Showing papers on "Cellular differentiation published in 1984"

Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines

Abstract: The recent availability in culture of embryo-derived pluripotential cells which exhibit both a normal karyotype and a high differentiative ability has encouraged us to assess the potential of these cells to form functional germ cells following their incorporation into chimaeric mice We report here the results of blastocyst injection studies using three independently isolated XY embryo-derived cell lines (EK CP1 , EK CC1 1 and EKCC1 2) which produce a very high proportion (greater than 50%) of live-born animals that are overtly chimaeric Seven chimaeric male mice, derived from these three lines, have, so far, proved to be functional germ-line chimaeras

Cellular Biology and Biochemistry of the Retinoids

Abstract: Publisher Summary This chapter discusses the cellular biology and biochemistry of the retinoids In isolated 9-day-old rat embryos, retinoic acid prevents the formation of the pharyngeal arches, which are derived from cephalic mesenchyme; these structures later form the maxilla and the mandible Another mesenchymal derivative, whose formation is markedly suppressed by retinoic acid in rat embryos, is the yolk sac circulation Retinoids can exert a powerful influence on cell differentiation in many different types of epithelia The effects of retinoids on differentiation of epithelia in organ culture result from a combination of complex cellular responses and interactions of different cell types in the explant One of the most illuminating examples of the ability of retinoids to promote differentiation is the effect of retinoids on mouse embryonal carcinoma cells These undifferentiated stem cells of teratocarcinomas are multipotential, that is, they can differentiate into a multiplicity of somatic cell types Another example of the ability of retinoic acid to promote terminal differentiation of neoplastic cells to nonneoplastic cell types is the effect of retinoic acid on human promyelocytic leukemia cells

Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate.

Abstract: The human colonic cancer cell line HT29 is undifferentiated in standard culture conditions (Dulbecco's medium:10% fetal bovine serum). These cells were cultured in 5 mM sodium butyrate for 9 days; then they were trypsinized and subcultured in sodium butyrate for an additional 14 days. Multinucleation occurred during this second phase of the treatment. The cells were then transferred to standard medium and multinucleation disappeared. Morphological changes appeared 10 to 12 days after return to standard culture conditions; some cells flattened and became more adherent to the bottom of the flasks. These altered cells divided actively and formed "flat foci" interspersed among the densely packed undifferentiated HT29 cells. This altered phenotype persists after more than 24 months of culture in standard medium. Clonal cell lines were established from these flat foci-forming cells and characterized. These clonal lines exhibited morphological cell polarity defined by an apical cell surface separated by junctional complexes from the basolateral cell surface. Functional differentiation did also occur since some clonal lines formed domes representing active transepithelial transport, and others exhibited massive mucus secretion. In conclusion, our findings indicate that permanently differentiated cell populations emerged in a colonic cancer cell line after sodium butyrate treatment. These new clonal lines will be useful in future models for the study of differentiation programs of both normal and cancerous colonic cells.

Cell death during differentiation of the retina in the mouse

Abstract: A reproducible pattern of cell death associated with differentiation of the retina in mice was analyzed quantitatively by microscopy. Cell death occurs primarily during the first 2 weeks after birth and is essentially complete by the end of the third week. Death of individual cells involves nuclear condensation and pyknosis (apoptosis), followed by phagocytosis of the cellular remains by adjacent cells or motile phagocytes. From birth through 4 days, an increasing incidence of cell death is observed among ventricular cells. Ganglion cell degeneration is prominent during the first 11 days, peaking on days 2-5. Many presumptive amacrine cells die within the inner plexiform and inner nuclear layers, particularly between 3 and 8 days. Among adjoining bipolar and Muller cells, degeneration reaches a peak at 8-11 days. On day 5, formation of the outer plexiform layer separates the rods into two groups. Rod nuclei situated on the inner side of that layer immediately move across it to enter the outer nuclear layer, but numerous cells die during nuclear migration. Sporadic death of rods continues during the following 2 weeks. Cell death associated with cell differentiation (histogenetic death) is considered to represent a normal developmental process. Possible mechanisms resulting in cell degeneration are discussed. It is suggested that genetically regulated cell death serves to fine-tune neuronal networks during the terminal stages of development.

Expression of myb, myc and fos proto-oncogenes during the differentiation of a murine myeloid leukaemia.

Abstract: It is widely thought that c-onc genes (or proto-oncogenes)—the cellular progenitors of retroviral transforming genes—are involved in cellular differentiation and/or proliferation. Such ideas originate primarily from the ability of v-onc genes and ‘activated’ c-onc genes to induce uncontrolled cellular proliferation, and their capacity to arrest or interfere with differentiation processes in some systems. Haematopoietic cell populations provide additional support for these ideas as c-myb RNA is present in cell lines corresponding to immature, but not mature, cell types1,2, and elevated levels have been found in tissues that are active in haematopoiesis2,3. We have now examined the effects of induced differentiation on c-onc gene expression in a murine myeloid leukaemia cell line, WEHI-3B (‘D+’ subline)4. Our results show that the expression of c-myb and c-myc, at the level of transcription, decreases only at late stages in the monocytic differentiation of WEHI-3B cells, while expression of c-fos increases markedly. We suggest that c-myb and c-myc do not themselves control myeloid differentiation, but that they function in the maintenance of the proliferative state of myeloid cells. The induction of c-fos may reflect its role in some macrophage-specific functions.

Cell and Molecular Biology: Concepts and Experiments

Abstract: Introduction to the Study of Cell Biology The Chemical Basis of Life Bioenergetics, Enzymes, and Metabolism The Structure and Function of the Plasma Membrane Aerobic Respiration and the Mitochondrion Photosynthesis and the Chloroplast Interactions Between Cells and Their Environment Cytoplasmic Membrane Systems - Structure, Function, and Membrane Trafficking The Cytoskeleton and Cell Motility The Nature of the Gene and the Genome Expression of Genetic Information - From Transcription to Translation The Cell Nucleus and the Control of Gene Expression DNA Replication and Repair Cellular Reproduction Cell Signaling - Communication Between Cells and Their Environment Cancer The Immune Response Techniques in Cell and Molecular Biology Glossary Answers to Odd-Numbered Analytic Questions Index

Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils.

Abstract: Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.

1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells

Abstract: The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 57 X 10(-9) M HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3 The receptor in both lines was characterized as a DNA-binding protein that migrated at 33S on high-salt sucrose gradients Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events

Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: Clonal analyses, growth kinetics, and cell cycle studies

Abstract: The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G1, S, G2, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (greater than 30 population doublings) and neonatal prokeratinocytes (greater than 50 population doublings) is associated with senescence and the G1 arrest of noncycling cells.

Interleukin 3: A differentiation and growth factor for the mouse mast cell that contains chondroitin sulfate E proteoglycan.

Abstract: Mouse mast cells were differentiated and grown by culturing bone marrow cells in medium containing 2 X 10(-10) M purified interleukin 3 (IL 3). The cells obtained were similar in ultrastructure, membrane antigen phenotype, proteoglycan type, and lipid products generated upon immunologic activation to mast cells differentiated in culture by WEHI-3-conditioned medium (WEHI-3-CM) and by concanavalin A (Con A) splenocyte-conditioned medium. Phenotypically, these cells expressed IgE receptors and H-2 antigens and were recognized by a monoclonal antibody (B23.1) that did not react with mouse serosal heparin-containing mast cells. The classic phenotypic markers of mouse T cells or macrophages were not detected. The mouse mast cells differentiated with IL 3 as well as those differentiated in WEHI-3-CM incorporated [35S]sulfate into a nonheparin proteoglycan of 150,000 to 200,000 m.w. Most of the 35S-labeled macromolecules were degraded by chondroitinase ABC to yield only two disaccharides, which co-chromatographed on ascending thin layer chromatography with delta Di-4S and delta Di-diSE; thus, the proteoglycan in these cells is composed of chondroitin sulfate E glycosaminoglycans. After sensitization with monoclonal IgE, washing, and antigen activation, the IL 3 differentiated cells released the preformed mediator beta-hexosaminidase and generated and released two major classes of lipid mediators. The quantities of leukotriene C4 (LTC4), leukotriene B4 (LTB4), and platelet-activating factor (PAF-acether) generated/10(6) cells were 17, 3.0, and 3.1 ng, respectively. The ratio of these three lipid mediators was similar to that obtained from mast cells differentiated in WEHI-3-CM and in Con A-conditioned medium. Thus, T cell-derived IL 3 is the component present in the conditioned media that is required for differentiation and growth of the subclass of mast cells containing chondroitin sulfate E proteoglycan, designated E-MC. The IL 3-dependent E-MC may represent the in vitro counterpart of the T-cell-dependent mucosal mast cell, suggesting in turn that the production of LTC4 and LTB4 and of PAF-acether may play a role in adaptive intestinal immunity to helminthic parasites.

Cell recognition during neuronal development.

Abstract: Insect embryos, with their relatively simple nervous systems, provide a model system with which to study the cellular and molecular mechanisms underlying cell recognition during neuronal development. Such an approach can take advantage of the accessible cells of the grasshopper embryo and the accessible genes of Drosophila. The growth cones of identified neurons express selective affinities for specific axonal surfaces; such specificities give rise to the stereotyped patterns of selective fasciculation common to both species. These and other results suggest that early in development cell lineage and cell interactions lead to the differential expression of cell recognition molecules on the surfaces of small subsets of embryonic neurons whose axons selectively fasciculate with one another. Monoclonal antibodies reveal surface molecules in the Drosophila embryo whose expression correlates with this prediction. It should now be possible to isolate the genes encoding these potential cell recognition molecules and to test their function through the use of molecular genetic approaches in Drosophila.

Expression of c-myc changes during differentiation of mouse erythroleukaemia cells

Abstract: The transforming gene of avian myelocytomatosis virus MC29, v-myc, causes a variety of malignancies in chickens. A cellular homologue, c-myc, has been implicated in B-cell malignancies in mice and humans but is also expressed in many normal cell types and may be important in the control of normal cell proliferation. c-myc is highly conserved in vertebrates. We have been investigating the relationship between c-myc expression and the terminal differentiation of cultured mouse erythroleukaemia (MEL) cells. We find that the level of c-myc messenger RNA shows a rapid biphasic change in MEL cells induced to differentiate by dimethyl sulphoxide or hypoxanthine. The changes occur during the first few hours of the differentiation programme and require active protein synthesis. These data suggest that changes in c-myc expression may be important in the irreversible commitment of MEL cells to terminal erythroid differentiation.

Epithelial mouse mammary cell line exhibiting normal morphogenesis in vivo and functional differentiation in vitro

Abstract: An epithelial cell line, designated COMMA-1D, was derived from mammary tissue of BALB/c mice in the middle of pregnancy. This line, in continuous cell culture for 12 months, exhibits several characteristics distinctive of normal mammary epithelial cells, including induction of casein synthesis in vitro and normal duct morphogenesis in the cleared mammary fat pads of syngeneic mice. The cells also form domes in high density culture and are positive for keratin intermediate filaments by indirect immunofluorescence. COMMA-1D cells have a near diploid number of chromosomes and do not grow in suspension culture or produce tumors in syngeneic hosts. This cell line should prove useful for studies examining the regulation of normal cellular differentiation of mammary cells as well as transformation of epithelial cells to the preneoplastic and neoplastic phenotypes.

Cell type-specific activation of actin genes in the early amphibian embryo.

Abstract: Muscle actin genes are the earliest yet described to show cell type-specific activation in amphibian embryos Gene-specific probes show that α-skeletal and α-cardiac actin genes start to be transcribed simultaneously at the end of gastrulation, but only in those regions of the mesoderm that subsequently form embryonic muscle Their expression provides a molecular marker for early cell determination

B cell origin of non-T cell acute lymphoblastic leukemia. A model for discrete stages of neoplastic and normal pre-B cell differentiation.

Abstract: The expression of B cell associated and restricted antigens on tumor cells isolated from 138 patients with non-T cell acute lymphoblastic leukemia (non-T cell ALL) was investigated by flow cytometric analysis by means of a panel of monoclonal antibodies. Tumor cells from these patients could be assigned to one of four subgroups: human leukocyte antigen-DR-related Ia-like antigens (Ia) alone (4%, stage I); IaB4 (14%, stage II); IaB4CALLA (33%, stage III); and IaB4CALLAB1 (49%, stage IV). The expression of B cell-restricted antigens (B4 and B1) and rearrangements of Ig heavy chain genes provided strong evidence for the B cell lineage of stages II, III, and IV tumors. The lineage of the Ia alone group is still unknown. The B4 antigen was expressed on approximately 95% of all non-T cell ALLs tested, and given its absence on T cell and myeloid tumors, it appears to be an exceptional marker to define cells of B lineage. The demonstration that Ia alone, IaB4, IaB4CALLA, and IaB4CALLAB1 positive cells can be readily identified by dual fluorescence analysis in normal fetal and adult bone marrow provided critical support for the view that these leukemic pre-B cell phenotypes were representative of the stages of normal pre-B cell differentiation. It was interesting that the IaB4+ cell was more frequently identified in fetal bone marrow than in adult marrow, whereas the predominant cell found in adult marrow expressed the IaB4CALLAB1 phenotype. These data suggest that the leukemogenic event may be random, since the predominant pre-B cell leukemic phenotype appears to correspond to the normal pre-B cell phenotype present in these hematopoietic organs. Our observations provide an additional distinction between adult and childhood ALL, since these studies show that most non-T cell ALLs seen in children less than 2 yr old are of stage II phenotype, whereas the majority of non-T ALLs in adults are of stage IV phenotype. Finally, it should be noted that the present study suggests that the analysis of leukemic B cell phenotypes and their normal counterparts can provide a mechanism for the investigation and orderly definition of stages of pre-B cell differentiation in man.

Isolation of murine pluripotent hemopoietic stem cells.

Abstract: A method described to purify pluripotent hemopoietic stem cells ( PHSC ) from adult mouse bone marrow. The method consists of three separation steps. First, bone marrow cells are centrifuged in a discontinuous metrizamide gradient and simultaneously labeled with wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC). Second, the low density cells are analyzed by a fluorescence-activated cell sorter (FACS) and the WGA-positive cells with medium forward and low perpendicular light scatter intensities are sorted. The WGA-FITC is removed from the cells by incubation with N-acetyl-D-glucosamine. Finally, the sorted cells are incubated with anti-H-2K-biotin and avidin-FITC and sorted a second time to enrich cells with high H-2K density. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 d and 6.6 colonies per 100 cells at 12 d after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony-forming unit/spleen) was 135 (range, 90--230; n = 15) and was similar to that for the cell type that provides radioprotection (180 +/- 70), indicating that these functional properties were copurified. Indirect evidence suggests that the spleen-seeding efficiency (f factor) of these cells is 0.10 and, therefore, the average purity of the sorted PHSC was 65% (range in 15 experiments, 35--110%). The sorted cells were all in the G1 or G0 phase of the cell cycle. They appeared to be undifferentiated blasts by morphological criteria. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. 32% of the sorted cells could be induced to form myeloid progeny in vitro. This procedure should be useful for direct studies on the regulation of hemopoietic cell differentiation.

Differentiation of F9 teratocarcinoma stem cells after transfer of c-fos proto-oncogenes.

Abstract: Transfer of mouse or human c-fos proto-oncogenes into F9 teratocarcinoma stem cells results in expression of c-fos mRNA and protein. This is accompanied by the appearance of morphologically altered cells which express several specific markers characteristic of differentiated cells, suggesting that c-fos plays a role in cellular differentiation.

Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal antibody.

Abstract: We have examined human B lymphocytes at different stages of differentiation for the expression of surface receptors for the C3d fragment of complement. C3d receptors (C3dR) were identified by indirect immunofluorescence using the HB-5 monoclonal antibody, which recognizes a 145,000 m.w. C3dR molecule on B lymphocytes. Pre-B and immature B cells from fetal bone marrow and liver did not express C3dR, whereas a small subpopulation (25%) of B cells in fetal spleen were C3dR+. Approximately 50% of the B cells in adult bone marrow were C3dR+, whereas the more mature B cells in the blood of newborns and adults and in peripheral lymphoid tissue of adults uniformly expressed the C3dR. Activated B cells responsive to T cell-derived differentiation factors were C3dR+, whereas plasma cells rarely expressed C3dR. T cells, NK cells, erythrocytes, and myelomonocytic cells did not express detectable surface C3dR. These results suggest that in hematopoietic and lymphoid tissues, the expression of C3dR is a specific feature of relatively mature lymphoid cells of B lineage.

Meiosis and differentiation of mouse germ cells.

Abstract: The female pathway of germ cell development is characterized by early entry into meiotic prophase, before birth in the mouse. This pathway is followed by all germ cells in the ovary and in the mesonephric region of the urogenital ridge in female embryos, and by all germ cells in the adrenals of both sexes. The male pathway, with meiosis delayed until well after birth, is taken by all germ cells within the testis cords, all or almost all of those within the testis but outside the cords, and most of those in the mesonephric region. Results of culture and co-culture experiments are described. No conclusive evidence has yet been obtained to discriminate between the hypothesis that all germ cells spontaneously enter meiosis before birth unless prevented from doing so, and the alternative hypothesis that germ cells only enter meiosis under the inducing influence of somatic cells. The pathways of development that radiate from an egg cell all lead to a state of terminal differentiation, with the single exception of the pathway that leads to another egg cell. This pathway is capable of cyclic repetition, even without the intervention of a fertilizing sperm: if a normal mouse embryo is aggregated with one derived from a parthenogenetically activated egg, the parthenogenetic component may contribute to the oocyte population as well as to all other cell types examined (Stevens, 1978).

Induction of Macrophage Differentiation of Human Normal and Leukemic Myeloid Stem Cells by 1,25-Dihydroxyvitamin D3 and Its Fluorinated Analogues

Abstract: This study shows for the first time that 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] and two fluorinated analogues of 1,25-(OH)2D3 [24,24-F2-1,25-(OH)2D3 and 26, 26, 26, 27, 27, 27-F6-1,25-(OH)2D3] induce macrophage differentiation of human normal and leukemic myeloid stem cells. The addition of either 1,25-(OH)2D3 or one of the two fluorinated analogues of 1,25-(OH)2D3 at concentrations as low as 10(-9) M to culture plates containing normal human marrow cells stimulated myeloid stem cells to preferentially differentiate to colonies of monocytes and macrophages. Over 80% of the normal human myeloid colonies were composed of only monocytes and macrophages in culture plates containing 10(-7) M 1,25-(OH)2D3 or one of the fluorinated analogues. In contrast, control plates not containing 1,25-(OH)2D3 had less than 35% macrophage colonies. Likewise, 1,25-(OH)2D3 and the two vitamin D analogues induced macrophage differentiation of leukemic colony-forming cells taken from patients. In plates containing 10(-7) M 1,25-(OH)2D3 or one of the analogues at 10(-8) M, 80% of chronic myelogenous leukemia and approximately 50% of acute nonlymphocytic leukemia colony-forming cells differentiated to macrophage-like cells. In contrast, control plates had about 30 and 20% macrophage colonies in cultures from chronic myelogenous leukemia and acute nonlymphocytic leukemia patients, respectively. Our findings suggest that 1,25-(OH)2D3 may play a role in hematopoiesis and that the compound or a related analogue may possibly have a therapeutic role in some leukemias.

Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells.

Abstract: Two monoclonal antibodies (TRA-1-60 and TRA-1-81) recognizing distinct cell surface antigens on human embryonal carcinoma (EC) cells were produced and characterized. These antibodies reacted strongly with undifferentiated human EC cells in indirect radioimmunoassays (RIA) and immunofluorescence (IF) assays, but only weakly or not at all with cells derived from pluripotent EC cells differentiating in vitro or in xenograft tumors, nor with other germ cell tumor cell lines that did not also express the typical features of human EC cells. They did not react with murine teratocarcinoma cell lines. A survey of other human tumor cell lines and normal human tissues disclosed that molecules recognized by these antibodies are not confined to human EC cells but that cross-reacting epitopes appear on several neoplastic and normal tissues, although in a different anatomical pattern for each antibody. Both antibodies immunoprecipitated a major polypeptide (apparent molecular weight approximately 240,000) and a minor polypeptide (apparent molecular weight approximately 415,000) from lysates of 125I surface-labeled human EC cells, in this respect resembling another monoclonal antibody, 8-7D, previously described by Blaineau et al. (1,2) However, sequential immunoprecipitation revealed that each of the three antibodies reacted with different molecules of slightly different molecular weights. The epitopes defined by the present antibodies differ from those recognized by the other human EC cell-specific monoclonal antibodies that have been described and provide new markers for studying the differentiation of pluripotent human EC cells.