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Isolation and Purification of Primary Oligodendrocyte Precursors

Oliver Bögler¹

¹Medical College of Virginia, Richmond, Virginia

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 3.4

DOI:

10.1002/0471142301.ns0304s00

Online Posting Date:

May, 2001

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Abstract

Oligodendrocytes are the myelinating cells of the central nervous system, and are avidly studied by investigators interested in neural cell lineages, myelination, multiple sclerosis, and oligodendroglioma. This unit presents a strategy for manipulating the division and differentiation of oligodendrocyte progenitors by isolating O-2A progenitors from optic nerve and then purifying and expanding them by culture in a defined medium supplemented with platelet-derived growth factor (PDGF) plus basic fibroblast growth factor (bFGF). Cells are then allowed to differentiate through removal of the growth factors. A protocol is also provided for monitoring the cell type composition of the cultures by immunohistochemistry. In addition to making it possible to generate large numbers of cells, this approach allows access to intermediate differentiation stages. Cells can thus be studied as they differentiate into oligodendrocytes, making the actual process of differentiation open to examination.

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Unit Introduction
Basic Protocol: Isolation of O-2A Progenitors From Rat Optic Nerve
Support Protocol: Immunohistochemistry of Optic Nerve Cell Cultures
Reagents and Solutions
Commentary
Bibliography
Tables

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Materials

Basic Protocol: Isolation of O-2A Progenitors From Rat Optic Nerve

Materials

L-15 medium with penicillin and streptomycin
7-day-old rat pups
PBS/EDTA (see recipe)
DMEM-BS medium (see recipe)
75-cm² tissue culture flasks (Falcon) coated with polylysine (appendix 2A)
Platelet-derived growth factor (PDGF; see recipe )
Basic fibroblast growth factor (bFGF; see recipe)
0.05% trypsin/0.5 mM EDTA (e.g., Life Technologies)
Soybean trypsin inhibitor (SBTI)/DNase (see recipe)
A2B5 and anti-GalC monoclonal antibodies (e.g., Boehringer Mannheim)
Anti-GFAP polyclonal antibody (e.g., Dako)

60-mm plastic petri dishes, sterile
15-ml conical plastic tubes (Falcon)
Dissecting instruments:
- Small dissecting scissors, ¾-in. (~2-cm) blade
- Dissecting scissors, 1½-in. (~4-cm) blade
- No. 7 Dumont forceps, straight and curved
- Curved microdissecting scissors, ¼-in. (7-mm) blade
- Scalpel with no. 10 blade
Dissection board (e.g., Styrofoam block covered with aluminum foil)
21-, 25-, and 27-G needles
2-ml tissue culture pipet
5-ml plastic snap-cap tube
1-ml syringe
Humidified 37°C, 7.5% CO₂ incubator
12-mm glass coverslips coated with polylysine (appendix 2A)
24-well plastic tissue culture dishes

Support Protocol: Immunohistochemistry of Optic Nerve Cell Cultures

Materials

Cells on coverslips (see Basic Protocol)
HBSS/5% serum (see recipe)
Antibodies: A2B5, anti-GalC, and anti-GFAP (Table 3.4.1)
Secondary antibodies (typically anti-rabbit-biotin, anti-mouse-IgM-FITC, and anti-mouse-IgG₃-TRITC diluted 1:100 in HBSS/5% serum; e.g., Southern Biotechnology)
Methanol, chilled on dry ice
Streptavadin-coumarin (e.g., Southern Biotechnology), diluted 1:100 in HBSS/5% serum
PBS (appendix 2A or, e.g., Life Technologies)
Antifade: 2.5% (w/v) 1,4-diazabicyclo[2.2.2]octane (Sigma) in glycerol
Nail varnish
Bright-field phase-contrast microscope (unit 2.1) equipped for epifluorescence, with selective filters for fluorescein, rhodamine, and coumarin

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Literature Cited

Literature Cited
	Bignami, A., Eng, L.F., Dahl, D., and Uyeda, C.T. 1972. Localization of glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res. 43:429-435.
	Bögler, O. and Noble, M. 1994. Measurement of time in oligodendrocyte-type-2 astrocyte (O-2A) progenitors is a cellular process distinct from differentiation or division. Dev. Biol. 162:525-538.
	Bögler, O., Wren, D.R., Barnett, S.C., Land, H., and Noble, M.D. 1990. Cooperation between two growth factors promotes extended self-renewal and inhibits differentiation of O-2A progenitor cells. Proc. Natl. Acad. Sci. U.S.A. 87:6368-6372.
	Bottenstein, J.E. and Sato, G.H. 1974. Growth of a rat neuroblastoma cell line in serum free supplemented medium. Proc. Natl. Acad. Sci. U.S.A. 76:514-517.
	Eisenbarth, G.S., Walsh, F.S., and Nirenberg, M. 1979. Monoclonal antibody to plasma membrane antigen of neurons. Proc. Nat. Acad. Sci. U.S.A. 76:4913-4917.
	Noble, M., Murray, K., Stroobant, P., Waterfield, M.D., and Riddle, P. 1988. Platelet derived growth factor promotes the division and motility, and inhibits premature differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell. Nature 333:560-562.
	Raff, M.C. 1989. Glial cell diversification in the optic nerve. Science 243:1450-1455.
	Raff, M.C., Miller, R.H., and Noble, M.D. 1983. A glial progenitor that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 303:390-396.
	Raff, M.C., Lillien, L.E., Richardson, W.D., Burne, J.F., and Noble, M.D. 1988. Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture. Nature 333:526-565.
	Ranscht, B., Clapshaw, P.A., Price, J., Noble, M., and Seifert, W. 1982. Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside. Proc. Natl. Acad. Sci. U.S.A. 79:2709-2713.
Key References
	Raff et al., 1983. See above.
Seminal paper describing the isolation of O-2A progenitor cells from rat optic nerve, their differentiation, and the antigenic phenotype of the glial cells found in optic nerve cultures.
	Bögler et al., 1990. See above.
Paper showing that the combination of PDGF and bFGF inhibits the differentiation of O-2A progenitors indefinitely while promoting their division, which is exploited in the method provided here.