Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives.

BACKGROUND: Teratocarcinoma is a malignant male germ cell tumour, which contains stem cells and differentiated cancer tissues. DNMT3B has been shown to be highly expressed in human teratocarcinoma stem cells, and to mediate cytotoxicity of Aza-deoxycytidine (Aza-dC) in a pluripotent stem cell line NTERA2. METHODS: We have established DNMT3B or POU5F1 (hereafter referred to as OCT4) knockdown in teratocarcinoma stem cells N2102Ep and TERA1 and in the pluripotent NTERA2 by a doxycycline-inducible system, and tested the cytotoxicity induced by Aza-dC. RESULTS: Silencing of DNMT3B led to apoptosis of human teratocarcinoma stem cells N2102Ep and TERA1. Further, we found that induction of apoptosis or differentiation in NTERA2 and human embryonic stem cells by Aza-dC requires DNMT3B. To test whether Aza-dC inhibits proliferation of differentiated teratocarcinoma cells, we depleted OCT4 expression in N2102Ep and TERA1 cells treated with Aza-dC. Treatment with Aza-dC reduced cell number of differentiated cells to a lesser extent than their undifferentiated parental stem cells. Moreover, in contrast to the stem cells, Aza-dC failed to induce apoptosis of differentiated cells. CONCLUSIONS: Our finding suggests that DNMT3B acts as an antiapoptotic gene in teratocarcinoma stem cells, and mediates apoptosis and differentiation of human pluripotent stem cells induced by Aza-dC, and that Aza-dC specifically induces apoptosis of teratocarcinoma stem cells.

Mapping the stem cell state: eight novel human embryonic stem and embryonal carcinoma cell antibodies.

The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state', yet it appears that this categorization may be an oversimplification, because a number of sub-states appear to exist within this state. To increase the resolution of the undifferentiated state, we have generated eight novel monoclonal antibodies, all capable of recognizing undifferentiated human ES and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment.

Human cytomegalovirus induces stage-specific embryonic antigen 1 in differentiating human teratocarcinoma cells and fibroblasts.

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.

Mineral resources, economic growth, and world populatic.

World population and world income can grow at any likely rate for the next 50 to 75 years, probably for longer, and mineral supplies will continue to keep pace with demand. Not, however, without environmental costs, without affecting Third World development, and, perhaps most important, without ignoring critical questions of power. In what might be termed the revisionist form of the limits to growth thesis, Aurelio Peccei and Alexander King, cofounders of the Club of Rome, seem to be saying that the forecasts of doom themselves are unimportant but they symbolize critical problems of the nature and uses of power in the modern world (30): . . . the Club of Rome is questioning the quality of growth and its distribution around the world. . . . We know that the present structure of the world is obsolete. . . . Both private and state capitalism are stale . . . we have to develop something else. Surely, continually increasing rates of mineral production are symptoms of this obsolete power structure, a result of the fact that, ultimately, population growth and monetary income growth lead to demands for natural resources that necessitate their being found and produced regardless of the implications. Since such higher rates of production are geologically and economically sustainable, we should choose among alternative paths of growth, and hence among alternative rates of mineral resource development, according to what we like or dislike about these implications. The key information will not be found in tables comparing reserves and consumption but in preferences and ethics.

Cytomegalovirus replicates in differentiated but not in undifferentiated human embryonal carcinoma cells.

To study the mode of action of human cytomegalovirus, an important teratogenic agent in human populations, the susceptibility of a pluripotent human embryonal carcinoma cell line to the virus was investigated. Viral antigens were not expressed nor was infectious virus produced by human embryonal carcinoma cells after infection, although the virus was able to penetrate these cells. In contrast, retinoic acid-induced differentiated derivatives of embryonal carcinoma cells were permissive for antigen expression and infectious virus production. Replication of human cytomegalovirus in human teratocarcinoma cells may therefore depend on cellular functions associated with differentiation.

Acquired Genetic and Epigenetic Variation in Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) can acquire non-random genomic variation during culture. Some of these changes are common in tumours and confer a selective growth advantage in culture. Additionally, there is evidence that reprogramming of human induced pluripotent stem cells (hiPSCs) introduces mutations. This poses a challenge to both the safety of clinical applications and the reliability of basic research using hPSCs carrying genomic variation. A number of methods are available for monitoring the genomic integrity of hPSCs, and a balance between practicality and sensitivity must be considered in choosing the appropriate methods for each use of hPSCs. Adjusting protocols by which hPSCs are derived and cultured is an evolving process that is important in minimising acquired genomic variation. Assessing genetic variation for its potential impact is becoming increasingly important as techniques to detect genome-wide variation improve.

Characterisation of large-conductance calcium-activated potassium channels (BK(Ca)) in human NT2-N cells.

Large-conductance calcium-activated potassium (BK(Ca)) channels were studied in inside-out patches of human NTERA2 neuronal cells (NT2-N). In symmetrical (140 mM) K(+) the channel mean conductance was 265 pS, the current reversing at approximately 0 mV. It was selective (P(K)/P(Na)=20:1) and blocked by internal paxilline and TEA. The open probability-voltage relationship for BK(Ca) was fitted with a Boltzmann function, the V((1/2)) being 76.3 mV, 33.6 mV and -14.1 mV at 0.1 muM, 3.3 muM and 10 muM [Ca(2+)](i), respectively. The relationship between open probability and [Ca(2+)](i) was fitted by the Hill equation (Hill coefficient 2.7, half maximal activation at 2.0 muM [Ca(2+)](i)). Open and closed dwell time histograms were fitted by the sum of two and three voltage-dependent exponentials, respectively. Increasing [Ca(2+)](i) produced both an increase in the longer open time constant and a decrease in the longest closed time constant, so increasing mean open time. "Intracellular" ATP evoked a concentration-dependent increase in NT2-N BK(Ca) activity. At +40 mV half-maximum activation occurred at an [ATP](i) of 3 mM (30 nM [Ca(2+)](i)). ADP and GTP were less potent, and AMP-PNP was inactive. This is the first characterisation of a potassium channel in NT2-N cells showing that it is similar to the BK(Ca) channel of other preparations.

Ultrastructural differentiation of a clonal human embryonal carcinoma cell line in vitro.

A cloned human embryonal carcinoma (EC) cell line 2102Ep derived from a testicular teratocarcinoma was characterized by means of electron microscopy and immunohistochemistry. These EC cells when plated at high cell density grow mostly as undifferentiated cells displayed relatively little pleomorphism. Eighty-five to 90% of these cells contain keratin in the form of peridesmosomal tonofilaments. Cell populations of the same clonal line plated at a low cell density contain, in addition to undifferentiated EC cells, large cells displaying complex cytoplasmic architecture, more complex junctions, and intracytoplasmic keratin in the form of bundles. Some of these cells also react with antibodies to human chorionic gonadotropin indicative of trophoblastic differentiation. Furthermore, some cells form "morules" which are multicellular aggregates composed of a core of EC cells and an attenuated, more differentiated outer cell layer. These data thus point out not only some similarities but also even more prominent differences between human and mouse EC cells.

The human embryonal carcinoma marker antigen TRA-1-60 is a sialylated keratan sulfate proteoglycan.

Human embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas, and they are key components of germ cell tumors (GCTs). They express several high molecular weight glycoprotein antigens that are down-regulated upon differentiation. One of these antigens, defined by monoclonal antibody TRA-1-60, can be detected in the serum of GCT patients and provides a useful complement to the established serum markers human chorionic gonadotropin and alpha-fetoprotein, especially in those patients without elevated serum human chorionic gonadotropin or alpha-fetoprotein. To examine the relationship of the TRA-1-60-defined antigen to similar antigens defined by other monoclonal antibodies, we have carried out comparative Western blot and immunoprecipitation analyses of human GCT-derived cell lines with monoclonal antibodies TRA-1-60, TRA-1-81, GCTM2, and K21. The TRA-1-60 antigen was detected by Western blot analysis in extracts of all human EC cell lines and in clinical specimens of GCT tested as a diffuse band with a molecular weight of >200,000. A similar but noticeably fainter band was detected in GCT composed of seminoma only. The antigen was not expressed by GCT-derived lines without an EC phenotype. Affinity bead-purified TRA-1-60, TRA-1-81, GCTM2 and K21 antigens reacted in Western blot analysis with each of the other antibodies tested, indicating that the epitopes recognized by each antibody are carried by the same molecular species. This molecule could be metabolically labeled with inorganic [35S]sulfate and was degraded by keratanase. Glycopeptides produced from affinity-purified TRA-1-60 antigen by extensive digestion with Pronase exhibited a molecular weight in excess of 10,000 and were degraded by keratanase. The TRA-1-60 epitope was destroyed by digestion with neuraminidase, but the epitopes defined by TRA-1-81, GCTM2, and K21 were not. Our results indicate that human EC cells generally express a cell surface sialylated keratan sulfate proteoglycan that is subject to modification to yield a variety of epitopes, one of which is recognized by the monoclonal antibody TRA-1-60. Sensitivity to milk alkaline digestion suggests that the oligosaccharides of this proteoglycan are O-linked to a core polypeptide.

Induction of class I major histocompatibility complex antigens in human teratocarcinoma cells by interferon without induction of differentiation, growth inhibition, or resistance to viral infection.

The behavior of human teratocarcinoma cells, and especially their stem cells (embryonal carcinoma cells), may provide insights into the properties of human early embryonic cells. We report here that human recombinant gamma-interferon (IFN-gamma) induced the expression of major histocompatibility complex Class I (HLA-A, B, C) antigens and beta 2-microglobulin in the two human embryonal carcinoma cell lines, 2102Ep cl.4D3 and NTERA-2 cl.D1, and in the yolk sac carcinoma cell line, 1411H; human recombinant IFN-alpha and IFN-beta were less effective inducers of these cell surface molecules. No induction was observed in the gestational choriocarcinoma cell line, JAR. Neither IFN-alpha, IFN-beta, nor IFN-gamma caused growth inhibition, expression of major histocompatibility complex Class II (HLA-DR) antigens, resistance to viral (vesicular stomatitis virus) infection, or expression of 2',5'-oligo(A)synthetase in any of the cells. Also, IFN-gamma neither induced differentiation of NTERA-2 cl.D1 cells, which are pluripotent human stem cells, nor influenced their differentiation induced by retinoic acid. However, developmental regulation of responsiveness to IFN was evident, since IFN-gamma induced higher levels of surface expression of HLA-A, B, C and beta 2-microglobulin in the retinoic acid-induced differentiated NTERA-2 cl.D1 cells than in the undifferentiated parental cells. Also, 2',5'-oligo(A)synthetase was inducible in the NTERA-2 cl.D1 differentiated cells by IFN-alpha and -beta, although not by IFN-gamma, and slight resistance to vesicular stomatitis virus infection was evident in aged cultures of differentiated cells exposed to IFN-alpha. The effect of recombinant mouse IFN-gamma on major histocompatibility complex expression by several murine teratocarcinoma cells was also examined: H-2 Class I (H-2Db), but not class II (I-Ab), antigens were induced in the parietal yolk sac carcinoma lines, PYS and F9Ac cl.9; in cultures of PCC3/A/1 containing both embryonal carcinoma (EC) and differentiated cells; and in cultures of the EC cells, PCC4azaR and PCC4AO, without evidence of differentiation. No induction was observed in the murine EC cell lines, F9 or FA (H-2Kk). Our results indicate that human EC cells, like murine EC cells, exhibit only a partial response to the interferons, and that the extent of this response is developmentally regulated.

Cell line derived from a metastasis of a human testicular germ cell tumor.

A cell line, designated 833K-E, has been established from a metastasis of a human testicular germ cell tumor that consisted of four histological types of tumor cells. The 833K-E cells have morphological and ultrastructural characteristics of epithelial cells and a hyperdiploid karyotype indicative of their human male origin. The cells grow in agar cultures and produce in nude mice tumors which have the hstological features of embryonal carcinoma without differentiated elements. Many of the cells express a stage-specific mouse embryonic antigen, and low levels of the major histocompatibility antigens and beta 2-microglobulin also were detected on a large percentage of the cells. A lymphoblastoid cell line (833K-LC) established from the same tumor specimen expresses major histocompatibility antigens and beta 2-microglobulin but does not express the embryonic antigen.

Human Wnt-13 is developmentally regulated during the differentiation of NTERA-2 pluripotent human embryonal carcinoma cells.

The Wnt gene family encodes a series of conserved glycoproteins that regulate pattern formation during embryogenesis, in a variety of tissues including the nervous system. As with other genes that control embryonic cell differentiation, members of the Wnt family have also been implicated in tumourigenesis. To search for Wnt genes involved in human teratocarcinomas, with a possible role in human embryogenesis, we used RT-PCR primed with degenerate oligonucleotides to analyse mRNA from differentiating cultures of the pluripotent human embryonal carcinoma (EC) cell line NTERA-2. NTERA-2 EC cells differentiate into neurons and other cell types when induced with retinoic acid. Wnt gene expression was not detected in the undifferentiated EC cells, but Wnt-related PCR fragments were amplified from differentiating cultures, 4-14 days after induction with retinoic acid. The RT-PCR products were composed primarily of DNA fragments corresponding to the recently identified human Wnt-13 gene. No other Wnt-related genes were identified. Northern analysis confirmed induction of Wnt-13 as a 2.4 kb mRNA during the early phases of retinoic acid-induced differentiation, and during differentiation along a non-neural pathway induced by hexamethylene bisacetamide (HMBA), but not in the terminally differentiated neurons. Wnt-13 remained expressed in non-neural differentiated NTERA-2 cells, even several weeks after the induction of differentiation. The time course of induction, its induction by HMBA, and its persistence in differentiated cells indicate that Wnt-13 expression is not dependent upon direct activation by retinoic acid. Wnt-13 was not detected, or only detected at low levels, in other human EC cells. However, it was found to be expressed at a high level in one malignant teratoma cell line, 577MF, that does not exhibit an EC phenotype although it was derived from a testicular teratocarcinoma. At least two members of the human frizzled gene family, thought to encode receptors for Wnt proteins, were also expressed in the NTERA-2 cells, suggesting the presence of a mechanism by which endogenously expressed Wnt-13 could modulate the histogenesis of teratocarcinomas by mediating interactions between sub-populations of differentiating EC cells. We note that Wnt-13 maps to chromosome 1p13, a region reported to be subject to relatively frequent loss of heterozygosity in germ cell tumours. Further analysis indicated that 465 bp of the published Wnt-13 sequence, within the predicted 5' UTR, is incorrect and is possibly derived from a human mitochondrial DNA sequence.

Human teratocarcinomas.

Teratocarcinomas are one of the commonest forms of cancer in young adult men. Cell lines derived from these tumors, and particularly the cell lines composed of their embryonal carcinoma (EC) stem cells, may provide useful information concerning the development and subsequent pathology of teratocarcinomas in humans. In addition, it is likely that human EC cells resemble early embryonic cells and can be used as an in vitro counterpart of such cells from the human embryo. Several common properties of human EC cells have been identified, and a human EC cell line, TERA-2, that is capable of extensive somatic differentiation has been cloned. In nude mice, TERA-2 EC cells form tumors containing neural elements and glandular structures that resemble primitive gut. In culture, these EC cells can be induced to differentiate by exposure to retinoic acid and hexamethylenebisacetamide (HMBA). Differentiation is marked by the disappearance of several cell surface antigens characteristic of human EC cells, and the appearance of other antigens on the various subsets of differentiated derivatives. In retinoic acid-induced cultures, these differentiated derivatives include neurons and cells permissive for the replication of cytomegalovirus, a virus that can cause birth defects in humans. On the other hand, HMBA appears to activate an alternative pathway of differentiation for TERA-2 EC cells, although the identity of the resulting cells remains to be elucidated. In addition to providing a tool for analyzing the evolution of teratocarcinomas in human patients, the TERA-2 EC cells may provide us with insights into the mechanisms of cellular differentiation in the human embryo and a model in which to investigate how teratogenic agents such as HCMV can disrupt these processes.

The expression and function of cadherin-mediated cell-to-cell adhesion in human embryonal carcinoma cells.

Human embryonal carcinoma (EC) cells typically require high cell densities to maintain their characteristic phenotype; they are generally subject to differentiation when cultured at low cell densities, marked by changes in morphology and expression of the surface antigen, SSEA-1. To test whether cadherin mediated cell-to-cell adhesion may be responsible for maintaining an EC phenotype we ascertained that human EC cells generally express E- and P-cadherins, and are subject to cadherin mediated, Ca2+ dependent aggregation. However, in the NTERA2 human EC cell line, inhibition of cadherin mediated adhesion by culture in low levels of Ca2+ did not result in the changes typically seen under low cell density conditions. Low Ca2+ levels also did not affect the pattern of differentiation in these cells following induction with retinoic acid. Therefore, cadherin-mediated cell adhesion does not appear to play a role in maintaining an EC phenotype. On the other hand, culture at both low cell density and in the absence of Ca2+ did result in changes in the patterns of cadherin expression suggesting a feedback regulatory effect of cell-to-cell adhesion. Further, lithium which inhibits the cytoplasmic kinase GSK3beta and hence influences beta-catenin levels did cause differentiation of NTERA2 cells. However, consideration of the phenotype of the resultant cells suggested that this effect may be because of lithium mimicking activation of a Wnt signalling pathway, rather than an effect on signalling consequent upon cadherin mediated cell to cell adhesion.

Rejection of C57BL/6 skin grafts by (C57BL/6 X Mus musculus castaneus)F1 hybrids.

Hybrid offspring from C57BL/6(B6) females mated to males of the subspecies Mus musculus castaneus received B6 skin grafts. No strong Y chromosome-linked histocompatibility genes were detected, although occasional rejection of parental grafts by both male and female hybrids was observed after long periods. Rejection was attributed to interaction of B6 and Castaneus-derived genes in the hybrids.

Gene regulation during neuronal and non-neuronal differentiation of NTERA2 human teratocarcinoma-derived stem cells.

We constructed cDNA libraries from a clonal human teratocarcinoma-derived cell line and two retinoic acid-induced derivatives: a homogeneous population of neurons and a FACS-isolated, non-neuronal population. These libraries are large and representative of the cells from which they were derived, as determined by colony hybridization. PCR analysis indicates that the transcripts encoding P- and E-cadherin are down-regulated whereas the the prion protein (PrP) transcript is up-regulated in neurons. These cells offer a promising system for investigations of human prion infection and the cDNA libraries provide a source of neuron-specific genes.

Teratocarcinomas and human embryology: pluripotent human EC cell lines. Review article.

The histogenesis of germ cell tumours (GCT) reflects the normal processes of gametogenesis, fertilisation and subsequent embryonic cell differentiation. Understanding the mechanisms that control the differentiation of embryonal carcinoma (EC) cells into a variety of embryonic and extraembryonic cell types is pertinent to understanding the progression of GCT. EC cells also provide a tool for analysing the mechanisms that control differentiation during embryonic development, and particularly the mechanisms that control differentiation along alternative cell line, NTERA2, into neurones and other cell types in response to agents such as retinoic acid, HMBA and the bone morphogenetic proteins. We have also compared the pluripotent NTERA2 EC cells with other human EC cell lines that exhibit a much reduced capacity for cell differentiation. A variety of genes are activated during NTERA2 differentiation. In particular we have identified a novel human member of the wnt family. This wnt gene is activated following retinoic acid induction of differentiation but is later down-regulated as the cells mature into neurones and other cell types. We have also observed expression of genes belonging to the Frizzled family, which is likely to include genes encoding receptors for the wnt gene products. Thus in the NTERA2 system, genes pertinent to regulating cell differentiation during embryonic development are activated and appear to play a role in modulating how these pluripotent human EC cells differentiate.

The psychology of social chess and the evolution of attribution mechanisms: explaining the fundamental attribution error.

Theory of mind is the field devoted to understanding how organisms discern the mental states of others. Because mental states are not directly observable, they can only be inferred from observable features of the actor (such as behavior) and the situational context that the actor is in. Social psychologists, who study theory of mind processes under the rubric of attribution research, have shown that people often make a logical error of inference: The "fundamental attribution error" (FAE) is the tendency to assume that an actor's behavior and mental state correspond to a degree that is logically unwarranted by the situation. The social environment in which theory of mind capacities evolved may have influenced attributional processing in ways that could explain the error. In particular, the error could be caused by a psyche that is designed (1) to consider only those noncorresponding mental states (such as deception) that could have fitness consequences to the mind reader; (2) to bias inferences in a way that reduces the costs of erroneous inferences; or (3) to bias inferences in a way that yields reputational benefits. The existing literature is reviewed in light of these hypotheses.

Embryonal carcinoma cells differentiate into parietal endoderm via an intermediate stage corresponding to primitive endoderm.

F9 embryonal carcinoma (EC) cells were used as a model system to study endoderm formation during mammalian embryogenesis. F9 cells treated with retinoic acid (RA) or RA plus dibutyryl cyclic AMP (cAMP) were examined for the expression of stage-specific embryonic antigen-3 (SSEA-3), a cell surface marker of primitive and visceral endoderm. SSEA-3 was not detected by indirect immunofluorescence on the surface of undifferentiated stem cells; however, a subset of SSEA-3-positive cells appeared with time in culture, amounting to 20% of cells 10 days after plating. When cultured in the presence of RA, the percentage of SSEA-3-positive cells increased to 70% of cells 10 days after plating. In contrast, treatment of cells with RA plus cAMP yielded differentiated cells that were SSEA-3-negative. These SSEA-3-negative cells exhibited ultrastructural features of parietal yolk sac endoderm. In contrast, SSEA-3-positive cells appearing in cultures treated with RA alone exhibited ultrastructural features of primitive endoderm on day 3, switching to ultrastructural features of parietal endoderm on day 10. Cells with hybrid features, resembling both visceral and parietal yolk sac, were also seen. We suggest that differentiation of F9 EC cells into parietal yolk sac-like cells can occur along two distinct pathways: 1) direct under the combined influence of RA and cAMP; and 2) indirect, under the influence of RA alone, in which cells first differentiate into primitive endoderm. Parietal yolk sac-like cells induced through the latter pathway continue to express SSEA-3, a cell surface marker of primitive endoderm that is not normally found on parietal endodermal cells in vivo.

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

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.