Reproducible Expansion and Characterization of Mouse Neural Stem/Progenitor Cells in Adherent Cultures Derived from the Adult Subventricular Zone

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Endogenous neural stem/progenitor cells (NSPCs) residing in the subventricular zone (SVZ) of the adult mouse forebrain have been shown to enhance their neurogenic potential in response to CNS injury. Mechanisms involved in regulating adult neurogenesis under naïve or stressed conditions can be studied using a monolayer cell?culture system of the nestin?expressing NSPC lineage to analyze proliferation, survival, and differentiation. Here, a protocol for the expansion of NSPCs for studies aimed at understanding the functional role of NSPCs in maintaining adult neurogenic processes is described. This unit outlines detailed procedures for: (1) isolation, maintenance, and culture of the NSPC component of the SVZ niche from the lateral wall of the lateral ventricle; (2) characterization of NSPC functions by examining proliferation, survival, and differentiation; and (3) efficient siRNA transfection methods in 96?well format. Curr. Protoc. Stem Cell Biol. 20:2D.8.1?2D.8.10. © 2012 by John Wiley & Sons, Inc.

Keywords: neural progenitor cell; subventricular zone; cultures; proliferation; survival; differentiation; siRNA

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Introduction
Basic Protocol 1: Isolation and Expansion of NSPCs from the Adult SVZ
Basic Protocol 2: Analysis of Proliferation, Survival, and Differentiation of Mouse NSPCs in 96‐Well Format
Basic Protocol 3: Efficient siRNA Transfection of Mouse NSPCs Using HiPerfect System in 96‐Well Format
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Isolation and Expansion of NSPCs from the Adult SVZ

Materials

Monolayer cell culture medium (MCM‐E/F; see recipe )
Epidermal growth factor (EGF; Millipore)
Basic fibroblast growth factor (bFGF; Millipore)
Dissociation solution (see recipe )
Five adult male mice (2 to 4 months old)
Leibovitz L‐15 medium (Invitrogen)
0.05% trypsin/EDTA (Invitrogen)
Sterile phosphate buffered saline (PBS; Invitrogen)
Freezing medium (see recipe )

0.22‐µm syringe filter
37°C water bath
Stereomicroscope
100 mm × 20–mm uncoated tissue culture–treated dishes (BD Biosciences)
5‐ml pipets
40‐µm cell strainer (Becton Dickinson)
15‐ and 50‐ml conical tubes
37°C incubator
Hemacytometer
Cryovials (Corning)
Mr. Frosty cryo‐freezing container (Nalgene, cat. no. 5100‐0001)

Basic Protocol 2: Analysis of Proliferation, Survival, and Differentiation of Mouse NSPCs in 96‐Well Format

Materials

SVZ‐derived NSPCs
MCM‐E/F (see recipe )
Bromodeoxyuridine (BrdU, Sigma)
1× PBS
10% buffered formalin (Azer Scientific)
1× PBST (see recipe )
2 N HCl/0.2% Triton X‐100
Borate buffer (see recipe )
Anti‐BrdU antibody (Roche)
Secondary antibody
Hoechst (Invitrogen)
Live/dead assay (Invitrogen)
Differentiation medium (see recipe )
4% paraformaldehyde

96‐well plates
37°C, 5% CO₂ humidified incubator
Fluorescence microscope

Basic Protocol 3: Efficient siRNA Transfection of Mouse NSPCs Using HiPerfect System in 96‐Well Format

Materials

Mouse neural stem/progenitor cells
MCM‐E/F (see protocol 1 )
MCM‐E/F without FBS
10 µM fluorescein‐labeled signal silence control siRNA (cell signaling)
HiPerfect (Qiagen, cat. no. 301704)
GAPDH control siRNA (Dharmacon)
Anti‐GAPDH antibody
1× phosphate buffered saline (PBS)

96‐well plates
37°C incubator
37°C water bath
Fluorescence microscope

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Figures

Figure 2.D0.1 Adherent cultures of NSPCs during normal growth, differentiation, and siRNA knockdown. (A and B ) Bright field images at 10× magnification of NSPCs grown on 100‐mm plastic dishes at 24 hr (A) and 5 days (B) after plating. (C ) Double immunofluorescence labeling using anti‐nestin (green) and anti‐glial fibrillary acidic protein (GFAP; red) is used to identify neural progenitor cells and visualized under fluorescence at 10× magnification. (D ) Differentiation of NSPCs into β‐tubulin III‐positive neurons (green) and GFAP‐positive astrocytes (red) following 7 days growth factor removal is visualized under fluorescence at 40× magnification. Representative images at 20× magnification of adherent NSPCs that were cultured for 24 hr in the presence of fluorescein‐labeled control siRNA (F ; green) at 50 nM. Cells were co‐labeled with Hoechst nuclear stain (H ; blue). Scales bars = 200 µM.

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Literature Cited

Literature Cited
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