lewis-x-antigen and Teratoma

Embryonal carcinoma (EC) cells, the malignant stem cells of teratocarcinomas, resemble early embryonic cells morphologically, developmentally, and with respect to several cell surface characteristics. EC cells often differentiate into a variety of cell types when treated with chemical agents such as retinoic acid, or when placed in a normal embryonic environment. Developmentally regulated surface antigens of EC cells and their differentiated derivatives have been defined using monoclonal antibodies. Many of these "differentiation antigens" have proved to be oligosaccharide chains carried on membrane glycolipids and/or glycoproteins. Our analyses of glycolipid composition in a pluripotent human EC cell line, NTERA-2, have revealed a complex set of glycoslylation changes that occur during cellular differentiation. Like early embryos, undifferentiated NTERA-2 EC cells express predominantly globo-series glycolipids. However, once differentiation is initiated by addition of retinoic acid there is a marked shift of cellular glycolipids from globo-series to lacto- and ganglio-series. Distinct subsets of differentiated cells are characterized by their expression of different patterns of glycolipid antigens. These changes in cell surface phenotype may serve to mark cellular identity and facilitate morphogenetic cell interactions during embryonic development.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Carbohydrate Sequence; Cell Differentiation; Fluorescent Antibody Technique; Glycoconjugates; Glycolipids; Glycosylation; Humans; Lewis X Antigen; Mice; Molecular Sequence Data; Teratoma

Topics: Animals; Antibodies, Monoclonal; Drug Carriers; Drug Delivery Systems; Evaluation Studies as Topic; Humans; Lewis X Antigen; Methotrexate; Mice; Teratoma

Topics: alpha-Fetoproteins; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Dysgerminoma; Glycolipids; Humans; Lewis X Antigen; Mice; Teratoma

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; Carbohydrates; Embryo, Mammalian; Embryonic Induction; Forssman Antigen; Galactose; Glycolipids; Glycopeptides; Humans; Lewis X Antigen; Mice; Polysaccharides; Rats; Receptors, Mitogen; Teratoma

Various cell surface markers, such as SSEA-1, SSEA-3, Forssman, I, i, brushin, FT-1, PNA receptors, FBP receptors, and DBA receptors, are expressed in certain subpopulations of early embryonic cells. They are useful in monitoring the process of in vitro differentiation of embryonal carcinoma (EC) cells. For example, SSEA-1 is a marker of EC cells, whereas DBA receptors are markers of endoderm cells and quasi-nullipotent EC cells. An important carrier of the developmentally regulated cell surface markers is embryoglycan, which is a class of glycoprotein-bound large carbohydrates and has the lactosaminoglycan-type structure.

Topics: Antigens, Neoplasm; Antigens, Surface; Cell Transformation, Neoplastic; Glycolipids; Glycoproteins; Lewis X Antigen; Polysaccharides; Receptors, Mitogen; Teratoma

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Surface; Carbohydrate Sequence; Cell Differentiation; Clone Cells; Embryo, Mammalian; Epitopes; Forssman Antigen; Globosides; Glycolipids; Humans; Lewis X Antigen; Mice; Phenotype; Teratoma

Leptin, a metabolic hormone, regulates the reproductive functions responding to both nutritional and body conditions. Embryonic stem cells play important roles in reproductive technology, but their derivation can be challenging. In this study, we evaluated the derivation rates of mouse embryonic stem cell (mESC) line from blastocysts developing in embryo culture media supplemented with different leptin concentrations. The results showed that addition of leptin into the embryo culture medium supported the in vitro development of mouse embryo. The mESC line derivation rates for media treated with 0, 10, 50, and 100 ng/ml of leptin were 61.24 % (54/88), 84.96 % (42/50), 81.79 % (61/76), and 85.78 % (56/67), respectively. In addition, leptin treatment of blastocysts upregulated the expression levels of the trophectoderm marker Cdx2, whereas inner cell mass markers Oct-4 and Nanog were not affected. mESC lines derived after leptin treatment demonstrated hallmarks of pluripotency, such as alkaline phosphatase activity, expression of, OCT4, NANOG, and SSEA1, as well as the ability to form embryoid bodies and well-differentiated teratomas. In conclusion, leptin has a positive effect on the derivation rate of mouse embryonic stem cell lines which may be, in part, due to its effects on the development of the trophectoderm cell lineage in the embryo.

Topics: Animals; Blastocyst; CDX2 Transcription Factor; Cell Differentiation; Cell Line; Cell Lineage; Cell Proliferation; Culture Media; Embryo Culture Techniques; Embryoid Bodies; Leptin; Lewis X Antigen; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mouse Embryonic Stem Cells; Nanog Homeobox Protein; Octamer Transcription Factor-3; Teratoma

Reprogramming somatic cells into a pluripotent state involves the overexpression of transcription factors leading to a series of changes that end in the formation of induced pluripotent stem cells (iPSCs). These iPSCs have a wide range of potential uses from drug testing and in vitro disease modelling to personalized cell therapies for patients. While viral methods for reprogramming factor delivery have been traditionally preferred due to their high efficiency, it is now possible to generate iPSCs using nonviral methods at similar efficiencies. We developed a robust reprogramming strategy that combines episomal plasmids and the use of commercially available animal free reagents that can be easily adapted for the GMP manufacture of clinical grade cells.

Topics: Alkaline Phosphatase; Amides; Animals; Antigens, Surface; Cell Culture Techniques; Cell Differentiation; Cellular Reprogramming; Dermis; Fibroblasts; Gene Expression; Hermanski-Pudlak Syndrome; Humans; Induced Pluripotent Stem Cells; Intercellular Signaling Peptides and Proteins; Lewis X Antigen; Mice; Models, Biological; Octamer Transcription Factor-3; Plasmids; Pyridines; Serial Passage; Teratoma; Transgenes

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

Topics: Animals; Biomarkers; Bone Morphogenetic Protein 2; Cell Differentiation; Culture Media; Embryoid Bodies; Gene Expression; Induced Pluripotent Stem Cells; Karyotyping; Leukemia Inhibitory Factor; Leukemia Inhibitory Factor Receptor alpha Subunit; Lewis X Antigen; Mice; Mice, Knockout; Microarray Analysis; Myoblasts; Myostatin; Nanog Homeobox Protein; Octamer Transcription Factor-3; Primary Cell Culture; SOXB1 Transcription Factors; Teratoma

We recently discovered a novel population of stem cells from the injured murine skeletal muscle. These injury induced muscle-derived stem cell-like cells (iMuSCs) are partially reprogrammed from differentiated myogenic cells and display a pluripotent-like state. The iMuSCs exhibit stem cell properties including the ability to differentiate into multiple lineages, such as neurogenic and myogenic differentiations; they also display a superior migration capacity that demonstrating a strong ability of muscle engraftment in vivo. IMuSCs express several pluripotent and myogenic stem cell markers; have the capability to form embryoid bodies and teratomas, and can differentiate into all three germ layers. Moreover, blastocyst microinjection showed that the iMuSCs contributed to chimeric embryos but could not complete germline transmission. Our results indicate that the iMuSCs are in a partially reprogrammed state of pluripotency, which are generated by the microenvironment of injured skeletal muscle.

Topics: Animals; Biomarkers; Blastocyst; Cell Differentiation; Cell Movement; Cellular Reprogramming; Embryo, Mammalian; Embryoid Bodies; Gene Expression; Germ Layers; Lewis X Antigen; Mice; Mice, Inbred C57BL; Microinjections; MSX1 Transcription Factor; Muscle, Skeletal; Myoblasts, Skeletal; Octamer Transcription Factor-3; Pluripotent Stem Cells; Receptors, CXCR4; SOXB1 Transcription Factors; Teratoma

Induced pluripotent stem (iPS) cells have great potential for personalized regenerative medicine. Although several different methods for generating iPS cells have been reported, improvement of safety and efficiency is imperative. In this study, we tested the feasibility of using a triple tyrosine mutant AAV2 (Y444+500+730F) vector, designated AAV2.3m, to generate iPS cells. We developed a polycistronic rAAV2.3m vector expressing three reprogramming factors, Klf4, Oct4, and Sox2, and then used this vector to infect mouse adipose-derived mesenchymal stem cells (AT-MSCs) to induce the generation of iPS cells. We demonstrated that (1) the triple tyrosine mutant AAV2 vector is able to reprogram mouse adult adipose tissue-derived stem cells into the pluripotent state. Those rAAV2.3m-derived iPS (rAAV2.3m-iPS) cells express endogenous pluripotency-associated genes including Oct4, Sox2, and SSEA-1, and form teratomas containing multiple tissues in vivo; (2) c-myc, an oncogene, is dispensable in rAAV2.3m-mediated cellular reprogramming; and (3) transgene expression is undetectable after reprogramming, whereas vector DNA is detectable, indicating that transgenes are silenced. These results indicated the rAAV vector may have some advantages in generating iPS cells.

Topics: Adipose Tissue; Animals; Cellular Reprogramming; Dependovirus; Genetic Vectors; Humans; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lewis X Antigen; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Transgenic; Octamer Transcription Factor-3; Pluripotent Stem Cells; Proto-Oncogene Proteins c-myc; SOXB1 Transcription Factors; Teratoma; Transduction, Genetic

Embryonic stem cell (ESC) derivatives offer promise for generating clinically useful tissues for transplantation, yet the specter of producing tumors in patients remains a significant concern. We have developed a simple method that eliminates the tumorigenic potential from differentiated ESC cultures of murine and human origin while purifying lineage-restricted, definitive endoderm-committed cells. A three-stage scheme utilizing magnetic bead sorting and specific antibodies to remove undifferentiated ESCs and extraembryonic endoderm cells, followed by positive selection of definitive endoderm cells on the basis of epithelial cell adhesion molecule (EpCAM) expression, was used to isolate a population of EpCAM(+)SSEA1(-)SSEA3(-) cells. Sorted cells do not form teratomas after transplantation into immunodeficient mice, but display gene and protein expression profiles indicative of definitive endoderm cells. Sorted cells could be subsequently expanded in vitro and further differentiated to express key pancreas specification proteins. In vivo transplantation of sorted cells resulted in small, benign tissues that uniformly express PDX1. These studies describe a straightforward method without genetic manipulation that eliminates the risk of teratoma formation from ESC differentiated derivatives. Significantly, enriched populations isolated by this method appear to be lineage-restricted definitive endoderm cells with limited proliferation capacity.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Cell Differentiation; Cell Lineage; Cell Separation; Cells, Cultured; Embryonic Stem Cells; Endoderm; Gastrointestinal Tract; Homeodomain Proteins; Humans; Lewis X Antigen; Male; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Stage-Specific Embryonic Antigens; Teratoma; Trans-Activators

Studies have shown that male germline stem cells (mGSCs), which are responsible for maintaining spermatogenesis in the male, could be obtained from mouse and human testis. However, the traditional cultural methods were mostly dependent on serum and feeder, and the initial mGSCs were either obtained from neonatal mice or the detailed description of its potency and origin was not provided. Here we reported a novel (retinol (RE) serum-free and feeder-free) system for the successful culture of adult germline stem cells from adult Kunming mice (8-24 weeks) testis. The isolated mGSCs cultured in RE serum-free and feeder-free medium maintained the typical morphology of undifferentiated embryonic stem cells (ESCs), and they proliferated well in RE medium analyzed by proliferation assay, RT-PCR, microarray, and Western blotting. These cells also showed typical properties of ESCs (alkaline phosphatase (AP) positive, expressions of Oct4, Sox2, Nanog, and SSEA1, with the capacity to form teratomas and differentiate into various types of cells within three germ layers). Taken together, we conclude that RE promotes the self-renewal of mGSCs and maintains the pluripotency of mGSCs, the RE serum-free and feeder-free system may be useful for the culture of pluripotent stem cell lines from adult testis tissues, which provides a new resource for tissue engineering and therapy for infertility.

Topics: Alkaline Phosphatase; Animals; Blotting, Western; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cells, Cultured; Culture Media, Serum-Free; Glial Cell Line-Derived Neurotrophic Factor; Homeodomain Proteins; Humans; Lewis X Antigen; Male; Mice; Myocytes, Cardiac; Nanog Homeobox Protein; Neurons; Octamer Transcription Factor-3; Pluripotent Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; SOXB1 Transcription Factors; Spermatozoa; Teratoma; Testis; Vitamin A; Vitamins

Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming through the ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. However, it is unclear whether postmitotic neurons are susceptible to direct reprogramming. Here, we show that postnatal cortical neurons, the vast majority of which are postmitotic, are amenable to epigenetic reprogramming. However, ectopic expression of the four canonical reprogramming factors is not sufficient to reprogram postnatal neurons. Efficient reprogramming was only achieved after forced cell proliferation by p53 suppression. Additionally, overexpression of repressor element-1 silencing transcription, a suppressor of neuronal gene activity, increased reprogramming efficiencies in combination with the reprogramming factors. Our findings indicate that terminally differentiated postnatal neurons are able to acquire the pluripotent state by direct epigenetic reprogramming, and this process is made more efficient through the suppression of lineage specific gene expression.

Topics: Animals; Animals, Newborn; Biomarkers; Blastocyst; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cells, Cultured; Coculture Techniques; Embryo Transfer; Fibroblasts; Genes, Reporter; Green Fluorescent Proteins; Homeodomain Proteins; Induced Pluripotent Stem Cells; Kruppel-Like Factor 4; Lewis X Antigen; Mice; Nanog Homeobox Protein; Neurons; Octamer Transcription Factor-3; Promoter Regions, Genetic; Repressor Proteins; Teratoma; Transplantation Chimera

Embryonic stem cells (ESC) have the unique ability to differentiate into a variety of tissue types. However, the realization of regenerative medicine will require the production of large quantities of ESC which subsequently have to be differentiated into the final phenotype. Thus, we sought to develop a simple and scaleable bioprocess to increase densities of ESC to achieve this goal. Using mouse embryonic stem cells (mESC) as a model, by combining automated feeding and culture of mESC on petriperm dishes, cell densities were enhanced up to 6.4 x 10(6) cells/cm2 compared to conventional petri dish culture which only reached 0.2 to 1.4 x 10(6) cells/cm2. It was found that mESC from all experiments maintained excellent viability, pluripotency, and genetic stability after growing for 6 days in petriperm cultures with automated feeding. The expression of Oct-4 transcription factor was observed in all cultures, mESC formed embryoid bodies in differentiated cultures and teratomas in SCID mice, confirming their pluripotency, and karyotype of the cultures was normal. This culture method was stable for routine passaging and a second mESC cell line was shown to perform in a similar manner on petriperm with automated feeding. This work represents an important step towards achieving high density cultures of ESC.

Topics: Alkaline Phosphatase; Animals; Biomarkers; Bioreactors; Cell Culture Techniques; Cell Cycle; Cell Differentiation; Cell Line; Cell Survival; Cells, Cultured; DNA-Binding Proteins; Embryo, Mammalian; Flow Cytometry; Fluorescent Dyes; Indoles; Karyotyping; Lewis X Antigen; Male; Mice; Mice, SCID; Octamer Transcription Factor-3; Pluripotent Stem Cells; Stem Cell Transplantation; Stem Cells; Teratoma; Transcription Factors

Previous reports have indicated that extracellular matrices (ECMs) affect the developmental fate of human embryonic stem cells (hESCs). Specially, type IV collagen and laminin, which belong to a group of macromolecular proteins with a substantial proportion of ECMs, are known to influence the proliferation and differentiation of hES cells. In this study, we evaluated the effects of type IV collagen and laminin in freezing medium on the survival and differentiation rates of hES cells after slow freezing and rapid thawing. The addition of type IV collagen (1 microg/ml) to the freezing medium significantly increased the survival rate of hES cells after thawing compared with that of a control group. The spontaneous differentiation rates of groups treated with type IV collagen (1 microg/ml) or laminin (1 microg/ml) were significantly lower than those of the control group. Frozen-thawed hES cells have currently been cultured for more than 70 passages and retain key properties of hES cells such as morphological characteristics, normal karyotype, marker expression (alkaline phosphatase, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Rex-1, and Oct-4), basement membrane-related gene expression, and the potential to differentiate into derivatives of all three germ layers. This new slow freezing method by ECM treatment is a reliable and effective cryopreservation method for pluripotent hES cells.

Topics: Alkaline Phosphatase; Animals; Antigens, Surface; Antigens, Tumor-Associated, Carbohydrate; Basement Membrane; Cell Culture Techniques; Cell Differentiation; Cell Line; Cell Proliferation; Cell Separation; Cell Survival; Cell Transplantation; Collagen Type IV; Cryopreservation; Culture Media; DNA-Binding Proteins; Embryo, Mammalian; Flow Cytometry; Gene Expression; Glycoproteins; Glycosphingolipids; Guanine Nucleotide Exchange Factors; Humans; Karyotyping; Laminin; Lewis X Antigen; Mice; Mice, SCID; Octamer Transcription Factor-3; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; Stage-Specific Embryonic Antigens; Stem Cells; Temperature; Teratoma; Time Factors; Transcription Factors

A 27-year-old man complaining of cough was admitted to our hospital because of a giant mediastinal tumor on the chest radiograph. Chest CT and MRI revealed a giant polycystic mediastinal tumor. Chest radiographs on admission showed left pleural effusion due to perforation of the cyst. Laboratory data showed high serum and pleural fluid concentrations of CA 125, CA 19-9, SLX and others. The mediastinal mass was resected and diagnosed pathologically as a mature teratoma. It is reported that patients with mediastinal teratomas often have pleural fluid as a result of self-digestion by pancreatic enzymes, and some mediastinal teratomas have high serum tumor marker levels. We suspected that the high serum tumor marker levels in our case were caused by the high concentrations of tumor markers in the pleural fluid. We suggest that serum tumor marker levels may be useful in the preoperative differential diagnosis of anterior mediastinal cystic tumors.

Topics: Adult; Biomarkers, Tumor; CA-125 Antigen; CA-19-9 Antigen; Humans; Lewis X Antigen; Male; Mediastinal Neoplasms; Teratoma

We previously proposed specific interaction of Le(x) (Gal beta 1-->4 [Fuc alpha 1-->3]-GlcNAc beta 1-->3Gal) with Le(x) as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggens et al., J. Biol Chem (1989) 264:9476-9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le(x) or other epitopes, and affinity chromatography on Le(x)-columns of multivalent lactofucopentaose III (Le(x) oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le(x)-expressing tumour cells vs their Le(x)-non-expressing variants showed that only Le(x)-expressing cells adhere to Le(x)-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le(x) determinant in F9 cells is not GSL but rather polylactosaminoglycan ('embryoglycan'), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca2+, and reversible dissociation in the absence of Ca2+ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le(x)-Le(x) interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le(x)-Le(x) interaction based on minimum energy conformation with involvement of Ca2+ is presented.

Topics: Carbohydrate Sequence; Cell Adhesion; Dipeptides; Glycosphingolipids; Lewis X Antigen; Liposomes; Models, Molecular; Molecular Sequence Data; Nephelometry and Turbidimetry; Polysaccharides; Teratoma; Tumor Cells, Cultured

The glycolipids of human teratocarcinoma-derived cell line NCCIT were compared with those of 5 murine teratocarcinoma-derived cell lines. Glycolipid antigens were identified by cell surface immunofluorescence and high-performance thin-layer chromatography (HPTLC) immunostaining with a panel of monoclonal anti-carbohydrate antibodies. Human NCCIT embryonal carcinoma (EC) cells contained extended globo-series glycolipids Gb5 (galactosyl globoside) and GL7 (sialyl galactosyl globoside) recognized by antibodies to stage-specific embryonic antigens 3 and 4 (SSEA-3 and -4). SSEA-4 was not detected by immunofluorescence on the surface of any of the 5 murine teratocarcinoma-derived cell lines examined; however, SSEA-3 was detected on the surface of murine cell lines resembling primitive endoderm (JC44, NF-PE) and trophectoderm (E6496D). HPTLC analysis revealed a large amount of globoside (Gb4) in these differentiated cells, which may account for their labeling with anti-SSEA-3 antibody. Globo-series glycolipids were also detected in murine EC cells; however, differences were noted between the 2 cell lines examined. F9 cells contained primarily Gb4 and Forssman glycolipid, whereas NF-1 cells contained only minor amounts of Gb4 and lacked Forssman glycolipid entirely. Our results, coupled with the known distribution of Forssman antigen in the egg cylinder-stage mouse embryo, suggest that F9 and NF-1 murine EC cells are replicas of cells at different stages of development of the embryonic ectoderm. Glycolipids of normal mouse embryos were examined for comparison. Gb4 and Forssman glycolipid were presents in both embryonic and extra-embryonic tissues, whereas Gb5 and GL7 were restricted to visceral yolk sac and placenta. Our results demonstrate that human and murine teratocarcinoma-derived cells both synthesize extended globo-series glycolipids; however, oligosaccharide chain elongation takes different pathways in the 2 species. These differences reflect species-related and cell type-specific patterns of glycosylation.

Topics: Animals; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Chromatography, High Pressure Liquid; Fluorescent Antibody Technique; Forssman Antigen; Globosides; Glycolipids; Glycosphingolipids; Humans; Lewis X Antigen; Mice; Stage-Specific Embryonic Antigens; Teratoma; Tumor Cells, Cultured

A 38-year-old man was admitted with persistent productive cough and right anterior chest pain. Chest X-ray showed two large masses connected with each other, one in the right lung field and the other in the anterior mediastinum. A tentative diagnosis of either lung abscess or bronchogenic carcinoma was initially made, because of elevated serum tumor markers (SLX and SCC) and persisting refractory inflammatory sings. However, open chest drainage revealed a few fine hairs and atheromatous materials within the masses, and the diagnosis of teratoma was made. We removed these masses, and investigated the reason for the elevation of tumor markers. Staining with SLX monoclonal antibody demonstrated that the pancreatic tissue in the masses contained SLX. Although this is the first reported case of teratoma producing tumor marker (SLX), it is highly possible that tumor markers may be elevated in the majority of patients with teratoma because of the genesis of this tumor.

Topics: Adult; Carcinoma, Bronchogenic; Diagnosis, Differential; Humans; Lewis X Antigen; Lung Abscess; Lung Neoplasms; Male; Mediastinal Neoplasms; Teratoma

Differentiation of Tera-2 human embryonal carcinoma cells by exposure to 2.1 mM alpha-difluoromethylornithine resulted in changes in morphology, a decrease in growth rate, and changes in the expression of SSEA-1 differentiation antigen. While the binding of 125I-insulin-like growth factor I (IGF-I) remained relatively constant during differentiation, binding of 125I-IGF-II increased 2-3-fold. Further, the binding of IGF-II was 87 times greater than IGF-I in both undifferentiated and differentiated cells. Undifferentiated Tera-2 cells exhibited a single class of binding sites for both IGF-I (KD = 1.2 nM, 7.0 x 10(3) sites/cell) and IGF-II (KD = 8.3 nM, 3.4 x 10(5) sites/cell). Following differentiation, IGF-I continued to bind to a single class of binding sites (KD 1.0 nM, 6.7 x 10(3) sites/cell) whereas IGF-II bound to both high-affinity sites (KDH 0.3 nM, 2.2 x 10(5) sites/cell) and low-affinity sites (KDL 15.1 nM, 1.6 x 10(7) sites/cell). The binding of iodinated IGF-II was blocked by unlabeled IGF-II but not IGF-I. In contrast, 125I-IGF-I binding was prevented by either IGF-I or IGF-II. Affinity cross-linking experiments demonstrated the presence of both type I and type II IGF receptors along with a number of IGF binding proteins. IGF-I failed to stimulate the incorporation of [3H]thymidine in both undifferentiated and differentiated cells. Although IGF-II caused a significant increase in [3H]thymidine incorporation in both undifferentiated and differentiated Tera-2 cells, the magnitude of the response and the sensitivity of the cells to IGF-II stimulation was diminished following differentiation. The observed changes in IGF-II binding, which occur in conjunction with cellular differentiation, may be an important feature of the expression of the differentiated phenotype by human germ cell tumors.

Topics: Binding Sites; Binding, Competitive; Cell Differentiation; Cell Division; Cell Line; DNA Replication; Eflornithine; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Kinetics; Lewis X Antigen; Recombinant Proteins; Teratoma; Thymidine

To investigate the possible involvement of topoisomerases in embryonal differentiation, we examined the effect of topoisomerase inhibitors on the in vitro differentiation of mouse embryonal carcinoma F9 cells. We found that camptothecin, teniposide (VM-26), or genistein, specific inhibitors of topoisomerases, induced morphological as well as biochemical changes (production of tissue plasminogen activator, synthesis of laminin, and disappearance of stage-specific embryonic antigen 1) specific to F9 cell differentiation. Since these changes were indistinguishable from those observed in F9 differentiation induced by retinoic acid (plus dibutyryl cyclic AMP), it was suggested that inhibition of cellular topoisomerase activities triggered F9 cell differentiation into parietal endoderm-like cells in the same manner as retinoic acid (plus dibutyryl cyclic AMP). Experiments using differentiation-resistant mutant F9 cell lines, however, indicated that the molecular cascade involved in topoisomerase inhibitor-induced differentiation involves different steps from those functioning in the retinoic acid-induced differentiation cascade.

Topics: Animals; Blotting, Western; Camptothecin; Cell Differentiation; Cell Line; DNA Topoisomerases, Type I; Dose-Response Relationship, Drug; Genistein; In Vitro Techniques; Isoflavones; Laminin; Lewis X Antigen; Mice; Protein-Tyrosine Kinases; Teniposide; Teratoma; Tissue Plasminogen Activator; Topoisomerase I Inhibitors; Tretinoin

A pluripotent human EC cell line (NEC14) could be induced to morphologically differentiate by treatment with 10(-2) M HMBA for 3 days in vitro. The changes in various differentiation-related markers (cell surface antigens, lectin binding sites, intermediate filaments, secreted products and extracellular matrix proteins) after induction of differentiation were examined in order to clarify the differentiation lineage. The results were as follows: 1) The most conspicuous changes in cell surface antigens after differentiation were the expression of major human histocompatibility antigens (HLA-A,B,C) and the changes in stage specific embryonic antigens (SSEA-1-/SSEA-3(+)----SSEA-1+/SSEA-3-). 2) Vimentin, mesenchymal intermediate filament, was only detected after the differentiation. 3) Tenascin, an extracellular matrix protein produced in mesenchymal cells, was produced after the differentiation. These results indicate that HMBA can induce NEC14 cells to differentiate into mesenchymal elements of embryonal mesoderm.

Topics: Acetamides; Antibodies, Monoclonal; Antigens, Surface; Binding Sites; Cell Differentiation; HLA Antigens; Humans; Intermediate Filaments; Lectins; Lewis X Antigen; Teratoma; Tumor Cells, Cultured

P19 embryonal carcinoma cells can be induced to differentiate with a pulse of only 4 hr in retinoic acid (RA). The efficiency of RA-induced differentiation is independent of the position of P19 cells in the cell cycle but is critically dependent on the ratio between the number of cells and the number of moles of RA in the culture medium. P19 cultures at lower cell density are more efficiently induced to differentiate than cultures containing cells at higher cell densities. This effect is not mediated by cell-to-cell contact but may be related to the rapid metabolism of RA by the cells. Individual clones of differentiating P19 cells can develop into at least three different cell types suggesting that each cell in the population of embryonal carcinoma cells retains pluripotency.

Topics: Antigens, Differentiation; Antigens, Surface; CD57 Antigens; Cell Cycle; Cell Differentiation; Contact Inhibition; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Glycolipids; In Vitro Techniques; Lewis X Antigen; Neurons; Teratoma; Tretinoin; Tumor Cells, Cultured

F9 teratocarcinoma stem cells differentiate into parietal endoderm-like cells when given retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (DB-cAMP). It is generally accepted that the stem cells are resistant to the action of cAMP alone and need to be primed by RA in order to respond to cAMP. In this report, we demonstrate that F9 stem cells differentiate into parietal endoderm-like cells in the absence of exogenous RA when treated with cholera toxin and 1-methyl,3-isobutyl xanthine (CT/MIX) or 8-bromo-cAMP/MIX (8B2-cAMP/MIX). Cells treated with CT/MIX or 8B2-cAMP/MIX were morphologically similar to parietal endoderm-like cells, produced high amounts of plasminogen activator, and synthesized both type IV collagen and laminin mRNA. Conversely, markers made in abundance by stem cells such as stage-specific embryonic antigen (SSEA-1) and an mRNA species of 6.8 kb (pST6-135) were markedly reduced in CT/MIX-treated cells. To prove that cAMP alone could induce differentiation Lipidex-1000, a hydrophobic gel, was used to remove 80-90% of the endogenous serum retinoids. F9 cells grown in this retinoid-depleted serum and treated with 8B2-cAMP/MIX differentiated to parietal endoderm-like cells as shown by both dramatic changes in morphology and induction of type IV collagen mRNA. Our results indicate that the differentiation of F9 to parietal endoderm-like cells can be induced by increased intracellular cAMP and is not strictly dependent on the addition of RA.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Cell Differentiation; Collagen; Cyclic AMP; Fluorescent Antibody Technique; Glycolipids; Humans; Laminin; Lewis X Antigen; RNA, Messenger; Teratoma; Tissue Plasminogen Activator; Tretinoin

The level of c-myc expression rapidly decreases during in vitro induced differentiation of mouse F9 embryonic teratocarcinoma to endoderm cells, raising the question of whether down regulation of c-myc expression is a part of the mechanism regulating differentiation. We have investigated the effect of enforced increases or decreases in c-myc RNA expression in F9 cells on growth and differentiation. The enforced expression of c-myc RNA in clones transfected with sense c-myc did not inhibit their terminal differentiation. Dramatically decreased c-myc RNA expression in antisense c-myc transfected clones also did not substantially alter the differentiation pathway, although the transformed cells withdraw from the cell cycle slightly earlier than control cells during the differentiation induction. These results suggest that the mechanism controlling differentiation operates independently of the level of c-myc RNA expression in F9 teratocarcinoma cells.

Topics: Animals; Cell Differentiation; Cell Division; Cloning, Molecular; Collagen; Gene Expression; Glycolipids; Lewis X Antigen; Mice; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Proto-Oncogenes; RNA; RNA, Antisense; RNA, Messenger; Teratoma

A scheme has been designed to synthesize a homologous series of new bifunctional chelating agents (BFCAs), which may increase the thermodynamic stability of metal chelates and conjugate at the specific sites on the monoclonal antibody molecule (MoAb) permitting us to analyze the structure-activity relationships of the series of compounds. Four such compounds have been prepared and characterized by FT-i.r. and NMR spectroscopy. One of them has been used to label an antibody with 111In, the stability and distribution of which has been examined in tumor-bearing mice and compared to that of the 111In-MoAb prepared using cyclic anhydride of DTPA. Enhanced tumor/blood ratios (9 vs 6.5), tumour to muscle ratios (7 vs 3), and decreased liver uptake (4 vs 12%) have been obtained.

Topics: Animals; Antibodies, Monoclonal; Chelating Agents; Glycolipids; Indium Radioisotopes; Isotope Labeling; Lewis X Antigen; Mice; Mice, Inbred BALB C; Radionuclide Imaging; Teratoma

Differentiation of the F9 cell line was induced by treating the cells with retinoic acid (10(-6) M) and dibutyryl cycloadenosinemonophosphate (10(-4) M). The population doubling time and the portion of cells in G1-phase increase and saturation density falls as the result of this treatment. Differentiated F9 cells demonstrate a decreased capacity of forming colonies in the soft agar, lose their capacity of proliferating at the clonal density, and acquire the limited life-span in culture after reseeding at a high density. Some cells in the differentiated population retain their capacity of forming colonies in the soft agar and (or) of binding antibodies against the stem cell marker SSEA-1. Cells with the stem cell morphology were found in the course of passaging of differentiated cells after reseeding at a high density. These cells were able to differentiate after the standard procedure of the induction of differentiation with retinoic acid and dibutyryl cAMP. Causes of the rising and supporting of heterogeneity of the differentiated F9 cells are discussed.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Surface; Biomarkers, Tumor; Bucladesine; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Glycolipids; Lewis X Antigen; Mice; Phenotype; Teratoma; Tretinoin; Tumor Cells, Cultured

A possible role of cell surface glycoconjugates in cell recognition has been envisioned based on recognition of carbohydrates by cell surface proteins such as endogenous lectins, glycosyltransferases, and hydrolases (refs. 18-22 in text). A new possibility that a specific carbohydrate at the cell surface could be recognized by the same or similar carbohydrate on the counterpart cell surface is now suggested by specific interaction between Lex and Lex, but not between Lex and sialylated or non-substituted type 2 chain. A new hypothesis is hereby proposed for carbohydrate-carbohydrate interactions as recognition signals during embryogenesis and organogenesis.

Topics: Antigens, Surface; Calcium; Carbohydrate Metabolism; Cell Aggregation; Embryonic and Fetal Development; Glycolipids; Lactose; Lewis X Antigen; Liposomes; Magnesium; Polysaccharides; Teratoma; Tumor Cells, Cultured

Cell surface expression of stage specific embryonic antigen 1 (SSEA-1), or Lex (III3 FucnLC4), was induced in differentiated human teratocarcinoma cells and in human diploid fibroblasts 3-6 d after infection with human cytomegalovirus (HCMV). In parallel, fucosylated lactoseries glycolipids bearing the SSEA-1/Lex epitope were readily detected in the infected cells but not in the uninfected cells. HCMV infection also results in altered expression of several glycosyltransferases. SSEA-1/Lex induction is probably a consequence of both increased expression of beta 1----3N-acetylglucosaminyltransferase, which catalyzes the rate-limiting step in lactoseries core chain synthesis, and subtle alterations in the relative competition for common precursor structures at key points in the biosynthetic pathway. Since SSEA-1 has been suggested to play a role in some morphogenetic cell-cell interactions during embryonic development, the induction of this antigen at inappropriate times might provide one mechanism whereby intrauterine infection with HCMV can damage the developing fetal nervous system.

Topics: Antigens, Viral; Cell Differentiation; Cells, Cultured; Cytomegalovirus; Fibroblasts; Glycolipids; Hexosyltransferases; Humans; In Vitro Techniques; Lewis X Antigen; Membrane Lipids; Teratoma

Methotrexate (MTX) was coupled to an IgM monoclonal antibody specific for stage-specific embryonic antigen-1 (SSEA-1), and the resulting immunoconjugate (MTX-anti-SSEA-1) was used for in vivo drug targeting in mice bearing MH-15 teratocarcinoma. Immunoconjugates having an average of 65 mol MTX/mol antibody retained full antigen-binding capacity. Mice bearing well-established tumors (approx. 1 g) were treated i.v. using the immunoconjugate. MTX-anti-SSEA-1 at 15 mg/kg of drug had significant antitumor activity with no significant systemic toxicity. Neither an irrelevant isotype-matched conjugate, MTX-MOPC-104E, prepared from the MOPC 104E myeloma protein, nor free MTX injected alone or with either antibody had any significant antitumor effect. These results indicate that IgMs can be effective drug carriers for tumor targeting in spite of their high molecular mass, and that antigens that are selectively accessible in tumors, even though present in normal tissues, can be suitable targets for in vivo chemoimmunotherapy.

Topics: Antibodies, Monoclonal; Antigen-Antibody Reactions; Antigens, Neoplasm; Glycolipids; Immunoglobulin M; Immunotherapy; Immunotoxins; Lewis X Antigen; Methotrexate; Teratoma

The 12S gene of the E1A region of adenovirus induces the production of a growth factor that is necessary for the immortalization of primary epithelial cells. This growth factor is able to replace the strict serum requirement of F9 cells, a teratocarcinoma-derived embryonal carcinoma cell line. Furthermore, the growth factor induces a morphological alteration in the F9 cells such that they resemble visceral endoderm cells and express the surface antigens, SSEA-1 and SSEA-3, characteristic of these differentiated cells.

Topics: Adenoviruses, Human; Animals; Antigens, Surface; Cell Differentiation; Cell Division; Fluorescent Antibody Technique; Glycolipids; Growth Substances; In Vitro Techniques; Lewis X Antigen; Mice; Teratoma; Tumor Cells, Cultured

Embryonal carcinoma(EC) cells, the undifferentiated stem cells of teratocarcinomas, have many properties in common with pluripotent embryonic cells, and thus provide an excellent system for studying the early events involved in embryonic development and stem cell differentiation. We have isolated three novel mutants with temperature-sensitive(ts) cell growth that were able to differentiate at a non-permissive temperature for cell growth. These mutations affect the progression of the cell cycle, leading to the transient accumulation of cells in a specific phase, the S phase, of the cell cycle, which is likely to be the primary cause of stem cell differentiation of EC cells at non-permissive temperature. Isolation of these mutants strongly supports the notion that there is a close association between the inhibition of DNA synthesis and EC cell differentiation.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Cell Differentiation; Cell Division; Cell Line; Glycolipids; Lewis X Antigen; Mice; Mutation; Plasminogen Activators; Temperature; Teratoma; Tumor Cells, Cultured

Most developmentally regulated epitopes identified on embryonal carcinoma cells and murine preimplantation embryos are associated with a glycoprotein-bound large glycan called embryoglycan. To prepare monoclonal antibodies recognizing other, less immunogenic stage-specific embryonic epitopes, we used embryoglycan-negative embryonal carcinoma cells P19XT.1.1 as immunogen. One monoclonal antibody prepared by this strategy was found to react specifically with mouse embryonal carcinoma and embryo-derived stem cell lines. The target epitope, TEC-4, was found to be expressed on eggs and two-cell embryos but was undetectable on later stages of mouse embryos and adult mouse tissues. NaDodSO4/PAGE of immunoaffinity-isolated antigen revealed that TEC-4 epitope is associated with glycoproteins of apparent Mr 120,000 and 240,000. The epitope was resistant to oxidation by sodium periodate and to digestion by endoglycosidase F but was sensitive to treatment with protein-denaturing agents and proteases, which suggested that the epitope is located in the protein moiety of the molecule. In the course of retinoic acid-induced differentiation of embryonal carcinoma cells the epitope disappeared before the onset of morphological differentiation. The combined data indicate that TEC-4 is an unusual stage-specific embryonic antigen that may be amenable to direct genetic analysis.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Cell Differentiation; Cell Line; Cytotoxicity, Immunologic; Fluorescent Antibody Technique; Gene Expression; Glycolipids; Lewis X Antigen; Mice; Molecular Weight; Polysaccharides; Teratoma; Tretinoin; Tumor Cells, Cultured

The understanding of differentiation in human teratomas requires a better definition of the phenotype and developmental potential of the stem cells in these tumours. We describe the characterization of 6 new cell lines from human testicular teratomas which are representative of 3 distinct cell types. Cell lines GCT 27, GCT 35, and GCT 48, identified as embryonal carcinoma, comprise epithelial cells which express cytokeratin intermediate filaments and desmoplakins, as determined by indirect immunofluorescence microscopy. A minority of the cells also express vimentin. Most cells in these cultures show surface staining with monoclonal antibodies (MAbs) to stage-specific embryonic antigens SSEA 3 and SSEA 4 but not SSEA 1. Staining with MAb W6/32, which recognizes HLA A,B and C chains in the presence of beta-2 microglobulin, is not above background level. When injected into nude mice, GCT 27 cells form tumours consisting of embryonal carcinoma, somatic tissues, and cells positive in immunocytochemical assays for alphafoetoprotein (AFP) and human chorionic gonadotrophin (HCG); GCT 35 cells form embryonal carcinomas with foci of AFP and HCG-positive cells; and GCT 48 cells form embryonal carcinoma only. A second type of cell (GCT 72) displays some properties of rodent visceral endoderm. GCT 72 cells contain cytokeratin intermediate filaments, but not vimentin, and show very strong staining at cell borders with anti-desmoplakin I + II antibody. At the cell surface, GCT 72 cells express the epitopes recognized by antibodies to SSEA 3, SSEA 4, and SSEA 1; staining with W6/32 is negligible. Levels of AFP in supernatants from GCT 72 cultures are in excess of 500 KIU/I. The tumours formed following inoculation of nude mice with GCT 72 cells are solid yolk-sac tumours, with all cells strongly positive for AFP. A third cell type (GCT 44 and GCT 46), resembles in some ways rodent parietal endoderm. These cells uniformly coexpress keratin and vimentin intermediate filaments, but not desmoplakins. The determinants recognized by MAbs to SSEA 3, 4, or 1 are not detected on the majority of cells in these cultures. In striking contrast to the other teratoma lines, these cells can attach to untreated tissue culture plastic in serum-free medium and may be serially cultivated under these conditions. The tumours formed in nude mice by these 2 cell lines are yolk-sac carcinomas with endodermal sinus tumour histology. Thus, human testicular teratomas consist of epithelial cells whi

Topics: alpha-Fetoproteins; Animals; Antigens, Surface; Cell Differentiation; Cell Line; Chorionic Gonadotropin; Dysgerminoma; Embryonal Carcinoma Stem Cells; Glycolipids; Humans; Lewis X Antigen; Male; Mice; Neoplastic Stem Cells; Teratoma; Testicular Neoplasms

The rate at which P19 embryonal carcinoma cells in monolayer culture become anchorage dependent during differentiation induced by retinoic acid (RA) was investigated. In both nonsynchronized cultures and cultures synchronized by mitotic selection, the ability to grow in semisolid medium, characteristic of the malignant stem cell, decreased after a lag period of about 12 hr in the continuous presence of RA, prior to an increase in cell generation time. However, striking differences between synchronized and nonsynchronized cultures were observed in their commitment to differentiation following RA removal. After only 2 hr of exposure to RA, synchronized cells continued a program of differentiation in which they became anchorage dependent, while at least 24 hr of exposure was required for exponentially growing cells to become similarly committed. Induction of anchorage dependence by RA was also strikingly cell cycle dependent; 2 or 4 hr of exposure of synchronized cells to RA in G1 phase, when the intrinsic capacity for soft agar growth is low, was sufficient to commit cells to anchorage dependence, but a similar exposure in S phase was not. Together, these results suggested that interactions between cells in different cell cycle phases in asynchronous cultures influenced commitment since exposure to RA for more than one cycle (13 hr) was required for all cells to become anchorage dependent. Increased plasminogen activator secretion and epidermal growth factor binding, markers of certain differentiated cell types, increased only 3 and 5 days after RA addition, respectively, and were not induced by pulsed exposure to RA of less than 24 hr, even in synchronized cells.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Cycle; Cell Differentiation; Cell Line; Culture Media; Cytoskeleton; Epidermal Growth Factor; ErbB Receptors; Glycolipids; Laminin; Lewis X Antigen; Mice; Plasminogen Activators; Teratoma; Time Factors; Tretinoin

The mouse embryonal carcinoma cell line F9 differentiates into parietal endoderm cells after a 3-day exposure to retinoic acid and dibutyryl cyclic AMP. Using the experimental metastases assay, we investigated the organ colonization properties of RA-treated and -untreated populations of F9 cells. The results show that untreated F9 cells colonize the liver with a high degree of specificity while the treated populations colonize the lungs. Cells derived from a lung colony colonized only the liver unless they were treated with RA. However, removal of the inducer from culture of differentiated cells did not cause reversal of the lung colonization potential. Our observations also indicate that it is unlikely that lung colonization is due to cell aggregation or to interaction between differentiated and undifferentiated cells. Taken together, these results suggest that RA induces the observed changes of organ colonization properties of F9 cells.

Topics: Animals; Bucladesine; Cell Line; Fucose; Glycolipids; Glycosylation; Lewis X Antigen; Liver Neoplasms; Lung Neoplasms; Mice; Neoplasm Metastasis; Teratoma; Tretinoin

Teratocarcinoma stem cells can be used to study certain events occurring during early mouse embryogenesis. We report that the outgrowth of parietal endoderm from teratocarcinoma stem-cell embryoid bodies in vitro is analogous to the same process in vivo in terms of the spatial distribution of endoderm types: only parietal endoderm migrates away from the aggregate, whereas visceral endoderm remains associated with the embryoid body. The outgrowths generated on a substrate of type-I collagen from PSA-1 and retinoic-acid-treated F 9 embryoid bodies were found to be comparable, even though these aggregates express different endoderm types. We demonstrated that retinoic-acid-treated F 9 embryoid bodies that contain essentially only visceral endoderm in suspension culture can nonetheless generate parietal-endoderm outgrowth when plated on type-I collagen, suggesting that substrate interaction plays an important role in inducing parietal-endoderm differentiation. These data indicate the usefulness and relevance of studying endoderm differentiation and outgrowth in vitro employing the teratocarcinoma model system.

Topics: alpha-Fetoproteins; Animals; Cell Differentiation; Cell Line; Collagen; Embryonal Carcinoma Stem Cells; Endoderm; Fluorescent Antibody Technique; Glycolipids; Laminin; Lewis X Antigen; Mice; Neoplastic Stem Cells; Teratoma

Both murine and heterotransplanted human nonseminomatous germ-cell tumors have been successfully located by external scintigraphy using radioiodinated anti-SSEA-1, a monoclonal IgM, and its pepsin-derived antigen-binding fragment, F(ab')2 mu. Antibody localization in the tumor is mainly due to antigenic specificity, rather than to nonspecific trapping, and depends strongly on the amount of time after injection. The antibody has been used for drug targeting in vitro and in vivo.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Antineoplastic Agents; Choriocarcinoma; Female; Glycolipids; Humans; Immunoglobulin Fab Fragments; Iodine Radioisotopes; Lewis X Antigen; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Pregnancy; Radionuclide Imaging; Teratoma; Transplantation, Heterologous

The X hapten (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) may play an important role in the adhesion of blastomeres during compaction. Therefore, we have investigated more thoroughly developmental changes in the fucosylation of lactoseries carbohydrate chains and the enzymatic basis of these fucosylation changes using well-characterized monoclonal antibodies. The Y hapten (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3]GlcNAc) and polymeric X haptens were detected by fluorescence-activated flow cytometry on murine embryonal carcinoma cells. In paraffin sections of postimplantation mouse embryos, the Y hapten was detected in the embryonic ectoderm and visceral endoderm on Days 5.5-7.5; this pattern of antigen expression is identical to that previously reported for the X hapten (SSEA-1). Thus, the Gal:alpha 1----2 (H) and GlcNAc:alpha 1----3 (X) fucosyltransferases appear to be co-regulated during embryogenesis. Reciprocal changes in X and Y hapten expression were observed, however, during preimplantation development. Unlike the X hapten, the Y hapten is expressed maximally on 16-cell morulae and 32- to 64-cell blastocysts. Eight-cell embryos cultured to the blastocyst stage in vitro did not acquire the Y hapten, however, suggesting a role for the uterine environment in carbohydrate antigen expression. Homogenates of F9 embryonal carcinoma cells were found to possess a potent GlcNAc:alpha 1----3 fucosyltransferase activity, as well as a weaker Gal:alpha 1----2 fucosyltransferase activity, using paragloboside as a substrate. The results suggest that embryonic cell surface carbohydrate phenotypes represent a balance in the competition between glycosyltransferases for available substrates. Rapid changes in carbohydrate expression during development may reflect intermediate states of cellular commitment and determination that are critical for lineage formation and morphogenesis.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Antigens, Neoplasm; Antigens, Surface; Blastocyst; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Embryo, Mammalian; Female; Flow Cytometry; Fluorescent Antibody Technique; Glycolipids; Lewis X Antigen; Mice; Pregnancy; Teratoma

Methotrexate (MTX) conjugates of a monoclonal antibody, anti-SSEA-1, containing an average of 45 mol MTX/mol of immunoglobulin M, were prepared by a carbodiimide coupling reaction. Binding experiments indicate that conjugation does not decrease the affinity of the antibody for its antigen. The conjugate strongly inhibits the growth of SSEA-1-bearing F-9 teratocarcinoma cells, with 50% inhibitory dose of 4.5 nM MTX, which makes it more active than free MTX (50% inhibitory dose of 15 nM). The drug-free antibody is not cytotoxic to F-9 cells at the concentrations used. The high efficacy of the conjugated drug may be due in part to the fact that anti-SSEA-1 antibody is an immunoglobulin M. MTX conjugated to nonspecific immunoglobulin M has little inhibitory effect (50% inhibitory dose of 150 nM). When acting on SSEA-1 negative cells, the two conjugates have only a small but identical effect. Thiamine pyrophosphate, an inhibitor of MTX transport, can prevent the cytotoxicity of the free MTX but not that of the anti-SSEA-1 conjugate. Leupeptin, an inhibitor of lysosomal protease, can partially protect F-9 cells against the antibody conjugate but not against free MTX. These results indicate that the MTX antibody conjugate binds specifically to F-9 cells, and is internalized and intracellularly degraded to release a small molecular active drug. Pretreatments of F-9 cells for 1 h with unlabeled antibody inhibits the subsequent uptake of identical concentration of labeled conjugate. The rate of internalization, however, regains almost normal values within 4 h, indicating a rapid reappearance of free antigenic sites at the cell surface.

Topics: Animals; Antibodies, Monoclonal; Glycolipids; Immunoglobulin G; Leucovorin; Leupeptins; Lewis X Antigen; Lysosomes; Methotrexate; Mice; Mice, Inbred BALB C; Teratoma; Thiamine Pyrophosphate; Tritium

Six embryonal carcinoma (EC) cell lines that are resistant to the cytotoxic, galactose-specific lectin abrin were isolated from mutagenized populations of either PSA-1 or F9 cells. The LD10 for each of the variant lines was at least 150-fold greater than that for parental cells. Indirect cytotoxicity tests demonstrated that all of the variant cell lines lacked both Stage Specific Embryonic Antigen-1 (SSEA-1, less than 1% of wild-type levels) and Forsmann antigen (less than 5% of wild-type levels). When abrin-resistant cells were fused to previously isolated SSEA-1-negative cells (M. J. Rosenstraus (1983), Dev. Biol. 99, 318-323) that express Forsmann antigen, the resulting hybrids expressed SSEA-1. This implies the mutation conferring abrin resistance is in a different gene than that defined by the previously isolated mutation. Thus, we have identified two genes that are required for SSEA-1 expression, one of which also appears to be required for Forsmann antigen expression. The F9-derived variants differentiated into visceral-like or parietal-like endoderm when treated with retinoic acid in the absence or presence of 8-bromo-cAMP, respectively. PSA-1-derived variants formed differentiated teratocarcinomas containing derivatives of all three germ layers. Thus the SSEA-1 and Forsmann haptenic determinants are not required for EC cells to differentiate into a broad spectrum of cell types; nor do they appear to be involved in the cell-cell interactions that are postulated to regulate visceral versus parietal endoderm differentiation.

Topics: Abrin; Animals; Antigens, Heterophile; Antigens, Neoplasm; Cell Differentiation; Drug Resistance; Embryonal Carcinoma Stem Cells; Forssman Antigen; Genes; Genetic Complementation Test; Glycolipids; Lewis X Antigen; Mutation; Neoplastic Stem Cells; Stem Cells; Teratoma

A monoclonal IgM antibody (anti-SSEA-1) and its divalent antigen-binding peptic fragment [F(ab')2 mu] were compared as in vivo tumor localization reagents in mouse teratocarcinomas. F(ab')2 mu is cleared more rapidly than whole antibody from the whole body, blood, and all tested organs (t1/2 for whole antibody approximately 18 hr; t1/2 for F(ab')2 mu, 12 hr). A corresponding average improvement in tumor-to-tissue ratio is observed 48 hr after injection and earlier. However, the affinity of the F(ab')2 mu for antigen is much lower, and a smaller fraction of the antibody fragment is retained in the tumor than with whole antibody. The fragment was not retained by animals bearing nonantigenic tumors.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Glycolipids; Immunoglobulin Fab Fragments; Immunoglobulin M; Iodine Radioisotopes; Kinetics; Lewis X Antigen; Mice; Mice, Inbred BALB C; Neoplasms, Experimental; Plasmacytoma; Radionuclide Imaging; Teratoma; Time Factors

Molecules carrying SSEA-1 were isolated from [3H]galactose-labeled embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. The antigenic molecules were degraded by extensive pronase digestion or mild alkaline treatment, and the majority of the products thus formed were so large as to be excluded from a column of Sephadex G-50. Therefore, the major carbohydrate constituent of the antigenic molecule was embryoglycan, the glycoprotein-bound large glycan in early embryonic cells. Furthermore, the binding of isolated embryoglycan with anti-SSEA-1 was directly shown by a modified Farr's assay. From these results we concluded that SSEA-1 determinant was carried by the large glycan.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Cell Line; Electrophoresis, Polyacrylamide Gel; Glycolipids; Glycoproteins; Lewis X Antigen; Mice; Molecular Weight; Polysaccharides; Teratoma

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.

Topics: Acetylation; Animals; Antigens, Neoplasm; Butyrates; Butyric Acid; Carrier Proteins; Cell Differentiation; Cell Line; Embryonal Carcinoma Stem Cells; Glycolipids; Histones; Lewis X Antigen; Mice; Neoplastic Stem Cells; Receptors, Retinoic Acid; Stem Cells; Teratoma; Tretinoin

On the assumption that a high specificity would be required for in vivo tumor location and drug targeting, much labor has been expended to produce monoclonal antibodies directed to antigens found primarily on tumors. However, for at least one tumor-associated antigen, stage-specific embryonic antigen 1 (SSEA-1), the concentration of the target antigen need not be higher in the tumor than that found in normal tissues for successful use of the corresponding monoclonal antibody in in vivo tumor location; rather, accessibility and antibody metabolism are more important.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Antigens, Neoplasm; Antigens, Surface; Brain; Glycolipids; Kidney; Lewis X Antigen; Liver; Mice; Salivary Glands; Teratoma

The human embryonal carcinoma cell lines NT2 /D1 and NT2 /B9, clonally derived from Tera-2, differentiate extensively in vitro when exposed to retinoic acid. This differentiation is marked by the appearance of several morphologically distinct cell types and by changes in cell surface phenotype, particularly by the disappearance of stage-specific embryonic antigen-3 (SSEA-3), which is characteristically expressed by human EC cells. Among the differentiated cells are neurons, which form clusters interconnected by extended networks of axon bundles, and which express tetanus toxin receptors and neurofilament proteins. These observations constitute the first instance of extensive somatic differentiation of a clonal human EC cell line in vitro.

Topics: Antigens, Surface; Cell Differentiation; Cell Line; Clone Cells; Embryonal Carcinoma Stem Cells; Glycolipids; Humans; Intermediate Filament Proteins; Lewis X Antigen; Membrane Proteins; Neoplastic Stem Cells; Neurons; Receptors, Cholinergic; Stem Cells; Teratoma; Tretinoin

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.

Topics: Acetamides; Animals; Antigens, Surface; Carrier Proteins; Cell Differentiation; Cell Line; Embryonal Carcinoma Stem Cells; Glycolipids; Hybrid Cells; Lewis X Antigen; Male; Mice; Mutation; Neoplastic Stem Cells; Plasminogen Activators; Receptors, Retinoic Acid; Stem Cells; Teratoma; Tretinoin

Cell lines corresponding to various cell lineages of the mouse embryo have been isolated from murine teratocarcinomas. Embryonal carcinoma cell lines are developmentally equivalent to the embryonic ectoderm or inner cell mass. Most of these cell lines have a modal chromosome number equal or close to 40, the normal mouse complement. However, cell lines corresponding to more advanced cell lineages (e.g., endoderm) are tetraploid or hypotetraploid and display multiple chromosomal rearrangements. This paper describes the isolation of a near-diploid differentiated cell line (LT-D) from an LT teratocarcinoma. The modal chromosome number of LT-D is 40, and this number is stable during at least 12 mo of continuous culture. LT-D cells are morphologically distinct from embryonal carcinoma cells and no longer express the SSEA-1 cell surface antigen or high alkaline phosphatase activity characteristic of embryonal carcinoma cells. LT-D cells can be induced to fuse into structures resembling myotubes. The formation of these structures is accompanied by expression of the muscle-specific isozyme of creatine phosphokinase and desmin, a muscle-specific component of intermediate filaments. Lastly, LT-D cells do not form tumors in syngenetic mice.

Topics: Alkaline Phosphatase; Animals; Cell Differentiation; Cell Line; Cell Separation; Creatine Kinase; Desmin; Female; Fluorescent Antibody Technique; Glycolipids; Intermediate Filament Proteins; Isoenzymes; Lewis X Antigen; Male; Mice; Muscles; Phenotype; Teratoma

Previous work has shown that murine embryonal carcinoma cells are refractory to infection with various viruses, including simian virus 40. Thus, large T and small t antigens, the products of the simian virus 40 early region, are not produced when the virus infects embryonal carcinoma cells, in contrast to other cell types. We show, by qualitative and quantitative analyses, that embryonal carcinoma cell hybrids, containing a simian virus 40 early region integrated into human DNA, are capable of producing viral large T antigen.

Topics: Animals; Antigens, Neoplasm; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Cell Line; Cell Transformation, Neoplastic; Embryonal Carcinoma Stem Cells; Fluorescent Antibody Technique; Genes; Genes, Viral; Glycolipids; Humans; Hybrid Cells; Kinetics; Lewis X Antigen; Mice; Neoplastic Stem Cells; Simian virus 40; Teratoma; Viral Proteins

Mice immunized with teratocarcinoma F9 cells or human blood group substances A, B or H exhibited significant inhibition of spermatogenesis comparable to the inhibition induced by immunization with testicular cells. All these immunization schemes resulted in the production of antibodies which recognize antigens common to F9 and spermatogenic cells. In addition to these antigens, both anti-F9 and anti-ABH sera also recognize antigens which are specific for F9 cells. One of them was identified as SSEA-1. These results support the hypothesis that oncofetal F9 antigens are carbohydrate structures, which may play an important role in spermatogenic cell differentiation.

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; Blood Group Antigens; Cell Line; Cricetinae; Cricetulus; Female; Fluorescent Antibody Technique; Glycolipids; Humans; Lewis X Antigen; Male; Mice; Ovary; Radioimmunoassay; Spermatogenesis; Teratoma; Testis

A murine embryonal carcinoma (EC) cell line heterozygous for t0 recessive lethal mutation has been established from an embryo-derived transplantable teratocarcinoma TC1Ph of the genotype (129-T/t0 X C3H/Di)t0/+. The EC cell line, designated EC1Ph, and two cloned sublines, EC1Ph/a and EC1Ph/b, maintain the diploid karyotype (40, XY) and give rise to teratocarcinomas with differentiated derivatives of EC cells after inoculation into syngeneic recipients. The cloned sublines express low or zero amounts of SSEA-1 and ECMA-7 stage-specific antigens. At some passages, the EC1Ph line and the cloned subline EC1Ph/b express a significant quantity of class I H-2 antigens. This unusual EC phenotype resembles that of human teratocarcinoma cell lines.

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; Cell Line; Cell Separation; Cell Transformation, Neoplastic; Clone Cells; Cytological Techniques; Glycolipids; Lewis X Antigen; Mice; Mice, Inbred Strains; Mutation; Neoplasm Transplantation; Phenotype; Radioimmunoassay; Teratoma

Topics: Animals; Carrier Proteins; Cell Differentiation; Cell Line; Clone Cells; Glycolipids; Humans; Lewis X Antigen; Mice; Receptors, Retinoic Acid; Teratoma; Tretinoin

Stage-specific embryonic antigen 1 (SSEA-1) is a glycoconjugate that is expressed on embryonal carcinoma cells but not on their differentiated derivatives. To determine if SSEA-1 is required for the expression of the EC cell phenotype, a variant cell line lacking this antigen was isolated by exposing mutagenized OTF9-63 (an F9 subclone) cells to anti-SSEA-1 antiserum plus complement. Indirect cytotoxicity tests demonstrated that the variant cell line exhibits at least a 500-fold reduction in SSEA-1 expression. Somatic cell hybrids constructed between variant and wild-type cells expressed SSEA-1, suggesting that the lack of SSEA-1 expression in the mutant is probably due to loss, rather than masking, of the antigenic site. The variant cell line was tumorigenic in syngeneic mice, forming teratocarcinomas composed exclusively of embryonal carcinoma cells. Monolayer cultures of the variant cell line formed parietal endodermal (laminin-positive) cells following exposure to retinoic acid whereas aggregates grown in suspension culture formed visceral endoderm. Also, like their parental cells, the variant cells sorted out to form simple embryoid bodies when mixed in suspension with parietal-like endodermal cells. Thus, the expression of a number of aspects of the embryonal carcinoma phenotype is not dependent on the presence of SSEA-1.

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; Cell Differentiation; Cell Line; Cell Survival; Genetic Variation; Glycolipids; Lewis X Antigen; Mice; Teratoma

Two monoclonal antibodies (MC631 and MC813-70) raised against 4- to 8-cell stage mouse embryos and a human teratocarcinoma cell line, respectively, detect the stage-specific embryonic antigens, the previously defined SSEA-3 and SSEA-4, described herein. These antibodies were both reactive with a unique globo-series ganglioside with the structure shown below: (formula; see text) The antibodies were found to recognize sequential regions of this ganglioside, i.e., MC813-70 recognizes the terminal 'a' structure whereas antibody MC631 recognizes the internal 'b' structure. Thus, a set of two antibodies defines this unique embryonic antigen. During differentiation of human teratocarcinoma 2102Ep cells, the globo-series glycolipids defined by these antibodies decrease and the lacto-series glycolipids, reacting with the SSEA-1 antibody appear. This antigenic conversion suggests that a shift of glycolipid synthesis from globo-series to lacto-series glycolipids occurs during differentiation of human teratocarcinoma and perhaps of pre-implantation mouse embryos.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Antigens, Neoplasm; Antigens, Surface; Blastocyst; Carbohydrate Conformation; Carbohydrate Sequence; Erythrocytes; Female; Glycolipids; Granulocytes; Humans; Lewis X Antigen; Lymphocytes; Mice; Neoplasms; Oocytes; Species Specificity; Teratoma

Two hybridoma antibodies (VEP8 and VEP9) raised against the promyelomonocytic leukemia cell line HL60 have previously been shown to distinguish human granulocytes and monocytes from other cells of the peripheral blood. We report here that both antibodies recognize the carbohydrate structure 3-fucosyl-N-acetyllactosamine with the following sequence: (formula; see text) This structure is the same as that recognized by a hybridoma antibody against mouse teratocarcinoma cells (anti-SSEA-1) which recognizes an early embryonic antigen in the mouse. Until recently this carbohydrate structure was considered to be rare among glycoproteins and glycosphingolipids. However, there is a growing list of human and animal glycoproteins in which this sequence has been detected by chemical and immunochemical methods. In this article we survey this information and discuss how this and other carbohydrate structures behave as differentiation- or tumor-associated antigens.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Epitopes; Granulocytes; Humans; Lewis X Antigen; Mice; Monocytes; Oligosaccharides; Orosomucoid; Teratoma

The carriers of the carbohydrate differentiation antigens I, i and SSEA-1 were investigated in embryonal carcinoma cell lines of mouse and differentiated cell lines derived from them. Glycoproteins were studied by immunostaining ('Western blotting') of total cell lysates and immunoprecipitation from lysates of galactose oxidase/NaB3H4-labelled cells; glycolipids were investigated by immunostaining of thin layer chromatograms. The antigenic activities detected by immunofluorescence of cell smears were reflected in the antigenicities of high-molecular-weight glycoproteins. These were polydisperse and markedly susceptible to digestion with endo-beta-galactosidase. Only the I antigen was detected on minor glycolipids. These observations indicate that glycoproteins rather than glycolipids are the major carriers of carbohydrate differentiation antigens I, i and SSEA-1 in the teratocarcinoma cell lines.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; beta-Galactosidase; Carbohydrates; Cell Line; Cell Membrane; Epitopes; Fluorescent Antibody Technique; Glycolipids; Glycoproteins; Glycoside Hydrolases; Humans; Lewis X Antigen; Mice; Molecular Weight; Teratoma

The isolation and characterization of variant embryonal carcinoma (EC) cells possessing altered cell-surface structures is described. The lectin peanut agglutinin (PNA), which binds to EC cells but not their differentiated derivatives, was used to select the variants. Clones resistant to the cytotoxic effect of PNA were isolated at a frequency of 4 X 10(-5) following mutagenesis. The resistant phenotype was stable in the absence of selection in all eight clones tested. The increased frequency of resistant clones following mutagenesis and the stability of the phenotype suggests a mutational origin. Somatic cell hybrids constructed between wild-type cells and two different PNA-resistant cell lines were sensitive to PNA; this suggests that the resistant phenotype is recessive. Binding assays demonstrated that resistant cells exhibited a twofold to fourfold reduction in the total amount of PNA bound. Together with the recessive behavior of the phenotype, this suggests that resistant cells are deficient for PNA receptors. The PNA-resistant cells also showed reduced binding of monoclonal antibody against stage-specific embryonic antigen 1 (SSEA-1) in indirect cytotoxicity tests. All eight PNA-resistant lines isolated were tumorigenic in syngeneic mice and gave rise to well-differentiated teratocarcinomas. The PNA-resistant cells behaved like their wild-type parents in a cell recognition assay; when incubated in suspension with endodermal cells, they sorted out to form simple embryoid bodies (a core of EC cells surrounded by an endodermal rind). Thus, EC cells can form tumors, differentiate, and recognize differentiated cells in a sorting assay despite a reduction in expression of the embryo-specific cell surface structures (s) that bind PNA and anti-SSEA-1 antibody.

Topics: Animals; Arachis; Cell Communication; Cell Differentiation; Cell Line; Endoderm; Genes, Recessive; Glycolipids; Lectins; Lewis X Antigen; Mice; Mutation; Neoplasms, Experimental; Peanut Agglutinin; Plant Lectins; Receptors, Mitogen; Teratoma

Topics: Animals; Antigens; Brain; Embryonic Development; Endometrium; Epididymis; Fallopian Tubes; Female; Fetus; Germ Layers; Gestational Age; Glycolipids; Kidney; Lewis X Antigen; Male; Mice; Pregnancy; Teratoma

Topics: Animals; Antibodies; Antigen-Antibody Complex; beta-Galactosidase; Blood Group Antigens; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Embryo, Mammalian; Glycolipids; Glycosphingolipids; I Blood-Group System; Immunodiffusion; Lewis X Antigen; Mice; Neoplasms, Experimental; Teratoma