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

Michelle H. Theus1, Jerome Ricard1, Daniel J. Liebl1

1 The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami, Florida
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.8
DOI:  10.1002/9780470151808.sc02d08s20
Online Posting Date:  March, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • 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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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% CO 2 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
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Alvarez‐Buylla, A. and Garcia‐Verdugo, J.M. 2002. Neurogenesis in adult subventricular zone. J. Neurosci. 22:629‐634.
   Del Valle, K., Theus, M.H., Bethea, J.R., Liebl, D.J., and Ricard, J. 2011. Neural progenitors proliferation is inhibited by EphB3 in the developing subventricular zone. Int. J. Dev. Neurosci. 29:9‐14.
   Doetsch, F., Garcia‐Verdugo, J.M., and Alvarez‐Buylla, A. 1997. Cellular composition and three‐dimensional organization of the subventricular germinal zone in the adult mammalian brain. J. Neurosci. 17:5046‐5061.
   Doetsch, F., Caillé, I., Lim, D.A., García‐Verdugo, J.M., and Alvarez‐Buylla, A. 1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703‐716.
   Jin, K., Mao, X.O., Sun, Y., Xie, L., and Greenberg, D.A. 2002. Stem cell factor stimulates neurogenesis in vitro and in vivo. J. Clin. Invest. 110:311‐319.
   Mirzaden, Z., Doetsch, F., Sawamoto, K., Wichterle, H., and Alvarez‐Buylla, A. 2010. The subventricular zone en‐face: Wholemount staining and ependymal flow. J. Vis. Exp. 39:e1938.
   Morshead, C.M., Reynolds, B.A., Craig, C.G., McBurney, M.W., Staines, W.A., Morassutti, D., Weiss, S., and van der Kooy, D. 1994. Neural stem cells in the adult mammalian forebrain: A relatively quiescent subpopulation of subependymal cells. Neuron 13:1071‐1082.
   Phelan, M.C. 2007. Basic techniques in mammalian cell tissue culture. Curr. Protoc. Cell Biol. 36:1.1.1‐1.1.18.
   Ricard, J., Salinas, J., Garcia, L., and Liebl, D.J. 2006. EphrinB3 regulates cell proliferation and survival in adult neurogenesis. Mol. Cell. Neurosci. 31:713‐722.
   Scheffler, B., Walton, N.M., Lin, D.D., Goetz, A.K., Enikolopov, G., Roper, S.N., and Steindler, D.A. 2005. Phenotypic and functional characterization of adult brain neuropoiesis. Proc. Natl. Acad. Sci. U.S.A. 102:9353‐9358.
   Shen, Q., Wang, Y., Kokovay, E., Lin, G., Chuang, S.M., Goderie, S.K., Roysam, B., and Temple, S. 2008. Adult SVZ stem cells lie in a vascular niche: A quantitative analysis of niche cell‐cell interactions. Cell Stem Cell 3:289‐300.
   Theus, M.H., Ricard, J., Bethea, J.R., and Liebl, D.J. 2010. EphB3 limits the expansion of neural progenitor cells in the subventricular zone by regulating p53 during homeostasis and following traumatic brain injury. Stem Cells 28:1231‐1242.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library