The use of neural stem cells (NSCs) in biomedical research is becoming increasingly popular, resulting in breakthrough studies rejected the longstanding belief that neuronal tissue is incapable of regeneration. The discovery that neurons, astrocytes, and oligodendrocytes arise from neural stem cells located in specific regions of the brain has revealed important clinical applications for treating central nervous system diseases, including Parkinson's disease, Alzheimer’s, and spinal cord repair.
Frequently Asked Questions
- What are neural stem cells?
Neural stem cells are multipotent stem cells from the central nervous system that can self-renew, and differentiate into neurons, astrocytes and oligodendrocytes1.
- Where are neural stem cells found?
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
- What are the steps to isolate neural stem cells from brain?
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.
- 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.
- 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.
- Immunopanning: Cells are isolated based on their differential binding to the cell culture plates previously coated with cell-surface antibody.
- Fluorescence activated cell sorting (FACS): Cells are sorted according to the expression of either nuclear or cytoplasmic markers.
- What are neurospheres?
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.
- How to characterize neural stem cells?
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.
- What is the media used to propagate neural stem cells?
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.
- How to maintain and propagate neural stem cells?
Both, neurospheres6 and monolayer cultures7 are used to culture and expand the neural stem cells. However, each has its own advantages and disadvantages: