Primary suspension culture of calf anterior pituitary cells on a microcarrier surface

In order to achieve a steady-state primary culture system for mammalian cells, with the potential to eventually correlate and control cell function and growth, a serious evaluation of various suspension systems was made. Calf anterior pituitary cells were employed as a differentiated cell type and successfully cultivated in a microcarrier suspension culture system. DEAE-Sephadex was demonstrated to be a satisfactory type of microcarrier. The cells readily attached to the bead and, after a short lag period, they actively proliferated on the bead surface to yield growth of a predominantly epithelial cell type. Under specific conditions the microcarrier supported primary cell growth up to levels of 2 × 10⁶ cells per ml. High bead concentrations inhibited cell growth. The inhibition could be overcome by using proportionately higher cell inoculum so that a concentrated culture with 5 × 10⁶ cells per ml was achieved. The inhibitory effect of high bead concentration was found to be due to the absorption of serum protein and certain growth enhancing factors. The fact that the growth enhancing factors were released from cells during the period of trypsinization and were both thermostable and nondialyzable, seems to suggest one approach to a dialysis culture system. In addition, relatively trauma-free primary cell cultures can be achieved by using explant culture without prior trypsinization. In microcarrier suspensions direct growth of primary rat mammary tumor cells was also demonstrated.     

Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

Influence of Viable Cells on the Resuscitation of Dormant Cells in Micrococcus luteus Cultures Held in an Extended Stationary Phase: the Population Effect

A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). In the present work, such cultures were studied by both flow cytometry and conventional microbiological methods and were found to contain various numbers of viable cells. Pretreatment of such cultures with penicillin G, and subsequent dilution, was used to vary this number. When the initial number of colony-forming cells per 30-ml flask was approximately nine (±five) or more, resuscitation of 10 to 40% of the cells, and thus culture growth, was observed. The lag period before the appearance of a population of cells showing significant accumulation of the fluorescent dye rhodamine 123 (i.e., of cells with measurable membrane energization) decreased from 70 to 27 h when the number of viable cells was increased from 30 to 10⁵ per flask, while the lag period before an observable increase in the number of colony-forming cells occurred was almost constant (at some 20 h). Provided there were more than nine (±five) initially viable cells per flask, the number of initially viable cells did not affect the final percentage of resuscitable cells in the culture. The lag period could be ascribed in part to the time taken to restore the membrane permeability barrier of starved cells during resuscitation, as revealed by flow cytometric assessment of the uptake of the normally membrane-impermeant fluorescent DNA stain PO-PRO-3 {4-[3-methyl-2, 3-dihydro-(benzo-1, 3-oxazole)-2-methylidene]-1-(3′-trimethylammonium propyl)-pyridinium diiodide}. Although cell populations which contained fewer than nine ±five viable cells per flask failed to grow, 4 to 20% of the cells (of 1.2 X 10⁶) were able to accumulate rhodamine 123 after 80 to 100 h of incubation, showing the ability of a significant number of the cells in the population at least to display “metabolic resuscitation.” Resuscitation and cell growth under such conditions were favored by the use of a 1:1 mixture of fresh lactate medium and supernatant from late-logarithmic-phase M. luteus cultures as the resuscitation medium. We conclude that the presence of a small fraction of viable cells at the onset of resuscitation facilitates the recovery of the majority of the remaining (dormant) cells. The cell density dependence of the kinetics, or population effect, suggests that this recovery is due to the excretion of some factor(s) which promoted the transition of cells from a state in which they are incapable of growth and division to one in which they are capable of colony formation.     

Proliferating cell nuclear antigen (PCNA) is expressed in activated rat skeletal muscle satellite cells

Skeletal muscle satellite cells from uninjured muscle of adult animals are generally found to be in a quiescent state, and when cultured, they remain quiescent in vitro for a period of time which is directly related to the age of the donor animal. A technique for studying the activation of satellite cells in primary cultures has been developed and employs proliferating cell nuclear antigen (PCNA) as a marker for entrance into the S phase of the cell cycle. PCNA is a protein involved in DNA replication and is maximally expressed in S phase of the cell cycle. We monitored PCNA expression in satellite cells isolated from young (3 week) and adult (9 month) rats, and our results indicate that satellite cells begin to accumulate PCNA prior to changes in cell number in both age groups. Using ELISA techniques, we demonstrated that addition of an extract of crushed muscle (CME) activated satellite cells and significantly reduced the length of the lag phase in cells from both age groups. Addition of bFGF shortened the lag phase of PCNA synthesis in satellite cells from 3-week-old rats but had no effect on the kinetics of PCNA expression in cells from 9-month-old rats. Based on our experiments, PCNA expression can be used as a marker to follow the entry of satellite cells into the cell cycle in primary mass cultures. © 1993 Wiley-Liss, Inc.     

Three-dimensional in vitro cell biology models of ovarian and endometrial cancer

Objectives:  This study aims to establish three-dimensional (3D) cell culture models of human ovarian and endometrial cancers and to compare biological and morphological characteristics of these models with those of two-dimensional (2D) models of the same cell lines and the primary tumours.
Methods:  3D models of ovarian and endometrial cancer cell cultures were established using a Rotary Cell Culture System. Immunohistochemical profiling and differential proteomics were used to characterize biological characteristics of multicellular spheroids (MCS) formed from these cultures. These were compared to characteristics of the same cells established in 2D and of the primary tumours from which the cell lines were derived.
Results:  MCSs from 3D cell cultures appeared histologically similar to the primary tumours. Immunohistochemical profiling of multiple markers, including CA125, BCL2 and p53, showed that patterns of protein expression in MCSs resemble those of the primary tumours. Proteomic profiling identified several differentially expressed protein markers between 2D and 3D cultures. These included prohibitin, which was down-regulated in 3D cultures suggesting cells proliferate less compared to 2D cultures; and VDAC1 and annexin 4, which were up-regulated in 3D cultures suggesting greater levels of apoptosis in 3D compared to 2D models.
Conclusion:  Establishing 3D models of cancer cell lines is likely to be of value for studying the molecular and biological mechanisms of ovarian/endometrial tumour progression and for testing novel molecular targets for cancer therapy.     

In vitro studies of the rabbit immune system: I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits

The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro. This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.     

Role of different lymphocyte subsets in human anti-viral T cell cultures

We have systematically studied uncloned human cell lines derived from anti-influenza A virus or anti-Epstein-Barr virus (EBV) bulk cultures, or from cultures highly enriched for CD4+ or CD8+ lymphocytes. The most noteworthy results are the following: (1) Anti-viral bulk cultures consisted of more than 90% of CD8+ cells in all cases. In contrast, anti-HLA cell lines are composed of approximately 50% CD8+ and 50% CD4+ cells. All of the CD8+ and CD4+ cells present in the culture were also 4B4+/2H4-. (2) In anti-viral bulk cultures, the cytolytic activity was restricted by HLA class I molecules and almost exclusively through a single HLA class I molecule. (3) Positively or negatively selected CD8+ lines showed the same restriction pattern. They grew less efficiently than bulk cultures but could be maintained in the absence of CD4+ cells. The CD4+ cells were however necessary at the beginning of the culture for the development of cytolytic anti-influenza virus CD8+ cells, whereas they were not required for the development of cytolytic anti-EBV CD8+ cells. (4) The CD4+ cell lines grew more actively than bulk cultures. A cytolytic activity for virus-infected cells was constantly detected in these culture from the third passage onward and it was always restricted by HLA class II molecules. This activity was maintained throughout the culture period. However, class II-restricted cytolytic cells were not detected during primary or secondary responses in vitro.     

Shear sensitivity of hybridoma cells in batch, fed-batch, and continuous cultures.

Previously, we observed that CRL-8018 hybridoma cells were more sensitive to well-defined viscometric shear during the lag and stationary phases than during the exponential phase of batch cultures. Some potential hypotheses for explaining the increase in shear sensitivity are (1) nutrient limitations that result in a decrease in production of specific cellular components responsible for the mechanical strength of the cell, (2) nutrient limitations that lead to synchronization of the culture in a cell cycle phase that is more sensitive to shear, or (3) a link between cell growth and shear sensitivity, such that slowly growing cells are more sensitive to shear. Here, the duration of the exponential phase was increased with use of fed-batch, and the effect on shear sensitivity of the cultures was measured with a viscometric technique. Extension of exponential growth resulted in an increased period during which the cells were insensitive to shear. Additionally, the shear sensitivity of the cells was constant over a wide range of growth rates and metabolic yields in chemostat cultures. These observations suggest that as long as the cells are actively (exponentially) growing, their shear sensitivity does not depend on the growth rate or metabolic state of the cell as expressed by metabolic yields. Thus, hypothesis 3 above can be dismissed.     

Expression of inducible nitric oxide synthase in human umbilical vein endothelial cells during primary culture

The adaptive response of endothelial cells to stress may lead to the upregulation of nitric oxide (NO) production. Herein, we report inducible nitric oxide synthase (iNOS) induction in primary cultures of human umbilical vein endothelial cells (HUVEC). The enzyme expression was earlier observed in 12-h cultures, reaching maximal levels after 3 days and decreasing when cells become confluent. The time course of NO production by HUVEC paralleled iNOS expression during the whole culture period, indicating that enzyme was functionally active. Conversely, iNOS induction could not be further detected in HUVEC subcultures passed once from cells presenting maximal levels of iNOS expression in the primary culture. Induction of iNOS in HUVEC was not related to lipopolysaccharide contamination, since the enzyme expression was not affected in the presence of polymyxin B added to primary cultures. Further analysis showed that aminoguanidine, a specific iNOS inhibitor, did not affect cell proliferation, suggesting that the NO produced by HUVEC may not be directly related to cell growth. Platelet endothelial cell adhesion molecule-1 expression was upregulated during cell confluence, in contrast to the decrease of iNOS expression and activity. The data suggest that iNOS expression may be a molecular mechanism mediating the adaptive response of endothelial cells to culture environment.     

The relation between protein synthesis and lipide accumulation in L strain cells and Ehrlich ascites cells

It has long been known that fat accumulates in old injured cells both in tissue culture and in many mammalian disease states. The use of L cells grown in suspension tissue culture permitted the opportunity to study conditions in which lipide accumulation could be retarded or accelerated. These cultures exhibit a three-phase growth curve which is similar to that previously found with bacteria and consists of a lag period, logarithmic growth period, and stationary period. Daily aliquots were removed from cultures going through these phases and protein and cholesterol content correlated with cell division. It was found that L cells gradually accumulated lipide in the cell concurrent with retardation of cell division and protein synthesis. Conversely old lipide-laden cells, placed in fresh media and encouraged to active division with net protein synthesis progressed from a high to a low lipide/cell ratio over a period of 2 to 4 days. An amino acid analogue p-fluorophenylalanine and a mitotic inhibitor, colchicine, also markedly increased the lipide/cell ratio. Similar results were found in in vitro experiments with Ehrlich ascites cells.     

Fine needle aspiration as a tool to establish primary human breast cancer cultures in vitro

OBJECTIVE: To verify the potential role of fine needle aspiration (FNA) cytology in obtaining malignant cells from primary breast cancer for establishment of a primary breast cancer cell line. STUDY DESIGN: In four patients with primary breast cancer subjected to FNA for diagnostic purposes, we attempted to establish primary cultures. We successfully obtained one primary cell line, originating in micropapillary invasive breast carcinoma. FNA material obtained under sterile conditions was centrifuged, and the cell pellet was washed with Dulbecco Modified Medium. The resulting suspension was seeded in 25-cm2 tissue culture flasks. The flasks were maintained with released caps in a 37 degrees C incubator with a humidified atmosphere of 5% CO2 in air. After one week, cells attached to the bottom of the flasks and began proliferating. When a culture became confluent, the cells were treated with 0.05% trypsin/0.02% EDTA in a PBS solution and subcultured. The flasks were observed daily with an inverted microscope, and culture passages were performed weekly. RESULTS: The cell line obtained was named I2FPRW and exhibited morphologic and immunohistochemical features of epithelial cells of mammary origin. The cells were positive for cytokeratins (AE1/AE3 and CK 7), EMA and c-erbB-2. At this writing, this cell line was in the 15th passage of subculturing in the flasks with 10% FBS. CONCLUSION: In the present study we demonstrated that is possible to establish a breast cancer cell line from material obtained by FNA cytology. FNA seems to be a valuable method of obtaining malignant cells from breast cancer able to grow free of fibroblasts in cell cultures.     

Proliferation kinetics of rabbit articular chondrocytes in primary culture and at the first passage

The in-vitro proliferation kinetics of young rabbit articular chondrocytes were compared in primary culture and at the first passage. The growth curves labelling and mitotic indices, percentage labelled mitosis (PLM) curves and DNA content distributions by flow-microfluorometric analysis during a 7-day growth period were determined in both cases. The length of the cell cycle and the doubling time calculated from the exponential part of the growth curve were quite similar: Tc = 19 hr and Td = 20 hr for the primary culture, Tc = 17 X 3 hr and Td = 20 hr for the first passage. However, the growth curve and the DNA distribution during the 7-day period showed some differences. The duration of the lag period studied by the growth curve was longer in the primary culture than at the first passage. This phenomenon was also observed using the FCM analysis. The growth fraction determination on the second day of culture was in accordance with the lower proliferation capacity of the cells in primary culture. These data suggest that it would be better to study growth kinetics and drug modifications in articular chondrocytes at the first passage than in primary culture.     

Maintenance of adult hamster pancreas cells on fibroblastic cells

Summary The maintenance of primary cultures of adult hamster pancreatic cells on layers of irradiated C3H/10T1/2 cells was studied. Various types of pancreatic cells, acinar, islet and ductular cells could be identified in the cultures by light and electron microscopy. Morphologically the various pancreatic cells retained many differentiated characteristics of their respective in vivo cell types. Insulin production was maintained at near Day 1 levels for the 16 d in culture for which it was measured. Colonies of epithelial cells continued to grow during a 20 d culture period. It is believed that this procedure for maintaining functional and growing pancreas cells in culture may be a useful in vitro model for studying the initiation of pancreatic carcinogenesis. Supported by Grant R01 CA 20022 and Contract N01 CP33278 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland.     

Chlorophyll synthesis in chlorella: regulation by degree of light limitation of growth

The degree of light limitation of growth is the primary controlling factor of chlorophyll synthesis during photoautotrophic growth of Chlorella. The chlorophyll content of the cells increases when light is limiting for growth as occurs in dense cultures, or in cultures under low incident light, or when the light is used less efficiently through partial inhibition of photosynthesis by 3-(p-chlorophenyl)-1, 1-dimethylurea. The chlorophyll content decreases when light is not limiting for growth, as occurs in cells in high light intensity and in dilute suspensions. The initial lag in rate of chlorophyll synthesis in a freshly inoculated culture can be attributed to light at first not being growth limiting, and then becoming growth limiting as the cell suspension becomes denser. Continuous culture experiments support the above conclusions by showing that under steady state conditions the chlorophyll content is inversely related to the relative available light.     

Cellular microenvironment influences the ability of mammary epithelia to undergo cell cycle

The use of cell culture models is a principal and fundamental technology used in understanding how mammalian cells work. However, for some cell types such as mammary epithelia, the lines selected for extended culture are often transformed or have chromosomal abnormalities, while primary cultures have such a curtailed lifespan that their use is restricted. For example, mammary luminal epithelial cells (MECs) are used to study mechanisms of breast cancer, but the proliferation of primary cell cultures is highly limited. Here we describe the establishment of a new culture system to allow extended analysis of cultures of primary mouse MECs. In 2D monolayer culture, primary MECs showed a burst of proliferation 2-3 days post isolation, after which cell cycle decreased substantially. Addition of mammary epithelial growth factors, such as Epidermal Growth Factor, Fibroblast Growth Factor-2, Hepatocyte Growth Factor, and Receptor Activator for Nuclear Factor κB Ligand, or extracellular matrix proteins did not maintain their proliferation potential, neither did replating the cells to increase the mitogenic response. However, culturing MECs directly after tissue extraction in a 3D microenvironment consisting of basement membrane proteins, extended the time in culture in which the cells could proliferate. Our data reveal that the cellular microenvironment has profound effects on the proliferative properties of the mammary epithelia and is dominant over growth factors. Moreover, manipulating the cellular environment using this novel method can maintain the proliferative potential of primary MECs, thus enabling cell cycle to be studied as an endpoint after gene transfer or gene deletion experiments.     

The Relation between Protein Synthesis and Lipide Accumulation in L Strain Cells and Ehrlich Ascites Cells

It has long been known that fat accumulates in old injured cells both in tissue culture and in many mammalian disease states. The use of L cells grown in suspension tissue culture permitted the opportunity to study conditions in which lipide accumulation could be retarded or accelerated. These cultures exhibit a three-phase growth curve which is similar to that previously found with bacteria and consists of a lag period, logarithmic growth period, and stationary period. Daily aliquots were removed from cultures going through these phases and protein and cholesterol content correlated with cell division. It was found that L cells gradually accumulated lipide in the cell concurrent with retardation of cell division and protein synthesis. Conversely old lipide-laden cells, placed in fresh media and encouraged to active division with net protein synthesis progressed from a high to a low lipide/cell ratio over a period of 2 to 4 days. An amino acid analogue p-fluorophenylalanine and a mitotic inhibitor, colchicine, also markedly increased the lipide/cell ratio. Similar results were found in in vitro experiments with Ehrlich ascites cells.     

Fibronectin expression during myogenesis

The biosynthesis and localization of fibronectin during chick muscle differentiation are described. This study employed two monoclonal antibodies, one that selectively killed mononucleated cells and one specific for avian fibronectin. These antibodies allowed precise analyses of fibronectin expression in well-defined cultures of myoblasts or myotubes and avoided the complications of exogenous fibronectin and contamination by fibroblasts or unfused myoblasts. Fibronectin synthesis, as a fraction of total protein synthesis, remains constant at 0.3-0.4% before and after myoblast fusion, suggesting that the absolute rate of fibronectin synthesis may increase somewhat when myotubes synthesize and accumulate myofibrillar proteins. The pattern of fibronectin arrangement does change during myogenesis. In myotube cultures, the appearance of pulse-labeled fibronectin at the cell surface and its secretion into the medium begin after a 2-3-h lag period, in contrast to the 30-min lag period observed in fibroblast cultures. This lag between polypeptide biosynthesis and the exteriorization of the new protein is thus a characteristic of each cell type rather than the protein. All of the major secretory proteins of myogenic cells, including fibronectin and collagenous components, share this 2-3-h intracellular transit time.     

Initiation of primary cell cultures from human intracranial tumors on extracellular matrix from bovine corneal endothelial cells

Tissue specimens from 105 human gliomas and 57 human meningiomas were obtained at surgery, dissociated into single cells and small cell aggregates and then plated onto plain plastic tissue culture dishes and dishes which had been precoated with an extracellular matrix (ECM) derived from bovine corneal endothelium. In 80% of the glioma cases we observed a marked improvement in initial plating efficiency, colony formation and speed of attachment when cells were plated on ECM. In 5 cases cells attached only to the ECM-coated dishes but remained afloat in the untreated dishes. In addition it could be noted that over the first 2 days, those cells which had been initiated on ECM showed more signs of morphological differentiation, i.e., extension of cytoplasmic processes or formation of fiber networks between cell groups. If adaptation occurred and proliferation began in vitro, either immediately or after a several days' lag phase, both the ECM-cultured cells as well as those which slowly had adapted to culture on plastic could be passed on to untreated culture ware and perpetuated thereon. In the case of well-differentiated low-grade gliomas where no growth in culture took place, the cultures on ECM could at least be used for initial experiments in the primary cultures (P0). Meningiomas usually attached well to both, plastic or ECM. In 50% of our cases the plating efficiency was higher on ECM but after successful initial culture, the delay until the cells on plastic reached confluence in comparison with those on ECM was 1 or 2 days. Again there were 2 cases in which the cells would not plate on plastic. Here the cells which after 1 day were still afloat plated to more than 80% within the first 2 h after transfer to ECM. In all cases the cells from plastic and ECM cultures were indistinguishable and could be passed onto untreated dishes henceforth. In later culture stages ECM offers several advantages: It is easier to shift cells to serum-free defined culture conditions, the cells will grow at a faster rate on ECM when in higher passages and the maximal number of passages possible is higher on ECM.     

Proliferation and differentiation potential of rat forebrain oligodendroglial progenitors both in vitro and in vivo

We have followed the development of the O-2A progenitor cell from the neonatal rat forebrain, both in dissociated cell culture and in cryostat sections, using immunocytochemical techniques employing a panel of antibodies that recognise the cells at different stages of their development. This included the monoclonal antibody LB1, which binds to the surface ganglioside GD3 expressed on O-2A progenitor cells. In secondary cultures enriched for O-2A progenitors maintained in a serum-free chemically defined medium, a large proportion of the cells are primed to differentiate into oligodendroglia and go on to express the oligodendroglial specific surface glycolipid galactocerebroside (GC) and then the myelin proteins CNP and MBP. However, a significant proportion of immature bipolar GD3+ cells remained after 6 days in secondary culture. It appears that not all the O-2A progenitors in our cultures differentiate immediately and some cells remain in an undifferentiated state and divide to replenish progenitor numbers. We have also identified in our cultures a small apolar GD3- cell, which when isolated differentiated into a GD3+ bipolar O-2A progenitor cell. We have termed this cell type a preprogenitor. The differentiation of this cell type into O-2A progenitors may be the source of the immature GD3+ cells present at the later stages of our secondary cultures. The proliferative profile of the cultures was studied using 5'bromo-2-deoxyuridine (BrdU) incorporation as an index of mitosis. Only the immature, bipolar O-2A progenitors were seen to divide at any time in serum-free culture. Neither the more mature multipolar O-2A cells nor the oligodendroglia were seen to divide. The developmental profile of the O-2A cells in the rat forebrain in vivo showed a largely similar progression to that in culture, with a time lag of at least 6 days between GD3 expression and the onset of myelination. BrdU incorporation studies in vivo also showed that the GD3+ progenitor cell is mitotic whereas the GC(+)-expressing oligodendroglia is not. We have shown that there are several significant alterations in the timing of antigen expression in both O-2A progenitors and oligodendroglia in vitro compared to that seen in vivo.