Neuromics’ hNP1 Neural Progenitors, DJ-1 and Stroke

Dr. Cesar V. Borlongan, University of South Florida and a team of researchers have successfully identified DJ-1 as a potential therapeutic target for treating stroke. They used our hNP1TM Human Neural Progenitors to confirm the neuroprotective properties of the DJ-1 protein: Yuji Kaneko, Hideki Shojo, Jack Burns, Meaghan Staples, Naoki Tajiri, Cesar V. Borlongan, DJ-1 ameliorates ischemic cell death in vitro possibly via mitochondrial pathway, Neurobiology of Disease, Available online 21 September 2013, ISSN 0969-9961, http://dx.doi.org/10.1016/j.nbd.2013.09.007.

hNP1 Human Neural Progenitors in Culture

hNP1 Human Neural Progenitors in Culture

Highlights

•DJ-1 translocation was assayed in oxygen–glucose deprived human neural progenitor cells.
•Immunofluorescent microscopy and ELISA were used to measure DJ-1 translocation.
•DJ-1 translocated preferentially into polarized mitochondria.
•DJ-1 translocation is associated with the preservation of functional mitochondria.
•DJ-1 exhibits antioxidative stress effects following ischemic stroke.

I will continue to post updates on Research success with our Cell Based Assay Solutions.

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.

Update-Cell Based Assay Solutions

We continue to expand our cell centric strategy. With the addition of Cancer Associated Fibroblasts (CAFs), we are now positioned to develop highly engineered customer specific co-cultures. These engineered cells will inculde our hMSCs, Osteoblasts, Chondrocytes and Fibroblasts. Our goal is to develop in vivo like cell based assays to lower the costs of discovery and increase your “hit” rate.

New Products
Cancer Associated Fibroblasts
Image: Culture of Adeno-Carcinoma CAFs at Day 2.
 

We will keep you posted as we add capability to our cell based assay solutions.

Musculoskeletal Disorders-Stem Cell Based Drug Discovery

A common Neuromics’ theme is harnessing the power of cellsTM. The raw cost of the cells are often the biggest consideration. We encourage our customers to focus on true costs. These include the # of cells (how many times can they be passaged?), culture viability (how long do the cells live) and bioactivity (how closely do cultures mimic in vivo behavior?). I would like to present a presentation and publication confirming our competitive advantage when analyzing true costs.

Setting a higher bar for Neuron-Glial Based Assays!

Dr. Randen Patterson and his team at UC Davis have developed new culturing techniques using our e18 Rat Primary Hippocampal Neurons. They have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons. This is good news indeed: Todd GK, Boosalis CA, Burzycki AA, Steinman MQ, Hester LD, et al. (2013) Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons. PLoS ONE 8(4): e58996. doi:10.1371/journal.pone.0058996.

We will continue to raise the bar. Better cultures=lower costs and better outcomes!

hN2 Human Neurons & hNP1 Neural Progenitors in Action

I have been promoting Dr. Steve Stice and his team. They are the brains behind our  hN2TM Human Neuron and hNP1TM Human Neural Progenitor Discovery Kits. I would like to share 2 recent publication referencing use of these discovery kits. These validate the postings on capabilities. They are the best solutions available for researchers searching for Neuron or Neural Progenitor Based Assays for basic research, toxicology studies or drug discovery.

Xiugong Gao, Hsiuling Lin, Radharaman Ray, Prabhati Ray. Toxicogenomic Studies of Human Neural Cells Following Exposure to Organophosphorus Chemical Warfare Nerve Agent VX. Neurochemical Research. February 2013…Human hN2 neurons were obtained from Neuromics…

Abstract: Organophosphorus (OP) compounds represent an important group of chemical warfare nerve agents that remains a significant and constant military and civilian threat. OP compounds are considered acting primarily via cholinergic pathways by binding irreversibly to acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Many studies over the past years have suggested that other mechanisms of OP toxicity exist, which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis. Here we performed a microarray study in which cultured human neural cells were exposed to 0.1 or 10 μM of VX for 1 h. Global gene expression changes were analyzed 6, 24, and 72 h post exposure. Functional annotation and pathway analysis of the differentially expressed genes has revealed many genes, networks and canonical pathways that are related to nervous system development and function, or to neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In particular, the neuregulin pathway impacted by VX exposure has important implications in many nervous system diseases including schizophrenia. These results provide useful information valuable in developing suitable antidotes for more effective prevention and treatment of, as well as in developing biomarkers for, VX-induced chronic neurotoxicity.

Protocol: Human hN2 neural cellswere obtained from Neuromics (Edina, MN). The hN2 cells were fully differentiated

normal human neural cells derived as adherent cells from human embryonic stem cell (hESC) WA09 line [34] and thus are considered as ‘‘matured’’ neuronal cells. It should
be noted that the universal neural cell marker Tuj (beta tubulin III) indicates that [80 % of the hN2 cells are neural. The other cell types which constitute\20 % of the cell population are mostly astrocytes and microglia, which are common glial cells found in the brain and spinal cord. The inclusion of the small amount of glial cells in the cell population better mimics real life situation in the central nervous system. The hN2 cells were seeded in 12-well plates at *500,000 cells/well in the AB2 Basal Medium complemented with ANS Supplement (cholinesterase free) provided by Neuromics and cultured at 37OC under
humidified 5 % CO2 for 48 h (without changing media) before VX exposure.

Xiufang Guo, Severo Spradling, Maria Stancescu, Stephen Lambert, James J. Hickman. Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials, In Press, Corrected Proof,Mar 2013.doi:10.1016/j.biomaterials.2013.02.061 ...hNP1, were obtained from Neuromics (Edina, Minnesota)…

Abstract: Although sensory neurons constitute a critical component for the proper function of the nervous system, the in vitro differentiation of functional sensory neurons from human stem cells has not yet been reported. This study presents the differentiation of sensory neurons (SNs) from a human neural progenitor cell line, hNP1, and their functional maturation in a defined, in vitro culture system without murine cell feeder layers. The SNs were characterized by immunocytochemistry and their functional maturation was evaluated by electrophysiology. Neural crest (NC) precursors, as one of the cellular derivatives in the differentiation culture, were isolated, propagated, and tested for their ability to generate sensory neurons. The hSC-derived SNs, as well as the NC precursors provide valuable tools for developing in vitro functional systems that model sensory neuron-related neural circuits and for designing therapeutic models for related diseases.

Images: Generation of Schwann cells from the differentiated culture. Immunostaining of a day 38 culture with the Schwann cell marker S100 demonstrating a significant number of Schwann cells in the culture. Schwann cells were located either within the neuronal clusters (A) or along the axonal bundles (B). The neuronal clusters and axonal bundles were marked by Peripherin immunostaining. doi.org/10.1016/j.biomaterials.2013.02.061

I will continue to post more proof regarding the capabilties and value of our human neurons & neural progenitors as pubs/data/images becomes available

New Osteoblasts

In conjuction with our manuacturing partner, Vitro Biopharma” , we are pleased to announce we now have commercially available unlabeled and FITC-labeled Osteoblasts. These are differentiated from our Umbilical Cord Blood derived Human Mesenchymal Stem Cells.

They are designed for:

  • Osteogenesis/Bone Formation Studies
  • Compound/Small Molecule Testing
  • Gene Expression Analysis
 

Images: (A) Human cord-blood MSCs were expanded in low-serum MSC-GroTM to confluence as shown here. (B) were differentiated in osteogenic MSC-GroTM. Early stage osteoblasts are shown here; the arrow shows early formation of mineralized matrix. (C)&(D) Mature osteoblasts stained positive for Alizarin red. Phase contrast image at 200 x, scale bar is 50 mmeters.

I will continue to update you on new Cell Based Assay Solutions.

QC Study of hMSC Derived Chondrocytes

Check out the latest images and data of Neuromics‘-Vitro Biopharma’s Human Chondrocytes in Culture Days1-5: MSC-Derived Human Chondrocytes in Culture-01-2013.

Phase contrast image (200x) of chondrocyte and associated cells in day five of culture as described (SC00A7 Protocol). Scale bar = 50 m. Chondrocyte is darker multicellular structure overlying attached cells, ~ 70 m in diameter.

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.