Intraspinal Transplantation of Mouse and Human Neural Precursor Cells

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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% CO₂ 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% CO₂ 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% CO₂ 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

Figure 2.D1.1 Fluorescent microscopy of hNPCs at time of dissociation for preparation for intraspinal transplantation. PAX6 and Nestin are markers for multipotent neural precursor cells and Dapi stains cell nuclei. Images are shown at 40× magnification.

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Figure 2.D1.2 (A ) Setup of stereotaxic apparatus with universal holder with needle support foot on right and electrode holder with removable open side clamp on left of dual small animal stereotaxic platform and tri‐fold paper towels. (B ) The image shows the standard settings for the stereotaxic apparatus to align the needle with the spinal cord at a 70° angle.

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Figure 2.D1.3 Coronal section of a C57Bl/6 mouse transplanted intraspinally with GFP‐mNPCs. Mouse was sacrificed at day 21 post‐transplant, 4% paraformaldehyde perfused, and 10‐µm coronal sections were cut. Transplanted GFP‐mNPCs survived and migrated into the white matter. Engrafted GFP‐mNPCs are green and Dapi‐stained cell nuclei are blue.

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Videos

Literature Cited

Literature Cited
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Intraspinal Transplantation of Mouse and Human Neural Precursor Cells

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited