Serum-free Mouse Embryo Cells Research Articles

Bone Morphogenetic Proteins Inhibit Proliferation, Induce Reversible Differentiation and Prevent Cell Death in Astrocyte Lineage Cells

Bone Morphogenetic Proteins (BMPs) induce the differentiation of Serum-free Mouse Embryo (SFME) cells into astrocytes (D'Alessandro et al., 1994) as demonstrated by change in morphology, increase in Glial Fibrillary Acidic Protein (GFAP) content and classification as both type 1 and 2 astrocytes. Further analyses showed that in the presence of BMP, cells which had differentiated into astrocytes were inhibited from proliferation. Moreover, removal of BMP resulted in a resumption of proliferation accompanied by a loss of GFAP expression over time, indicating that under these in vitro conditions the differentiation was reversible. Since EGF is absolutely required for the survival of SFME cells, we examined the effect of its removal in the presence of BMP. Cell survival was > 80% in the presence of BMP-2, 7 or 2/7 and < 10% in the presence of TGF-beta 1. These data demonstrate that BMPs have effects on the proliferation, differentiation and survival of cells in the astrocyte lineage.

Bone Morphogenetic Proteins Induce Differentiation in Astrocyte Lineage Cells

Serum-free mouse embryo (SFME) cells express Glial Fibrillary Acidic Protein (GFAP), a specific marker of the astrocyte lineage, when treated with either Transforming Growth Factor Beta (TGF-beta) or calf serum. We examined the effects of the related Bone Morphogenetic Proteins (BMPs) which are expressed in the developing murine nervous system. Treatment with the heterodimers BMP-2/6 and 2/7 followed by the homodimers BMP-2, 4, 5, 6, and 7 induced higher levels of GFAP in these cells than either TGF-beta 1 or activin when tested at the same concentration. The BMP-induced cells resembled classically described astrocytes and were characterized by antibody markers as type 1 and type 2. In addition, these astrocytes also showed increased levels of the cell adhesion molecules CD44 and neural cell adhesion molecule (N-CAM), both known to be expressed by this cell type. These data clearly demonstrate that the BMPs function as differentiation factors as well as regulators of adhesion molecule expression for cells of the astrocyte lineage and suggest a key role in glial development in the nervous system.

Regulation of glial fibrillary acidic protein in serum-free mouse embryo (SFME) cells by leukemia inhibitory factor and related peptides

The serum-free mouse embryo (SFME) cell line, derived in serum-free medium from 16-day-old mouse embryos, exhibits unique properties. SFME cells grow indefinitely in culture without senescence, require epidermal growth factor (EGF) or fibroblast growth factor (FGF) for survival and are growth-inhibited by serum. The cell line expresses glial fibrillary acidic protein (GFAP) in response to transforming growth factor β or serum and cells with similar properties can be isolated directly from brain. Culture of SFME cells with leukemia inhibitory factor (LIF), a peptide implicated in neural tissue development, also resulted in expression of GFAP. Other peptides that share signal transduction mechanisms with LIF - ciliary neurotropic factor, oncostatin M and interleukin-6 - also caused expression of GFAP in these cells. These effects were inhibited by concentrations of EGF or FGF that promoted rapid cell growth.

Characterization of human plasma growth inhibitory activity on serum-free mouse embryo cells.

Serum-free mouse embryo (SFME) cells are a cell line derived in medium in which serum is replaced with growth factors and other supplements. These cells display unusual properties: a) they do not lose proliferative potential or show gross chromosomal aberration upon extended culture, b) they depend on epidermal growth factor (EGF) for survival, and c) they are reversibly growth inhibited by plasma and serum. Transfection of SFME cells with oncogenes (ras, neu, SV40 T antigen) results in cells that grow in serum-supplemented medium and no longer require EGF for survival. The growth inhibitory activity of human plasma on SFME cells was investigated. The activity was present in delipidated plasma and was not dialyzable against 1M acetic acid. The activity precipitated in 33% methanol, bound to concanavalin A-agarose and was retarded by Sephadex G-50 in 200 mM acetic acid. A fifty- to one-hundred-fold purification was achieved, although most of the differential inhibition of untransformed vs. transformed cells was lost in the course of the purification.

Expression of a TGFβ regulated, brain-specific mRNA in serum-free mouse embryo (SFME) cells

The serum-free mouse embryo (SFME) cell line was isolated from 16-day-old Balb/c mouse embryos in medium in which the usual serum supplement to the culture medium was replaced by purified growth factors and other components. SFME is an unusual line that does not undergo senescence in vitro, maintains an apparently normal karyotype, and is growth inhibited by serum. Transforming growth factor β (TGFβ) or calf serum induces expression of the astrocyte marker glial fibrillary acidic protein (GFAP) in these cells, and similar cells can be isolated directly from brain. By differential screening of a cDNA library derived from SFME cells, a calf serum- and TGFβ-regulated 8.5 kb mRNA was identified in SFME cells and the cDNA partially sequenced. This mRNA was detected only in RNA preparations from brain among a number of tissues examined, and may provide an additional marker of TGFβ-regulated differentiation in these cells.

Non-transformed, but notras/myc-transformed, Serum-free Mouse Embryo Cells Recover from Growth Suppression by Azatyrosine

The anti‐proliferative effect of azatyrosine, a newly discovered antibiotic from Streptomyces, was examined in Balb/c‐originated serum‐free mouse embryo (SFME) cells and transformed ras/myc SFME cells which have activated human c‐Ha‐ras genes. Azatyrosine suppressed their growth in a concentration‐dependent manner. Growth suppression in both cells was detectable within 2 days after culture with 250 μg/ml azatyrosine. Non‐transformed SFME cells, however, regained rapid growth after 6 days even in the presence of azatyrosine, whereas ras/myc SFME cells did not recover from the suppression. Despite the growth inhibition of ras/myc SFME cells, expression of human ras in the cells was not inhibited by azatyrosine. Meanwhile, SFME cells have the ability to express glial fibrillary acidic protein (GFAP). This expression is induced by serum‐supplemented medium, though the serum inhibits the growth of SFME cells. Azatyrosine did not induce GFAP in ras/myc SFME cells, but inhibited growth. Furthermore, azatyrosine did not induce GFAP in SFME cells, and had no effect upon the expression of GFAP induced by serum in these cells. These results suggest that azatyrosine inhibited the growth of ras/myc SFME cells through a mechanism independent of those involved in growth inhibition and induction of GFAP expression by serum in SFME cells.

Death of serum-free mouse embryo cells caused by transforming growth factor beta 1 and effects of nutritional factors.

Transforming growth factor beta 1 (1 ng/ml) caused death of serum-free mouse embryo cells cultured in a medium consisting of a 1:1 mixture of Dulbecco's Modified Eagle's medium and Ham's F12 medium supplemented with fibronectin, insulin, transferrin, epidermal growth factor, and high density lipoprotein. Cell death occurred in the presence of polyunsaturated fatty acids including linoleic acid in the absence of selenium. The death could be reversed by adding alpha-tocopherol to the culture indicating a mechanism involving fatty acid peroxidation. Butylated hydroxytoluene was a poor suppressor of cell death in contrast to alpha-tocopherol. High density lipoprotein and fatty acid-free albumin also suppressed cell death at the level of 20 micrograms/ml and 1 mg/ml, respectively. Transforming growth factor beta 1 also caused a low rate of cell growth after heat treatment of the cells at 45 degrees C.

Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation.

Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, are proastroblasts that are acutely dependent on epidermal growth factor (EGF) for survival. Ultrastructurally, an early change found in SFME cells deprived of EGF was a loss of polysomes which sedimentation analysis confirmed to be a shift from polysomes to monosomes. The ribosomal shift was not accompanied by decreased steady-state level of cytoplasmic actin mRNA examined as an indicator of cellular mRNA level. With time the cells became small and severely degenerate and exhibited nuclear morphology characteristic of apoptosis. Genomic DNA isolated from cultures undergoing EGF deprivation-dependent cell death exhibited a pattern of fragmentation resulting from endonuclease activation characteristic of cells undergoing apoptosis or programmed cell death. Flow cytometric analysis indicated that cultures in the absence of EGF contained almost exclusively G1-phase cells. Some of the phenomena associated with EGF deprivation of SFME cells are similar to those observed upon NGF deprivation of nerve cells in culture, suggesting that these neuroectodermal-derived cell types share common mechanisms of proliferative control involving peptide growth factor-dependent survival.

Karyotypic stability of serum-free mouse embryo (SFME) cells.

Mouse embryo cultures derived in serum-containing medium undergo growth crisis or senescence after fewer than 20 population doublings, followed by the emergence of genetically altered, polyploid 'immortalized' cells capable of growing indefinitely. Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, do not exhibit growth crisis or gross chromosomal aberrations when cultured for well over 100 population doublings and display other unique properties. We examined culture conditions and physiological factors affecting karyotypic stability in long term cultures of SFME cells derived from several mouse strains. Cloning SFME cells consistently isolated colonies with altered karyotype, even when the clones were derived from parent cultures with no karyotypic alterations. After 140-200 population doublings in vitro, the percentage of SFME cells showing hyperdiploidy or structural chromosomal abnormalities increased, although the modal chromosome number remained diploid. SFME cells transformed with molecularly cloned oncogenes did not show alterations in karyotype beyond that expected from the clonal origins of these cells, indicating that malignant transformation of SFME cells does not result in general karyotypic instability.

Serum inhibition of proliferation of serum-free mouse embryo cells

Serum-free mouse embryo (SFME) cells, derived in medium supplemented with insulin, transferrin, high density lipoprotein, epidermal growth factor, and fibronectin, do not undergo crisis, maintain a predominately diploid karyotype with no detectable chromosomal abnormalities for well over 100 population doublings in vitro, and are growth inhibited by concentrations of serum that are growth-stimulatory for most cell lines in culture. Serum inhibition of SFME cell proliferation was reversible and was not prevented by addition of the supplements of the serum-free medium, even when added repeatedly during the culture period. The serum effect on SFME cell proliferation could be detected after incubation in serum-containing medium for as little as 8 h. SFME cells in serum-containing medium were arrested in the G1 phase of the cell cycle with a greatly reduced rate of incorporation of precursors into DNA and thymidine kinase activity, while a reduction in rate of incorporation of amino acids into protein was not observed. SFME cultures maintained for extended periods in serum-containing medium underwent a crisis-like period followed by the appearance of variant cells capable of growing in serum-supplemented medium. These cells exhibited abnormal karyotype and were resistant to several inhibitors of proliferation active on the parent SFME cell type.

Serum and transforming growth factor beta regulate glial fibrillary acidic protein in serum-free-derived mouse embryo cells.

Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, display distinctive properties: (i) SFME cells do not lose proliferative potential or show gross chromosomal aberration upon extended culture, (ii) these cells depend on epidermal growth factor for survival; and (iii) SFME cell proliferation is reversibly inhibited by serum. Treatment of SFME cells with serum or transforming growth factor beta led to the appearance of glial fibrillary acidic protein, a specific marker for astrocytes. The appearance of glial fibrillary acidic protein in cultures was reversed upon removal of transforming growth factor beta or serum. Cells with properties similar to SFME cells were also isolated from adult mouse brain. These results suggest a role for transforming growth factor beta in astrocyte differentiation in developing organisms and in response to injury and identify the cell type that has the unusual properties of SFME cells.

Transforming growth factor beta regulates cystatin C in serum-free mouse embryo (SFME) cells

Differential screening of a cDNA library derived from mRNA of TGF beta-treated serum-free mouse embryo (astrocyte precursor) cells isolated a strongly TGF beta-regulated mRNA that codes for cystatin C, a cysteine protease inhibitor. Increase in cystatin C mRNA level was observed within four hours after treatment with picomolar concentrations of TGF beta. The increase was reversible upon removal of TGF beta and was not prevented by cycloheximide. These results suggest that cystatin C expression may represent a developmentally regulated differentiated function of astrocytes, and also suggest that cystatin C expression may be involved in the response of brain cells to platelet release of TGF beta after trauma or injury.

Ras and neu oncogenes reverse serum inhibition and epidermal growth factor dependence of serum‐free mouse embryo cells

Serum-free mouse embryo cells, cultured in basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibronectin, and high-density lipoprotein, do not exhibit growth crisis, lack detectable chromosomal aberrations, are nontumorigenic in vivo, are dependent on epidermal growth factor for survival, and are growth inhibited by serum or platelet-free plasma. These cells after transfection with the human Ha-ras or rat neu oncogenes no longer required epidermal growth factor for survival, were tumorigenic in vivo, and also proliferated in serum-containing medium. Autocrine activity capable of replacing epidermal growth factor was detected in conditioned medium from ras-transformed cultures, but little such activity was detected in medium from neu-transformed cultures. In addition, the capability of ras or neu-transformed cells to grow in serum-containing medium could not be mimicked in untransformed cells by the addition of growth factors or conditioned medium from transformed cells. These results suggest that the known structural similarity of the neu gene product to the EGF receptor is also reflected in a functional similarity by which the mutationally activated neu protein can replace the ligand-activated EGF receptor. These results also suggest that the ability of ras- and neu-transformed cells to escape the effect of the inhibitory serum activity is a nonautocrine property distinct from the acquisition of EGF autonomy.

Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation is prevented by cycloheximide, 12-O-tetradecanoylphorbol-13-acetate, or vanadate

Serum-free mouse embryo cells cultured in medium supplemented with insulin, transferrin, high-density lipoprotein, and fibronectin are dependent on epidermal growth factor for survival. Cycloheximide or actinomycin D prevented cell death caused by growth factor deprivation, suggesting that cell death required the synthesis of RNA and protein, a phenomenon similar to that reported for neuronal cell death in the absence of nerve growth factor. Orthovanadate, an inhibitor of phosphotyrosine phosphatases, and 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, also prevented serum-free mouse embryo cell death in the absence of epidermal growth factor.

Effects of herbacitrin, a flavonoid, on the growth of normal and transformed serum-free mouse embryo cells.

Effects of herbacitrin, a flavonoid, on the growth of normal and transformed serum-free mouse embryo cells.

Serum-free mouse embryo cells: growth responses in vitro.

We have derived serum-free mouse embryo (SFME) cultures in a basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin. These cells are nontumorigenic, lack gross chromosomal aberrations, and exhibit several other unique properties, including dependence on EGF for survival and growth inhibition by serum. We have examined the concentration dependence of the growth stimulatory effects of protein supplements used in the SFME medium formulation and surveyed other supplements that might act as alternative or complementary additions to the culture medium. Insulin could be replaced by insulin-like growth factor I and EGF could be replaced by transforming growth factor alpha in the same concentration range. Transferrin could be replaced by higher concentrations of lactoferrin. Deterioration of cultures in the absence of EGF began within 8 hours of the removal of the growth factor, and could be prevented by the addition of fibroblast growth factor/heparin-binding growth factor. Attachment proteins other than fibronectin were effective on SFME cells, but limited success was obtained when substituting other lipid preparations for HDL. These data introduce a precise system for exploring the unusual characteristics of SFME cells and contribute additional information that may be useful in the extension of these approaches to other cell types and species.