Slice Culture Method for Studying Migration of Neuronal Progenitor Cells Derived from Human Embryonic Stem Cells (hESC)

Radmila Filipovic1, Saranya Santosh Kumar1, Ben A. Bahr2, Joseph Loturco1

1 Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut, 2 Biotechnology Research and Training Center, William C. Friday Laboratory, University of North Carolina Pembroke, Pembroke, North Carolina
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.7
DOI:  10.1002/9780470151808.sc01h07s29
Online Posting Date:  May, 2014
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Abstract

In this unit we describe an overlay brain slice culture assay for studying migration of transgenic neurospheres derived from human embryonic stem cells (hESC). Neuronal progenitor cells were generated from hESC by derivation of embryoid bodies and rosettes. Rosettes were transfected using the PiggyBac transposon system with either control plasmids (GFP) or plasmid encoding a gene important for migration of neuronal progenitor cells, Doublecortin (DCX). Transfected cells were subsequently grown in low‐adhesion plates to generate transgenic human neurospheres (t‐hNS). Organotypic slice cultures were prepared from postnatal rat forebrain and maintained using the interface method, before transfected t‐hNS were overlaid below the cortex of each hemisphere. After 1 to 5 days, forebrain slices were fixed and processed for immunofluorescence. The distance at which cells migrated from the center of neurospheres to the host forebrain tissue was measured using Image J software. This protocol provides details for using the slice culture method for studying migration and integration of human neuronal cells into the host brain tissue. Curr. Protoc. Stem Cell Biol. 29:1H.7.1‐1H.7.14. © 2014 by John Wiley & Sons, Inc.

Keywords: organotypic slice cultures; overlay essay; human embryonic stem cell; neuronal progenitors; piggyBac; migration

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of Transgenic Neurospheres from hESC‐ (t‐hNS)
  • Basic Protocol 2: Organotypic Forebrain Rat Slice Cultures
  • Basic Protocol 3: Slice Culture Overlay Assay to Study Migration of t‐hNS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of Transgenic Neurospheres from hESC‐ (t‐hNS)

  Materials
  • γ‐irradiated mouse embryonic fibroblasts (MEF; GlobalStem)
  • 0.1% gelatin (see recipe)
  • MEF medium (see recipe)
  • CT‐2 hESC line (Stem Cell Core, University of Connecticut Health Center)
  • H9 cell line (WA09, Stem Cell Core, University of Connecticut Health Center)
  • Human embryonic stem cell (hESC) medium (see recipe)
  • Collagenase IV (Invitrogen)
  • Neural induction medium (NIM; see recipe)
  • Poly‐D‐lysine (Sigma)
  • Phosphate‐buffered saline (PBS; Ca2+, Mg2+‐free; Invitrogen, cat. no. 14190‐250)
  • Laminin (Invitrogen)
  • Accutase (Invitrogen)
  • Amaxa human stem cell Nucleofector kit (Lonza) containing:
    • Human stem cell nucleofection solution
  • Plasmids (all plasmids are made in the laboratory):
    • pPBCAG‐GFP
    • pPBCAG‐DCX‐IRES‐GFP
    • pCAGPBase
  • ROCK inhibitor (EMD Chemicals)
  • Low‐adhesion 6‐ and 12‐well plates (Thermo Fisher Scientific)
  • Fisher Scientific water bath ISOTEMP 110
  • Forma Series II water‐jacketed CO 2 incubator
  • Nuare Class II biological safety cabinet
  • Inverted microscope (Nikon Eclipse TE300)
  • Dissecting microscope (Nikon)
  • 1000‐ and 200‐µl tips (Fisher)
  • Protecting surgical masks (Fisher)
  • 5‐ml glass pipets (Fisher)
  • 50‐ml conical tubes
  • 2‐ml aspirating pipets (Fisher)
  • 6‐well plates (Thermo Fisher Scientific)
  • Coverslips (12 mm, Electron Microscopy Sciences)
  • Microcentrifuge (Eppendorf)
  • Hemacytometer (Cole‐Parmer)
  • 0.5‐ml microcentrifuge tubes
  • Amaxa II nucleofector (Lonza, Program setting A‐023)
  • Fluorescent microscope

Basic Protocol 2: Organotypic Forebrain Rat Slice Cultures

  Materials
  • Slice buffer (see recipe)
  • Wistar, male, postnatal day 3 (PD3) rats
  • Anesthetic reagent (Isoflurane)
  • Phosphate‐buffered saline (PBS; Ca2+, Mg2+‐free; Invitrogen, cat. no. 14190‐250)
  • Low‐temperature‐melting agarose (Invitrogen)
  • Distilled water
  • Ice
  • SuperGlue
  • NIM (see recipe)
  • Slice medium (see recipe)
  • 4% paraformaldehyde (PFA; see recipe)
  • Glass flask (Fisher)
  • −80°C freezer
  • Scissors (Bio Precision Instruments)
  • 60‐mm petri dishes (Fisher)
  • Spatulas (Bio Precision Instruments)
  • Scalpels (Fisher)
  • Embedding molds (Thermo Electron)
  • Gas dispenser
  • Oxygen tank (Airgas)
  • Vibratome (Leica VT 1200S)
  • 6‐well plates (Corning)
  • Inserts (0.4‐µm porosity; Millipore)
  • Forceps, sterile
  • Class II biological safety cabinet
  • 37°C, 5% CO 2 incubator

Basic Protocol 3: Slice Culture Overlay Assay to Study Migration of t‐hNS

  Materials
  • Rat brain slices (see protocol 2)
  • Transgenic hNS (see protocol 1)
  • Slice medium (see recipe)
  • Phosphate‐buffered solution (PBS; Ca2+, Mg2+‐free; Invitrogen, cat. no. 14190‐250)
  • 4% paraformaldehyde (PFA; see recipe)
  • Rabbit anti‐GFP antibody (Invitrogen)
  • Mouse anti‐Nestin human specific (Millipore)
  • Goat anti‐mouse IgG 568
  • Goat anti‐rabbit IgG 488
  • DAPI (Hoechst 33342 stain, 2 µg/ml; Sigma)
  • Blocking solution (see recipe)
  • 30%, 50%,70%,100% ethanol (Fisher)
  • Benzyl alcohol (Sigma)
  • Benzyl benzoate (Sigma)
  • Nail polish
  • Prolong‐Gold anti‐fade reagent (Invitrogen)
  • 37°C humidified incubator
  • 1.5‐ml microcentrifuge tubes
  • 60‐mm petri dishes
  • Dissecting microscope
  • 200‐µl pipet tips
  • 6‐well plates
  • Glass slides (Fisher)
  • 0.75‐thick cover glass (Fisher)
  • Spatula
  • Zeiss Axio imager M2 microscope equipped with Apotome
  • Image J 1.45 software
  • 5‐ml glass pipets (Fisher)
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Figures

Videos

Literature Cited

Literature Cited
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