Neural stem cells are characterized by their ability to 1) self-renew and to 2) generate the different cell types found in the central nervous system including both neural and glial subtypes. Isolation and in vitro analysis of neural progenitor cell populations have been important for deciphering the cellular and molecular mechanisms underlying neurogenesis, and for optimizing stem cell-based treatment of neurological disorders and injuries. In the adult mammalian brain, NSCs exist mainly in two neurogenic regions: the subgranular zone of the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. Recently, the use of pluripotent stem cells to make patient derived neural progenitors have aided to generate more relevant “disease-in-a-dish” cellular models of many age related neurological diseases.
Figure 1. Regions of neurogenesis in the mammalian brain. Proliferating neural stem cells are found in the subgranular zone (SGZ) of the hippocampal dentate gyrus and subventricular zones (SVZ) of the adult and developing mammalian brain.
Isolation of NSCs from Neural Tissue
Differentiation of Human iPSCs into NSCs
Dual SMAD inhibition is a well-established method to derive neural progenitor cells from human ES/iPS cells in monolayer cultures. This protocol uses two SMAD inhibitors, Noggin (SRP4675) and SB431542 (S4317), to drive the rapid differentiation of ES/iPS cells into a highly enriched population of NPCs. Noggin acts as a BMP inhibitor and SB431542 inhibits the Lefty/Activin/TGFβ pathways by blocking the phosphorylation of ALK4, ALK5, and ALK7 receptors. In an effort to make a more defined and optimized neuronal differentiation protocol, Li and colleagues modified the original protocol to establish a completely small molecule-based differentiation method, which relies on three small molecules to inhibit GSK-3β, CHIR99021 (SML1046), TGFβ, SB431542 (S4317), and Notch, Compound E (565790) signaling pathways, along with human LIF (LIF1010). This new small molecule-based neural differentiation protocol increased neural differentiation kinetics and allowed the derivation of truly multipotent neural stem cells that respond to regional patterning cues specifying forebrain, midbrain, and hindbrain neural and glial subtypes.
Application Note: Robust Differentiation of Human iPSCs into Lineage-Specific Neuronal and Glial Cells Utilizing Dual-SMAD Inhibition
Characterization of Neural Stem Cells
Presently, neural stem cells are often identified based upon the presence of molecular markers that are correlated with the stem and/or progenitor state along with the absence of a more differentiated phenotype as assessed through marker analysis. NSCs positively express stem cell markers Nestin (ABD69, MAB353), Sox-2 (AB5603) and Musashi (MABE268) and lack the more differentiated lineage markers including βIII-tubulin (MAB1637) for neurons, GFAP (AB5804) for astrocytes and O1 (MAB344) for oligodendrocytes.
Culture of NSCs in 3D Neurosphere Culture
Culture of NSCs in 2D Monolayer Culture
We recommend coating tissue culture plastic or glassware with poly-L-ornithine and laminin. Poly-L-ornithine and laminin provide optimal matrix for adhesion and growth of the NSCs.
Figure 2. Neural stem cell culture characteristics. NSCs can be grown as floating 3D neurospheres (A) or attached 2D monolayers (B) on poly-L-ornithine/laminin coated plates. Multipotent neural stem cells express Nestin (C) and Sox-2 (D).
Figure 3. Differentiation of Neural Stem Cells. Multipotent NSCs express Nestin/Sox-2 and can differentiate into BIII-Tubulin positive neurons (B) or GFAP positive astrocytes (C) under appropriate culture conditions.
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