Release of angiotensin I-converting enzyme by endothelial cells in vitro

Bovine fetal aortic endothelial cells cultured in serum-containing medium accumulate angiotensin I-converting enzyme (ACE) activity and also release it into the culture medium. Following subcultivation of a confluent culture using trypsin-EDTA, cellular ACE activity falls 50% within 8 h, but no ACE activity is detected in the medium, suggesting intracellular loss of the enzyme activity. ACE activity reappears in both the cell lysate and culture medium after the culture becomes confluent. The rate of accumulation of ACE activity released into the medium is always greater than that for cellular activity. For example, 21 days following subcultivation 80-85% of the total culture activity is detected in the medium. Both cellular and medium-associated ACE decrease proportionately as the culture progresses through its in vitro lifespan.     

The effects of division rate-limiting amounts of fetal bovine serum on the proliferation and aging of cultured chick cells

Chick embryo fibroblasts serially propagated in media containing division ratelimiting amounts of fetal bovine serum underwent premature culture senescence as illustrated by accelerated declines in the number of cells incorporating ³H-thymidine, increased population doubling times, reduced cell densities at subcultivation, and reduced replicative life-spans compared to cells grown in medium containing non-rate-limiting amounts of serum. Low serum serially propagated “senescent” cultures returned to 10% serum containing medium had proliferative rates, incorporated ³H-thymidine, and attained saturation densities at confluency similar to younger cells. “Senescent” cells serially propagated in low serum and returned to 10% serum achieved life-spans similar to cells continuously grown in the presence of 10% serum. The results of these and other studies show that cells serially propagated in the presence of division rate-limiting amounts of fetal bovine serum, or at high inoculation densities, accumulate a substantial number of cells in the population during exponential growth conditions that are not senescent but are prevented from entering DNA synthesis becuase of mitogen limitations. Our results indicate that the amount of serum mitogen in the growth medium affects only the rate at which cells express their genetically predetermined replicative potential and not the replicative lifespan per se. These results are discussed in relation to the techniques that should be employed for studying cellular aging and the mechanism of senescent cell formation.     

Establishment and characterization of porcine aortic endothelial cell cultures with prolonged replicative lifespan by a non-enzymatic method

The aim of this work was the establishment and characterization of porcine aortic endothelial cell cultures with prolonged lifespan. Endothelial cells where isolated from porcine thoracic aorta and established in primary culture; after sub cultivation, the cells showed typical morphology of endothelial phenotype with cobblestone appearance and growth in monolayer; they were positive against anti-CD31 and anti-CD54 immunostaining and Ac-LDL-Dil uptake. The cells were able to migrate in culture and showed a normal growth curve. The phenotype of our in vitro model of endothelial cells was stable through subcultivation; so, it should be a valuable tool for diverse studies of the endothelial response against physiological and pathological stimulus, particularly it could be useful to study interactions of endothelium with pathogenic bacteria causing diseases in pigs, as they are difficult to study in vivo.     

Paraquat depresses the activity of angiotensin converting enzyme expressed by human umbilical vein endothelial cells in culture

The activity of angiotensin converting enzyme (ACE) in cell lysate of cultured human umbilical vein endothelial cells (HUVEC) after a 24-hour incubation with 10(-3) and 10(-4)M of paraquat (PQ) was decreased. However, LDH released into the culture medium of HUVEC during the 24-hour incubation with PQ was not increased. Many investigators show that the change in serum ACE activity reflects the impairment of vascular endothelial cells. We showed in this report that ACE was decreased even at an early stage of endothelial injury induced by PQ, when LDH release is not yet increased.     

Bovine endothelial cells transformed in vitro by benzo(a)pyrene

A cloned strain of bovine vascular endothelial cells with a finite in vitro lifespan was treated with benzo(a)pyrene (BP) after approximately 75% of its lifespan was completed. Untreated cultures of this strain senesced upon serial subcultivation and contained large, nondividing cells. In three out of seven trials, BP treatment produced transformed cells appeared in the cultures concomitant with the senescence of the parent cells. All transformed cell lines examined exhibited indefinite lifespans and altered karyotypes. Two of the lines retained most of the characteristics of normal endothelial cells, except that one became aneuploid and the other polyploid, Neither of these lines formed tumors when inoculated into nude mice. The remaining two lines retained mostly diploid kayotypes, but a high percentage of cells contained Robertsonian translocations. In one line cell volume was markedly reduced. In addition, these lines grew in multilayers, were anchorage independent, and proliferated in medium containing 0.5% serum. When 10⁷ cells of these lines were injected into nude mice, tumors appeared within 1 week and were identified as malignant hemangioendotheliomas of bovine origin.     

Enzymatic responses to radiation in cultured vascular endothelial and smooth muscle cells

Confluent monolayers of bovine aortic endothelial and smooth muscle cells were exposed to 0-5.0 Gy of 60Co gamma rays. From 0 to 72 hr after irradiation, the monolayer and culture medium were analyzed for cell (nuclei) number, DNA and protein content, the activities of angiotensin converting enzyme (ACE), lactate dehydrogenase (LDH), and superoxide dismutase (SOD), and LDH isoenzyme profile. Irradiated endothelial cells exhibited a time- and dose-dependent increase in cell detachment, decreased DNA and protein content and reduced ACE active per attached cell, increased LDH and SOD activities per microgram of DNA, and increased LDH activity in the culture medium. The latter was accompanied by a shift from LDH 1 to LDH 4 and 5. The release of LDH activity, observed after 0.5 Gy, was the most sensitive endothelial response, and occurred independent of or preceding cell detachment. Vascular smooth muscle cells contained two to three times more SOD activity than did endothelial cells and exhibited no significant responses to 5.0 Gy.     

Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts.

Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.     

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.     

Impact of culture conditions on the proliferative lifespan of human T cells in vitro

BACKGROUND: In human T cells, telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control the lifespan of T cells. To improve T-cell expansion it is of critical importance to understand the effects of culture parameters on telomerase activity and lifespan. METHODS: We investigated the influence of culture condition (FCS, human AB serum and autologous serum) and stimulation (PHA/feeder cells, anti-CD3/CD28 beads) on the lifespan, clonogenicity (number of positive wells), cell cycle, telomerase activity and telomere length of T cells in vitro. RESULTS: The proliferative lifespan of T cells expanded with PHA/feeder cells and autologous serum from different donors was doubled compared with stimulation with PHA/feeder cells and AB serum. No or only a small difference was found for T cells expanded with anti-CD3/CD28 beads and autologous or AB serum. The use of autologous serum also increased the clonogenicity to about three-fold compared with the use of AB serum or FCS, without any signs of differences in the fractions of cycling cells. Interestingly, T cells cultured with autologous serum exhibited a significantly higher telomerase activity at day 6 after stimulation and a reduced decline of telomerase activity compared with cultures with AB serum. DISCUSSION: The use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity and increases the telomerase activity of human T cells in vitro. This might be useful for applications where large numbers of specific T cells are required.     

Anchorage independent growth and plasminogen activator production by bovine endothelial cells

Endothelial cells obtained from the aortae of 1- to 2-d-old calves were cloned at high efficiency using fibrin-coated dishes. Primary cultures as well as clones derived from them produced high fibrinolytic activity when grown on 125I-fibrin-coated dishes which was 90% dependent upon the presence of plasminogen. High plasminogen-dependent proteolytic activity was also demonstrated in endothelial cell lysates and in the culture medium of the cells. The production and secretion of the plasminogen activator(s) were found to increase during the log phase of cell growth and to reach a maximum level at confluence. These endothelial cells exhibited morphological phenotypes comparable to those of transformed cells when grown in the presence of acid-treated fetal calf, dog, or human serum. Furthermore, they demonstrated anchorage independent growth, and large colonies were formed in semisolid media. Spontaneous neoplastic transformation of these cells was excluded by karyotypic analysis, lack of tumorigenicity in athymic nude mice, and limited lifespan in culture. Cell clones isolated from colonies grown in agarose demonstrated the same growth characteristics and proteolytic activity as before plating in agarose. High fibrinolytic activity, morphological changes in the appropriate serum, and growth in semisolid media may therefore be indicative of the migratory and/or invasive capacity of both nontransformed endothelial cells as well as tumor cells.     

Adrenergic enzymes in cultured mouse neuroblastoma: absence of detectable aromatic-L-amino-acid decarboxylase.

The relative activities of tyrosine hydroxylase, aromatic-l -amino-acid decarboxylase and dopamine beta-hydroxylase were established in a number of clones of neuroblastoma cells isolated from the uncloned mouse C-1300 tumor. One clone, NBD-2, was chosen for further analysis on the basis of its relatively high activities of tyrosine hydroxylase and dopamine beta-hydroxylase. The levels of these enzymes, and monoamine oxidase and catechol O-methyltransferase, were at least 20-80 fold lower in the neuroblastoma culture than in mouse superior cervical ganglion. More importantly, aromatic-l -amino-acid decarboxylase activity was not even detectable in any neuroblastoma clone examined. Based on the relative sensitivities of the tyrosine hydroxylase and aromatic-l -amino-acid decarboxylase assays and on the ratio of these two enzymes in the mouse ganglion, decarboxylase activity is more than 10 fold lower in the cultured cells than would be predicted on the basis of tyrosine hydroxylase activity. Dialysis and mixing studies with neuroblastoma extracts and partially purified aromatic-l -amino-acid decarboxylase did not reveal the presence of any endogenous inhibitors that could account for the low level of decarboxylase activity in the cultured cells. During growth of the neuroblastoma cells to confluency, only one enzyme, monoamine oxidase, exhibited an elevated specific activity on the basis of cell number. However, when based on the amount of protein, the specific activity of all measurable enzymes increased in culture-because cell protein decreased 5 fold during growth to confluency. These findings are discussed with respect to individual cell function.     

Density-dependent regulation of cell surface gamma-glutamyl transpeptidase in cultured glial cells.

A decline in cell surface gamma-glutamyl transpeptidase specific activity was previously observed to be concomitant with C6 glial cell proliferation. To elucidate the underlying factor(s) mediating gamma-glutamyl transpeptidase down-regulation, the effects of C6 cell density and culture conditions on cell surface transpeptidase activity levels were investigated. After 24 h of culture, the transpeptidase specific activities were inversely related to the initial plating densities. The lower-density cultures showed an induction within 24 h of plating. As the cultures proliferated, the specific transpeptidase activities declined to a common low level at post-confluency. The gamma-glutamyl transpeptidase down-regulation was unrelated to cell growth rate and was most pronounced during logarithmic proliferation. Induction and down-regulation of gamma-glutamyl transpeptidase activity at low cell densities were not a result of trypsinization. Supplementation of low-density cultures with conditioned medium, use of matrix-coated wells, or periodic replacement of growth media to prevent conditioning had minor effects on the decline of cell surface activity. Kinetic analysis showed that the Michaelis constants and the reaction mechanism were unaltered by cell density, indicating that down-regulation was not due to allosteric factors or an alteration in enzyme character. A reduction in the maximal velocity of cell surface transpeptidation at higher cell densities suggested that gamma-glutamyl transpeptidase down-regulation is related to the concentration of enzyme at the cell surface. Immunocytochemical localization of gamma-glutamyl transpeptidase demonstrated that gamma-glutamyl transpeptidase antigen levels decrease as C6 cell density increases. These results led us to propose that cell-cell contact stimulates the disappearance of gamma-glutamyl transpeptidase from the surface of cultured C6 glial cells.     

Phospholipase C and D in the commitment to survival or death in the early lag phase of tetrahymena cultures

We made three kinds of experiments in order to elucidate aspects of physiological mechanisms involved in a series of specific events leading to either cell death or survival in the lag phase of culture growth. We studied the fate of newly inoculated Tetrahymena cells in small droplets at 'high' (more than 1000 cells ml(-1)) and 'low' cell densities (less than 600 cells ml(-1)) in a nutrionally complete, synthetic nutrient medium. Confirming previous results we found that the cells in high-density cultures multiplied to final densities around 500,000 cells ml(-1) and that cells in low-density cultures died before multiplying. The appearance of the cells was recorded with a video camera at 20 frames per second for 6 h or until they died. The results indicated that the death process took place within milliseconds. We also studied the effects of U 73122, an inhibitor of the phosphatidylinositol-specific phospholipase C, on cell survival at low densities. At low inhibitor concentrations low-density cells were rescued from dying. At high inhibitor concentrations all cells died, and phosphatidylinositol - but not phosphatidylserine and phosphatidylcholine - saved them. The results indicate that the paths leading to either cell death or to cell proliferation separate within the first few minutes after subcultivation into a new medium, since the first cells in each culture died within 4-30 min after inoculation. Our results also indicate that some PLC activity was required for stimulation of phospholipase D, and that cell death during the early lag phase is caused by a shortage in phosphatidylinositol before the phospholipase D activity is upregulated. These experiments are shedding light on the lethal consequences of a cellular depletion of the important signalling compound phosphatidylinositol in an in vivo system, and may help to elucidate mechanisms behind the century-old fact that eukaryote cells die when inoculated at too low a cell density to survive.     

Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Summary Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.     

Enhanced proliferation of WI38 cells in the presence of glucocorticoid-conditioned medium

The addition of hydrocortisone (HC) or dexamethasone (DEX) to WI38 cells at subcultivation is known to result in increased saturation densities (20-40%). We report that maximal increases in saturation density are, however, only observed if HC is added to the culture shortly after subcultivation. We have found that the proliferative response of WI38 cells to glucocorticoids is mediated by a secondary stimulatory factor(s) present in medium-conditioned by cells in the presence of the hormone. Control cultures refed with medium conditioned in the presence of HC for the first 24 h after seeding (24 h HC-CM) achieve saturation densities 20-40% higher than cultures refed with either medium conditioned in the absence of the hormone (24 h CM) or 24 h CM supplemented with fresh HC. Furthermore, WI38 cultures remain responsive to the stimulatory activity present in 24 h HC-CM throughout the growth cycle. The stimulatory effects of 24 h HC-CM are enhanced by repeatedly refeeding cultures; WI38 cells refed every 2 days with 24 h HC-CM demonstrate an extended period of logarithmic growth and achieve densities 2-3 times higher than controls. A preliminary characterization of this activity shows it to be of low molecular weight (MW) (dialyzable using 12 000 MW cut-off tubing) and heat-stable (75 degrees C).     

Culture of human blood vessel endothelial cells--a simple and inexpensive culture method

The purpose of this review is to introduce a simple and inexpensive culture method for human umbilical blood vessel endothelial cells. The medium used is MCDB-104 supplemented with 10% fetal bovine serum, 70 ng/ml endothelial cell growth factor from new-born bovine brains, 10 ng/ml murine epidermal growth factor, and 100 micrograms/ml heparin. The culture dishes are coated with gelatin. Under these conditions, endothelial cells from human vessels were grown with doubling times of 18-22 hrs and reached saturation densities of 8-12 x 10(4) cells/cm2. To determine the lifespan of the endothelial cells, the cells were serially subcultivated weekly at an inoculum size of 1,000 cells/cm2. Human endothelial cells from umbilical vein and artery were grown for 21 to 37 passages with 55 to 125 population doublings. This culture method seems to be useful for studying cell proliferation and functions of human endothelial cells.     

Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control

Markers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII-related antigen. Treatment of IVEC cells with Interleukin-1β (IL-1 β) at 10 U.ml^?1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 μM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT-PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium. © 1993 Wiley-Liss, Inc.     

Enzyme activity during the growth and aging of human cells in vitro

Acid phosphatase, alkaline phosphatase, and lactic dehydrogenase activities have been compared in normal human diploid cell strains and in SV₄₀-transformed heteroploid cell lines derived from them. A higher level of acid phosphatase activity was observed in diploid cultures derived from adult lung than in cultures derived from fetal lung of similar passage levels. The alkaline phosphatase activity of normal diploid fibroblasts was significantly higher than that of SV₄₀-transformed cell lines derived from them. Generally, the lactic dehydrogenase activities of all these cell cultures were similar. Human diploid cells in culture “age,” in the sense that their ability to proliferate decreases with time during serial subcultivation. Evaluation of the activities of these three enzymes during the “aging” process showed that, although alkaline phosphatase and lactic dehydrogenase activities were similar in “young” and “senescent” cells, acid phosphatase showed a small but significant increase in the senescent cells.     

The effect of population density on phenylalanine hydroxylase activity in rat-hepatoma cells in culture

The expression of phenylalanine hydroxylase activity in rat-hepatoma cells in culture (line H4-II-E-C3) is a function of culture density: under normal growth conditions in the presence or in the absence of exogenously added hydrocortisone, the levels of this enzyme are low in subconfluent cell populations, but increase steeply as cultures attain confluency. This phenomenon (i) is not an artifact of the subcultivation process and (ii) is not produced by some alteration in the growth medium effected by high-density cultures. The levels of phenylalanine hydroxylase in high-density cultures of H4-II-E-C3 cells in the presence of serum plus added hydrocortisone are at least 80% of those seen in adult-rat liver. It is concluded that this density-associated phenomenon is the result of an intrinsic property of H4-II-E-C3 cells and possibly constitutes a form of epigenetic control governing the sensitivity of these cells to stimulation by serum or by serum plus hydrocortisone.     

Induction of an oligodendroglial enzyme in C-6 glioma cells maintained at high density or in serum-free medium.

The relationship between cell density and the activity of 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP), an enzyme believed to be specific to oligodendroglial cells and myelin in the brain, has been studied in cultured C-6 glioma cells. Over a 12-day period, the specific activity of CNP underwent a 4-fold increase in conjunction with an increase in the cell density (total protein/flask) and a decline in the growth rate of the cultures. In contrast, the specific activity of Na+,K+-ATPase was not influenced by cell density. Experiments with cultures seeded at different initial densities indicated that the increase in CNP activity coincided with the attainment of a specific cell density rather than with the length of time that the cells were maintained in culture. Arrest of cell proliferation in non-confluent C-6 cells by means of thymidine blockade was not sufficient to cause an increase in the activity of CNP; however, removal of serum from the culture medium resulted in a 3-fold induction of the enzyme in the absence of a high degree of cell contact. The induction of CNP in cells maintained in serum-free medium paralleled the development of a series of distinct morphological changes reminiscent of glial differentiation, which occurred within 48 hours after removal of the serum. Inhibition of protein synthesis by cycloheximide prevented the induction of CNP in serum-free cultures. The demonstration that an enhancement of an oligodendroglial characteristic in C-6 glioma cells can be obtained by growing the cells to high density or by removing serum from the medium, provides further support for the suggestion that these cells may be analogous to the glial stem cells present in the developing brain.