A rapid and sensitive method for detecting human cells in xenogeneic hosts.

Xenografts, specifically transplantation of human cells into other species, are a valuable tool in preclinical transplantation experiments. A central issue is accurate identification of the grafted cells, particularly in cases in which cellular migration has occurred. We report that detection of grafted human cells can be achieved by in situ hybridization techniques using human centromeric probes which result in unambiguous nuclear labeling. The resulting reaction can be combined with immunocytochemical or histochemical techniques for cell-type characterization.

O-2A glial progenitors from mature brain respond to CNS neuronal cell line-derived growth factors.

During development, myelin-forming oligodendrocytes and type 2 astrocytes are believed to arise from bipotential (O-2A) glial progenitors. Previously we found that conditioned medium (CM) from the B104 rat CNS neuronal cell line promotes growth of neonatal rat O-2A progenitors in serum-free culture conditions with subsequent increases in differentiated progeny. We now report that O-2A progenitors are present in mature rat brains and that this CM promotes the growth, motility, and bipolar morphology of these cells from 30- and 65-day-old rat brains, as shown by quantitative studies using double immunostaining and [3H]thymidine-autoradiography. In addition, the growth-promoting action of B104 CM is not neutralized by antibodies to platelet-derived growth factor, a proposed progenitor mitogen. Subsequent to the proliferation of these O-2A progenitors, increases in oligodendrocytes and type 2 astrocytes occur. These data suggest a novel therapeutic strategy for some demyelinating diseases, e.g., multiple sclerosis, where there is a deficit in oligodendrocytes. Although it has been proposed by others that mature brain O-2A progenitors are less proliferative and thereby incapable of adequately replenishing lost oligodendrocytes in these diseases, we present in vitro evidence for continued response of mature brain O-2A progenitors to this neuronal cell line-derived mitogen.

Growth factor responses of enriched bipotential glial progenitors.

Responses of oligodendrocyte/type 2 astrocyte (O-2A) glial progenitors from neonatal rat brains to different growth factors were studied by a new, serum-free method. Enriched tertiary cultures of O-2A progenitors were produced after 6-7 days in vitro using the growth-promoting factors from the B104 CNS neuronal cell line, heparin, and mechanical separation. These cultures contained about 75-90% A2B5+ cells with less than 10% type 1 astrocytes, and the yield was 4.4 x 10(5) cells/brain. B104 conditioned medium (CM) factors increased both O-2A progenitor number and [3H]thymidine-labeling indices after three days. However, type 1 astrocyte CM was required for continued survival of enriched progenitors beyond 1 day in tertiary culture. Platelet-derived growth factor (PDGF) and glia maturation factor also showed growth-promoting action, but were less effective than B104 CM at tested doses. PDGF-neutralizing antibodies had no effect on progenitor survival or response to B104 CM factors. Thus, type 1 astrocyte-derived PDGF was not required for this response, B104 CM is not likely to contain PDGF, and B104 CM factors act directly on O-2A progenitors. Fibroblast growth factor, transforming growth factor beta, interleukin 2, epidermal growth factor, and triiodothyronine showed no growth-promoting activity; moreover, interleukin 2, epidermal growth factor, transforming growth factor beta, and 0.5% fetal bovine serum inhibited B104 CM action. Enriched progenitors exhibited bipotentiality by slowly differentiating into oligodendrocytes in serum-free medium, whereas culture in 10% fetal bovine serum increased type 2 astrocytes. Thus, this new method selects or produces progenitors which are similar to those from mature brains.

Bipotential glial progenitors are targets of neuronal cell line-derived growth factors.

The growth-promoting activity of conditioned medium (CM) from the B104 CNS neuronal cell line was studied in glial cultures from neonatal rat brain. This CM at 33% (v/v; 8-12 micrograms protein/ml) produced large numbers of oligodendrocytes and multipolar glial progenitors after an 8 to 12-day treatment. At all times studied, cells of the oligodendrocyte/type 2 astrocyte (O-2A) lineage were increased due to CM-treatment, while type 1 astrocytes, microglia, and other cell types were not. Furthermore, we observed a large decrease in the percentage of oligodendrocytes in the O-2A lineage, suggesting a delay in differentiation of the progenitors. By 8 days in vitro (DIV), dose-dependent increases in numbers of galactocerebroside (GalC)-positive cells (oligodendrocytes) and A2B5-positive cells (immature oligodendrocytes and glial progenitors) occurred. In contrast, at 4 DIV only A2B5-positive cells were increased in a dose-dependent manner. The latter cells can differentiate primarily into oligodendrocytes or type 2 astrocytes depending on the culture conditions. Complement lysis studies confirmed that the A2B5-positive, but not the GalC-positive, population at 4 DIV was required for increases in oligodendrocytes to occur by 8 DIV. The [3H]thymidine labeling index of the A2B5-positive population also increased in response to CM in a dose-dependent manner, but the GalC-positive labeling index showed only small increases at 4 DIV and none at later times. Our results suggest that the delayed differentiation coupled with the selective stimulation of the bipotential glial progenitors produces the large increases in numbers of oligodendrocytes observed at 8-12 DIV.

CNS neuronal cell line-derived factors regulate gliogenesis in neonatal rat brain cultures.

Recent studies suggest that heterotypic cell-cell interactions influence gliogenesis in the developing rat central nervous system. CNS neuron-derived factors have been hypothesized to exist, and several have been identified and partially characterized which affect the number of oligodendrocytes in vitro. In order to study further the role of neurons in gliogenesis, we have used serum-free culture conditions, the B104 CNS neuronal cell line as a source of soluble factors, and dissociated neonatal rat brain cells as a source of glial cells. We have analyzed the response of the glial cells to serum-free B104 conditioned medium using morphological, immunocytochemical, autoradiographic, and enzymatic methods. Dose-dependent increases in the number of morphologically identified oligodendrocytes occur in response to this conditioned medium. Galactocerebroside (GalC) is a specific marker for oligodendrocytes, and the A2B5 antigen marks bipotential glial progenitor cells and their progeny: immature oligodendrocytes and type 2 astrocytes. In the presence of conditioned medium, the number of cells expressing GalC and/or A2B5 antigen increases over time when measured at 4, 8, and 12 days in vitro. A significantly weaker effect is seen if serum is also present. Since the vast majority of A2B5-positive cells in conditioned medium treated cultures lack glial fibrillary acidic protein (GFA), indicative of type 2 astrocytes, they represent glial progenitors and immature oligodendrocytes. Double immunostaining combined with autoradiography suggests that the latter cell types are the target cells for the oligodendrocyte-promoting activity. In addition, the conditioned medium markedly increases 2',3' cyclic nucleotide 3'-phosphodiesterase (an oligodendrocyte marker) and to a lesser extent enhances glutamine synthetase activity (an astrocyte marker). Type 1 astrocytes are also more morphologically differentiated in this condition, and their percentage is decreased simultaneously. Conditioned medium from other donor neural cells either has no activity or is much less effective than B104 conditioned medium. The active factors are soluble, sensitive to both trypsin and 100 degrees C treatment for 20 min, and appear to be 30-100 kilodaltons by stirred cell ultrafiltration. In summary, we have identified a potent source of growth-stimulating factors that produce increased numbers of glial progenitor cells and oligodendrocytes; the same conditioned medium also appears to inhibit type 1 astrocyte proliferation.

Pure astrocyte cultures derived from cells isolated from mature brain.

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.

Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes.

I have defined the basic requirements for the proliferation of cell lines expressing oligodendrocyte properties and for the survival of galactocerebroside-positive oligodendrocytes derived from neonatal rat brains. Conventional serum-containing medium can be replaced by 01 medium, a chemically defined medium supplemented with insulin, transferrin, sodium selenite, and biotin. Thyroid hormone is not required. When cells are plated directly into O1 medium, the substratum has to be modified by precoating with polylysine and adding fibronectin to the medium prior to the cells. Both cell lines and brain cells can be subcultured numerous times in O1 medium without initial culture in serum-containing medium. Brain cultures can be maintained in O1 medium for several months and contain a significantly higher percentage of mature oligodendrocytes, a lower number of astrocytes, and no fibroblasts as compared to cells maintained in serum-containing medium.

Factors influencing astrocyte growth and development in defined media.

A previously described serum-free, defined medium (G2 medium) containing transferrin, selenium, hydrocortisone, biotin, fibroblast growth factor (FGF) and fibronectin developed for the growth of human and rat derived glioma cells was investigated for its ability to support proliferation of astrocytes in primary cultures of neonatal rat cerebrum. These cells were able to grow in G2 medium. Enhanced proliferation and repeated subcultivation were obtained after adding insulin and/or epidermal growth factor (EGF) to the G2 medium at concentrations of 5 µg/ml and 10 ng/ml, respectively. In these modified media (called G4 and G5 medium) astrocytes showed a higher degree of morphological differentiation as compared to serum supplemented medium. Cell type specificity was determined by immunocytochemical staining of glial fibrillary acidic (GFA) protein, which could already be demonstrated 5 days after plating cells. G4 and G5 represent the first serum-free defined media in which astrocytes proliferate and differentiate without preceding or intermediate contact to serum supplemented medium. Modification of the culture substratum by adding hyaluronic acid and chondroitin sulfate A to G4 medium (G2 medium + insulin) enhanced proliferation of astroglial cells by a factor of about 1.5. In the presence of epidermal growth factor no response to the altered culture dish surface was observed and the addition of fibronectin, otherwise a stringent plating requirement, was no longer necessary.

Proliferation of glial-derived cells in defined media.

A serum-free defined medium has been formulated that supports proliferation and morphologic differentiation of U-251 MGsp human and C6-2BD rat glioma cells. This defined medium consists of a basal medium supplemented with transferrin, fibroblast growth factor, hydrocortisone, selenium, biotin, and fibronectin (G2 medium). When U-251 cells were plated in G2 medium on poly-D-lysine precoated dishes, their growth rate was 77% and final cell density was 82% of serum-grown counterparts. The growth rate of C6 cells in G2 medium was 67% compared to cells cultured in serum supplemented medium. Although G2 medium supported the growth of human and rat glioma cells, LA-N-1 human neuroblastoma and WI-38 human fibroblast cells showed no increase in cell number when grown in G2 medium compared to basal medium. A similar formulation (G3 medium), lacking fibroblast growth factor and hydrocortisone, supported the proliferation of RN-22 rat schwannoma cells. Morphologic differences were observed between cells grown in the presence of serum and in defined media. All three glial cell lines changed from a flattened shape in serum supplemented medium to a more spherical appearance in defined medium. In addition, both U-251 and C6 cells developed numerous processes, some reaching several cell diameters in length. These defined media will facilitate studies of the growth and differentiation of glial-derived cells.

Proliferation of glioma cells in serum-free defined medium.

The requirement for a serum supplement to basal culture medium to support the growth and viability of cells in vitro has been a major hindrance to reducing and thereby controlling experimental variables. The complexity and undefined nature of serum as well as the variability of serum lots make it difficult to reproduce data and to interpret results of experiments. This paper presents preliminary results obtained with C6 glioma cell cultures in which serum was replaced by growth factors and a hormone. A brief review of the use of serum-free defined medium for the culture of tumor cells and the potential uses of defined media that are cell-type specific are also presented.

Growth of a rat neuroblastoma cell line in serum-free supplemented medium.

The rat neuroblastoma B104 cell line, which originated in the central nervous system, was able to proliferate in the absence of serum in synthetic medium supplemented with insulin, transferrin, progesterone, selenium, and putrescine. When added individually, each supplement had little or no effect; however, in combination there was a marked synergistic effect on cell number. The cells attained the same saturation density in this medium as in medium with 10% fetal calf serum added. More extensive process formation was observed in the supplemented medium, and other differentiated properties were retained as well.