StemPro™ Neural Stem Cells - FAQs

NSCs do not stain with neuron-specific markers. If staining was observed, this could have resulted from antibody non-specific staining. We recommend checking antibody specificity and the staining protocol:

- Titrate primary antibody to find the optimal antibody working concentration.
- Change the permeabilization protocol, because a high concentration of permeabilization reagent(s) could result in high background.
- Use enough blocking solution such as 5-10% serum to block non-specific staining.
- Include isotype control.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Here are some reasons why your cell culture could have failed:

- Did not store cells correctly: These cells should be stored in liquid nitrogen.

- Medium used was not the recommended one: The complete medium of Gibco H9-derived NSCs (Cat. No. N7800) and Gibco StemPro NSCs (Cat. No. A15654 or A15655) have different formulations. Extra components (heparin and ascorbic acid) need to be added into the medium to culture StemPro NSCs.

- Did not thaw cells correctly: Do not thaw the cells for longer than 2 mins at 37 degrees C. After cells have thawed, transfer to 50-mL tube first, then add pre-warmed complete medium drop-wise (approximately 1 drop per second) while swirling the tube.

- Did not count cell viability after thawing cells and did not seed them appropriately: You need to count cell viability with trypan blue. At least 1 x 10E6 viable cells/mL are provided in each vial. For H9-derived NSCs, recommended seeding density is > 1 x 10E5 viable cells/cm2. For StemPro NSCs, recommended seeding density (suspension) is >7 x 10E4 viable cells/mL.

- Plate is not coated or has been coated incorrectly: For H9-derived NSCs and adherent culture, you need to properly coat the tissue culture plate with Gibco Geltrex Matrix, fibronectin, or poly-L-ornithine/laminin, following instructions for coating. For StemPro NSCs, we recommend that you grow them in suspension culture, as adherent culture would trigger differentiation.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

We offer two types of human NSCs:

- Gibco StemPro Neural Stem Cells (Cat. No. A15654 or A15655): Isolated from human fetal cortex brain and manufactured under good manufacturing practice (GMP). Each lot is generated from the same master bank (same donor) so that the lot-to-lot variability is low. Cell doubling time is ~ 100 hours.

- Gibco Human Neural Stem Cells (H9-derived) (Cat. No. N7800): Induced from H9 human ESC using a proprietary method. Cell doubling time is ~ 40-50 hours and increases with increasing passage number.

Both cells are tested for their ability to retain their proliferation and differentiation potential for at least 3 passsages after thawing. Both are able to differentiate into neurons, astrocytes, and oligodendrocytes. However, StemPro Neural Stem Cells are recommended to grow in suspension culture as adherent culture would trigger differentiation, whereas Human Neural Stem Cells (H9-derived) can grow under both suspension and adherent conditions.

For NSC expansion, the following growth factors are used: recombinant EGF (Cat. No. PHG0314), recombinant FGF-basic (Cat. No. PHG0024), and recombinant VEGF (Cat. No. PHC9394). In addition, several neurotrophins such as BDNF Cat. No. 10908010), NT-3, CNTF (Cat. No. PHC7015), and GDNF (Cat. No. PHC7044)are also used in the related studies.

Information pertaining to whether a specific product has been tested against the WHO Reference Standard can typically be located on the product page or Certificate of Analysis (COA).

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Human NSCs can grow in Gibco StemPro NSC SFM (Cat. No. A1050901) on dishes pre-coated with Gibco Geltrex Matrix or Gibco CELLstart substrate. Alternatively, if the goal is to obtain neurons, NSCs can also be grown on Neurobasal medium supplemented with Gibco B-27 supplements without vitamin A on a pre-coated dish.

NSCs are generally characterized by their ability to form neurospheres when plated at cloning density (Nat Methods 2:333 (2005)). NSCs can also be characterized by (1) RT-PCR of Sox1, Sox2, and Nestin or (2) immunohistochemical staining for nestin, Pax6, Sox2, and Ki67.

Neural stem cells (NSCs) are self-renewing multipotent cells of the nervous system capable of differentiating into neurons, oligodendrocytes, and astrocytes. NSC can be generated by induced differentiation from embryonic stem (ES) cells, or isolated from various regions of the brain including the cortex, the subventricular zone (SVZ), and the ventricular zone, or generated from bone marrow-derived mesenchymal stem cells (MSCs) (J Cell Biochem 114:764 (2013)). NSCs are valuable tools for the study of neurogenesis and neurotransmitter and receptor function. NSCs were used in the investigation of different CNS disorders such as PD and Huntington's disease in various animal models (J Cell Biochem 114:764 (2013)).