Neural Stem Cell FAQs

During embryonic development, NSCs are ubiquitously found in all regions of CNS, including cortex, thalamus, spinal cord and septum. However, in the adult mammalian brain they are restricted to two regions:

• Subventricular zone (SVZ) of the lateral ventricles²
  
• Subgranular zone (SGZ) within the dentate gyrus of hippocampus³

The isolation of neural stem cells includes four steps.⁴

Dissection: Thin slices of tissue from appropriate location of brain are made and they are further minced into small pieces for enzymatic digestion.

Enzymatic digestion: The isolated tissue is surrounded by extracellular matrix and it is digested with proteases like trypsin and papain.

Mechanical disaggregation:  Disaggregation either through trituration or passing the cell suspension through syringes and needles remove neural stem cells from the remaining tissues.

Enrichment: Cells obtained from above steps are heterogeneous and they are subsequently enriched based on the phenotype of cells.

1. Adherence to the cell culture plate: Subtypes of neural cells are distinguished based on the adherence to the surface of cell culture plate coated with different substrates.
   
   
2. Differential gradient centrifugation: Neural stem cells are distributed in density gradients and can be collected separately. The gradient is formed by using different reagents, including percoll, sucrose solutions and bovine serum albumin.
   
   
3. Immunopanning: Cells are isolated based on their differential binding to the cell culture plates previously coated with cell-surface antibody.
   
   
4. Fluorescence activated cell sorting (FACS): Cells are sorted according to the expression of either nuclear or cytoplasmic markers.

Neural stem cells, when plated at appropriate densities in suitable low attachment culture plates, will divide continuously to generate non-adherent spherical clusters of cells, referred to as neurospheres. Neurospheres contain small percentage of true neural stem cells, while the remaining cells are in different phases of differentiation⁵.

Neural stem cell antibodies can identify neural stem cells based on the expression of three neural stem cell markers: Nestin, Sox2 and Musashi. In addition astrocytes and oligodendrocytes can also be identified by detecting differentiated lineage markers, beta-III-tubulin, GFAP and O1 respectively by immunocytochemistry.

Serum-Free NSC expansion media are used to maintain neural stem cell cultures when supplemented with L-Glutamine and FGF-2. Under appropriate conditions, neural stem cells should expand indefinitely. However, routine karyotyping is recommended.

Both, neurospheres⁶ and monolayer cultures⁷ are used to culture and expand the neural stem cells. However, each has its own advantages and disadvantages:

Neural progenitors are progeny of neural stem cells that can differentiate into more than one neural cell type. However, unlike neural stem cells they have limited proliferative and self-renewal ability.

Dual-SMAD inhibition is a common technique used to differentiate NSCs into neurons. These Neural Induction Media rely on both small molecules neural inducers and supplements to generate a highly enriched population of terminally differentiated TUJ-1/MAP2ab positive end stage neurons.

Transplantation of exogenous neural stem cells or terminally differentiated neural cells into a host brain may cure several neurodegenerative diseases like Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, spinal cord injury, amyotrophic lateral sclerosis and brain ischemia⁸.