrocytic markers as early as 5days of differentiation, with near complete suppression of neuronal differentiation.


Images: Morphology and gene expression after 15 and 30 days of differentiation of cells with astrocytic treatment. Bright field images of hNP cells differentiated (A) with or (B) without astrocytic treatment. A and B compare morphology of cultured cells in treated vs. untreated differentiation at 15 days. Treated and untreated cells were cryopreserved at d6 and subsequently thawed and cultured for an additional 9 days. Flow cytometry analysis to determine percent of GFAP+ and S100B+ cells at d15 of differentiation. Data is presented as histograms for (C) GFAP and (D) S100B with corresponding immunoreactive cells in insets from a parallel culture. Immunocytochemistry detects expression of (E) GFAP with S100B (inset showing distinct staining for both markers), (F) GFAP with GLAST, and (G) GFAP with ALDH1L1 at d30 of differentiation.

The Protocol:  For astrocytic differentiation of hNP cells, neuronal differentiation media were supplemented with BMP2 (20 ng/mL) and combinations of Aza-C and TSA; Aza-C (500 nM), TSA (100 nM) and BMP2 (20 ng/mL) for 2 days, with one complete media change in between, followed by differentiation media supplemented with BMP2 but not with Aza-C or TSA. Cells were harvested prior to analysis at 5, 15 or 30 days of treatment or for cryopreservation to d6 or d10 of differentiation. For cryopreservation, cells were
dissociated with Accutase™ and frozen in differentiation media containing 10% DMSO. Viability was assessed at 30 days in Aza-C and TSA treated cultures by trypan blue exclusion, and datawas acquired using a Cellometer Auto T4® (Nexcelom Biosciences).

I will keep you updated on new differentiation protocols for our potent, pure and widely used hNP1 Human Neural Progenitors to new phenotypes.

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.

Ion Channels and Neuromics’ STEMEZ Cells

In my conversation with neuro-drug discover researchers, I am frequently being asked about the potential of using our STEMEZ(TM) hNP1 Human Neural Progenitors Expansion Kits for studying ion channels. How effective are these cells as a source for studying neurodegenerative diseases and for drug screening assays?  There is good news from Dr. Steve Stice and my friends from ArunA and UGA.

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.
hNP1_Gene_Expression

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).

hNP1_Electrophysiology

Images: Sodium channel activity in differentiated hNP cells was measured using whole cell voltage clamp. 81 total hNP cells cultured in the absence of bFGF from 4 to 27 days were analyzed. Of these, 34 exhibited no fast inward currents in response to a step depolarization indicating the 348 absence of functional voltage gated sodium channels (Figure 4G). The remaining cells yielded between 0.04 – 1.5 nA of inward current in response to the step depolarization (Figures 4B and 4G). These currents inactivated rapidly in all cases (Figures 4B and 4C) and could be abolished with the addition of 1 μM TTX (n = 3 cells; Figure 4C). Voltage-dependent steady state inactivation (n = 11 cells; Figure 4D) and recovery from fast inactivation (n = 5 cells; Figure 4E) were also observed on several positive cells. A subset of these cells was subjected to current clamp and action potentials were elicited by current injection (n = 8 cells, Figure 4F). In support of this, increasing concentrations of a sodium channel activator veratridine in a FLIPR assay on differentiated hNP cells show an increasing calcium response (Figure 4H). This probably resulted from voltage-gated sodium channel depolarization of cells that subsequently allowed calcium influx through calcium channels. These data indicate that differentiation of hNP cells by removal of bFGF can lead to a neuronal cell that can generate action potentials and depolarize the cell. The 58% hit rate for voltage-gated sodium channel function (Figure 4G), does not reflect the true proportion of sodium channel positive cells in our differentiated hNP cells, but rather our ability to morphologically distinguish these cells from negative cells by eye. An example of the morphology of a sodium channel positive cell is shown in Figure 4A. The positive cells were phase bright with a few long processes.

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.

STEMEZ hN2 Human Neurons Data

I have been working with Dr. Steve Stice and Aruna Biomedical to deliver human stem and neural cells to identified niche research areas related to drug discovery.  Neuromics rolled out STEMEZTM hN2 Human Neurons Discovery Kits several months ago. Applications for these include: cellular model studies, high content screening, developmental studies, RNAi studies and genetic manipulation.

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

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.

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. 

Isolating and Maintaining Embryonic Stem Cells

I have featured Steve Stice and his team at ArunA Biomedical and UGA. They are pioneers in developing Embryonic Stem Cell Based Cultures and Assays for Drug Discovery and Basic Research. Given the importance of their work, I am commited to keeping my finger on the pulse of their advances and discoveries.

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.

Steve Stice-The Professor Entrepreneur

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

Steve embodies a rare blend of entrepreneurship and scientific curiosity. He has been referred to in the press as “part professor; part entrepreneur”. This uniquely positions Steve to take his inventions from the lab directly to the marketplace by forming Biotechnology Companies. The DNA for ArunA comes from several of his earlier start-ups: Advanced Cell Technology and Cytogenesis (now part of BresaGen).

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

As co-founder and CSO of Advanced Cell Technology, he has helped commercialize discovery platforms that could enable the application of stem cell technologies to the field of regenerative medicine to bring effective therapies to patients suffering from degenerative diseases like age-related macular degeneration. The company recently passed the milestone of  successfully. restoring visual function in rats through the implantation of RPE cells derived from human embryonic stem cells and in early 2008, completed pre-IND meetings with the FDA. Yes, Human Stem Cell based therapies have the potential to make the blind  see.

This bring us to ArunA. I am excited about their current and future products because their is a pent up need for them by the Neuroscience Research community which includes many of Neuromics’ Customers.

The ArunA Biomedical Story
Steve started ArunA in 2003. It actually sprung from a frustrating aspect of using Stem Cells for research. They are infinitely useful but hard to grow in cultures and differentiate into the research required cell types. Steve became acutely aware of this from his work starting in 2001 including a 5 day course he taught at NIH. Steve understood that most researchers do not want to spend the time and related frustrations associated with  this exercise. It is kind of like building a computer so you could enjoy the benefits of the web. In other words, Neuroscientists could care less about undifferentiated stem cells. At the very least, they want pure and healthy Neural Progenitors. These can then be expanded and differentiated into specific neurons. For example an ALS Researcher would be interested in making Motor Neurons; a Parkinson’s Researcher, Dopamanergic Neurons and a Pain Researcher, GABAmanergic. Nirvana for these researchers would be having pure cultures of these Neuron types at their fingertips.

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.