Isolation and culture of neural stem cells from adult mouse subventricular zone for genetic and pharmacological treatments with proliferation analysis

Abstract

Neural stem cells (NSCs) from the subventricular zone (SVZ) of the mouse brain can be expanded in vitro and grown as neurospheres, which can be stored long-term in liquid nitrogen. Here, we present a protocol for isolation and culture of NSCs from the adult mouse SVZ. We describe how to grow and expand primary NSCs to neurospheres, followed by differentiation and nucleofection/pharmacological treatments. Finally, we describe RNA extraction, EdU labeling of the cells, and immunofluorescent analysis to examine their proliferation. For complete details on the use and execution of this protocol, please refer to Radecki et al. (2020).

Keywords: Cell Biology; Cell culture; Cell isolation; Neuroscience; Stem Cells.

Graphical abstract

Figure 1

Preparation for dissection and slicing the brain (A) Tools utilized for gross dissection and brain removal, from left to right: Large scissors for cervical dislocation, medium scissors for skin removal, small scissors for skull dissection, pointed tip forceps (×2) for skull removal, flat tip forceps for brain removal. (B) Acrylic brain matrice and tools for slicing the brain and transferring the slices. (C) Beaker and plate setup on ice for cooling tools and dissection media. (D) Mouse brain placed in a coronal acrylic brain matrice with the dorsal surface facing up. (E) Brain with tissue slicer blades placed every 1 mm from the olfactory bulbs to the anterior edge of the hippocampus.

Figure 2

Slicing the brain and dissecting the SVZ (A) 1mm thick brain slices numbered from rostral (#1) to the caudal slice (#7). (B) Tools for SVZ microdissection including 2 pairs of fine point non-serrated forceps with straight and angled iris scissors. (C) Enlarged image of brain slice #4 showing the speckled appearance of the striosomes in the striatum and the SVZ outlined by the red dotted line. (D) Image showing the dissected SVZ with the incision from the ventral surface of the brain along the lateral and medial edges of the SVZ, outlined by the red dotted line.

Figure 3

Growth of neurospheres (A) RFP expressing fate-mapped neurospheres imaged 5 days after dissociation of tertiary neurospheres. (B) Graph of the diameters of secondary neurospheres during the first 5 days after dissociation shows that neurosphere growth rates are consistent across experiments. The size of neurospheres were 24.3 ± 2 μm on day 1, 46.7 ± 0.9 μm on day 2, 75.3 ± 4.1 μm on day 3, 116.6 ± 0.7 μm on day 4, and 153.7 ± 0.9 μm on day 5. N = 3 independent cultures, data ± SEM. (C) Images of secondary neurospheres comparing their size at each day after dissociation. On day 5, neurospheres begin to show a dark center which expands as they grow larger with a necrotic center. The bottom right is an example of neurospheres attached to the bottom of the plate, and beginning to differentiate.

Figure 4

Differentiation of neural stem cells Bright field images of neural stem cells (NSCs) superimposed on fluorescent images of RFP expressing NSCs after plating on GFR-Matrigel coated coverslips for differentiation. NSCs were imaged 1, 3, 5, 7, 9, and 11 days following plating. The cells continue to proliferate during the first 4–5 days of differentiation, and elaborate processes as they differentiate into the astrocytic, neuronal, and oligodendroglial lineage. Scale = 100 μm.

Figure 5

Immunofluorescent labeling of differentiated neural stem cells Tertiary neurospheres were dissociated and plated on GFR-Matrigel coated coverslips in a 24-well plate for differentiation. Cells were immunolabeled after 14 days to detect co-expression of RFP in oligodendrocyte progenitor cells (OPCs) with NG2 antibody, oligodendrocytes with MBP antibody, astrocytes with GFAP antibody and proliferation with EdU labeling. BF = Brightfield, RFP = Red Fluorescent Protein, MBP = Myelin Basic Protein, GFAP = Glial Fibrillary Acidic Protein, Scale=10 μm.