Neural Stem Cell FAQs

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

1. 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.
   
   
2. 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

3. 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.
   
   
   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.

4. 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.
   
   
5. 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.
   
   
6. 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.
   
   
7. 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:

	Neurosphere Culture	Monolayer Culture
Advantages	Recapitulates in-vivo conditions, through cell-cell interactions to allow survival of stem cells	Ease to study properties at individual cell level Consistent performance
Disadvantages	Variable sphere formation The diffusion of nutrients and growth factors can be compromised within 3D structures. Inability to track individual cells	Cells are exposed to homogeneous environment Does not mimic in-vivo conditions

8. What are neural progenitor cells?
   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.
   
   
9. How to differentiate neural stem cells into neurons?
   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.
   
   
10. What are the potential therapeutic applications of neural stem cells?
    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.

Figure 1. A,B) Human iPSC derived neural stem cells grown in monolayer cultures express stem cell markers Nestin and Sox-2. C) NSCs can be further differentiated into b-III-tubulin positive neurons.

References

1. Galli R, Gritti A, Bonfanti L, Vescovi AL. 2003. Neural Stem Cells. Circulation Research. 92(6):598-608. https://doi.org/10.1161/01.res.0000065580.02404.f4

2. Sanai N, Tramontin AD, Quiñones-Hinojosa A, Barbaro NM, Gupta N, Kunwar S, Lawton MT, McDermott MW, Parsa AT, Manuel-García Verdugo J, et al. 2004. Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature. 427(6976):740-744. https://doi.org/10.1038/nature02301

3. Gonzalez-Perez O, Chavez-Casillas O, Jauregui-Huerta F, Lopez-Virgen V, Guzman-Muniz J, Moy-Lopez N, Gonzalez-Castaneda RE, Luquin S. 2011. Stress by noise produces differential effects on the proliferation rate of radial astrocytes and survival of neuroblasts in the adult subgranular zone. Neuroscience Research. 70(3):243-250. https://doi.org/10.1016/j.neures.2011.03.013

4. la Cruz JO, Ayuso-Sacido A. Neural Stem Cells from Mammalian Brain: Isolation Protocols and Maintenance Conditions. https://doi.org/10.5772/32766

5. Reynolds BA, Rietze RL. 2005. Neural stem cells and neurospheres?re-evaluating the relationship. Nat Methods. 2(5):333-336. https://doi.org/10.1038/nmeth758

6. Reynolds B, Weiss S. 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 255(5052):1707-1710. https://doi.org/10.1126/science.1553558

7. Theus MH, Ricard J, Liebl DJ. 2012. Reproducible Expansion and Characterization of Mouse Neural Stem/Progenitor Cells in Adherent Cultures Derived from the Adult Subventricular Zone. Current Protocols in Stem Cell Biology. 20(1):2D.8.1-2D.8.10. https://doi.org/10.1002/9780470151808.sc02d08s20

8. Casarosa S, Bozzi Y, Conti L. 2014. Neural stem cells: ready for therapeutic applications?. Molecular and Cellular Therapies. 2(1):31. https://doi.org/10.1186/2052-8426-2-31