Glia-Induced Morphological Differentiation in Neuroblastoma Cells

Abstract

Glial cells release a factor into their culture medium that induces a high 4egree of morphological differentiation 'in neuroblastoma cells under normal growth conditions. This phenomenon is not correlated with a change; in intracellular adenosine 3': 5'-cyclic'monophosphate or in the rate of cell growth. Media from other cell lines tested induce less morphological differentiation or have no effect.Process formation by neuroblastoma cells in tissue culture has been termed morphological differentiation, and its importance as a model for neural differentiation has been pointed out by many authors (1-4). X-Irradiation (5), removal of serum (2), and addition of N6,02'-ibutyryl adenosine 3': 5'-cyclic monophosphate (But2cAMP) (3, 4) or 5-bromodeoxyuridine (BrdU) (6) can induce this process formation. All these treatments are unphysiological, even at low degree of morphological differentiation, and they all interfere with the growth rate of the cells. Considering the theoretical importance of cell to cell interactions in the central nervous system, we tried to influence process formation by using medium from glial cell cultures, We report that such a glial conditioned medium induces a high percentage of morphological differentiation under physiological conditions. MATERIALS AND METHODSNeuroblastoma' cells, clone NB-2a, derived from the C-1300 tumour cell line (7), and glial cells, clone C-6, derived from a N-nitrosomethylurea-induced rat glial tumour (8) To determine the percentage of cells undergoing morphological differentiation, 50,000 trypsinized neuroblastoma cells were plated per 60-mm dish. After 16 hr of incubation, the medium was replaced by the medium to be tested, and 48 hr later all cells with and without processes were counted in at least five randomly selected areas of each culture plate. Cells having processes longer than the diameter of the cell body were considered morphologically differentiated. A total of 300-400 cells were counted per dish. The values reported here represent the percentage of differentiated cells determined in three parallel cultures.For intracellular cAMP determination, the cells were quickly washed with Dulbecco's phosphate buffered saline (pH 7.2, 37°) then fixed with 1 ml of 0.05 N HCl. They were scraped with a rubber policeman and the suspension was heated for 10 min in boiling water. The samples were then neutralized with 0.1 N NaOH to pH 7.3 and treated with 50 Ml of 0.17 M BaSO4 and 50 Mu of 0.15 M Ba(OH)2. The solution was clarified with a glass-fiber filter and cAMP was determined in triplicate by the method of Gilman (14). For the early time points the cells of two or three plates were extracted in the same 1 ml of 0.05 N HCl. At each time point, three parallel cultures were washed, then solubilized in 0.5% sodium dodecyl sulfate for protein determinations (15). RESULTS AND DISCUSSIONThe induction of morphological differentiation of neuroblastoma cells by glial conditioned medium is illustrated by the micrographs in Fig. 1 (Table 1). Unlike those treatments, condi...

Morphological differentiation of neuroblastoma cells in medium supplemented with delipidated serum.

“…Thus, the effect Finally, the fact that serum-free glial-conditioned medium can antagonize the effect of oleic acid bears directly on studies in this laboratory on the possible biological relevance of the previously described glial factor (2,3). Our be carefully evaluated in tissue culture studies that attempt to link biochemical parameters to membrane properties.…”

“…The study of such a model, which in its most fundamental form can be considered as one of morphological differentiation, should also give direction to an understanding of the biochemical mechanisms underlying neuronal plasticity. We have previously shown that glial cells in culture release a macromolecular factor that can induce morphological differentiation of neuroblastoma cells without affecting the growth rate of these cells (2). It has also been demonstrated (3) that this glial factor is distinct from the well-documented nerve growth factor.…”

“…Mouse neuroblastoma cells, clone NB2A, and rat glioma cells, clone C-6, were grown as described (2) with the exception that, if not specifically stated otherwise, 35-mm dishes from Falcon or Corning were inoculated with 20,000 EDTAdislodged neuroblastoma cells per dish containing 2 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Colorado). After 16 hr, the medium was replaced by 2 ml of the medium to be tested.…”

Olfactory ensheathing cells: Historical perspective and therapeutic potential

“…Both OECs and astrocytes within the NFL of the olfactory bulb also express protease nexin-1 (PN-1; Reinhard et al, 1988;Scotti et al, 1994), whereas astrocytes elsewhere in the CNS do not express this enzyme. PN-1 has been demonstrated to function as a chemotropic factor for neurites in vitro (Monard et al, 1973;Guenther et al, 1985;Zurn et al, 1988) and could potentially be functioning in the same manner within the primary olfactory pathway.…”

In Vitro Astrocytic Differentiation From Embryoid Bodies of an Experimental Mouse Testicular Teratoma

“…In cultures older than 24 hours, a third cell tvpe ( Figure 12) was occasionally found in the periphery but never within the neuroepithelial cell populations. Its pale expansive cytoplasm had indistinct borders, abundant perinuclear filamentous material, and occasionallv, long delicate processes contacting adjacent cells.…”