A simple approach for mouse embryonic stem cells isolation and differentiation inducing embryoid body formation

Stem cells were derived from hatched blastocyst-stage mouse embryos of the C57BL/6 strain employing a knockout serum replacement instead of the traditional fetal calf serum, thereby avoiding the use of immunosurgery. Although fetal calf serum was not good for isolation of stem cells, a combination of this serum plus knockout serum increased the expansion rate of the cell culture. The derived cells were capable of maintaining an undifferentiated state during several passages, as demonstrated by the presence of alkaline phosphatase activity, stage-specific embryonic antigen 1 (SSEA-1), and octamer binding protein 4 (Oct-4). Suspension culture in bacteriological dishes gave better results than the hanging drop method for differentiation by means of embryoid body formation. Mouse embryonic stem cells showed spontaneous differentiation into derivatives of the 3 germ layers in culture media supplemented with fetal calf serum but not with knockout serum.     

Down-regulation of Fas-mediated apoptosis by plasma transglutaminase factor XIII that catalyzes fetal-specific cross-link of the Fas molecule

The Fas antigen, also designated as APO-1 or CD95, is a member of the tumor necrosis factor receptor superfamily and can mediate apoptotic cell death in various cells. We report here that blood coagulation factor XIII (plasma transglutaminase, fibrin stabilizing factor) inhibits apoptosis induced by a cytotoxic anti-Fas monoclonal antibody in Jurkat cells. When cells were treated with the antibody in fetal calf serum-containing media, higher-molecular-weight (180 K) polypeptides containing Fas molecule were detected by immunoblotting. Under conditions where the transglutaminase activity was eliminated or suppressed, the cross-link of Fas was not observed, and concurrently cell death was hastened. Moreover, an antibody against factor XIII strongly accelerated the Fas-mediated apoptosis. Furthermore, addition of partially purified factor XIII neutralized the apoptosis-promoting effect of anti-factor XIII antibody, indicating that this enzyme is involved in cross-link of Fas and down-regulates Fas-mediated apoptotic cell death. Significantly, the cross-link of Fas was seen only in fetal calf serum but not in newly-born calf serum, 1-year-old calf serum or adult bovine serum. These data suggest that plasma transglutaminase factor XIII may play a key role in fetal development of vertebrates via cross-link of Fas antigen.     

Characterization of a fetal calf serum-derived molecule reactive with human natural antibodies: its occurrence in tissue culture-grown type C RNA viruses.

By means of a sensitive radioimmunoprecipitation (RIP) assay, simian sarcoma virus-simian sarcoma-associated virus (SSV-SSAV), purified from culture fluids of infected normal rat kidney (NRK) cells, was shown to acquire a surface antigen from serum used in the tissue culture medium. This antigen, which was acquired when serum from either fetal calf, horse, swine, rabbit, or chicken origin was used, accounted for a substantial portion (but not all) of the total precipitating activity exhibited by natural human antibodies for membrane-associated antigens of these viruses. By 1) alcohol precipitation, concanavalin A chromatography, and Sephadex G-150 filtration of fetal calf serum (FCS) proteins or 2) chromatography of serum proteins over a human IgG-containing immunoaffinity column, a glycoprotein of approximately 55,000 daltons has been identified which is a minor constituent of FCS (less than 0.1% of total protein) and has the antigenic capacity of whole FCS.     

Factors affecting cytotrophoblast cell viability and differentiation: Evidence of a link between syncytialisation and apoptosis

A relationship between cytotrophoblast differentiation (syncytialisation) and apoptosis is hypothesised to exist, but has not been clearly determined. To address this, we explored the effects of cAMP, an inducer of syncytialisation, on human choriocarcinoma cell differentiation and viability under three different culture conditions related to diverse survival status: no serum, 10% fetal calf serum or 10% charcoal-stripped fetal calf serum. 8-Br-cAMP increased BeWo cell viability in culture media without serum, but viability was decreased in a dose- and time-dependent manner when serum was present. The appearance of apoptotic nuclei fragments were only observed when BeWo cells were cultured in media containing serum combined with 8-Br-cAMP treatment. In addition, the ratio of FasL to Fas expression following treatment with 8-Br-cAMP increased by 20-fold in 10% charcoal-stripped fetal calf serum media and 65-fold 10% fetal calf serum media, and activation of caspase-3 also required media with serum. The markers of syncytialisation (syncytin 1 expression and human chorionic gonadotropin secretion) were induced significantly by 8-Br-cAMP, and were higher in 10% fetal calf serum media than in 10% charcoal-stripped fetal calf serum media, than in the absence of serum. Syncytia formation was stimulated by 8-Br-cAMP and this required serum in the media. We now show that factors contained within serum are necessary for cAMP-stimulated cytotrophoblast differentiation, that syncytialisation involves apoptotic events, and that a lack of serum based factors could switch the cellular program away from differentiation.     

The regulation of cell growth: II. Some characteristics of a fetal calf serum factor (FF2) stimulating ornithine decarboxylase in mouse liver

A heat stable, non-dialysable fetal calf serum factor (FF₂), capable of stimulating ornithine decarboxylase in mouse liver, kidney and spleen, has been detected in fetal calf serum and commercial preparations of 81% pure fetuin.The factor has a molecular weight of approx. 17 500, contains sulfhydryl groups necessary for its activity, and is protease resistant.Stimulation of hepatic ornithine decarboxylase by the fetal calf serum factor is dose and time dependent and is blocked by both cycloheximide and by actinomycin D, if the latter is administered within 1 h of the factor. Theophylline enhances the effect of the fetal calf serum factor on ornithine decarboxylase in the liver and the factor stimulate ornithine decarboxylase in hypophysectomized and adrenalectomized rats.     

Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.     

Reduced frequency of sister chromatid exchanges in human lymphocytes cultured with autologous serum

Summary The incidence of sister chromatid exchange (SCE) was determined in human lymphocytes cultured with fetal calf, human AB, and autologous serum. In each individual studied, cells grown in medium supplemented with fetal calf and human AB serums showed higher yields of SCE than those cultured with autologous serum. Increased concentration of fetal calf and human AB serum in the tissue culture medium resulted in elevated frequency of SCE. No such elevation in SCE frequency was observed with increased concentration of autologous serum. The results indicate the presence of extraneous SCE-inducing factors in fetal calf and human AB serum, the nature of which is not precisely known.Aided by C.S.I.R. Grant No. 7/45 (1052/77) EMR I     

A requirement for cholesterol for phorbol ester-induced adhesion of a human monocyte-like cell line

The human monocyte/macrophage-like cell line U937, which is a cholesterol auxotroph, is nonadherent. However, it becomes adherent after treatment with phorbol 12-myristate 13-acetate (phorbol ester). We investigated the effects of cellular cholesterol depletion and repletion on the effectiveness of phorbol ester to induce adhesion to substratum. Almost 70% of cellular cholesterol is depleted by incubation of the cells for 24 hrs in the growth medium in which delipidated fetal calf serum is substituted for fetal calf serum without affecting viability or the rate of growth. The use of delipidated fetal calf serum inhibited phorbol ester-induced adhesion by 40%. If the cells were preincubated in the medium containing delipidated fetal calf serum 6 hrs prior to addition of phorbol ester, adhesion was inhibited by 90%. Addition of cholesterol to the medium containing delipidated fetal calf serum, which replenishes cellular cholesterol, restored the ability of phorbol ester to induce adhesion to levels seen in cells cultured in the medium containing fetal calf serum. Epicholesterol was not as effective as cholesterol in supporting adhesion. Cholesterol depletion did not inhibit phorbol ester stimulation of superoxide anion production. These observations indicate a function for cholesterol in phorbol ester-induced adhesion that is independent of phorbol ester-induced superoxide anion production. It is proposed that cholesterol is required for synthesis and/or proper orientation and distribution, in the plasma membrane, of macromolecule(s) that mediate phorbol ester-induced adhesion.     

IL 1 requirement for B cell activation revealed by use of adult serum

Fetal calf serum is an essential component of the culture medium developed by Mishell and Dutton for the immunization of murine spleen cells in vitro. Serum from adult donors (mouse, human, rabbit) does not support antibody synthesis in this system. This "deficiency" of adult serum can be overcome with IL 1. Adult serum in the presence of IL 1 is as effective in stimulating a B cell response against xenogeneic red cells as fetal calf serum. We attribute the capacity of fetal calf serum to support an immune response in the absence of exogenous IL 1 to serum factors that cause macrophages to release IL 1 endogenously. Our findings strengthen the notion that IL 1 plays an essential role in the process of B cell activation and suggests that the use of fetal calf serum should be avoided in studies concerned with the function of interleukin 1.     

Differentiation in vitro of mouse embryos to the stage of early somite

Mouse blastocysts continuously differentiate in vitro to the early somite stage with reconstituted rat tail collagen as the substrate for the attachment. In order for this to occur, it appears that two differentiation barriers must be overcome. The first, the formation of egg cylinders from the inner cell mass, can be overcome by incubating embryos in heat-inactivated fetal calf serum. The second, the formation of the early somite from the presomite stage, can be overcome by replacing fetal calf serum with human cord serum.Mouse blastocysts were initially incubated with calf serum in Eagle's minimum essential medium. After shedding the zona pellucida, the denuded blastocysts lay flat on the surface of the collagen. Soon thereafter, trophoblastic cells invaded the underlying collagen leaving the rounded inner cell mass protruding from the surface of the collagen. By replacing calf serum in the medium with fetal calf serum the inner cell mass differentiated into endoderm and ectoderm to form an egg cylinder.The egg cylinder rapidly became elongated and formed extraembryonic and embryonic regions. However, the embryonic region shrank from this point on in the fetal calf serum, and the resulting yolk sac formation did not contain the embryo proper. When fetal calf serum was replaced with human cord serum at the end of the egg cylinder stage (equivalent to embryos of about 7.5 days gestation) neural tissue, cardiac chambers, and somites were formed.     

Asparaginase and glutaminase activities in culture media containing dialyzed fetal calf serum

Summary Degradation of asparagine in media containing dialyzed fetal calf serum has been shown to be the result of asparaginase, which is active even at 4°C. The asparaginase activity of undialyzed fetal calf serum is only one-tenth of that found in the dialyzed serum. Glutaminase activity was also demonstrated in the medium containing dialyzed fetal calf serum. Minor changes in some other amino acids were also observed. This work was supported in part by Grant CA02568, National Cancer Institute, National Institutes of Health.     

Serum effects on the response of mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae.

The response of mammalian cells to Pseudomonas and diphtheria exotoxins was studied. A method was developed whereby the sensitivity of cells to these two toxins could be quantitated. The method is versatile and can be used to study the effects of toxins on many cellular metabolic or transport processes. The type of serum used in the culture medium significantly influenced the response of cells to the toxins. Calf, horse, and human sera protected cells while fetal calf serum did not. Precipitation with (NH4)2SO4 demonstrated the probable presence of toxin-specific antibody in the protective calf serum while none was detected in the nonprotective fetal calf serum. The level of antibody in calf serum, as titrated by hemagglutination, was sufficient to account for all the observed protection. It is suggested that fetal calf serum be used for all future cell culture studies of bacterial toxins.     

Possible role of H1 histone in the differentiation of mouse erythroleukemia cells

Serum-starved mouse erythroleukemia cells, stationary phase cells or cells cultured in dibutyryl cAMP (1 mM) can be induced to differentiate by addition of 20% fetal calf serum plus cycloheximide. Culturing unstarved log phase cells in 20% fetal calf serum plus low levels of cycloheximide and histone H1 also causes a significant level of differentiation. These same concentrations of cycloheximide and H1 histone employed separately with 20% fetal calf serum do not induce differentiation. The role these procedures may have in causing an accumulation of histone H1 and cell differentiation is discussed.     

Isolation and characterization of mannose 6-phosphate/insulin-like growth factor II receptor from bovine serum.

A convenient means was devised for the purification of milligram quantities of a soluble form of the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF II receptor). The receptor was purified to near homogeneity from bovine serum by affinity chromatography on agarose-pentamannosephosphate in the absence of detergent. Approximately 2.5 mg of receptor were obtained from 500 ml of fetal calf serum. The concentration of receptor in serum decreased sharply with development. Fetal calf serum Man-6-P/IGF II receptor was immunologically similar to detergent-solubilized, membrane-bound Man-6-P/IGF II receptor from bovine liver. N-Terminal sequence analysis revealed that the purified serum receptor, but not the solubilized, membrane-associated receptor, contains stoichiometric amounts of bound IGF II. The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography studies suggest that the fetal calf serum receptor (in contrast to the solubilized, membrane-bound bovine testis receptor) does not aggregate. The affinity of the fetal calf serum receptor for bovine testis beta-galactosidase approximated one-half that observed for solubilized, membrane-bound bovine testis receptor.     

Microendocytosis in eosinophilic leukocytes

Rat peritoneal eosinophils were examined after intraperitoneal infusion either of a mixture of phosphate-buffered saline (PBS) and colloidal gold or of fetal calf serum. These cells characteristically contained vesiculotubular structures, cuplike structures, and small granules during centrifugation. The cup-shaped structures and elaborate labyrinths of vacuole-like spaces increased markedly after injection of the PBS-colloidal gold mixture, presumably as features of heightened microendocytic activity. The vesiculotubular structures increased greatly after infusion of fetal calf serum. A few cyrstalloid granules exhibited fine-structural changes after the PBS-colloidal gold injection, and more numerous crystalloid granules appeared altered after fetal calf serum. Infrequent small granules contained a lucent, crystal-like silhouette after the fetal calf serum injection. Eosinophils evidenced microendocytic uptake of gold spherules into coated vesicles, the cup-shaped structures, and the small granules, but not into the vesiculotubular structures or crystalloid granules after intraperitoneal infusion of the PBS-gold mixture. Strong unmasked acid phosphatase activity in small granules contrasted with the general lack of activity in normal-appearing crystalloid granules and moderate activity in apparently altered crystalloid granules, presumably reflecting active and latent forms of enzyme in the different granules.     

Inhibition of sperm motility by bovine serum components.

Various sources and components of mammalian sera were evaluated for their ability to maintain or inhibit sperm motility. Human, rabbit, hamster, and porcine sera were equal in ability to maintain motility of human sperm. Four sources of fetal calf serum and one source of neonatal calf serum were unable to maintain motility of human sperm or sperm-fertilizing potential. In the presence of human serum, fetal calf serum actually inhibited human sperm motility. Fetuin, a component of fetal calf serum, contained the inhibitory activity. An inhibitory effect of fetuin on porcine and caprine sperm motility was also observed. The inhibitory activity resided in the second peak when fetuin was separated by isoelectric focusing. The sperm head membranes remained impermeable to dye, and mitochondrial membrane potential was maintained after motility had been reduced to almost zero by incubation with fetuin and fetuin fractions. Fetuin or the active portion of the molecule may be a useful component of a vaginal contraceptive and in research where inhibition of motility is desirable.     

Cell spreading factor. Occurrence and specificity of action.

A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.     

Mitogenic effect of estradiol on MCF-7 human breast cancer cells can be modulated by serum

The MCF-7 human breast cancer cell line responds to estradiol stimulation in vitro by increased proliferation only if prolonged subcultures in dextran-coated charcoal-treated fetal calf serum have been made previously. This growth stimulation is not obtained when cells are grown in medium containing 5% untreated fetal calf serum. We describe here the culture conditions under which we obtain a reproducible estradiol effect on cell growth.     

Comparative effect of rat and fetal calf serum on measurement of the natural tumoricidal activity of rat lymphocytes,macrophages and polymorphonuclear cells

Summary The effect of rat serum versus fetal calf serum on the in vitro natural cytolytic activity of rat lymphocytes, macrophages and polymorphonuclear cells against syngeneic tumour cells was compared. The cytolysis level mediated by the three varieties of effector cells was lower when rat serum was used instead of fetal calf serum to supplement the culture medium. This could explain in part the discrepancies found between in vitro and in vivo studies.     

Sodium Citrate as a Substitute for Fetal Calf Serum in the Micronucleus Test

The micronucleus test developed recently by Schmid and coworkers (Boller and Schmid 1970, Ledebur and Schmid 1973, Schmid 1976) is a rapid, convenient, and sensitive procedure for the detection of induced chromosome aberrations in vivo. It is now widely used for evaluating the mutagenic potential of drugs and other chemicals. The test involves the demonstration of micronuclei which result from lagging of acentric chromosome fragments or even of whole chromosomes during mitosis due to spindle disruption in the anucleate young erythrocytes of bone marrow smears (for details see Schmid 1976). Success or failure of the technique largely depends on the quality of the smear. Cell clumping and cell damage render the smear valueless. Schmid (1976) recommends the use of fetal calf serum for preparing the best smears. However, as he also noted, fetal calf serum is very expensive. Moreover it is not readily available in certain countries, particularly developing ones. It is not easy to procure heat inactivated human AB serum either, which Schmid has suggested as a good substitute for fetal calf serum. Difficulty in obtaining these important elements of the procedure is overcome to a great extent by the brief use of 1% sodium citrate solution at 20-25 C as a substitute for fetal calf serum.