Culture System for Rodent and Human Oligodendrocyte Specification, Lineage Progression, and Maturation

Araceli Espinosa‐Jeffrey1, Dustin R. Wakeman2, Seung U. Kim3, Evan Y. Snyder2, Jean de Vellis1

1 David Geffen School of Medicine at UCLA, Los Angeles, California, 2 The Burnham Institute for Medical Research, La Jolla, California, 3 University of British Columbia Hospital, Vancouver, British Columbia, Canada
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.4
DOI:  10.1002/9780470151808.sc02d04s10
Online Posting Date:  September, 2009
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Abstract

Here we document protocols for the production, isolation, and maintenance of the oligodendrocyte phenotype from rodent and human neural stem cells. Our unique method relies on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of oligodendrocytes as they advance from oligodendrocyte progenitors to mature, myelinating oligodendrocytes. Curr. Protoc. Stem Cell Biol. 10:2D.4.1‐2D.4.26. © 2009 by John Wiley & Sons, Inc.

Keywords: neural stem cells; NSC; oligodendrocyte specification; oligodendrocyte maturation; lineage progression; oligospheres; neurospheres

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Rodent Neural Stem Cells
  • Support Protocol 1: Preparation of Anti‐PSA‐NCAM‐Coated Dishes for Selecting NSC by Immunopanning
  • Support Protocol 2: Assessing Cell Viability
  • Basic Protocol 2: Propagation of Rodent NSCs as Two‐Dimensional Cultures
  • Alternate Protocol 1: Formation, Propagation, and Maintenance of Neurospheres in Three‐Dimensional Cultures
  • Support Protocol 3: Cryopreservation/Thawing of NSC Stocks
  • Basic Protocol 3: Oligodendrocyte Commitment in Two‐ and Three‐Dimensional Cultures
  • Basic Protocol 4: Culturing Oligodendrocytes for Lineage Progression and Maturation
  • Basic Protocol 5: Propagation of Oligodendrocytes for In Vitro Myelination Assays
  • Support Protocol 4: Preparation of Cortical Neurons
  • Basic Protocol 6: Transplantation of OL Progenitors into Neonatal Rats
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Rodent Neural Stem Cells

  Materials
  • One timed‐pregnant, embryonic day 14 to 16 (ED14 to ED16) Sprague‐Dawley rat (Charles River Laboratories)
  • Basal stem cell medium (STM‐II; see recipe) supplemented with 1% (w/v) BSA (Sigma, cat no. A‐3156)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. P‐5368)
  • Complete stem cell medium (STMIIc; see recipe)
  • Dissection instruments, sterile (refer to numbers in Figure ):
    • No. 1. Mayo scissors (Fine Science Tools, cat. no. 14010‐17)
    • No. 2. Lister scissors (Fine Science Tools, cat. no. 14131‐14)
    • No. 3. blunt‐pointed forceps (Fisher, cat. no. 08‐887)
    • No. 4. iris scissors (Fine Science Tools, cat. no. 14060‐09)
    • No. 5. Moria iris forceps (Fine Science Tools, cat. no. 11373‐12)
    • No. 6. Dumont #7 forceps (Fine Science Tools, cat. no. 11297‐10)
    • No. 7. 140‐µm and 230‐µm sieves (Cellector, E‐C Apparatus Corp.; http://www.thermo.com)
    • No. 8. 20‐ml syringe (Kendall, cat. no. 520673; http://www.kendallhq.com)
    • No. 9. 18‐G Quincke spinal luer‐lock needle for dissociation (100‐mm length; Unimed; http://www.unimed.ch/)
  • 100 × 15–mm petri dish (bacterial grade, non TC‐treated; BD Falcon, cat. no. 351029)
  • 50‐ml and 15‐ml conical tubes
  • Centrifuge (e.g., IEC Clinical)
  • 100‐mm anti‐PSA‐NCAM coated dishes ( protocol 2)
  • 37°C, 4.5% CO 2 incubator (adjustable to 5% if growth is slow), 95% humidity
  • Additional reagents and equipment for isoflurane anesthesia of the mouse (unit 2.5), assessing cell viability ( protocol 3), and counting cells using a hemacytometer (unit 1.3)
NOTE: All dissection instruments, plasticware, and glassware must be sterile.NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for care and use of laboratory animals.

Support Protocol 1: Preparation of Anti‐PSA‐NCAM‐Coated Dishes for Selecting NSC by Immunopanning

  Materials
  • 50 mM Tris⋅Cl, pH 9.5
  • Bovine serum albumin (BSA; Sigma, cat. no. A‐3156)
  • Anti‐PSA‐NCAM antibody (Iowa DSHB, http://dshb.biology.uiowa.edu/, cat. no. 5A5)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. P‐5368)
  • 100 × 15–mm petri dish (bacterial grade, non‐TC‐treated; BD Falcon, cat. no. 351029)

Support Protocol 2: Assessing Cell Viability

  Materials
  • Tris‐buffered saline (TBS; see recipe)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. P‐5368)
  • 1 µM SYTOX blue nucleic acid stain (Invitrogen Molecular Probes, cat. no. S7020) in PBS
  • Microscope slides and coverslips
  • Fluorescence microscope

Basic Protocol 2: Propagation of Rodent NSCs as Two‐Dimensional Cultures

  Materials
  • Cultures of freshly isolated neural stem cells ( protocol 1) or their progenitors
  • Hanks' buffered salt solution (HBSS) without Ca2+ or Mg2+
  • Complete stem cell medium (STMIIc; see recipe)
  • Cell Freezing Medium, serum‐free, 1× (Sigma, cat. no. C2639)
  • Cell scraper
  • 15‐ml conical tubes
  • Centrifuge (e.g., IEC Clinical)
  • 20‐ml syringe
  • 18‐G Quincke spinal luer‐lock needle for dissociation (100‐mm length; Unimed; http://www.unimed.ch/)
  • 12.5‐cm2 and 75‐cm2 tissue culture flasks (Falcon), anti‐PSA‐NCAM‐coated ( protocol 2)
  • 1.2‐ml cryovials
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.3) and freezing cells ( protocol 6)
NOTE: All steps are performed at room temperature (20°C).

Alternate Protocol 1: Formation, Propagation, and Maintenance of Neurospheres in Three‐Dimensional Cultures

  Materials
  • Conditioned medium (see protocol 4)
  • Complete stem cell medium (STMIIc; see recipe)
  • Established NSC cultures ( protocol 4)
  • Glass Erlenmeyer flasks, 25‐ml or 50‐ml with cap
  • 37°C incubator with shaker
  • 20‐ml syringe
  • 18‐G Quincke spinal luer‐lock needle for dissociation (100‐mm length; Unimed; http://www.unimed.ch/)
  • 50‐ml conical tubes
  • Centrifuge (e.g., IEC Clinical)
  • 0.22‐µm sterile filters
NOTE: All steps are performed at room temperature (20°C).

Support Protocol 3: Cryopreservation/Thawing of NSC Stocks

  Materials
  • NSC cultures ready for freezing ( protocol 4)
  • Hanks' balanced salt solution (HBSS) without Ca2+ or Mg2+
  • Complete stem cell medium (STMIIc; see recipe)
  • Cell Freezing Medium, serum‐free, 1× (Sigma, cat. no. C2639)
  • Liquid nitrogen
  • Conditioned medium (CM; see protocol 4)
  • Cell scrapers
  • Centrifuge (e.g., IEC Clinical)
  • 20‐ml syringe
  • 18‐G Quincke spinal luer‐lock needle for dissociation (100‐mm length; Unimed; http://www.unimed.ch/)
  • 1.2‐ml cryovials
  • Cryogenic slow‐freezing chamber (Nalgene, cat. no. EW‐44400‐00)
  • 2‐ml tubes (Fisher)
  • Anti‐PSA‐NCAM coated tissue culture vessels ( protocol 2)
  • Additional reagents and equipment for testing cell viability ( protocol 3)
NOTE: All steps are performed at room temperature (20°C).

Basic Protocol 3: Oligodendrocyte Commitment in Two‐ and Three‐Dimensional Cultures

  Materials
  • NSC cultures (2‐D or 3‐D; protocol 4 or protocol 5)
  • Hanks' balanced salt solution (HBSS) without Ca2+ or Mg2+
  • OL specification medium (OSM‐II; see recipe)
  • Cell scraper
  • 15‐ml conical tubes
  • 20‐ml syringe
  • 18‐G Quincke spinal luer‐lock needle for dissociation (100‐mm length; Unimed; http://www.unimed.ch/)
  • Anti‐IgM coated 100‐mm petri dishes or tissue culture flasks: prepare as in protocol 2 but substitute goat anti‐IgM antibody (ABR, sold by Thermo Scientific, cat. no. PA1‐86106) for anti‐PSA‐NCAM antibody
  • 37°C, 4.5% CO 2 incubator
  • 12‐ml syringe (Tyco Healthcare, cat. no. 512852)
  • Additional reagents and equipment for maintaining cells ( protocol 1)
NOTE: All steps are performed at room temperature (20°C).NOTE: We recommended precalibrating the percentage of CO 2 1 day before plating the cells. If the incubator is shared with other people or needed at 5% for NSC propagation and maintenance, we recommend using tissue culture flasks for 2‐D cultures instead of petri dishes. Close the cap of the flask completely and then open it one‐quarter of a turn before placing in the incubator at 5% CO 2. For propagation and maintenance of OL spheres, the Erlenmeyer flask should also be kept open just enough to ensure O 2/CO 2 exchange. When using 4.5% CO 2, loosen the caps of the flasks until half‐way open.

Basic Protocol 4: Culturing Oligodendrocytes for Lineage Progression and Maturation

  Materials
  • OLPs ( protocol 7, step 6a)
  • OL specification medium (OSM‐II; see recipe)
  • GDM medium (see recipe)
  • Recombinant human basic fibroblast growth factor (bFGF; Invitrogen)
  • OLDEM medium (see recipe)
  • Poly‐D‐lysine‐coated wells/plates (see recipe)
  • Additional reagents and equipment for oligodendrocyte differentiation in two‐dimensional culture ( protocol 7)

Basic Protocol 5: Propagation of Oligodendrocytes for In Vitro Myelination Assays

  Materials
  • OL plated on (uncoated) plastic tissue culture dishes (from protocol 8, step 2; also see Fig. )
  • GDM medium (see recipe)
  • Conditioned medium (from GDM; protocol 8)
  • OLDEM medium (see recipe)
  • Cell scraper
  • Neuronal cultures ( protocol 10) plated on poly‐D‐lysine‐coated coverslips
  • Complete Neurobasal‐N medium for cortical neurons (see recipe)
  • 40‐µm cell strainers (BD Falcon, cat. no. 352340)
NOTE: All steps are performed at room temperature (20°C).

Support Protocol 4: Preparation of Cortical Neurons

  Materials
  • Complete Neurobasal‐N medium for cortical neurons (see recipe)
  • Poly‐D‐lysine‐coated (see recipe) coverslips in wells of 12‐ or 24‐well plates
  • 37°C 4.5% CO 2 incubator, 95% humidified
  • Combustion Test Kit (Bacharach, cat. no. 10‐500; http://www.bacharach‐inc.com)
  • Additional reagents and materials for isolation of rodent brain cells (see protocol 1)
NOTE: All steps are performed at room temperature (20°C).

Basic Protocol 6: Transplantation of OL Progenitors into Neonatal Rats

  Materials
  • Neonatal rat pup, post‐natal day 0 to 5 (P0 to P5)
  • 70% ethanol
  • Dulbecco's phosphate‐buffered saline (DPBS; without calcium or magnesium, e.g., Cellgro, cat. no. 21‐031‐CV), sterile
  • Microcentrifuge tube with cell sample (suspended in PBS; may be from various protocols in this unit depending on experimental question to be addressed)
  • Borosilicate glass (Sutter Instrument Co., cat. no. B100–75‐15)
  • Micropipet puller (Sutter Instrument Co., Model P‐87)
  • Aspirator tube assemblies for calibrated microcapillary pipets (Sigma‐Aldrich, cat. no. A5177–5EA)
  • Fiber‐optic light source for transillumination
  • Warming pad
  • Warm‐water glove balloon
  • Additional reagents and equipment for preparing injection micropipet (Lee et al., )
NOTE: Required materials may vary depending upon the grafting method of choice.
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Figures

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Literature Cited

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