Imaging Neural Stem Cell Fate in Mouse Model of Glioma

Khalid Shah1

1 Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5A.1
DOI:  10.1002/9780470151808.sc05a01s8
Online Posting Date:  March, 2009
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Abstract

This unit describes a protocol for following the fate of stem cells in real time in a mouse model of glioma. Stem cells and tumor cells can be transduced with lentiviral vectors bearing two different luciferases, firefly luciferase (Fluc) and Renilla (Rluc) luciferase, respectively. With the cells labeled in this manner, bioluminescence imaging can be used to study the fate of stem cells in glioma‐bearing brains in vivo. Curr. Protoc. Stem Cell Biol. 8:5A.1.1‐5A.1.11. © 2009 by John Wiley & Sons, Inc.

Keywords: neural stem cell; bi‐modal vector; luciferase; fluorescent proteins; glioma; in vivo imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Engineering Stem Cell and Glioma Lines
  • Support Protocol 1: Bioluminescence Imaging in Culture
  • Basic Protocol 2: Cell Transplantation and Imaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Engineering Stem Cell and Glioma Lines

  Materials
  • Human neural stem cells (NSC; Rubio et al., )
  • NSC culture medium (see recipe)
  • 0.25% (w/v) trypsin/EDTA (Invitrogen)
  • Human glioma cells (Gli36; Shah et al., )
  • Glioma cell culture medium: DMEM containing 10% FBS and 1× penicillin/streptomycin
  • Plasmid for NSC cells: lentiviral plasmid bearing a fusion between GFP and Fluc (GFP‐Fluc; Shah et al., )
  • Plasmid for glioma cells: lentiviral plasmid bearing a fusion between Rluc and DsRed2 (Rluc‐DsRed2; Shah et al., )
  • Phosphate‐buffered saline (PBS; e.g., Invitrogen)
  • NSC culture medium (see recipe) containing 8 µg/ml polybrene (add from 8 mg/ml polybrene stock in PBS; Fisher)
  • Glioma cell culture medium (see above) containing 8 µg/ml polybrene (add from 8 mg/ml polybrene stock in PBS; Fisher)
  • 5‐cm culture dishes (Corning)
  • Fluorescence microscope with appropriate filters for GFP and rhodamine
  • Cell sorter (e.g., FACScalibur from BD Biosciences)
  • Additional reagents and equipment for fluorescence‐activated cell sorting (Robinson et al., )

Support Protocol 1: Bioluminescence Imaging in Culture

  Materials
  • NSC and glioma cells bearing bioluminescent and fluorescent markers ( protocol 1)
  • NSC culture medium (see recipe)
  • 150 mg/ml D‐luciferin stock (firefly luciferase substrate; Biotium, cat. no. 10110‐1; http://www.biotium.com) in PBS
  • Glioma cell culture medium: DMEM containing 10% FBS
  • 1 mg/ml coelenterazine stock (substrate for Renilla luciferases; Biotium, cat. no. 10102‐2; http://www.biotium.com) in ethanol
  • 48‐ or 96‐well clear‐bottom black‐walled plate
  • Bioluminescence imaging system with IVIS‐200 or IVIS‐100 (Caliper; http://www.caliperls.com/) or similar bioluminescence imaging system

Basic Protocol 2: Cell Transplantation and Imaging

  Materials
  • SCID mice (6‐to 8‐weeks‐old; Charles River Laboratories)
  • Anesthetics: ketamine and xylazine (also see Donovan and Brown, )
  • Betadine solution (Bruce Medical; http://www.brucemedical.com/)
  • 70% isopropyl alcohol (Fisher)
  • Phosphate‐buffered saline (PBS), sterile
  • Gli36‐Rluc‐DsRed2 glioma cells ( protocol 1)
  • Bone wax (Ethicon)
  • Coelenterazine (100 µg/animal in 150 µl saline; Biotium, cat. no. 10102‐2)
  • D‐luciferin (150 µg/g body weight in 150 µl saline; Biotium, cat. no. 10110‐1)
  • Animal shaver
  • Stereotaxic frame (Harvard Apparatus, cat. no. 726049)
  • Stereo dissecting microscope: variable magnification (1 to 4.5; Nikon)
  • Fine scissors (Fine Science Tools, cat. no. 14084‐08)
  • Forceps, angled and straight and ultrafine angled (Fine Science Tools)
  • Cotton‐tipped applicators
  • Hand‐held micro‐drill (Fine Science Tools, cat. no. 18000‐17) with 0.45‐mm round drill burr (VWR)
  • 10‐µl Hamilton gastight 1701 syringe with 26‐G needle
  • 4–0 vicryl sutures or surgical staples
  • Bioluminescence imaging system with IVIS‐200 or IVIS‐100 (Caliper; http://www.caliperls.com/) or similar bioluminescence imaging system
  • Additional reagents and equipment for anesthesia of mice (Donovan and Brown, )
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Figures

Videos

Literature Cited

Literature Cited
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   Bhaumik, S. and Gambhir, S.S.. 2002. Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc. Natl. Acad. Sci. U.S.A. 99:377‐382.
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