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

Araceli Espinosa‐Jeffrey1, Dustin R. Wakeman2,3, Seung U. Kim4, Evan Y. Snyder3, Jean de Vellis1

1 David Geffen School of Medicine at UCLA, Los Angeles, California, 2 University of California at San Diego, La Jolla, California, 3 The Burnham Institute for Medical Research, La Jolla, California, 4 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: 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 2D.4.2):
    • 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 (Support Protocol 1)
  • 37°C, 4.5% CO2 incubator (adjustable to 5% if growth is slow), 95% humidity
  • Additional reagents and equipment for isoflurane anesthesia of the mouse (unit 2A.5), assessing cell viability (Support Protocol 2), and counting cells using a hemacytometer (unit 1C.3)
     FigureFigure 2D.4.2 Instruments required for dissection. (1) Scissors for decapitation; (2) scissors to cut the head skin to expose the skull; (3) forceps to hold the head in place as you cut the skin and cut the skull cartilage with scissors (4) to expose the brain. Some users prefer the curved scissors (2) instead of (4). Use the same scissors to transfer the brain to the petri dish containing PBS. Forceps (5) and (6) are to hold the brain in place and remove the meninges, respectively. After removal of the meninges, place the brains in HBSS while dissecting the rest of the brains. A filter mesh (7) is used to filter the cell suspension after dissociation. A 20-ml sterile syringe (8) and sterile 18-G dissociation needle (9) are used to dissociate the cells.

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 (Basic 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 (Support Protocol 1)
  • 1.2-ml cryovials
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1C.3) and freezing cells (Support Protocol 3)

NOTE: All steps are performed at room temperature (20°C).

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

 Materials
  • Conditioned medium (see Basic Protocol 2)
  • Complete stem cell medium (STMIIc; see recipe)
  • Established NSC cultures (Basic Protocol 2)
  • 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 (Basic Protocol 2)
  • 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 Basic Protocol 2)
  • 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 (Support Protocol 1)
  • Additional reagents and equipment for testing cell viability (Support Protocol 2)

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; Basic Protocol 2 or Alternate Protocol)
  • 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 Support Protocol 1 but substitute goat anti-IgM antibody (ABR, sold by Thermo Scientific, cat. no. PA1-86106) for anti-PSA-NCAM antibody
  • 37°C, 4.5% CO2 incubator
  • 12-ml syringe (Tyco Healthcare, cat. no. 512852)
  • Additional reagents and equipment for maintaining cells (Basic Protocol 1)

NOTE: All steps are performed at room temperature (20°C).

NOTE: We recommended precalibrating the percentage of CO2 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% CO2. For propagation and maintenance of OL spheres, the Erlenmeyer flask should also be kept open just enough to ensure O2/CO2 exchange. When using 4.5% CO2, loosen the caps of the flasks until half-way open.

Basic Protocol 4: Culturing Oligodendrocytes for Lineage Progression and Maturation

 Materials
  • OLPs (Basic Protocol 3, 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 (Basic Protocol 3)

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

 Materials
  • OL plated on (uncoated) plastic tissue culture dishes (from Basic Protocol 4, step 2; also see Fig. 2D.4.5)
  • GDM medium (see recipe)
  • Conditioned medium (from GDM; Basic Protocol 4)
  • OLDEM medium (see recipe)
  • Cell scraper
  • Neuronal cultures (Support Protocol 4) 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% CO2 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 Basic 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., 2008)

NOTE: Required materials may vary depending upon the grafting method of choice.
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Figures

  •  FigureFigure 2D.4.1 Oligodendrocyte specification and lineage progression. Oligodendrocytes undergo sequential morphological changes as they develop from uncommitted NSC to a committed OLP and acquire characteristics inherent in a functional OL. The list of OL markers below each developmental stage is not exhaustive but represents frequently used markers to identify OLs and their developmental stage. Media (also see Reagents and Solutions): stem cell medium (STM; Espinosa-Jeffrey et al., 2002); OL specification medium (OSM; Espinosa-Jeffrey et al., 2002); glia defined medium (GDM; Espinosa de los Monteros and de Vellis, 1996); OLDEM (Espinosa de los Monteros et al., 1988, 1997). Modified from Arenander and de Vellis (1995).
    View Image
  •  FigureFigure 2D.4.2 Instruments required for dissection. (1) Scissors for decapitation; (2) scissors to cut the head skin to expose the skull; (3) forceps to hold the head in place as you cut the skin and cut the skull cartilage with scissors (4) to expose the brain. Some users prefer the curved scissors (2) instead of (4). Use the same scissors to transfer the brain to the petri dish containing PBS. Forceps (5) and (6) are to hold the brain in place and remove the meninges, respectively. After removal of the meninges, place the brains in HBSS while dissecting the rest of the brains. A filter mesh (7) is used to filter the cell suspension after dissociation. A 20-ml sterile syringe (8) and sterile 18-G dissociation needle (9) are used to dissociate the cells.
    View Image
  •  FigureFigure 2D.4.3 Rodent neural stem cell preparation. Following dissection, the cell suspension is plated on anti-PSA-NCAM antibody–coated dishes and allowed to adhere. The process can be performed repeatedly to increase the numbers of neural stem cells, as two-dimensional cultures or three-dimensional “sphere” cultures (shown on the left side of the diagram). Every time cells are propagated, use anti-PSA-NCAM-coated dishes. Alternatively, cells can be propagated and immediately used for cell culture experiments (as shown on the right side of the diagram). While we prefer to use committed OL progenitors for cell transplants, other investigators also use uncommitted progenitors for grafting.
    View Image
  •  FigureFigure 2D.4.4 Oligodendrocyte specification. The transition of NSC to commit to the OL lineage is brief, but sequential rather than abrupt. In order for cells to survive, they must acclimate to their new environment. OLP can be propagated to create frozen stocks as three-dimensional “oligosphere” cultures (shown on the left of the diagram) or frozen without propagation (as shown in the sequence in the center of the diagram). OLP can also be propagated in two-dimensional cultures for cryopreservation, for specific cell culture experiments, or for cell replacement therapies (as shown on the right side of the diagram).
    View Image
  •  FigureFigure 2D.4.5 Oligodendrocyte lineage progression and maturation. After commitment of NSC to the OL lineage, cells are propagated at the OLP stage to create a frozen stock (steps indicated on the left portion of the flow chart) or processed further for transplantation studies (as shown by the middle arrow on the diagram). To allow OLP to further mature along the OL lineage and become myelinated, cells are transitioned into OLDEM for at least 48 hr. Once OL have reached this stage of maturation, they are excellent for cell culture studies but are not recommended for cell grafting. Detachment from the substrate can damage the numerous delicate cell processes; therefore these cultures are no longer a quality source for cell transplants.
    View Image
  •  FigureFigure 2D.4.6 Phase-contrast view of neural stem cells derived from embryonic day 16 rat brain at passage number 2 (P2). NSC were plated and maintained in OSM for 2 days (A), 3 days (B), or 3 days in OSM then switched to GDM for 1 day (C). Cells in OSM still proliferate while in OSM. When cells from (A or B) are plated and maintained in OSM on poly-d-lysine-coated coverslips for 1 day, they start to display a bipolar or multipolar morphology (D) and most express the immature precursor marker, nestin (green) but not Tf (red), an early marker for OL. After 2 days in OSM, bipolar nestin+ cells coexpress transferrin (Tf) (E). After 4 days in OSM, cells were switched to GDM for 1 day; they developed numerous cell processes and coexpressed sulfatides (recognized by the anti-O4 antibody, green) and myelin basic protein (MBP; red) (F). Panels G to I are human cells. (G) Phase-contrast view of human NSCs (HFB-2050) acclimated and expanded in STM, then replated and maintained in OSM for 2 days. (H) OL derived from human NSCs (HFB-2050) were specified to the OL lineage with OSM and maintained in GDM for 10 days. OL matured and started to express MBP (red). (I) Rat cortical neurons (NFM-200-red) were cultured for 10 days, then human OLP derived from NSC (HFB-2050) were added in coculture for 24 hr. These cells were labeled with human nuclei marker (HuNu, green).
    View Image
  •  FigureFigure 2D.4.7 Human OL derived from (HFB-2050) human fetal NSC were labeled with fluorescent fast blue (FB; Sigma, cat. no. F-5756). A total of 60,000 cells were grafted into the corpus callosum (CC) of P(5) rat pups born to a myelin-deficient (md) carrier mother. At a time point 23 days after grafting, samples were harvested and examined. Grafted NSC survived and migrated extensively within the host brain parenchyma extending along the corpus callosum (CC) and caudate putamen (CPu). In the sketch, dots represent the location where FB+ cells were found. The sketch represents a sagittal view of the transplanted rat brain at 28 days of age, IS indicates where cells were implanted.
    View Image

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Literature Cited

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