Fluorescence‐Based Sorting of Neural Stem Cells and Progenitors

Dragan Maric1, Jeffery L. Barker1

1 National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.18
DOI:  10.1002/0471142301.ns0318s33
Online Posting Date:  November, 2005
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Abstract

Neural stem cells (NSCs) are defined as undifferentiated cells originating from the neuroectoderm that have the capacity both to perpetually self‐renew without differentiating and to generate multiple types of lineage‐restricted progenitors (LRPs). LRPs can themselves undergo limited self‐renewal and ultimately differentiate into highly specialized cells that make up the nervous system. However, this physiologically delimited definition of NSCs and LRPs has become increasingly blurred due to lack of protocols for effectively separating these types of cells from primary tissues. This unit discusses recent attempts using fluorescence‐activated cell sorting (FACS) strategies to prospectively isolate NSCs from different types of LRPs as they appear in vivo, and details a protocol that optimally attains this goal. Thus, the strategy presented here provides a framework for more precise studies of NSC and LRP cell biology in the future, which can be applied to all vertebrates, including humans.

Keywords: central nervous system; development; cortex; neural stem cells; lineage‐restricted progenitors; fluorescence‐activated cell sorting; cell fate

     
 
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Table of Contents

  • Basic Protocol 1: Identification of Different Types of LRPs in a Telencephalic Tissue Section
  • Basic Protocol 2: Identification and Sorting of NSCs and LRPs from Telencephalic Dissociates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Identification of Different Types of LRPs in a Telencephalic Tissue Section

  Materials
  • Timed pregnant Sprague‐Dawley rats (Taconic Farms)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • 4% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS)
  • 30% (v/v) sucrose in PBS (see appendix 2A for PBS)
  • Isopentane (Fisher), cooled in liquid N 2
  • Antibodies and detection reagents (see Table 3.18.1), diluted in NPM with BSA (see recipe for NPM)
    • 10 µg/ml mouse IgM anti‐JONES
    • 10 µg/ml goat anti–mouse IgM–Alexa Fluor 546
    • Mixture of 1 µg/ml mouse IgG1 anti‐vimentin, 5 µg/ml mouse IgG2a anti‐PCNA, and 10 µg/ml TnTx/mouse IgG2b anti‐TnTx
    • Mixture of 10 µg/ml goat anti–mouse IgG1–Alexa Fluor 488, 10 µg/ml goat anti–mouse IgG2a–biotin, and 10 µg/ml goat anti–mouse IgG2b–Alexa Fluor 647
    • 10 µg/ml streptavidin–Alexa Fluor 750 (Molecular Probes)
    • 10 µg/ml 4,6‐diamidino‐2‐phenylindole (DAPI; Molecular Probes)
  • Normal physiological medium (NPM; see recipe) with and without bovine serum albumin (BSA)
  • 70% (v/v) ethanol
  • Aqua Mount solution (Lerner Laboratories)
  • Clear nail polish
    Table 3.8.1   Materials   Primary and Secondary Antibodies/Reagents to Identify Select CNS Cell Types   Primary and Secondary Antibodies/Reagents to Identify Select CNS Cell Types

    Cell phenotype Primary antibody/reagent Secondary antibody/reagent
    Immature precursors Mouse IgG1 anti‐nestin c Goat anti–mouse IgG1–biotin d and SA‐AMCA e
    Mouse IgG1 anti‐vimentin f Goat anti–mouse IgG1–AF 488 g
    Neurons ChTx‐biotin h SA‐PE/Cy5 d
    TnTx j/mouse IgG1 anti‐TnTx j Goat anti–mouse IgG1–PE/Cy5 d
    TnTx j/mouse IgG2b anti‐TnTx k Goat anti–mouse IgG2b–AF 647 g
    Mouse IgG2b anti‐TuJ1 h Goat anti–mouse IgG2b–AF 647 g
    Neuroglial progenitors Mouse IgM anti‐A2B5 f Goat anti–mouse IgM–PE e
    Mouse IgM anti‐A2B5‐PE l
    Mouse IgM anti‐JONES h Goat anti–mouse IgM–AF 546 g
    Oligodendrocytes Mouse IgM anti‐O4 f Goat anti–mouse IgM–FITC e
    Mouse IgM anti‐O4‐FITC l
    Astrocytes Rabbit IgG anti‐GFAP f Goat anti–rabbit IgG–TRITC m
    Microglia Mouse IgG2a anti‐CD11b n Goat anti–mouse IgG2a–FITC d
    Proliferating cells Mouse IgG2a anti‐PCNA f Goat anti–mouse IgG2a–biotin d and SA‐AF 750 g
    Mouse IgG1 anti‐BrdU‐FITC
    Apoptotic/necrotic Annexin V–FITC

     aDivide all unconjugated primary reagents in small stock aliquots (e.g., 20 µl) and store up to 1 year at –20°C. Thaw required quantity of reagent just before use and do not refreeze. Do not freeze fluorochrome‐conjugated antibodies and reagents (e.g., anti‐A2B5‐PE or secondary reagents). Rather, store these items protected from light up to 6 months at 4°C or until the expiration date supplied by the manufacturer.
     bAbbreviations (in alphabetical order): AF, Alexa Fluor; AMCA, aminomethylcoumarin; BrdU, bromodeoxyuridine; CD11b, cluster differentiation antigen 11b (clone OX42); ChTx, cholera toxin B subunit; FITC, fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; JONES, anti‐9‐O‐acetylated GD3 ganglioside; PE, phycoerythrin; PE/CY5, phycoerythrin/carbocyanine dye 5 conjugate; PCNA, proliferation cell nuclear antigen; SA, streptavidin; TnTx, recombinant tetanus toxin fragment C; TRITC, tetramethyl rhodamine isothiocyanate; TuJ1, tubulin β III.
     cFor availability contact Department of Biological Sciences, University of Iowa, Iowa City, Ia.
     dSupplier: Caltag.
     eSupplier: Jackson ImmunoResearch.
     fSupplier: Chemicon International.
     gSupplier: Molecular Probes.
     hSupplier: Sigma.
     JSupplier: Roche Diagnostics.
     kFor availability, contact Dr. William Habig, U.S. Food and Drug Administration, Bethesda, Md.
     lFor availability, contact Dr. Rick I. Cohen, Coriell Institute for Medical Research, Camden, N.J.
     mSupplier: Southern Biotechnology Associates.
     nSupplier: Serotech.
     Supplier: Becton Dickinson Immunocytometry Systems.
     Supplier: Trevigen, Inc.
  • Ruler or caliper
  • Dissecting microscope (e.g., Nikon TMS)
  • Dissecting instruments (e.g., Roboz Surgical Instrument Co.)
    • Dissecting scissors: 7 in. curved; 67‐mm blades
    • Microdissecting scissors: 3‐3/4 in. angular; 14‐mm blades
    • Microdissecting forceps: 3‐1/4 in. straight; fine points
  • Cryostat (e.g., Jung Frigocut Model 2800E, Leica Instruments)
  • Poly‐L‐lysine‐precoated 3 × 1–in. Poly‐Prep microscope slides (Sigma)
  • Humidified chamber
  • Coplin jars
  • 22 × 50–mm coverslips (e.g., Corning)
  • Fluorescence microscope (e.g., Axiovert 200, Carl Zeiss) equipped with:
    • 25× Plan‐Neofluar objective (Carl Zeiss)
    • High‐resolution cooled digital camera (e.g., 12‐bit ORCA‐ER, Hamamatsu Photonics)
    • 100‐W mercury arc lamp (Carl Zeiss)
    • Optimized excitation/emission filter sets (Omega Optical) for aminomethylcoumarin (AMCA), Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 750
    • 10 µg/ml 4,6‐diamidino‐2‐phenylindole (DAPI; Molecular Probes)
  • Image capture and processing software (e.g., Openlab from Improvision Ltd., and Adobe Photoshop)
  • Additional reagents and equipment for euthanasia of the rat ( appendix 4H) and cryostat sectioning (unit 1.1)

Basic Protocol 2: Identification and Sorting of NSCs and LRPs from Telencephalic Dissociates

  Materials
  • Timed pregnant Sprague‐Dawley rats (Taconic Farms)
  • Hanks' balanced salt solution (HBSS; Invitrogen), ice cold
  • HBSS/papain solution (see recipe)
  • Normal physiological medium (NPM; see recipe) with and without bovine serum albumin (BSA)
  • Primary antibodies and reagents (see Table 3.18.1), diluted in NPM with BSA (see recipe for NPM):
    • ChTx‐biotin
    • TnTx/mouse IgG1 anti‐TnTx
    • Mouse IgM anti‐A2B5
    • Mouse IgM anti‐JONES
    • PE‐conjugated mouse IgM anti‐A2B5
    • FITC‐conjugated mouse IgM anti‐O4 (optional)
    • Mouse IgG1 anti‐nestin (dilute supernatant 1:10 in NPM with BSA)
    • Mouse IgG1 anti‐BrdU‐FITC (dilute commercially available stock 1:10 in NPM with BSA)
    • Mouse IgG2b anti‐TUJl
    • Rabbit IgG anti‐GFAP
  • Secondary antibodies and detection reagents (Table 3.18.1), diluted in NPM with BSA (see recipe for NPM):
    • Streptavidin‐PE/Cy5
    • Goat anti–mouse IgG1–PE/Cy5
    • Goat anti–mouse IgM–PE
    • Annexin V–FITC
    • Goat anti–mouse IgG1–biotin
    • Streptavidin‐AMCA (Jackson ImmunoResearch)
    • Goat anti–mouse IgG2b–Alexa Fluor 647
    • Goat anti‐rabbit‐TRITC
  • Neurobasal (NB) medium (Invitrogen) supplemented with 1× B27 additives (from 50× stock; Invitrogen) and 1× penicillin/streptomycin/neomycin (PSN; from 100× stock; Sigma)
  • Medium for self‐renewing growth conditions: NB medium supplemented with 1× B27 additives and 1× PSN and containing 10 ng/ml basic fibroblast growth factor (bFGF; Intergen)
  • Medium for differentiating growth conditions: NB medium supplemented with 1× B27 additives and 1× PSN and containing 10 ng/ml basic fibroblast growth factor (bFGF; Intergen) and 10 ng/ml epidermal growth factor (EGF; Sigma)
  • 5‐Bromo‐2′‐deoxyuridine (BrdU, Sigma)
  • 4% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS)
  • 70% ethanol
  • 0.4% (v/v) Triton X‐100 in PBS (see appendix 2A for PBS)
  • 2 N HCl
  • 0.1 M Na 2O 4B 7 (Sigma), pH 8.5
  • 50 µg/ml unlabeled mouse IgG (Sigma)
  • Dissecting microscope (e.g., Nikon TMS)
  • Dissecting instruments (e.g., Roboz Surgical Instrument Co.)
    • Dissecting scissors: 7 in. curved; 67‐mm blades
    • Microdissecting scissors: 3‐3/4 in. angular; 14‐mm blades
    • Microdissecting forceps: 3‐1/4 in. straight; fine points
  • 35‐mm plastic petri dishes (Thomas Scientific)
  • 15‐ml (17 × 120–mm) conical polystyrene centrifuge tubes or 12 × 75–mm round‐bottom polystyrene tubes with cell‐strainer caps (Becton Dickinson)
  • Cell strainers (optional)
  • 37°C incubator without CO 2
  • Platform rocker (e.g., Bellco Biotechnology)
  • Wide‐mouth plastic transfer pipets (PGC Scientific)
  • Narrow‐mouth glass Pasteur pipets (Daigger Scientific)
  • Tabletop centrifuge, low speed (e.g., Sorvall Legend RT)
  • Fluorescence‐activated cell sorter (e.g., FACSVantage SE, Becton Dickinson) equipped with:
    • Argon ion laser tuned to 488‐nm excitation wavelength
    • Band‐pass filters set at 530 ± 30, 575 ± 25, and 675 ± 20 nm (Omega Optical)
  • CellQuest acquisition and analysis software (Becton Dickinson)
  • Poly‐D‐lysine and fibronectin‐coated photo‐etched coverslips in tissue culture dishes (see recipe)
  • 37°C humidified, 9% CO 2 incubator
  • Inverted fluorescence microscope (e.g., Axiovert 200, Carl Zeiss) equipped with:
    • 25× Plan‐Neofluar objective (Carl Zeiss)
    • High‐resolution cooled digital camera (e.g., ORCA‐ER, Hamamatsu Photonics)
    • 100‐W mercury arc lamp (Carl Zeiss)
    • Optimized excitation/emission filter sets (Omega Optical) for aminomethylcoumarin (AMCA), fluorescein isothiocyanate (FITC), phycoerythrin (PE), tetramethyl rhodamine isothiocyanate (TRITC), and Alexa Fluor 647
  • Additional reagents and equipment for euthanasia of the rat ( appendix 4H) and counting cells using a hemacytometer ( appendix 3B)
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Figures

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Literature Cited

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