Cell Differentiation, 15 (1984) 109-113
Elsevier Scientific Publishers Ireland, Ltd.
C D F 00266
Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal antibodies P. Dr~_ber and Z. P o k o r n ~ Institute of Molecular Genetics, Czechoslovak Academv of Sciences, 142 20 Prague 4, Czechoslovakia (Received 22 October 1984)
Three differentiation antigens of mouse teratocarcinoma stem cells are defined using a panel of ten IgM-class monoclonal antibodies raised against teratocarcinoma F9 cells. TEC-01 and four other antibodies define an antigen that corresponds to SSEA-I. TEC-02 antibody defines an antigen that is expressed on teratocarcinoma stem cells, parietal yolk sac cells PYS-2, unfertilized eggs including the zona pellucida and blastocysts. It is absent from all mouse adult tissues tested. Three other antibodies exhibit binding properties similar to TEC-02. TEC-03 antibody defines an antigen that is expressed on teratocarcinoma stem cells, PYS-2 cells and mouse blastocysts. It is absent from all mouse adult tissues except for lungs. cell surface antigen; monoclonal antibody; mouse embryo; teratocarcinoma
Materials and Methods
Various stages of mouse embryonic development are characterized by the expression of different gene products, some of them being expressed on cell surfaces. Products of early stages are also expressed on teratocarcinoma (TC) stem cell lines (Artzt et al., 1973). To study the biosynthesis and developmental aspects of these products, it is essential to have good tools for their identification, isolation and experimental manipulation of their expression. In this respect monoclonal antibodies against antigens common to embryonic and TC stem cells proved to be the most valuable (Solter and Knowles, 1978). Here we report the distribution and partial characterization of ten monoclonal antibodies raised against F9 TC cells. The data indicate that three developmentally regulated antigens are defined by these antibodies.
Cells" The origin, properties and culture conditions have been described (Dr/~ber et al., 1980; Drfiber and Stanley, 1984a).
Preparation of monoclonal antibodies B A L B / c mice were immunized intraperitoneally by 8 weekly injections (10 7 cells per dose) of irradiated F9 cells. The last injection was given intravenously 3 days before fusion. Spleen cells from the mouse with the highest titer of anti-F9 antibodies in the serum were fused with the P3X63-Ag8.653 mouse myeloma cells. Fusion, isolation of hybridoma cells, their cloning and production, and characterization of the antibodies were performed by standard procedures as described
0045-6039/84/$03.00 ':~ 1984 Elsevier Scientific Publishers Ireland, Ltd.
previously (Drhber et al., 1980). The antibodies contained in ascitic fluid were purified by ammonium sulfate precipitation and gel exclusion chromatography.
lmmunoassays Direct and indirect binding radioimmunoassays (RIAs) were performed as described (Drfiber and Stanley, 1984b). In an indirect immunofluorescence assay, the fluorescein isothiocyanate-labeled swine anti-mouse IgM was used as a second antibody. Carbohydrate-binding specificity of TEC-01 antibody was determined in a solid phase RIA in which human saliva served as a source of the antigen for immobilization (P. Drfiber and Z. Pokornfi, in preparation).
Results Monoclonal antibodies which are listed in Table I have been selected for their binding to teratocarcinoma F9 cells but not to 1 2 9 / S v mouse thymocytes. All of them are of IgM class with kappa light chain. Extensive testing against a panel of mouse cell lines has shown that these antibodies can be divided into three groups. TEC-01 antibody binds to an antigen, which was provisionally termed TEC-1 and which is expressed on TC stem cells but not on PYS-2 cells; T-04-T-07 antibodies exhibit reactivity similar to TEC-01. TEC-02 antibody binds to an antigen TEC-2, which is expressed strongly on PYS-2 cells and less on TC stem cells; an antigen with a similar distribution is also detected by T-08, T-09, and T-010 antibodies. TEC-03 antibody binds to an antigen TEC-3, which is expressed on both TC stem cells and PYS-2 cells. The reactivity of TEC-01, TEC-02, and TEC-03 antibodies with mouse adult tissues was tested by absorption. As shown in Table II, TEC-01 antibody was absorbed by the brain and kidney, and TEC-03 antibody by the lungs. Other combinations of antibodies and tissues (including heart, skeletal muscle, liver, spleen, thymus and testis; data not shown) did not give significant absorption. An analysis of the expression of TEC-1, -2,
Reactivity with cell lines Antibody
TEC-01 TEC-02 TEC-03 T-04 T-05 T-06 T-07 T-08 T-09 T-010
Binding (cpm × 10-2)
to the cells
19 11 50 27 23 27 25 8 11 12
54 9 35 44 32 44 45 2 3 8
0 42 6 0 0 0 0 80 38 3O
24 5 7 27 18 29 42 1 4 5
a Indirect binding RIA using ascitic fluid from hybridomabearing mice (dilution 1:10000). Results are the average of triplicate samples with cpm of normal mouse serum controls subtracted (200-300 cpm).
Reactivity with adult mouse tissues Absorption a
None Brain Lung
Binding (cpm × 10 2) to F9 cells TEC-01
30 1 27 0
37 33 31 32
27 24 5 21
a Equal volumes of packed cells and ascitic fluid (dilution 1 : 10000) were mixed and incubated 2 h at 4°C. The residual
antibody activity was tested in an indirect RIA. The results are the average of triplicate samples with background (300 cpm) subtracted.
Structure of the synthetic oligosaccharides used to determine the sugar specificity of TEC-01 antibody Trivial name
]~-D-GaI(1 ---, 4)- D-GlcNAc 713 a-L-Fuc fl-D-Gal (1 --~ 4) -D-GIcNAc
tl-2 a-L-Fuc H type 2
fl-D-Gal(l -~ 4)-D-GIcNAc T1-2
Fig. 1. Reactivity with unfertilized eggs and blastocysts from 129/Sv mice. TEC-02 on an unfertilized egg with (a,b) or without (c) the zona pellucida and early (d) or late (e,f) blastocyst; TEC-01 on early blastocyst (g): TEC-03 on early blastocyst (h). Fluorescence (a,c,d,f h) and phase contrast (b,e) photomicrographs. Bars, 50 p,m.
a n d -3 antigens on h u m a n cells has shown that TEC-1 antigen is expressed on peripheral blood granulocytes, sperm a n d myetoid leukemia cell line K 562. It is absent from peripheral blood lymphocytes and erythrocytes a n d T leukemia cell line MOLT-4. T E C - 2 a n d TEC-3 antigens are not
expressed on any of these cells and cell lines. TEC-1 is also present in h u m a n saliva (data not shown). The reactivity of the antibodies with mouse unfertilized eggs a n d blastocysts has been tested by an indirect i m m u n o f l u o r e s c e n c e assay (Fig. 1).
Percent inhibition 100 80
1 10 100 1000 ()JM) Oligosaccharide concentration Fig. 2. Sugar hapten inhibition of TEC-01 antibody binding to h u m a n saliva. Control binding (no inhibition) was about 4000 cpm. For the structure of X, Y and H type 2 oligosaccharides see Table III.
TEC-01 and TEC-03 antibodies react only with blastocysts, whereas TEC-02 antibody also reacts with unfertilized eggs including the zona pellucida. The binding of TEC-01 antibody to human saliva was inhibited by a synthetically prepared oligosaccharide X hapten, but not by Y or H type 2 hapten (Fig. 2); their structure is shown in Table III. Neither of these oligosaccharides nor simple sugars at concentrations up to 0.1 M could inhibit the binding of TEC-02 or TEC-03 antibodies to the target antigen.
Discussion A set of ten monoclonal antibodies described here define three developmentally regulated antigens. TEC-01 and four other antibodies recognize an antigen whose distribution is similar to SSEA-1 (Solter and Knowles, 1978) and ECMA-7 (Kemler, 1980). In addition, the binding of two of these antibodies (TEC-01 and anti-SSEA-1) to the target antigen is specifically inhibited by the same oligosaccharide galactose(~81 ---, 4)fucose(cd ~ 3)-Nacetylglucosamine (Fig. 2, Gooi et al., 1981). Competition experiments and analysis of the binding of
these antibodies to the Chinese hamster ovary cell mutants L E C l l and LEC12, which express different amounts of SSEA-1 (Campbell and Stanley, 1983) have shown, however, that the reaction patterns of these antibodies are not entirely identical (P. Drfiber, unpublished data). Such differences in fine specificity are a common feature of monoclonal antibodies against the same carbohydrate antigen (Gooi et al., 1983) and explain subtle differences in tissue specificity among anti-SSEAl-like antibodies (Gooi et al., 1983; Sato et al., 1983). TEC-02 and three other antibodies with similar binding properties and TEC-03 antibody define TEC-2 and TEC-3 antigens, respectively. Comparison of the distribution of these antigens with other antigens previously described as being expressed on murine embryonic cells and TC stem cell lines indicates that TEC-2 and TEC-3 are new stage-specific embryonic antigens. The biochemical nature of these antigens is not known but our unpublished data suggest that TEC-2 antigen is located in the carbohydrate portion of surface glycoconjugates. These monoclonal antibodies can be useful not only for tracing positive cells during differentiation in vitro or in vivo, but also for studying the biosynthesis and developmental aspects of the corresponding antigens. An example is the use of TEC-01 antibody conjugated to a toxic plant lectin ricin for a single-step selection of TC stem cell mutants from which TEC-1 antigen was selectively removed (Dr~ber and Vojtigkovfi, 1984).
Acknowledgements We thank Dr. F. Fran6k for kindly providing swine anti-mouse IgM, and Dr. P. Sinay for synthetic oligosaccharides.
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