Electrical Preconditioning of Stem Cells

Cool Science

The ability to manipulate hNPCs via a conductive scaffold creates a new approach to optimize stem cell-based therapy and determine which factors (such as VEGF-A) are essential for stroke recovery: Paul M. Georgea, Tonya M. Blissb, Thuy Huab, Alex Leed, Byeongtaek Oh, Alexa Levinson, Swapnil Mehta, Guohua Sun, Gary K. Steinberg. Electrical preconditioning of stem cells with a conductive polymer scaffold enhances stroke recovery. doi.org/10.1016/j.biomaterials.2017.07.020...anti βIII-tubulin (1:500, Neuromics, Edina, MN)...

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).

We have excellent Stem Cell Differentiation Markers. Check them out.

Staining Neurons and 3-D

Tuj-1 or Beta Tubulin Antibody in Action!

Our potent Neuron-Glia Markers are widely used and frequently published. We are also pleased with the many positive reviews.

In this study researchers use our Tuj-1/Beta Tubulin Antibody to stain neurons in 3-D Cultures: Gaoying Sun, Wenwen Liu, Zhaomin Fan, Daogong Zhang, Yuechen Han, Lei Xu1, Jieyu Qi, Shasha Zhang, Bradley T. Gao, Xiaohui Bai,Jianfeng Li,Renjie Chai, Haibo Wang. The three-dimensional culture system with matrigel and neurotrophic factors preserves the structure and function of spiral ganglion neuron in vitro...Tuj-1-β-tubulin (1:1000, Neuromics, USA)...

Images: Morphology of the SGNs growth cone cultured in 2D and 3D systems. Phalloidin, green; β-tubulin, red.

I will continue to post positive developments concerning use of our Neuron-Glia Markers.

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.

Neuron-specific class III beta-tubulin (TuJ1) Staining in hNT2.19 Neurons

Our Tuj-1 antibody is widely used and frequently published. It is proving a potent marker for confirming the differentiation of human neural progenitors to neurons.

In this study the authors use the marker for staining hNT2.19 Neurons: Mary J. Eaton, Yerko Berrocal, and Stacey Q. Wolfe. Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines. Pain Research and Treatment. Volume 2012 (2012), Article ID 356412, 31 pages. doi:10.1155/2012/356412. 
The results show great promise. They show hNT2 or hNT2-derived cell lines, such as hNT2.17 and hNT2.19, have great potential to permanently reverse symptoms of neuropathic pain following PNS and CNS injuries and can offer new hope to treat these intractable conditions to significantly improve human health. This includes neuropathic pain resulting from diabetic neuropathy and Spinal Cord Injury.

Images: Comparison of graft sites of hNT2.17 and hNT2.19 in the QUIS and severe contusive-SCI models, respectively, at 6 weeks after cell transplant. (a) Sagittal section of anti-GABA-immunostained QUIS + hNT2.17 transplant lumbar spinal cord 6 weeks after grafting. Easily detectible hNT2.17 cells stain for GABA (arrows) on the pial membranes. (b) Sagittal section of anti-NuMA-immunostained QUIS + hNT2.17 transplant lumbar spinal cord 6 weeks after grafting. Easily detectible hNT2.17 cells stain for NuMA (arrows) on the pial membranes in adjacent sections. The hNT2.19 were alternately injected into the subarachnoid space two weeks after severe contusive SCI. Cell graft sites were co-localized with 5HT (c) and the human-specific marker TuJ1(d) (neuron-specific class III β-tubulin). There are many surviving hNT2.19 (d) grafted cells visible on the pial surface, which stain for TuJ1 (arrows) at the end of the experiment, 56 days after SCI and about 6 weeks after cell transplant. Adjacent sections with the same grafted hNT2.19 (c) are labeled for 5HT (arrows). Magnification bar = 20 μm.

Protocol: Modified methods for staining spinal cord sections for the human neuron-specific class III beta-tubulin (TuJ1) to identify grafted hNT2.19 neurons after grafting have previously been described [51]. The sections were washed with 0.1 M PBS pH 7.4 and permeabilized with 0.4% Triton-X-100 in 0.1 M PBS, 10% normal goat serum (NGS) for one hour. The sections were then incubated overnight at 4°C in the primary anti-TuJ1 antibody (1 : 100 DPBS), and the permeabilizing solution, followed by a one-hour incubation at room temperature with the secondary antibody solution, biotinylated mouse IgG raised in goat (Vector; 1 : 200), a Peroxidase ABC reporter in 0.1 M PBS (Vector) and “VIP” substrate (Vector). Some sections were stained in the absence of primary antibody and served as the negative controls.

I will continue to post new applications for our stem cell research reagents.

Lineage Selection-Neural Stem Cells for SC Grafts

Our Neural Progenitor Markers keep moving up in the "hit parade". These markers are important for lineage selection. This selection is essential to circumvent the possibility of tumor formation and facilitate the safe translation of ES-based therapies to humans.

Here's a recent pub referencing use of several of our markers for selecting or confirming lineage: J. Simon Lunn, Crystal Pacut, Emily Stern, Stacey A. Sakowski, J. Matthew Velkey, Sue O'Shea, Eva Feldman. Intraspinal transplantation of neurogenin-expressing stem cells generates spinal cord neural progenitors. dx.doi.org/10.1016/j.nbd.2011.12.044...Tuj1 (Neuromics, 1:1000), Nestin (Neuromics, 1:500)...
Highlights: Expression of appropriate transcription factors is one approach to direct the differentiation of ES cells towards a specific lineage and stop proliferation. Neural differentiation can be initiated in ES cells by expression of Neurogenin1 (Ngn1). In this study we investigate the effects of controlled Ngn1 expression on mouse ES (mES) cell differentiation in vitro and following grafting into the rat spinal cord. In vitro, Ngn1 expression in mES cells leads to rapid and specific neural differentiation, and a concurrent decrease in proliferation. Similarly transplantation of Ngn1-expressing mES cells into the spinal cord lead to in situ differentiation and spinal precursor formation. These data demonstrate that Ngn1 expression in mES cells is sufficient promote neural differentiation and inhibit proliferation, thus establishing an approach to safely graft ES cells into the spinal cord.

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).

I will continue to post updates on the application of Neuromics' Stem Cell Markers

Mouse epiSCs Into Myelinating Cells

This study published recently in Nature Methods hit my radar scope becaused it referenced use of our widely used and frequently published stem cell marker Tuj 1 (Neuron-specific class III beta-tubulin): Fadi J Najm, Anita Zaremba, Andrew V Caprariello, Shreya Nayak, Eric C Freundt, Peter C Scacheri, Robert H Miller & Paul J Tesar. Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells. Nature Methods (2011) doi:10.1038/nmeth.1712.

Dr. Paul Tesar and his team at Case Western University demonstrated the ability to convert pluripotent epiblast stem cells into pure populations of myelinating cells, called oligodendrocyte progenitor cells (OPCs). First, stem cells in a petri dish are treated with molecules to direct them to become the most primitive cells in the nervous system. To produce OPCs, these primitive cells are treated with a defined set of proteins. The cells were cultured on laminin and treated withh apporopriate growth factors. The OPCs were nearly homogenous and could be multiplied to obtain more than a trillion cells.

The OPCs were treated with thyroid hormone, which is key to regulating the transition of the OPCs to oligodendrocytes. The result was the OPCs stopped proliferating and turned into oligodendrocytes within four days.

These methods could used to potentially produce stable and pure populations of human OPCs in a significant enough number to treat patients with demyelinating diseases such as multiple sclerosis and cerebral palsy.

Turning Placenta Into Brain

We are pleased to feature a recent publication featuring:

C. Bettina Portmann-Lanz PhD, Andreina Schoeberlein PhD, Reto Portmann PhD, Stefan Mohr MD, Pierre Rollini PhD, Ruth Sager1 and Daniel V. Surbek MD. Turning placenta into brain: placental mesenchymal stem cells differentiate into neurons and oligodendrocytes. doi:10.1016/j.ajog.2009.10.893... β-Tubulin III (Tuj-1), Mouse, Neuromics...

The researchers successfully induced neural stem (NSC) and progenitor cells (NPC) from human placental tissues.

Here are the highlights:

Study Design
Placental stem cells from first-trimester placental chorionic villi and term chorion were isolated. Neural differentiation was initiated with plating on collagen, retinoic acid, and/or human brain-derived neurotrophic factor and epidermal and fibroblast growth factor. Differentiation into neurons, oligodendrocytes, and astrocytes was monitored by immunohistochemistry. Two-dimensional polyacrylamide gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry were used to identify proteins involved in the differentiation.

Results
Differentiated cells were mostly immediately postmitotic with some more but not fully mature postmitotic neurons. Neurons had dopaminergic or serotonergic character. Some cells differentiated into predominantly immature oligodendrocytes. Upon differentiation, neuron-specific proteins were up-regulated, whereas placental proteins were reduced.

Conclusion
Stem cells derived from human placenta can be differentiated into neural progenitors.

Featured Antibody
Tuj 1 (Neuron-specific class III beta-tubulin)-Mouse

Related Reagents:
NSE (Neuron-Specific Enolase)

Nestin

Musashi-1

Neuron/Glial Markers

Stem Cell Research Reagents

Tuj-1-Neuronal Differentiation Marker

Our Tuj-1 antibodies are widely used and frequently referenced in customer publications. They are proven markers for Neural Progenitor and Neuronal Differentiation. Here's the latest reference:

Alonso M. Higuero, Lucía Sánchez-Ruiloba1, Laura E. Doglio, Francisco Portillo1, José Abad- Rodríguez, Carlos G. Dotti and Teresa Iglesias. Kidins220/ARMS modulates the activity of microtubule-regulating proteins and controls neuronal polarity and development. JBC Papers in Press. Published on November 10, 2009 as Manuscript M109.024703.

...Tuj 1 (Neuron-specific class III beta-tubulin)-Mouse (MO15013, Neuromics Antibodies, Edina, MN)...

Immunofluorescence Method:

Cells grown on coverslips were fixed for 5 min in 4% paraformaldehydecontaining 4% sucrose in phosphate buffer saline (PBS) at 37ºC. Cells were then permeabilized with 0.2% Triton X-100 in PBS during 5 min at room temperature. After blocking (5% bovine serum albumin in PBS for 1 h), cells were incubated with the corresponding primary antibodies, and immunoreactivity was detected with the suitable fluorophore-conjugated secondary antibody before mounting on slides with Mowiol4-88 (Harland Co., UK). Confocal images were acquired using an inverted Leica TCS SP5 laser confocal microscope with a 63X Plan- Achromatic oil immersion objective and processed with LAS AF Leica Application Suite and Adobe Photoshop CS2 (Adobe Systems
Inc., CA). All images correspond to the projection of sections from a ~50μm z-stack, except for colocalization analysis where they correspond to 0.5-0.7μm single sections.

Image: Rat hippocampal neurons were fixed at 1.5 DIV and immunostained for the neuronal marker βIII-Tubulin/Tuj1 (blue).

NSE and TUJ-1 and Parkinson's Disease Research

I would like to thank Meghan Coakley from University College Cork for alerting me to a new publication referencing our Chicken NSE (Neuron-Specific Enolase) and Tuj 1 (Neuron-specific class III beta-tubulin) antibodies.

Here's her feedback: "Just letting you know we published our paper using the beta-III-Tubulin and NSE antibodies you supplied to us – Timmons et al., Neuroscience Letters, Oct 1, 2009 [Epub ahead of print]. The antibodies were excellent and I’m sure we’ll be using Neuromics again in the future."

Timmons S, Coakley MF, Moloney AM, O' Neill C. Akt signal transduction dysfunction in Parkinson's disease. Neurosci Lett. 2009 Oct 1. [Epub ahead of print].

Abstract: Significant attention has been drawn to the potential role of defective PI3-kinase-Akt (PKB) signalling in Parkinson's disease (PD) neurodegeneration and to the possibility that activation of Akt may provide neuroprotection in PD. However, little knowledge exists on the integrity of the Akt system in PD. Results of the present study show diminished levels of both total and active phospho(Ser473)-Akt in the brain in PD. This was evident by western blot analysis of midbrain fractions from PD compared to non-PD control brain, but more specifically by immunofluorescence microscopy of the substantia nigra pars compacta (SNpc). Here, double immunofluorescence microscopy found Akt and phospho(Ser473)-Akt to be expressed at high levels in tyrosine hydroxylase (TH) immunopositive dopaminergic neurons in control human brain. Selective loss of these neurons was accompanied by a marked decrease of Akt and phospho(Ser473)-Akt expression in the PD brain, however Akt and active phospho(Ser473)-Akt are still evident in degenerating dopaminergic neurons in the disease. This suggests that it may be possible to target neuronal Akt in advanced PD. Converse to the marked loss of neuronal Akt in PD, increased Akt and phospho(Ser473)-Akt levels were observed in small non-TH positive cells in PD SNpc, whose increased number and small nuclear size indicate they are glia. These findings implicate defective Akt as a putative signalling pathway linked to loss of dopaminergic neurons in PD.

Other Reagents to Consider:
Tuj 1 (Neuron-specific class III beta-tubulin)-Mouse Monoclonal
Neuron/Glial Marker Antibodies
Neurotrophins-Neuron/Glial Marker Proteins
Neurodegenerative Disease Research Antibodies
Neurodegenerative Disease Research Proteins
Stem Cell Research Reagents

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