Growth Requirements in Vitro of Oligodendrocyte Cell Lines
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 1955-1959, March 1986 Neurobiology Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes (CO-13-7 cell line/insulin/transferrin/fibronectin/polylysine) JANE E. BOTTENSTEIN Marine Biomedical Institute and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550-2772 Communicated by Gordon H. Sato, November 15, 1985 ABSTRACT I have defined the basic requirements for the serum-supplemented medium these oligodendrocytes do not proliferation ofcell lines expressing oligodendrocyte properties divide in response to several known mitogens: fibroblast and for the survival of galactocerebroside-positive oligoden- growth factor, pituitary extract, myelin basic protein, epi- drocytes derived from neonatal rat brains. Conventional se- dermal growth factor, or insulin. However, autoradiographic rum-containing medium can be replaced by 01 medium, a and electron microscopic studies in vivo suggest that (i) the chemically defined medium supplemented with insulin, trans- percentage of mature oligodendrocytes should be greater and ferrin, sodium selenite, and biotin. Thyroid hormone is not (it) oligodendroblasts, even myelinating oligodendrocytes in required. When cells are plated directly into 01 medium, the some cases, are capable of further proliferation postnatally substratum has to be modified by precoating with polylysine (8-10) and in adult rodents after trauma (11). and adding fibronectin to the medium prior to the cells. Both Conventional culture methods appear to be suboptimal for cell lines and brain cells can be subcultured numerous times in the survival and proliferation of oligodendrocytes. Further- 01 medium without initial culture in serum-containing medi- more, the complexity, variability, and largely undefined um. Brain cultures can be maintained in 01 medium for several nature ofthe biological fluid supplements preclude definition months and contain a significantly higher percentage of mature of the molecular growth requirements of oligodendrocytes. oligodendrocytes, a lower number of astrocytes, and no fibro- Thus, I have attempted to define the growth requirements of blasts as compared to cells maintained in serum-containing oligodendrocytes by using serum-free culture methods. The medium. strategy of defining the requirements for growth of continu- ous cell lines expressing the phenotype of interest and Oligodendrocytes in the central nervous system (CNS) and applying this knowledge to culture of their normal cell Schwann cells in the peripheral nervous system are respon- counterparts has proven useful in the past for neurons and sible for forming multilayered myelin sheaths around astrocytes (12-19). Although these cell lines are of tumor neuronal axons, which increases their conduction velocity origin, they share some of the growth requirements of their and consequently the efficiency of information transfer. It normal cell counterparts. In addition, tumor cells often has been reported that some cultured oligodendrocytes are produce growth factors that act in an autocrine fashion to directly depolarized by y-aminobutyric acid (1) and exhibit stimulate their own growth (20). After determining the growth diaminobutyric acid-sensitive y-aminobutyric acid uptake requirements of the appropriate cell line, I then evaluate (2), suggesting an additional role in modulating neuronal whether normal cell counterparts have any additional re- function. Although vertebrate cell culture studies have yield- quirements. ed important information about Schwann cell division, mat- I now describe the growth requirements for oligodendro- uration, and interaction with neurons, much less is known cytes. I used the recently established CO-13-7 clonal cell line about oligodendrocytes. Clearly, an understanding of the for the initial phase of this investigation. It is a somatic cell molecular regulation of survival, growth, and maturation of hybrid of a normal calfbrain oligodendrocyte fused with a C6 oligodendrocytes is important for the analysis of develop- glial tumor cell (21). Unlike the normal oligodendrocyte mental and pathological processes. parent, the hybrid cells proliferate continuously as does the Oligodendrocytes from late-term embryonic, neonatal, and tumor cell parent. The C6 cell line expresses both astrocyte- adult CNS of several mammals have been maintained in vitro specific (glial fibrillary acidic protein) and oligodendrocyte- by using conventional culture methods (3). These consist of specific [2',3'-cyclic nucleotide 3'-phosphodiesterase (EC a synthetic culture medium supplemented with undefined 3.1.4.37), glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), biological fluids, such as serum, plasma, and/or embryo and the induction of glycerol-3-phosphate dehydrogenase by extract, and culture surfaces unmodified or coated with glucocorticoids] properties (22). In addition to the oligoden- collagen or polylysine. Most data reported describe dissoci- drocyte-specific markers present in C6 cells, CO-13-7 cells ated cultures of mixed cellular phenotypes, although there also contain galactocerebroside (GalCer; galactosylceram- are reports of ide), sulfatide [galactosyl(3-0-sulfate)-ceramide], and myelin enrichment for oligodendrocytes (3). In neo- basic protein (21). Thus, CO-13-7 cells exhibit a broad natal CNS cultures, although neurons are present in the initial spectrum and substantial levels ofexpression ofthe six major population of cells, they disappear within 1 week of in vitro biochemical properties of oligodendrocytes. The latter phase culture leaving a bed layer of phase-contrast gray cells of this investigation used dissociated cultures of neonatal rat (astrocytes and connective tissue cells) with <2-5% phase- brain. contrast dark, process-bearing cells (oligodendrocytes) on This study shows that sustained division of cell lines top (4-6). Thereafter, mature oligodendrocytes do not in- expressing oligodendrocyte properties and long-term surviv- crease in number and rarely if ever incorporate [3H]thy- al of neonatal rat brain oligodendrocytes is possible using a midine, suggesting that little or no cell division occurs (7). In chemically defined medium (CDM) and a permissive sub- stratum. A significant increase in the percentage of mature The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" I Abbreviations: CDM, chemically defined medium; CNS, central in accordance with 18 U.S.C. §1734 solely to indicate this fact. nervous system; FCS, fetal calf serum; GalCer, galactocerebroside. 1955 Downloaded by guest on September 28, 2021 1956 Neurobiology: Bottenstein Proc. Natl. Acad. Sci. USA 83 (1986) oligodendrocytes, a decrease in astrocytes, and no fibro- DMEM]. This is replaced with rabbit anti-GalCer (diluted blasts are obtained in 01 medium compared to serum- 1:50 with solution A) for 30 min. After washing with PBS, containing medium. goat anti-rabbit IgG coupled to rhodamine (diluted 1:50 with solution A) is added for 30 min. After washing again, cells are fixed with cold 5% (vol/vol) acetic acid/95% (vol/vol) EtOH MATERIALS AND METHODS for 10 min at -20°C. After extensive washing to regain pH Continuous Cell Lines. CO-13-7 and ROC-1 hybrid cell lines 7.3, cells are mounted in 30%o (vol/vol) glycerol in PBS, were obtained from F. A. McMorris (Wistar Institute of covered with a glass coverslip, and sealed with nail varnish. Anatomy and Biology, Philadelphia); 3T3 Swiss mouse cell Cells are examined with a Nikon fluorescence microscope line was provided by D. Carney of this institution. Stock with rhodamine filters, epi-illumination, and phase contrast cultures were maintained in Dulbecco's modified Eagle's optics. medium (DMEM) supplemented with 5% (vol/vol) fetal calf Materials. DMEM (430-2100), Ham's F12 medium serum (FCS); 5% (vol/vol) calf serum; 1.2 g of NaHCO3 per (430-1700), penicillin/streptomycin/neomycin (600-5640), liter; and 15 mM Hepes (pH 7.3). Medium was changed three and gentamycin (600-5710) were from GIBCO; fetal calf and times per week. Cells were grown in tissue culture flasks calf sera, HBSS (9222 and 9230), trypsin (9336), and EDTA (Falcon; 25 cm2) in 3-4 ml of medium in a humid atmosphere (9314) were from Irvine Scientific; biotin was from of 95% air/5% CO2 at 370C and were subcultured before Calbiochem-Behring; spectrographically pure sodium confluence with 0.05% trypsin/0.5 mM EDTA in phosphate- selenite was from Johnson Matthey (London); Hepes buffered saline (PBS; pH 7.0) without Ca2' or Mg2+. After (H-3375), NaN3 (S-2002), soybean trypsin inhibitor (T-9003- detachment, cells were enumerated with a Coulter counter. 1S), poly(D-lysine) (P-7886; Mr 30,000-70,000), human trans- Dissociated Neonatal Rat Brain Cultures. Sprague-Dawley ferrin (T-2242), bovine insulin (1-5500), and other materials rat littermates (1-3 days postnatal) were dipped in 70%o were from Sigma. Rabbit anti-GalCer (24) was generously ethanol and then decapitated. Whole brains were removed provided by K. Fields (Albert Einstein College of Medicine, and collected in a dish containing Ham's F12 medium with New York), and mouse monoclonal anti-Thy 1.1 (25) by R. 10% (vol/vol) FCS and antibiotics (50 jug ofpenicillin/ml, 50 Pruss (National Institute of Mental Health, Bethesda, MD); ,ug of streptomycin/ml, 10 ,ug of neomycin/ml and 50 ,ug of mouse monoclonal anti-glial fibrillary acid protein was pur- gentamycin/ml).