Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7768-7772, November 1987 Neurobiology Release of autocrine growth factor by primary and immortalized Schwann cells (cell culture/laminin) SETH PORTER*t, Luis GLASER*t, AND RICHARD P. BUNGEt Departments of *Biological Chemistry and tAnatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110; and tDepartments of Biology and Biochemistry, University of Miami, Coral Gables, FL 33156 Communicated by Gerald D. Fischbach, July 20, 1987 ABSTRACT Schwann cells derived from neonatal rat proliferation. Because Schwann cells secrete laminin and sciatic nerve are quiescent in culture unless treated with specific form a basement membrane at an initial stage of neuronal mitogens. The use of glial growth factor (GGF) and forskolin ensheathment (21), it is important to establish the role of has been found to be an effective method for stimulating these components in the control of proliferation and differ- proliferation of Schwann cells on a poly(L-lysine) substratum entiation. while maintaining their ability to myelinate axons in vitro. We We report that repetitive passaging of Schwann cells find that repetitive passaging of Schwann cells with GGF and derived from neonatal rat can result in a population of forskolin results in the loss of normal growth control; the cells immortalized cells that, depending upon their duration in are able to proliferate without added mitogens. The immor- culture, display all or most ofthe functional characteristics of talized cells grow continuously in the absence of added growth a primary Schwann cell. It was found that both immortalized factor and in the presence or absence of serum yet continue to and primary Schwann cells secrete growth-promoting activ- express distinctive Schwann cell-surface antigens. The cells can ity. Whereas the mitogen(s) secreted by primary cells are associate with axons in culture, deposit a basal lamina, and almost completely dependent upon supplementation with ensheath axons, but they gradually lose their capacity to laminin or fibronectin, the immortalized cells secrete a myelinate axons. The immortalized cells release growth-pro- product(s) whose activity is enhanced but not dependent moting activity into their culture medium. The released activity upon exogenous laminin or fibronectin. We describe here is effective in stimulating proliferation of primary Schwann some ofthe characteristics ofthe immortalized Schwann cells cells that retain normal growth properties. Extracellular ma- and the mitogenic activity that they secrete. trix molecules (laminin and fibronectin) augment the response of primary Schwann cells to the secreted mitogen. Quiescent MATERIALS AND METHODS primary Schwann cells also secrete a growth factor into their culture medium, but its activity is detectable only in the Materials. One- to 3-day-old Sprague-Dawley-derived rats presence of added laminin or fibronectin. The results suggest were obtained from Chappel Breeders (St. Louis), tissue- that both normal and immortalized Schwann cells secrete an culture plasticware from Falcon, fetal bovine serum (FBS) autocrine growth factor. Response to the autocrine factor from Hazelton Research Products (Denver, PA), and tissue appears to entail a multicomponent mechanism. Unlike pri- culture media from the Media Center at Washington Univer- mary cells, immortalized Schwann cells have the capacity to sity School of Medicine. [methyl-3H]Thymidine (6.7 Ci/ secrete all of the necessary components and to respond to them mmol; 1 Ci = 37 GBq) was acquired from New England constitutively. Nuclear. Collagenase (type I), L-1-tosylamido 2-phenylethyl chloromethyl ketone-treated trypsin (TPCK-trypsin), and In the absence of neurons, rat Schwann cells in culture are rabbit complement were from Cooper Biomedicals (Malvern, quiescent, even in serum-containing medium (1-3). Contact PA). Poly(L-lysine) hydrobromide (Mr 30,000-70,000) was with neurons (1, 3, 4) or with axolemma preparations derived from Sigma. Anti-Thy-1.1 hybridoma cells were from the from cultured neurons (4-6), PC12 pheochromocytoma cells American Type Culture Collection. Laminin was from (7), or brain (6, 8, 9) will induce Schwann cells to enter the GIBCO and human plasma fibronectin was a generous gift cell cycle and undergo mitosis in vitro. In addition, a limited from J. McDonald (Washington University, St. Louis). array of soluble growth factors are active in stimulating Schwann Cell Culture. Rat sciatic nerve Schwann cells proliferation ofcultured Schwann cells. A protein designated were prepared and purified according to the methods of glial growth factor (GGF), isolated from brain and pituitary Brockes et al. (2). In brief, sciatic nerves were dissected from (10-12), is mitogenic for Schwann cells and astrocytes. A neonatal rats, dissociated with 0.1% collagenase and 0.25% second protein purified from brain, glial maturation factor trypsin in Leibovitz' L-15 medium, and plated on tissue- (13, 14), affects the morphology of Schwann cells and central culture plastic in Dulbecco's modified Eagle's medium glial cells and has weak mitogenic activity. Agents that (DMEM) plus 10% FBS. After 18-24 hr, the medium was increase intracellular cAMP are also mitogenic for Schwann changed to one containing 10 ,uM 1-,/-D-arabinofuranosylcy- cells (10, 15, 16). It is not known whether soluble polypeptide tosine. Three to 5 days later, the cells were suspended with growth factors are relevant to the contact-mediated mito- 0.05% trypsin and 0.02% EDTA in calcium- and magnesium- genic stimulation of Schwann cells by neurons, and the role free Hanks' balanced salts solution and treated for 30 min ofcAMP in neuronal stimulation of Schwann cells remains to with anti-Thy-1.1 hybridoma culture supernatant and then be established (7, 17). In addition, the extracellular matrix rabbit complement to remove contaminating fibroblasts. components laminin (18) and fibronectin (19), when added in Treatment and passaging of cells in medium containing GGF soluble or substrate-bound (20) form, stimulate Schwann cell and forskolin have been described (16). In brief, Schwann The publication costs of this article were defrayed in part by page charge Abbreviations: GGF, glial growth factor; DMEM, Dulbecco's mod- payment. This article must therefore be hereby marked "advertisement" ified Eagle's medium; FBS, fetal bovine serum; CM, conditioned in accordance with 18 U.S.C. §1734 solely to indicate this fact. medium (media). 7768 Downloaded by guest on October 2, 2021 Neurobiology: Porter et al. Proc. Natl. Acad. Sci. USA 84 (1987) 7769 cells isolated as described and plated on a poly(L-lysine) in which the incubation with anti-laminin was done in the substratum were treated continuously with a crude GGF presence or absence of excess purified laminin, and the preparation (final concentration 10 ,ug/ml) and 2 ILM degree of staining obtained in the presence of added laminin forskolin in DMEM with 10% FBS. Cells were passaged was subtracted from the value obtained in the absence of every 7-10 days. added laminin. The rabbit anti-laminin antiserum for these Conditioned Medium (CM). Immortalized Schwann cells experiments was prepared in our laboratory by C. Eldridge (1.17 cells) were plated at 8.5 x 104 per cm2 in poly(L-lysine)- and M. Ard, and the rabbit anti-fibronectin antiserum was treated plastic tissue-culture dishes in serum-free medium provided by J. McDonald. (N2; ref. 22) without nerve growth factor. CM was collected three times per week and stored frozen at -70'C. Cells were RESULTS passaged once a week. Assay of Schwann Cell Proliferation. Primary Schwann cells We showed previously (16) that primary Schwann cell that had been treated with arabinofuranosylcytosine and cultures can be effectively expanded by using a combination anti-Thy-1.1 but that had never been treated with mitogens of GGF and forskolin [an activator of adenylate cyclase (29)] were plated into poly(L-lysine)-coated 24-well culture dishes on a poly(L-lysine) substratum. Schwann cells treated in this at 50,000 cells per well in DMEM plus 10% FBS. After 24 hr, way for 2-3 months (6-9 passages) retain normal growth the medium was replaced with 0.2 ml of CM diluted with control. After GGF and forskolin are removed for 4 days, the DMEM and FBS to obtain the desired final concentration of cells return to quiescence in 10% FBS and will respond to CM protein and 10% FBS. Cells were treated with mitogen re-addition of mitogens with the same kinetics as the original for 72 hr, fresh mitogen and medium being added every 24 hr, cell population. Further, after continuous passage for up to 8 and [3H]thymidine (2 ttCi/ml) was included for the last 24 hr. months (24 passages), the cells still retain their ability to Medium was then aspirated, and cells were washed with differentiate and to myelinate sensory axons in vitro (16). medium and then treated for 30 min with 10% trichloroacetic Although the cells are still capable of differentiating, acid. Cells were washed again, solubilized in 0.3 M NaOH/ continuous treatment with GGF and forskolin for longer than 1% NaDodSO4, and prepared for scintillation counting (8). 4 months (12 passages) causes the cells to become indepen- Immunocytochemistry. Cells plated on poly(L-lysine)-treat- dent of exogenous mitogens. Unlike the original cell popu- ed glass coverslips were washed once with L-15 medium plus lation, they proliferate in the absence of GGF and forskolin. 10% horse serum, incubated for 45 min with antibody [rabbit Their growth is independent of serum; the rates of prolifer- anti-laminin serum from J. Sanes, Washington University; ation are comparable in serum-containing and serum-free rabbit Ran-1 antiserum (23) from K. Fields, Albert Einstein media (Fig. 1). The doubling time of 2 days is the same as in University; and mouse monoclonal antibody 217C (24, 25) cultures containing GGF plus forskolin or GGF plus cholera from J.