Intraspinal Transplantation of Mouse and Human Neural Precursor Cells

Jason G. Weinger1, Lu Chen1, Ronald Coleman2, Ronika Leang1, Warren C. Plaisted1, Jeanne F. Loring2, Thomas E. Lane1

1 University of California, Irvine, California, 2 The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.16
DOI:  10.1002/9780470151808.sc02d16s26
Online Posting Date:  September, 2013
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Abstract

ABSTRACT

This unit describes the preparation and transplantation of human neural precursor cells (hNPCs) and mouse neural precursor cells (mNPCs) into the thoracic region of the mouse spinal cord. The techniques in this unit also describe how to prepare the mouse for surgery by performing a laminectomy to expose the spinal cord for transplantation. NPCs genetically labeled with eGFP transplanted into the spinal cord of a mouse following viral‐mediated demyelination can efficiently be detected via eGFP expression. Transplantation of these cells into the spinal cord is an efficacious way to determine their effects in neurological disorders such as multiple sclerosis, Alzheimer's disease, and spinal cord injury. Curr. Protoc. Stem Cell Biol. 26:2D.16.1‐2D.16.16. © 2013 by John Wiley & Sons, Inc.

Keywords: human neural precursor cells; mouse neural precursor cells; laminectomy; intraspinal transplantation; multipotency

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of hNPCs for Mouse Intraspinal Transplantation
  • Support Protocol 1: Verifying NPC Phenotype of hNPCs
  • Alternate Protocol 1: Preparation of mNPCs for Intraspinal Transplantation
  • Basic Protocol 2: Preparation of Mice for Intraspinal Transplantation
  • Basic Protocol 3: Intraspinal Transplantation of mNPCs or hNPCs and Post‐Operative Care
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of hNPCs for Mouse Intraspinal Transplantation

  Materials
  • Poly‐L‐ornithine (Sigma, cat. no. P3655)
  • Laminin (BD Biosciences, cat. no. 354239)
  • 1× HBSS (Cellgro, cat. no. 21‐022‐CM)
  • hNPCs
  • Complete hNPC medium (see recipe)
  • Accutase (Invitrogen, cat. no. 11330‐032)
  • 0.4% Trypan blue
  • Tissue culture hood (Biosafety Class II A/B3)
  • 6‐well tissue culture plates
  • 37°C, 5% CO 2 humidified tissue culture incubator
  • 50‐ml conical tubes
  • Refrigerated tabletop centrifuge
  • Hemacytometer with cover slip
  • Inverted phase‐contrast microscope
  • Light duty wipe (Kimwipe)
  • 1.7‐ml microcentrifuge tube

Support Protocol 1: Verifying NPC Phenotype of hNPCs

  Materials
  • hNPCs
  • Poly‐L‐ornithine
  • Laminin (BD Biosciences, cat. no. 354239)
  • Complete hNPC medium (see recipe)
  • 1× PBS (see recipe)
  • 1% paraformaldehyde
  • 1× PBS with 0.5% (v/v) BSA (filter through a 0.22‐µm sterile filter; store up to 4 weeks at 4°C)
  • Goat serum (Vector Laboratories, cat. no. Y0322)
  • Triton‐X100 (Sigma, cat. no. X100)
  • Antibodies:
    • Nestin (rabbit polyclonal; Millipore, cat. no. ABD69)
    • Pax6 (rabbit polyclonal; Covance, cat. no. PRB278P)
    • Goat‐anti‐rabbit IgG (Jackson ImmunoResearch Laboratories, cat. no. 111‐005‐144)
    • Goat‐anti‐rabbit secondary
      • Alexa 594‐conjugated (Invitrogen, cat. no. A11037)
      • Alexa 488‐conjugated (Invitrogen, cat. no. A11008)
  • Dapi Fluormount‐G mounting medium (SouthernBiotech, cat. no. 0100‐20)
  • 4‐well chamber glass slides (Lab‐Tek; cat. no. 154526)
  • 37°C, 5% CO 2 humidified tissue culture incubator
  • Chemical fume hood
  • Humidified chamber
  • Coverslips
  • Inverted fluorescent microscope with 40× objective

Alternate Protocol 1: Preparation of mNPCs for Intraspinal Transplantation

  Materials
  • GFP‐mNPCs
  • Complete mNPC medium (see recipe)
  • Trypsin‐EDTA (Gibco, cat. no. 25300‐054)
  • Dulbecco's modified Eagle's medium (DMEM)
  • 1× HBSS
  • Tissue culture hood (Biosafety Class II A/B3)
  • 75‐cm2 flasks
  • 37°C, 5% CO 2 humidified tissue culture incubator
  • 50‐ml conical tubes
  • Refrigerated table‐top centrifuge
  • Light‐duty wipe (Kimwipe)
  • 1.7‐ml conical tubes
  • Additional reagents and equipment to count cells (see protocol 1)

Basic Protocol 2: Preparation of Mice for Intraspinal Transplantation

  Materials
  • Hair removal cream (Nair)
  • Iodide solution (Betadine surgical scrub; Fisher cat. no. 19‐027132)
  • Petroleum jelly (e.g., Vaseline)
  • Mice
  • Ketamine/xylazine hydrochloride (see recipe)
  • Diluted dishwashing soap (e.g., Dawn, 1:50 in water)
  • 70% ethanol (optional)
  • Sterile saline
  • Weigh boats
  • 28‐G needles attached to 1‐ml syringes (Fisher, cat. no. 14‐829‐1B)
  • Electric hair clipper
  • Gauze‐tipped applicators, sterile and non‐sterile
  • Gauze squares, sterile and non‐sterile
  • Colored tape
  • Laminar flow cabinet
  • Tri‐fold paper towels
  • Fiber optic illuminator (Fisher Scientific, cat. no. 12‐562‐36)
  • Dry glass bead sterilizer (Steri 350; Simon Keller AG, cat. no. 06‐12287)
  • 50‐ml glass beaker (optional)
  • Small Graefe forceps (Fine Science Tools, cat. no. 11053‐10)
  • Scalpels with no. 10 and no. 15 blades
  • Micro‐scissors (World Precision Instruments, cat. no. 555500S)

Basic Protocol 3: Intraspinal Transplantation of mNPCs or hNPCs and Post‐Operative Care

  Materials
  • 70% ethanol
  • Sterile 1× HBSS
  • mNPCs or hNPCs
  • Laminectomized mouse (see protocol 4)
  • Lactated ringers (Hospira, cat. no. NDC 0409‐7953‐03)
  • Buprenorphine (Buprenex; Western Medical Supply, cat. no. 7292)
  • Stereotaxic apparatus (Kopf instruments) including: universal holder with needle support foot (cat. no. 1772), electrode holder with removable open side clamp (cat. no. 1773), dual small animal stereotaxic platform (cat. no. 902)
  • Tri‐fold paper towels
  • 50‐ml conical tubes
  • Test tube holder
  • 10‐µl Hamilton syringe with removable plunger (Hamilton Company, cat. no. 7635‐01)
  • Hamilton needles (30‐G needle, point style 4, 30° bevel; Hamilton Company, cat. no. 7803‐07)
  • Pipets and filtered tips
  • Hemostat (Fine Science Tools, cat. no. 13010‐12)
  • Olsen‐Hegar needle holder (Fine Science Tools, cat. no. 12502‐12)
  • Sutures (size 5‐0, 3/8 in. circle, 19‐mm needle, 45‐cm braided thread; Ethicon, cat. no. 1676G)
  • Reflex 7 wound clip applicator (Fine Science Tools, cat. no. 12031‐07)
  • 10‐ml syringes
  • 18‐G, ½‐in. needles
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Figures

Videos

Literature Cited

Literature Cited
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