Cells from Diseased Donors

Focus on Neuro Diseases

We now have the capability to provide cells of the central and peripheral nervous system from donors with Neuro diseases. These include cells from donors with ALS, AD, PD, and Brain Cancer Donors, to name a few.

We have provided cells to virtually all the large Pharmas and many small and mid-size Bio-techs. We have worked with Novartis to gain 21-CFR compliance for cells that they are using for their eye diseases drug discovery programs.

I am at your “beck and call” should you have interested in exploring specific capabilities further. You can e-mail or call me at 612-801-1007

Human Astroglia and Schwann Cells-BBB Model

Broadening our Capabilities

At the core of our solutions are many options for primary human cells. We are especially pleased that we have growing capabilities to provide new cells to researchers studying autoimmune neuro-degenerative diseases like ALS and MS with the addition of:
Schwann Cells

Human Schwann Cells (HSwC) are isolated from human spinal nerve. HSwC are cryopreserved at passage one and delivered frozen. Each vial contains >5 x 10^5 cells in 1 ml volume. HSwC are characterized by immunofluorescence with antibodies specific to S100, GFAP, and CD90. HSwC are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. HSwC can to further expand for 10 population doublings in our Schwann Growth medium (cat # SGM001).
Human Astrocytes

 Human Brain Astrocytes cultured with AlphaBioCoat.
Human Blood Brain Barrier Model
I will continue to post updates here.

Mouse and Human Motor Neurons

Designed for Neuro-muscular Diseases Research

Clients have been using our easy to culture and research proven GFP Labeled Mouse Motor Neurons for Neuro-muscular disease research. This includes the inclusion of the cells in several ALS drug discovery programs being conducted by large Pharma.

Image: GFP+ mMN Mouse Motor Neurons at 2 days post thaw 20X.

I am pleased to announce the addition of Human Motor Neurons to our Primary Human, Mouse and Rat Neurons, Astrocytes and Neuron-Astroglial co-culture solutions.

Image: Human alpha-Motor Neurons

Questions?  Do not hesitate to contact me directly, Pete Shuster, CEO and Owner, Neuromics, pshuster@neuromics.com and direct phone: 612-801-1007. Thank you.

Apoptosis and Neurodegeneration

Towards the Development of Disease Specific Assays
Neurodegenerative diseases are becoming increasingly prevalent, especially in the Western societies, with larger percentage of members living to an older age. They have to be seen not only as a health problem, but since they are care-intensive, they also carry a significant economic burden.

Apoptotic pathways are induced in many of these diseases and are key culprits in disease progression.

Figure: Schematic representation of apoptotic pathways. Apoptosis triggered by internal (intrinsic) or external (extrinsic) stress signals that is activated by binding of ligands (e.g. FasL, APO-2L, TRAIL, TNF) to cell surface receptors (e.g. Fas, DR4, DR5, TNF-R1). The intrinsic apoptosis pathway might be triggered by p53 upon DNA damage following exposure to cellular stress. In the intrinsic pathway, death signal reaches mitochondria, leading to release of cytochrome c, which can binds to Apaf1. The cytochrome c/Apaf1 make a complex with pro-caspase-9 (in the presence of dATP), activates caspase-9, which promotes caspase-3 activation, eventually leading to cell death. The extrinsic pathway is initiated through the stimulation of the members of tumor necrosis factor receptor (TNF-R) family (transmembrane death receptors) by their respective ligands. These receptors activate pro-caspases-8, -10 by recruiting the endogenous adaptor protein FADD. Procaspase-8, -10 cleave themselves to form activated caspase-8 or -10. Ultimately, effector enzymes such as caspase-3, -6, -7 are activated in this cascade to mediate apoptosis. Likewise, there can be cross-talk between the intrinsic and extrinsic pathways. For example caspase-8 may cleave Bid to form tBid that is a strong activator of the intrinsic/mitochondrial apoptotic pathway. The intrinsic pathway is usually activated by the recruitment of BAX and BAK to outer mitochondrial membrane, causing cytochrome c release formation of apoptosome and subsequent activation of caspase-9. Activated caspase-9 proteolytically activates caspases-3, -6, and -7. Moreover, some of the effector caspases also can activate caspase-8, forming a positive amplification loop. doi:10.1016/j.pneurobio.2013.10.004.

Working with the Apoptosis Experts at Immunochemistry Technologies and Human Astroglial-Neuron Biosensors Experts at ArunA Biomedical, we plan on  developing disease specific assays. Here's a map of the general process.

Larger Image

We welcome feedback and input on your interests. You can email rose@neuromics.com. We will continue to post updates on exciting new developments.

MEA and Motor Neurons

Plating Densities of Motor Neurons Matter!

I wanted to share some of the tips and data shared during the ArunA Biomedical's/Axion Biosystem's Webinar on MEA and our Mouse Motor Neurons

Plating Cells on Axion's MEA

• Surface Coating PEI-laminin for adhesion and uniform monolayer development 
• Dotting Constrains cells to the array 
• Requires fewer cells per well 
• Media changes every 2-3 days
• Variations on Cell Density

Detailed Protocol for Culturing Motor Neurons on MEA

Different Plating Densities

Plating Densities between 60,000-80,000 Optimal
At these densities there was the least variation in mean firing rates. This data shows as the density increased the cells moved towards firing in synchronicity.

Figure: Difference in Mean Motor Neurons Firing Rates vs Plating Densities

The demand for using these motor neurons for neuromuscular diseases drug discovery has been brisk and growing. Should you have question on how they would work for your unique applications, do not hesitate to contact me directly @ 612-801-1007 or pshuster@neuromics.com. Pete Shuster, CEO and Owner, Neuromics

GFP Labeled Motor Neurons and MEA

Big Upcoming Webinar

We have been strategically partnering with ArunA Biomedical to improve how we serve Neuro-drug discovery and Neurotox research community. I am especially excited about how well are our GFP Labeled Mouse Neurons are working in many different and unique research applications. They have proved an important tool in the study of neuro-muscular diseases like ALS, Parkinson's and Multiple Sclerosis.

I am pleased to announce a coming Webinar: “GFP+ Motor Neurons: Development and in-vitro Functional Assessment” Wednesday June 10th, 11:30 AM EDT-Register Today!

Download Flyer. I will continue to post unique assays developed for our Astro-glial Neuron solutions.

Solutions for Studying Neuro-degeneration

Data Rich and Frequently Published

The Neuromics' brand is built, in part, by our proven ability to provide solutions for the study of neuro-degeneration. These include:

• Neuronal-Glial Markers
• Primary Neurons and Astrocytes
• Apoptosis Research Reagents
• Transfection Reagents

A recent example shows the use of one of  MAP-2 markers to study hearing decline with age: Radtke-schuller S, Seeler S and Grothe B(2015) Restricted loss of olivocochlear but not vestibular efferent neurons in the senescent gerbil (Meriones unguiculatus). Front. Aging Neurosci. 7:4. doi:10.3389/fnagi.2015.00004.

Figure: Lipofuscin granules in MSO neurons of an aged gerbil. MSO neurons are MAP2 immunostained (Alexa Fluor 647, red). Lipofuscin granules have been excited with the DAPI excitation wavelengths and appear blue. Confocal images show a maximum projection of image stacks in A and a single optical image of 0.3 µm thickness in the enlargement in B. Scale bar in A: 50 µm and 20 µm in B.

We stand ready to serve you. Should you have interest or questions, do not hesitate to contact me directly: Pete Shuster-Owner/CEO-pshuster@neuromics.com or direct phone: 612-801-1007. Thank you.

OPC Markers!

Effective Oligodendrocyte, Oligodendroglial Oligodendrocyte Lineage Markers are important for determining the differentiate state of Oligodendrocyte Precursor Cells. This is important for the study of de and re-myelination of neurons and the discovery of potential therapeutic targets for diseases like MS and ALS.

Here researchers use our Olig2 antibody to study the differentiation state of Fetal Human Oligodendrocyte Progenitor Cells: Crystal R. McClain, Fraser J. Sim and Steven A. Goldman. Pleiotrophin Suppression of Receptor Protein Tyrosine Phosphatase-β/ζ Maintains the Self-Renewal Competence of Fetal Human Oligodendrocyte Progenitor Cells. The Journal of Neuroscience, 24 October 2012, 32(43): 15066-15075; doi: 10.1523/​JNEUROSCI.1320-12.2012.
Abstract: Oligodendrocyte progenitor cells (OPCs) persist in human white matter, yet the mechanisms by which they are maintained in an undifferentiated state are unknown. Human OPCs differentially express protein tyrosine phosphatase receptor β/ζ (PTPRZ1) and its inhibitory ligand, pleiotrophin, suggesting the maintenance of an autocrine loop by which PTPRZ1 activity is tonically suppressed. PTPRZ1 constitutively promotes the tyrosine dephosphorylation of β-catenin and, thus, β-catenin participation in T cell factor (TCF)-mediated transcription. Using CD140a/PDGFRα-based fluorescence-activated cell sorting to isolate fetal OPCs from the fetal brain at gestational ages 16–22 weeks, we asked whether pleiotrophin modulated the expansion of OPCs and, if so, whether this was effected through the serial engagement of PTPRZ1 and β-catenin-dependent signals, such as TCF-mediated transcription. Lentiviral shRNAi knockdown of PTPRZ1 induced TCF-mediated transcription and substantially augmented GSK3β inhibition-induced TCF-reporter luciferase expression, suggesting dual regulation of β-catenin and the importance of PTPRZ1 as a tonic brake upon TCF-dependent transcription. Pharmacological inhibition of GSK3β triggered substrate detachment and initiated sphere formation, yet had no effect on either proliferation or net cell number. In contrast, pleiotrophin strongly potentiated the proliferation of CD140a+-sorted OPCs, as did PTPRZ1 knockdown, which significantly increased the total number of population doublings exhibited by OPCs before mitotic senescence. These observations suggest that pleiotrophin inhibition of PTPRZ1 contributes to the homeostatic self-renewal of OPCs and that this process is mediated by the tonic activation of β-catenin/TCF-dependent transcription.

Images: To verify that GSK3β inhibition was effecting TCF activation through altering localization of β-catenin, the Wnt signaling intermediate, β-catenin, was localized by confocal imaging in OPCs, validated as such by their coexpression of Olig2.

Marker Options:

Name	Catalog #	Type	Species	Applications	Size	Price
CNPase	CH23013	Chicken IgY	H; M	ICC; IHC	100 ul	$89
Caspr2	SP15104	Sheep IgG	H; M	IHC; WB; E	100 ug	$365
HSP105	MO20028	Mouse IgG	H; M; R	IHC; WB	100 ul	$155
MAG/Siglec 4a	GT15152	Goat IgG	R	IHC; WB; E	100 ug	$365
MOG	GT15141	Goat IgG	H	IHC; WB; E	100 ug	$365
Mash1	GT15216	Goat IgG	M	IHC; WB; E	100 ug	$365
Mash1	MO15048	Rat IgG	H; M	ICC; WB; E	100 ug	$255
NOGO Receptor	GT15154	Goat IgG	H	IHC; WB; E	100 ug	$365
OMgp	GT15200	Goat IgG	H	WB; E	100 ug	$365
Olig1	RA14141	Rabbit IgG	R	IHC	100 ul 100 ul @ 1mg/ml	$350 $95
Olig1,2,3	MO15059	Mouse IgG	H; R	IHC	100 ug	$305
Olig2	GT15132	Goat IgG	H; M	IHC; WB; E	100 ug	$365
Olig2	RA25081	Rabbit IgG	H; M; R	ICC; IHC; WB; IP	100 ul	$395
Oligodendrocyte Marker O1	MO15001	Mouse IgM	H; M; R	IHC; FC	50 ug	$215
Oligodendrocyte Marker O4	MO15002	Mouse IgM	C; H; M; R	IHC	50 ug	$215
Oligodendrocyte Marker O4-Phycoerythrin Labeled	FC15013	Mouse IgM	H	FC	100 Tests	$305
PDGF R Alpha/CD140A	GT15150	Goat IgG	M	IHC; WB; E	100 ug	$365

ApoTransferrin and the fate of Neural Stem Cell/Progenitors

Implications for De-Myelinating Diseases Like MS and ALS

Dr. Juana María Pasquini and her team at the University of Buenos Aires are ongoing users of our Neural Stem Cell-Progenitor (NSC-NP) Markers. In this study, they use these markers to determine the states and fates of NSCs and NPs as they proliferate and differentiate and the related role of ApoTransferrin (aTF). Here we learn aTf exposure during differentiating conditions favours OL maturation from OPCs by promoting OL morphological development. This evidence supports a key role of Tf on the generation of OL from NSC/NPCs and highlights its potential in demyelinating disorder treatment: Silvestroff L , Franco PG , Pasquini JM (2012) ApoTransferrin: Dual Role on Adult Subventricular Zone-Derived Neurospheres. PLoS ONE 7(3): e33937. doi:10.1371/journal.pone.0033937.

Proliferation rates under different conditions are shown in A–C. BrdU incorporation (red) during proliferation (CTLP, A) or differentiation (CTLPCTLD, B). BrdU+ cells are expressed as a percentage of total nuclei for either condition in C. Free floating NS during proliferation express Nestin (D, green) and GFAP (E, green). After dissociation, NS-derived cells continue to express Nestin (F, green). PDGFRα+ (G, green) and NG2+ cells (H, green). Few MBP+ (I, green) cells were found under proliferative conditions. A large proportion of BrdU incorporating cells (J, red) co-expressed with NG2 (J, green). Some BLBP+ cells (K, green) incorporated BrdU (K, red). After differentiation (L–O), MBP+ cells were found with a highly branched and complex morphology (L, green). Cells expressed GFAP (M, green), as well as the neuronal NF200 marker (N, green). BrdU incorporating (O, red) cells were mostly NG2+ (O, green) during differentiation conditions. BrdU+ cells co-expressing NG2, as a proportion of total BrdU+ cells, are shown in P for either culture condition. A representative Western Blot membrane in Q shows how MBP levels increase in whole cell protein extracts as cells differentiate. The densitometric analysis of the MBP isoforms/GAPDH ratio of 5 independent experiments was semi-quantitated in R. All 4 MBP isoforms were pooled and considered as a single value before normalizing to GAPDH values. Blue colour in images indicates Höechst nuclear dye. Scale bar in A represents 250 µm for A and B. Scale bar in D equals 100 µm in D–I and L–N, scale bar in J equals 250 µm in J and O, and scale bar in K represents 50 µm. Bars in P represent mean values of 2 independent experiments. Bars in C and R represent Mean + SD of 4 and 5 individual cultures, respectively. Student's t Test was used to analyze data in C, while a One Way ANOVA with an SNK Post-test was used to analyze data in R. * p<0.05, ** p<0.01, *** p<0.001

Note:  PDGFRα+ is a marker for oligodendrocytes (OLs).

Here's the pathway model that sumarizes authors' findings

I will keep you posted on research that could implications for the discovery of de-myelinating disorder therapies.

Primary Neurons vs PC12 cells for Compound Testing

This publication compares PC12 Cells vs E18 Primary Cortical Neurons. The cells showed permeability to some key compounds where the Neurons did not. This demonstrates the importance of including primary neurons in compound testing assays for Neuro-disease research: Wei Zhang , Radhia Benmohamed, Anthony C. Arvanites, Richard I. Morimoto, Robert J. Ferrante, Donald R. Kirsch, Richard B. Silverman. Cyclohexane 1,3-diones and their inhibition of mutant SOD1-dependent protein aggregation and toxicity in PC12 cells. Bioorganic & Medicinal Chemistry. Elsevier Ltd. All rights reserved.doi:10.1016/j.bmc.2011.11.039.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2–3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC50 of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efficiently penetrate the blood–brain barrier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cell potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73)
were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar
potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel
therapeutic candidates for ALS.

see: Bioorg. Med. Chem. 2011, 19, 613. and J. Med. Chem. 2012, in press

Related Links: Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes.

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.

Glutaredoxin 2 prevents aggregation of mutant SOD1

Our PTEN-induced kinase, PINK1 or PARK6 Antibody is an excellent marker for Amyotrophic Lateral Sclerosis (ALS) and Parkinson's Disease (PD) researchers.

Here's  new publication referencing use of this antibody:  Alberto Ferri, Paolo Fiorenzo, Monica Nencini, Mauro Cozzolino, Maria Grazia Pesaresi, Cristiana Valle, Sara Sepe, Sandra Moreno, and Maria Teresa Carrì. Glutaredoxin 2 prevents aggregation of mutant SOD1 in mitochondria and abolishes its toxicity.
Hum. Mol. Genet., first published on Sep 20, 2010 as doi: doi:10.1093/hmg/ddq383

Abstract:
Vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS) arises from a combination of several mechanisms, including protein misfolding and aggregation, mitochondrial dysfunction and oxidative damage. Protein aggregates are found in motoneurons in models for ALS linked to a mutation in the gene coding for Cu,Zn superoxide dismutase (SOD1) and in ALS patients as well. Aggregation of mutant SOD1 in the cytoplasm and/or into mitochondria has been repeatedly proposed as a main culprit for the degeneration of motoneurons. It is, however, still debated whether SOD1 aggregates represent a cause, a correlate or a consequence of processes leading to cell death. We have exploited the ability of glutaredoxins (Grxs) to reduce mixed disulfides to protein thiols either in the cytoplasm and in the IMS (Grx1) or in the mitochondrial matrix (Grx2) as a tool for restoring a correct redox environment and preventing the aggregation of mutant SOD1. Here we show that the overexpression of Grx1 increases the solubility of mutant SOD1 in the cytosol but does not inhibit mitochondrial damage and apoptosis induced by mutant SOD1 in neuronal cells (SH-SY5Y) or in immortalized motoneurons (NSC-34). Conversely, the overexpression of Grx2 increases the solubility of mutant SOD1 in mitochondria, interferes with mitochondrial fragmentation by modifying the expression pattern of proteins involved in mitochondrial dynamics, preserves mitochondrial function and strongly protects neuronal cells from apoptosis. The toxicity of mutant SOD1, therefore, mostly arises from mitochondrial dysfunction and rescue of mitochondrial damage may represent a promising therapeutic strategy.

Related Reagents: Parkin Parkin-2 PARK2 Co-regulated (PACRG) PARK7 (DJ-1) LRRK2 (PARK8)                         Neurodegenerative Disease Research Antibodies

Neurodegenerative Disease Research Proteins Neurotransmission -Neurotransmission Research Antibody Categories                          Neurotrophins and Growth Factor Antibodies Neuron-Glial Expressed-Includes Neurotrophin Proteins

Apoptosis Research Reagents-Apoptosis Categories-includes: detection kits, antibodies and proteins Primary Neurons and Astrocytes -Primary human, rat and mouse neurons and astrocytes by Category