FGF and Stem Cells-Options Matter

Proven, Potent and Cost Effective Fibroblast Growth Factors (FGF).

Neuromics have a wealth of expertise in Stem Cells, Media and Growth Factors. FGF is an important component of Stem Cell Based Assays. Our goal is to provide an FGF that fits your requirements like "hand in glove".

Here's a small sampling:
ISO-kine bFGF-100% animal free and serum free-is produced in the endosperm tissue of barley grain (Hordeum vulgare), that exhibits up to 50 times less protease activity than E.coli or mammalian cells. Barley seed is void of any human or animal viral contaminants that could jeopardize your cell culture.

Images A: Expression of OCT4 (green) in the ORF group. B: Expression of TRA-1-60 (green) in the ORF group.

FGF basic (146 aa)-Anika Chowdhury, Louis W Bezuidenhout, Aillette Mulet-Sierra, Nadr M Jomha and Adetola B Adesida. Effect of interleukin-1β treatment on co-cultures of human meniscus cells and bone marrow mesenchymal stromal cells. BMC Musculoskeletal Disorders 2013, 14:216 doi:10.1186/1471-2474-14-216

...15 million MNCs were seeded per 150 cm2 tissue culture flask. Culture media was alpha MEM supplemented with 10% heat inactivated fetal bovine serum, 1 mM sodium pyruvate, 100 mM HEPES buffer, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml L-glutamine (all from Invitrogen, Mississauga, Ontario, Canada) and 5 ng/ml of basic FGF or FGF-2 (Neuromics, Edina, MN, USA). Plastic adherent MNCs were allowed to attach and proliferate for 7 days before the first media change under normal oxygen tension (21% O2; 95% air) at 37°C in a humidified incubator with 5% CO2...

Images: Histological characteristics of pellets formulated from mono-cultured MCs, mono-cultured BMSCs and co-cultures of MC and BMSCs after a total of 17 days culture in defined serum-free chondrogenic media. (A-B) Safranin O and collagen II immuno-histochemical staining of representative pellets from cells derived from the same donor. Magnification lens × 20; scale bar is 100 μm. Chowdhury et al. BMC Musculoskeletal Disorders 2013 14:216 doi:10.1186/1471-2474-14-216

We are working hard to provide unique and cost effective solutions for your Stem Cell Based Assay Requirements.

Stem Cell Markers

Our Stem Cell Reagents our widely used and frequently published. We are proud of the positive feedback on our Stem Cell Markers.

Image: Tuj-1 staining of Neuron-specific class III β-tubulin in differentiated human neural progenitor cells. Cells were stained using goat anti-mouse Alexa Fluor 488 (green) secondary antibody (Molecular Probe, A-11001) and counterstained with PI (red).

Image: Neural progenitors were labeled with anti-rat Nestin polyclonal antibody (Cat#:GT15114) and stained with conjugated donkey anti-goat secondary anti-body (green). Differentiated neurons were labeled with neuron-specific mouse anti-β-III tubulin/ Tuj1-(Cat#MO15013 monoclonal antibody (red). Nuclei were stained with DAPI (blue).

Tuj-1: Customer Publications and Data.

I will continue to post updates.

Protocol for Flow Cytometric Sorting of Enriched Neuronal Cultures from iPSCs

Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, the authors present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. They have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f^-/CD200^high surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology: Turaç G, Hindley CJ, Thomas R, Davis JA, Deleidi M, et al. (2013) Combined Flow Cytometric Analysis of Surface and Intracellular Antigens Reveals Surface Molecule Markers of Human Neuropoiesis. PLoS ONE 8(6): e68519. doi:10.1371/journal.pone.0068519

Figure 1. Experimental outline. Schematic illustrating the research strategy of identifying novel surface marker combinations on a target population in neural and other stem cell differentiation systems for which intracellular, standard immunocytochemical markers are well established. Following harvesting, the resulting single cell suspension is subject to surface antigen candidate staining, followed by gentle fixation, permeabilization and subsequent co-staining with known intracellular markers. CD markers co-labeling the target population serve as positive markers, those absent on the target population serve as negative markers. In a separate, subsequent step, a combination of the identified positive and/or negative CD markers enables the flow cytometric enrichment of the viable population of interest from a heterogeneous cell suspension for further study and biomedical applications. doi:10.1371/journal.pone.0068519.g001.

Figure 2. Accurate detection of intracellular antigens with optimized fixation-permeabilization conditions preserving surface antigens. Flow cytometric detection of TUJ1, MAP2 and nestin antigens in BJ fibroblasts and the neural SH-SY5Y cell line (A). TUJ1 and nestin are present in both cell lines, while the mature neuronal marker MAP2 was only detected in SH-SY5Y cells (arrows). Note stable fluorescent levels of the negative population, indicating low background staining using this protocol. Representative experiment of three independent repeats shown. (B) Corresponding validation by immunofluorescence analysis. (C) Quantitation of TUJ1, MAP2 and nestin intracellular antigen detection (n=3). Error bars indicate standard deviation. (D) Response of TUJ1 and MAP2 intracellular antigen expression to 6 DIV of 10 µM retinoic acid (RA) treatment of SH-SY5Y cells. Note disappearance/reduction of subsets negative for these markers (upward shift, green arrows), as well as a shift toward CD184low expression with differentiation (blue arrows). doi:10.1371/journal.pone.0068519.g003.

Please note: In addition to the our Human Mouse Monoclonal Nestin Antibody used in the study, we have an extensive catalog of stem cell solutions. I will continue to post highlights of new applications.

Everything You Wanted to Know About Stem Cells

Unforgettable way to learn about Stem Cells.

Rap on Dr. Jonathan Garlick Rap on.

If you are doing stem cell related research, check out our:

hNP1™ Human Neural Progenitor & hN2™ Neuron Discovery Kits Derived from H9 (WA09) ECSs-Consistent, Easy to Use & Cost Effective	Neural Stem Cells Media
hMPro™ Human Mesenchymal Progenitors (hMPCs) hESC Derived	Human Mesenchymal Stem Cells (hMSCs) hMSCs Derived from Umbilical Cord Blood
MSCGro™ Mesenchymal Stem Cell Media Proven and Potent Culture Growth and Differentiation Media for Mesenchymal Stem Cells (MSCs)	Cell Cryopreservation Media Maximizes cell recovery, attachment and growth
Expansion/Differentiation Kits SC to Oligodendrocytes and Dopaminergic Neurons Kits	Bioluminomics™ In Vitro Assays Brought to you by HemoGenix®
3-D Cell Based Assay Solutions Nanofibers, Hydrogels and Extracellular Matrix (ECM) Proteins	Stem Cell Research Antibodies
Stem Cell Research Proteins	Rat Neurosphere Neuroprogenitor Tissue Tissue provided live, unseparated, fresh from E18 rat cortex/hippocampus including subventricular zone.

Shiny, New GF nanoparticle labeled hMSCs

Human Mesenchymal Stem Cells-Green Fluorescent Nanoparticle-labeled-These cells are generated by chemical transfection of CdSe/ZnS nanoparticles, 100 nm in diameter and show a strong fluorescent signal (Excitation maximum= 515 nm; Emission maximum= 520-550 nm) detectable by standard FITC filters.

Image: FITC analysis of cells during fourth passage following transfection. Image obtaining using FITC filter on Olympus CKX41 microscope at 200 X equipped with a Retiga 2000 digital camera and Q-Capture software program. Scale bar is 25 micrometers. Fluorescent nanoparticles are less prevalent within the cytoplasm than earlier passages. Technical Information & Data.

They retain GFNP labeling through 3+ passages and can be differentiation into chondrogenic, adipogenic or osteogenic lineages. Excellent for regeneration, toxicity and related studies. Technical Information & Data.

Image: Osteogenic differentiation of GF nanoparticle-labeled MSCs. Image obtaining using phase contrast Olympus CKX41 microscope at 100 X equipped with a Retiga 2000 digital camera and Q-Capture Pro software program. Scale bar is 25 micrometers. Note the presence of mineral deposits indicating differentiation into osteoblasts. Chondrogenic and adipogenic differentiation was also apparent.

• All Stem Cell Research Reagents.

100% Animal Free FGF Basic

New ISO-kine^TM FGF basic-only 39 USD-10 ug.

Fibroblast Growth Factor (FGF) Recombinant Proteins are widely used as tools in Stem Cell Research. They have the capabilities to catalyze growth and differentation as well as maintain cell stasis. We continue to offer new FGFs to make sure we meet the unique demands of our cutsomers and collaborators  .

Our E. Coli derived Human FGF (146 aa) recombinant protein is our #1 seller. As Stem Cell research moves from the bench top to the bedside, there will be a growing need for 100% animal free proteins. We are please to announce we have added a 100% animal free FGF (146 aa) basic that has bio-activity comparable to our top selling options

Image: ISOKine^TM bFGF vs our potent and proven e-coli derived bFGF.

I anticipate offering more ISOKine^TM Bio-risk free Stem Cell Research growth factors to meet growing demand. This growth will be driven by their unique advantages. These include low protease activity and secondary metabolite content and simple protein content all of which aid in downstream processing. These products also have the G.R.A.S. (Generally Recognised As Safe) status from FDA.

Ready for Prime Time Stem Cell Markers

Researchers frequently reference use of Neuromics' Stem Cell Markers in publications. This is an important affirmation for us as these tools are critical for determining the differentiation state of Stem Cells. In this publication, the authors use our Mouse Monoclonal Nestin Antibody to understand the mechanisms underlying neural progenitor differentiation and neuronal fate. This understanding is an important precursor for using these cells in Regenerative Medicine: Serafí Cambray, Charles Arber, Graham Little, Antonios G. Dougalis, Vincenzo de Paola, Mark A. Ungless, Meng Li and Tristan A. Rodríguez. Activin induces cortical interneuron identity and differentiation in embryonic stem cell-derived telencephalic neural precursors. Nature Communications 3, Article number: 841 doi:10.1038/ncomms1817. Received 10 January 2011 Accepted 29 March 2012 Published 15 May 2012.

In this study, the authors show that Activin provides telencephalic neural precursors with positional cues that specifically promote the acquisition of a calretinin interneuron fate by controlling the expression of genes that regulate cortical interneuron identity. This work demonstrates a novel means for regulating neuronal differentiation and specification of subtype identity.

Images: (a)immunostaining (left panels) and quantifications (right panel) indicating that Shh promotes and cyclopamine inhibits proliferation in neural precursors (Nestin+/β-III-tubulin+ cells in cyclopamine 49±4.3/35.1±1.8%, Shh 80.3±3.2/14.8±0.7% and control cultures 68.4±7.2/20.1±2.8%; n=3, mean±s.e.m.). (b) Relative expression levels of Gli1 and Ptch1 during the first 5 days of Activin or control treatment. (c) Immunoblot analysis of Gli1 and Cyclin D1 levels during the first 4 days of Activin or control treatment illustrating how Activin represses the expression of these proteins. (d) Normalized mRNA levels of Gli1 and Ptch1 after 24 h exposure to the indicated conditions illustrating how Shh induces the expression of these genes and Activin inhibits their expression (n=3, mean±s.e.m. Student's t-test. *P<0.005 and **P<0.05). ESCs were differentiated for 5 days as a monolayer, then replated into poly-D-lysine/laminin-coated dishes and cultured in NBB27 media (controls), NBB27+10 ng ml−1 Activin, NBB27+100 ng ml−1 Shh, NBB27+10 μm cyclopamine or NBB27 + 10 ng ml−1 Activin + 100 ng ml−1 Shh. Scale bar=50 μm.

Image: 

Image: Model for how Activin induces the differentiation and CGE fate in telencephalic neuronal precursors.


The protocol described in this manuscript represents a method to obtain an enriched source of calretinin interneurons from both mouse and human ESCs. Therefore, our work significantly contributes to the aim of generating the diverse neuronal subtypes required for the safe and successful use of ESCs in regenerative medicine.

The capabilities of stem cells markers matter in developing protocols for use in Regenerative Medicine.

Mesechymal Stem Cell DifferentiationPathways

As a provider of Human Mesenchymal Stem Cells (hMSCs), MSCGro™ Mesenchymal Stem Cell Media and Stem Cell Markers, I receive questions related to differentiation. Specifically, researchers desire to drive these cells to specific progenitor and cell phenotypes like Osteocytes, Adipocytes and Chondroytes.

I would like to share a pathway map that gives a snapshot of these pathways:

Regenerative Biology of the Spine and Spinal Cord. Edited by: Rahul Jandial, Mike Y. Chen, Bihong T. Chen and Joseph Ciacci. ISBN: 978-1-4614-4089-5. Publication date: May 25, 2012. Series: Special Books

I will continue to post information that will enable the researchers to harness the power of Mesenchymal Stem Cells.

3-D Cell Based Assay Solutions

We want to help our customers, collaborators and friends bring their cell based assays to life! Neuromics began offering solutions in early 2011. We have extended our reach with our 3D Nanofibers. These 3-D environments are more biologically realistic leading to:
•more effective biomedical research.

•earlier breakthroughs.

•faster and cheaper time to market for drug development.

•improved stem cell expansion rates.

Here's a video showing green neurosphere (brain cancer cells)  migrating along our nanofibers aligned in the vertical direction. This enables cancer researchers to see cancer in ways never before possible!

Find more videos like this on TechLounge

I will update you as data and related publications become avaialable using these solutions.

hNP1 Neural Progenitors Thawing+ Plating Protocol

This is a great video that shows thawing + plating protocol for our eSC Derived Human NP1 Neural Progenitors.

Reagents that work for Drug Discovery and Basic Research.

We have redesigned our website! New capabilities easier for visitors to find products and related product content:

Reagents that work for Drug Discovery and Basic Research.

Posted using ShareThis

Rock Solid Tuj-1

The feedback from customers on our Tuj 1 (Neuron-specific class III beta-tubulin) is that it works!

This is confirmed by the growing list of references in key publications. Here's the latest:

S A Sakowski, S B Heavener, J S Lunn, K Fung, S S Oh, S K Spratt, N D Hogikyan and E L Feldman. Neuroprotection using gene therapy to induce vascular endothelial growth factor-A expression. Gene Therapy advance online publication 3 September 2009; doi: 10.1038/gt.2009.111.

...TUJ1 (Neuromics, Edina, MN, USA). ...

Tuj 1 (Neuron-specific class III beta-tubulin)
Related Reagents:
Nestin
Musashi-1
Other Reagents to Consider:
Stem Cell Reagents
Neuron/Glial Markers

Tracking STEMEZ hNP1 Progenitor Cell Fate

We would like to promote an important new discovery using our STEMEZ(TM) hNP1 Human Neural Progenitors Discovery Kit.

Human embryonic stem cell–derived neural progenitors (NP) present an important tool for understanding human development and disease. Optimal utilization of NP cells, however, requires an enhanced ability to monitor these cells in vitro and in vivo. Here we report production of the first genetically modified self-renewing human embryonic stem cell–derived NP cells that express fluorescent proteins under constitutive as well as lineage-specific promoters, enabling tracking and monitoring of cell fate. Nucleofection, transfection, and lentiviral transduction were compared for optimal gene delivery to NP cells. Transduction was most efficient in terms of transgene expression (37%), cell viability (39%), and long-term reporter expression (>3 months). Further, the constitutive gene promoters, cytomegalovirus, elongation factor 1α, and ubiquitin-C, exhibited comparable silencing (20–30%) in NP cells over a 2-month period, suggesting their suitability for long-term reporter expression studies. Transduced NP cells maintained their progenitor state and differentiation potential, as demonstrated by expression of endogenous NP markers and neuronal markers after differentiation. We also detected reporter expression in astrocytes generated from NP cells transduced with an astrocyte-specific gene promoter, glial fibrillary acidic protein, demonstrating the usefulness of this approach. The genetically manipulated NP cells described here offer great potential for live cell–tracking experiments, and a similar approach can as well be used for expression of proteins other than reporters.

Related Publication:
Sujoy K. Dhara, Brian A. Gerwe, Anirban Majumder, Mahesh C. Dodla, Nolan L. Boyd, David W. Machacek, Kowser Hasneen, Steven L. Stice. Genetic Manipulation of Neural Progenitors Derived from Human Embryonic Stem Cells. Tissue Engineering Part A. -Not available-, ahead of print. doi:10.1089/ten.tea.2009.0155

Neuromics' Stem Cell Markers Referenced:
Musashi-1, Rabbit, 1:100 , Catalog#:RA14128
Nestin, Mouse 1:500, Catalog#: MO15012
Tuj 1 (Neuron-specific class III beta-tubulin), Mouse, 1:500, Catalog#: MO15013
TH, Chicken, 1:80, Catalog#: CH23006

STEMEZ hNP1 Neural Progenitors Now Available

We are pleased to announce the introduction of our STEMEZ ^TMhNP1 Human Neural Progenitors Discovery Kit. The kit was developed by our partner, ArunA Biomedical.

Description/Data:
STEMEZ^TM hNP1 Human Progenitors are fully differentiated are derived as adherent cells from hESC WA09 line. The cells are shipped frozen in a vial with 1 x 10⁶ cells; once thawed they should be immediately plated on a Matrigel (or other suitable extracellular matrix protein)-coated dish and maintained in the accompanying serum-free medium. The neurons should be used within 14 days of thawing. These cells have the capabilities to:

• Display immunoreactive properties consistent with neural precursors and can be maintained in a proliferative state in monolayer cultures.
• Differentiate under serum-free conditions into neuronal phenotypes with functionally responsive transmitter receptors in vitro .
• Maintain multiple neural phenotypes in long term serum-free culture.
• Can be directed to alter phenotypic characteristics under different media conditions.

Applications: Gene Expression Analysis Western blotting, Flow Cytometry, Immunocytochemistry, FACS sorting, DNA Microarray, RT-PCR, Neurite Outgrowth assays, FLIPR calcium assays, cytotoxicity, and second messenger signaling.

Images: STEMEZ^TM hNP1 Human Neural Progenitor Cells are grown as monolayers (A), are karyotypically normal (B) and express NSC markers, Nestin Mouse and Sox-2 (C, D, E). Nuclei of the cells were visualized with DAPI (blue). The Sox-2 transcription factor is co-localized with the DAPI (blue) staining in the nucleus (F).

STEMEZ hNP1 FAQs

Note: STEMEZTM NP1 Progenitors and hN2 Human Neurons can be genetically manipulated with GFP reporters without altering the function and differentiation potential of these cells. In contrast to many stem cell sources, the reporter genes are not readily silence in the neural cultures. See: doi:10.1089/ten.tea.2009.0155.

DNA fingerprint cells: The loci match the DNA fingerprint pattern for the H9 (NIH designation, WA09) HESC line as published in http://stemcells.nih.gov/research/nihresearch/scunit/.

Viral tests cells: This lot was derived from the H9 hESC line that has been tested for Hepatitis B, Hepatitis C, HIV-1, HIV-2, HTLV-I/II, HSV1, HSV2, EBV, and CMV. The H9 cell line has been tested and shown to be negative. (Tests performed by GIVF Laboratories).

Related Reagents:

• STEMEZ(TM) hN2 Human Neurons Discovery Kit


• Neuron/Glial Marker Antibodies


• Neurotrophins and Growth Factor Antibodies


• Fibroblast Growth Factor Proteins


• Neurotrophins-Neuron/Glial Marker Proteins


• Stem Cell Reagents


• Transfection Reagents