Pluripotent Human Embryonal Carcinoma Cell Research Articles

Neuroprotective effects of boron nitride nanoparticles in the experimental Parkinson's disease model against MPP+ induced apoptosis.

Parkinson's disease (PD) is one of the most aggressive neurodegenerative diseases and characterized by the loss of dopamine-sensitive neurons in the substantia nigra region of the brain. There is no any definitive treatment to completely cure PD and existing treatments can only ease the symptoms of the disease. Boron nitride nanoparticles have been extensively studied in nano-biological studies and researches showed that it can be a promising candidate for PD treatment with its biologically active unique properties. In the present study, it was aimed to investigate ameliorative effects of hexagonal boron nitride nanoparticles (hBNs) against toxicity of 1-methyl-4-phenylpyridinium (MPP+) in experimental PD model. Experimental PD model was constituted by application of MPP+ to differentiated pluripotent human embryonal carcinoma cell (Ntera-2, NT-2) culture in wide range of concentrations (0.62 to 2mM). Neuroprotective activity of hBNs against MPP+ toxicity was determined by cell viability assays including MTT and LDH release. Oxidative alterations by hBNs application in PD cell culture model were investigated using total antioxidant capacity (TAC) and total oxidant status (TOS) tests. The impacts of hBNs and MPP+ on nuclear integrity were analyzed by Hoechst 33258 fluorescent staining method. Acetylcholinesterase (AChE) enzyme activities were determined by a colorimetric assay towards to hBNs treatment. Cell death mechanisms caused by hBNs and MPP+ exposure was investigated by flow cytometry analysis. Experimental results showed that application of hBNs increased cell viability in PD model against MPP+ application. TAS and TOS analysis were determined that antioxidant capacity elevated after hBNs applications while oxidant levels were reduced. Furthermore, flow cytometric analysis executed that MPP+ induced apoptosis was prevented significantly (p < 0.05) after application with hBNs. In a conclusion, the obtained results indicated that hBNs have a huge potential against MPP+ toxicity and can be used in PD treatment as novel neuroprotective agent and drug delivery system.

Abstract 2684: Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility

Abstract Testicular germ cell tumors (TGCT) are ideally suited to agnostic genome-wide association methods because of their known high heritability and homogeneous cell of origin. Here we report the most updated results from the international Testicular Cancer Consortium (TECAC) that extends published findings from our recent meta-analysis, which included 3,558 cases and 13,970 controls from five centers. To these existing samples, we added summary statistics from 300 cases and 151,991 controls provided by deCODE combined with imputed genotypes from an additional 5,602 cases and 5,006 controls assembled from 15 TECAC centers, almost all (97%) of which were centrally genotyped using the Illumina Human Core array. A total of 9,458 TGCT cases were included in our current meta-analysis, representing a 66% increase since our last publication. We identified 14 new and confirmed 41 previously reported loci that surpassed genome-wide significance (5 × 10-8). Fifteen previously reported loci did not reach this threshold, although one did so in subgroup analysis. In addition, 57 new loci attained nominal significance (5 × 10-8 ≤ p ≤ 1 × 10-5). As expected, a substantial proportion of top hits continue to map to genetic regions belonging to biological pathways essential to male germ cell development, sex determination, and chromosomal segregation. For example, newly identified genes include anaphase promoting complex subunit 2 (ANAPC2), BCL2 like 11 (BCL211), anti-Mullerian hormone receptor type 2 (AMHR2), and DEP domain containing mTOR interacting protein (DEPTOR). Our findings also further implicate genes located on the X chromosome, specifically the androgen receptor (AR). In analyses stratified by tumor subtype, we identified GATA-binding protein 4 (GATA4) as a susceptibility locus among men diagnosed with seminoma. Despite a similar effect size observed among men diagnosed solely with non-seminoma (i.e. excluding cases with a mixture of both seminoma and non-seminoma tumors), this marker did not surpass genome-wide significance in this subtype likely because of the smaller case sample size. GATA4 previously has been associated with overall risk of TGCT (Litchfield et al., Nat Genet. 2017). Biological mechanisms for all genome-wide significant loci are currently being explored using publicly available data sets, ATAC-seq of four TGCT cell lines, and SPATIaL-Seq (a novel high-resolution chromatin conformation capture (3C) assay) of the NTERA2 pluripotent human embryonal carcinoma cell line. Additional genotyping of top hits in an independent sample set of 1,103 TGCT cases and 1,210 controls is currently ongoing. Citation Format: Louise C. Pyle, John Pluta, Kevin T. Nead, Nandita Mitra, Javier Benitez, D. Timothy Bishop, Victoria Cortessis, Alberto Ferlin, Jourik Gietema, Mark Greene, Tom Grotmol, Ramneek Gupta, Rob Hamilton, Michelle A. Hildebrandt, Trine B. Haugen, Lambertus Kiemeney, Christian Kubisch, Davor Lessel, Paloma Martin, Thorunn Rafnar, Lorenzo Richiardi, Rolf Skotheim, Clare Turnbull, Fredrik Wiklund, Tongzhang Zheng, Ewa Rajpert-De Meyts, Stephen M. Schwartz, Katherine A. McGlynn, Peter A. Kanetsky, Katherine L. Nathanson, Testicular Cancer Consortium (TECAC). Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2684.

Activity of aaptamine and two derivatives, demethyloxyaaptamine and isoaaptamine, in cisplatin-resistant germ cell cancer

We analyzed the effects of all three marine alkaloids aaptamine, demethyloxyaaptamine and isoaaptamine in NT2-R, a cisplatin-resistant subline of the human embryonal carcinoma cell line NT2. All aaptamines were found to be equally effective in both cell lines, excluding cross-resistance between aaptamines and cisplatin in vitro. At the inhibitory concentration (IC50), aaptamine exerted an antiproliferative effect, whereas demethyloxyaaptamine and isoaaptamine were strong inducers of apoptosis. We analyzed the changes in the proteome of NT2-R cells treated with these compounds. 16-22 proteins were found to be significantly altered, of which several were validated by Western blotting and two-dimensional Western blotting analysis. Changes in the proteome pattern frequently resulted from post-transcriptional protein modifications, i.e. phosphorylation or hypusination in the case of eIF5A. Although the lists of altered proteins were heterogeneous and compound-specific, gene ontology analyses identified rather similar profiles regarding the affected molecular functions. Ingenuity pathway analysis by IPA put the following factors in a central position of the hypothetical networks: myc and p53 for aaptamine; tumor necrosis factor (TNF) for demethyloxyaaptamine; and all three, myc, p53, and TNF for isoaaptamine. Our results represent an important step towards a better understanding of the molecular basis underlying the observed bioactivity of these promising marine compounds. We characterized the mode of action of three aaptamines, marine natural compound with anti-tumor activity, using a functional proteomics approach and the cisplatin-resistant pluripotent human embryonal carcinoma cell line NT2-R. The manuscript is of particular scientific interest, as we could reveal the similarities and differences of the modes of action. Furthermore, we were able to identify several new targets of these promising compounds. We found hypusination of eIF5A to be a prominent feature exclusively of aaptamine treatment, as this was not observed upon treatment with demethyloxyaaptamine or isoaaptamine. Our results are a step towards unraveling the mode of action of these interesting compounds.

Human Breast Cancer Cells Are Redirected to Mammary Epithelial Cells upon Interaction with the Regenerating Mammary Gland Microenvironment In-Vivo

Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display ‘normal’ behavior when placed into ‘normal’ ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for ‘normal’ gland development in-vivo. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts) confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini) were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic) respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo.

Proteomic Profiling of Germ Cell Cancer Cells Treated with Aaptamine, a Marine Alkaloid with Antiproliferative Activity

Aaptamine is a marine compound isolated from the sponge Aaptos aaptos showing antiproliferative properties via an undefined mode of action. We analyzed the effects of aaptamine treatment on the proliferation and protein expression of the pluripotent human embryonal carcinoma cell line NT2. Effects on proliferation, cell cycle distribution, and induction of apoptosis were analyzed. At lower concentrations, including the IC50 of 50 μM, aaptamine treatment resulted in a G2/M phase cell cycle arrest, whereas at higher concentrations, induction of apoptosis was seen. Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of the most significantly up- and down-regulated proteins. Aaptamine treatment at the IC50 for 48 h resulted in alteration of 10 proteins, of which five each showed up- and down-regulation. Changes in the 2D map were frequently noticed as a result of post-transcriptional modifications, e.g., of the hypusine modification of the eukaryotic initiation factor 5A (eIF5A). Observed alterations such as increased expression of CRABP2 and hypusination of eIF5A have previously been identified during differentiation of pluripotent cells. For the first time, we describe changes in protein expression caused by aaptamine, providing valuable information regarding the mode of action of this compound.

Selective repression of retinoic acid target genes by RIP140 during induced tumor cell differentiation of pluripotent human embryonal carcinoma cells

BackgroundThe use of retinoids as anti-cancer agents has been limited due to resistance and low efficacy. The dynamics of nuclear receptor coregulation are incompletely understood. Cell-and context-specific activities of nuclear receptors may be in part due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We had previously shown that RIP140 limits RA induced tumor cell differentiation of embryonal carcinoma; the pluriopotent stem cells of testicular germ cell tumors. This implies that RIP140 represses key genes required for RA-mediated tumor cell differentiation. Identification of these genes would be of considerable interest.ResultsTo begin to address this issue, microarray technology was employed to elucidate in a de novo fashion the global role of RIP140 in RA target gene regulation of embryonal carcinoma. Subclasses of genes were affected by RIP140 in distinct manners.Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140. Hence, RIP140 appears to discriminate between different classes of RA target genes. In general, RIP140-dependent gene expression was consistent with RIP140 functioning to limit RA signaling and tumor cell differentiation. Few if any genes were regulated in a manner to support a role for RIP140 in "active repression". We also demonstrated that RIP140 silencing sensitizes embryonal carcinoma cells to low doses of RA.ConclusionTogether the data demonstrates that RIP140 has profound effects on RA-mediated gene expression in this cancer stem cell model. The RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation and the findings suggest that RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy. We discuss these data in the context of proposed models of RIP140-mediated repression.

Transcriptional program of bone morphogenetic protein-2-induced epithelial and smooth muscle differentiation of pluripotent human embryonal carcinoma cells

Pluripotent human embryonal carcinoma NTera2/cloneD1 (NT2/D1) cells respond to multiple vertebrate patterning factors and offer a unique model system to investigate the signaling events associated with lineage determination and cell differentiation. Here, we define the temporal changes in global gene expression patterns in NT2/D1 cells upon treatment with bone morphogenetic protein-2 (BMP-2). Exposure to BMP-2 rapidly induced the expression of several transcription factors involved in establishing non-neural ectodermal fate followed by the appearance of epithelial-specific markers. Subsequent loss of stem cell markers was coupled to gene expression changes associated with decreased proliferative activity. Temporal clustering of gene expression patterns revealed a concurrent down-regulation of multiple transcripts involved in neurogenesis, neurite outgrowth, and axonal guidance, suggesting that the BMP-mediated differentiation process involves pro-epithelial as well as anti-neurogenic mechanisms. In addition, increased expression of smooth muscle markers both by gene expression and immunohistochemistry was detected. Several neural crest markers were induced preceding such a differentiation, compatible with a neural crest origin of NT2/D1-derived smooth muscle cells. Comparison of changes in transcript expression between BMP-2-induced epithelial versus all-trans-retinoic acid (ATRA)-induced neural differentiation revealed potential candidates for regulation of BMP-2 signaling and suppression of neural fate by BMP-2. This study suggests that BMP-2-induced differentiation of NT2/D1 cells provides a powerful assay to study early human epithelial and smooth muscle development.

Surface antigens of human embryonic stem cells: changes upon differentiation in culture.

We have analysed the surface antigen phenotype of a human embryonic stem (hES) cell line (H7) and the changes that occur upon differentiation induced by retinoic acid, hexamethylene bisacetamide and dimethylsulphoxide. The undifferentiated stem cells expressed Stage Specific Embryonic Antigen-3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-8 but not SSEA1. In these characteristics they closely resemble human embryonal carcinoma (EC) cells derived from testicular teratocarcinomas, and are distinct from murine EC and ES cells. The undifferentiated cells also expressed the liver/bone/kidney isozyme of alkaline phosphatase detected by antibody TRA-2-54, the class 1 major histocompatability antigens, HLA-ABC, and the human Thy1 antigen. Differentiation of hES cells was induced by retinoic acid, HMBA and DMSO with the appearance of various cell types including neurons and muscle cells. The surface antigens characteristically expressed by hES cells were down-regulated following induction of differentiation and other antigens appeared, notably several ganglioside glycolipids detected by antibodies VIN-IS-56 (GD3 and GD2), VIN-2PB-22 (GD2), A2B5 (GT3) and ME311 (9-O-acetyl-GD3). Whereas the expression of HLA was slightly down-regulated upon differentiation, its expression was strongly induced by interferon-y in both the undifferentiated and the differentiated cells, although the induction in the differentiated cultures was considerably stronger than in the stem cells. In all of these features the human ES cells, and their pattern of differentiation, resembled the pluripotent human EC cell line NTERA-2 although clearly the range of cells generated by the hES cells was considerably greater.

Developmental regulation of neurogenesis in the pluripotent human embryonal carcinoma cell line NTERA-2.

Embryonal carcinoma (EC) cells provide a caricature of pluripotent embryonic stem (ES) cells and may be used as surrogates for investigating the mechanisms that regulate cell differentiation during embryonic development. NTERA-2 is a human EC cell line that differentiates in response to retinoic acid yielding cells that include terminally differentiated neurons. The expression of genes known to be involved in the formation of the vertebrate nervous system was examined during retinoic acid-induced NTERA-2 differentiation. Differentiation of these human EC cells into neurons could be divided into three sequential phases. During phase 1, in the first week of differentiation, hath1 mRNA showed a small transient increase that correlated with the rapid accumulation of nestin message, a marker of neuroprogenitors. Transcripts of nestin were quickly downregulated during phase 2 as expression of neuroD1, characteristic of neuroprogenitors exiting the cell cycle, was induced. A neural cell surface antigen, detected by the monoclonal antibody A2B5, was expressed by cells exiting the cell cycle, correlating with the expression of neuroD1 as the cells became post-mitotic. Markers of mature neural cells (e.g. synaptophysin and neuron-specific enolase) were subsequently increased during phase 3 and were maintained. This regulated pattern of gene expression and commitment to the neural lineage indicates that differentiation of NTERA-2 neurons in vitro follows a similar pathway to that observed by neural ectodermal precursors during vertebrate neurogenesis in vivo.

A mouse model for valproate teratogenicity: parental effects, homeotic transformations, and altered HOX expression.

Valproate (VPA) is one of several effective anti-epileptic and mood-stabilizing drugs, many of which are also potent teratogens in humans and several other mammalian species. Variable teratogenicity among inbred strains of laboratory mice suggests that genetic factors influence susceptibility. While studying the genetic basis for VPA teratogenicity in mice, we discovered that parental factors influence fetal susceptibility to induced malformations. Detailed examination of these malformations revealed that many were homeotic transformations. To test whether VPA, like retinoic acid (RA), alters HOX expression, pluripotent human embryonal carcinoma cells were treated with VPA or RA and Hox expression assessed. Altered expression of specific Hox genes may thus account for the homeotic transformations and other malformations found in VPA-treated fetuses.

Human embryonic stem cells.

Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.

Differential gene expression between human neurons and neuronal progenitor cells in culture: an analysis of arrayed cDNA clones in NTera2 human embryonal carcinoma cell line as a model system

To elucidate the highly complex expression pattern of the genes involved in human neuronal differentiation, differential gene expression between human neurons and neuronal progenitor cells was investigated by analysis of a cDNA expression array in a pluripotent human embryonal carcinoma cell line NTera2 (NT2), a model system of human neuronal differentiation. Among 588 arrayed cDNA clones, 87 genes showed a differential expression pattern between undifferentiated neuronal progenitor cells (NT2-U) and NT2-derived differentiated neurons induced by treatment with retinoic acid (RA) (NT2-N), while 26 genes could not be analyzed due to high background signals. The levels of expression of 76 genes, including those encoding a group of transcription factors, intracellular signal-transducing proteins, cell death-regulatory proteins, and growth factors/cytokines/neurotransmitters and their receptors, were elevated after neuronal differentiation, while the levels of 11 genes, including those coding for cellular proliferation-related proteins, were decreased. Among the differentially expressed genes following induction of neuronal differentiation, significant up-regulation of the growth-associated protein (GAP-43), low-affinity nerve growth factor receptor p75 (LNGFR), and defender against apoptotic cell death (DAD1) mRNAs and substantial down-regulation of the proliferation-associated gene (PAG), fibroblast growth factor receptor-1 (FGFR-1), and cellular RA-binding protein-II (CRABP-II) mRNAs were verified by Northern blot analysis. These results indicate that the analysis of cDNA expression arrays provides a useful approach for screening and identification of a set of distinct genes that undergo highly complex regulation during human neuronal differentiation.

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.

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 surface antigen phenotype of human embryonal carcinoma cells: modulation upon differentiation and viral infection.

The cell surface provides a rich source of developmentally controlled molecules that are of interest to several branches of basic cell biology and clinical medicine. Originating from studies of ontogeny and function in the immune system, the notion of cell surface antigen phenotype, defined by the pattern of expression of several “differentiation antigens” (Bennett et al. 1972; Boyse and Old 1978), also provides a valuable tool for following the progression of cell types in other complex differentiating systems such as those presented by teratocarcinomas. We have used the expression of a variety of surface antigens to provide objective criteria for the identification of human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas; we have also used changes in the expression of such antigens to study the differentiation of a pluripotent human EC cell line, TERA 2 (Andrews et al. 1984b). Extension of these observations to a clinical setting could provide the basis for improved histological classification of human germ cell tumors. Moreover, as we report here, the shedding of certain surface antigens characteristic of EC cells and their detection in the serum of patients could provide a useful additional means of monitoring the progress of therapy.

Human teratocarcinoma stem cells: glycolipid antigen expression and modulation during differentiation.

Teratocarcinomas are germ cell tumors in which pluripotent stem cells, embryonal carcinoma (EC) cells, undergo differentiation along the pathways resembling those occurring during early embryogenesis. Human EC cell lines established in vitro provide a model for studying embryonic cellular differentiation in a way that is pertinent to early human development. The predominant glycolipid antigens expressed by EC cells of both humans and mice have globoseries core structures; in humans they are terminally modified to yield the monoclonal antibody-defined stage-specific embryonic antigens SSEA-3 and SSEA-4, and also globo-ABH antigens; in the mouse terminal modification yields the Forssman antigen rather than SSEA-3 and -4. These observations focus attention on the possible role of the P-blood group system, which regulates synthesis of globoseries oligosaccharides, in the behavior of cells in the early embryo and in teratocarcinomas. Marked changes in the core structures of the cell surface glycolipids occur as the EC cells differentiate; thus globoseries structures rapidly diminish and are replaced by lactoseries and then by ganglioseries glycolipids. During differentiation of the NTERA-2 line of pluripotent human EC cells into neurons and other cell types, the various subsets of differentiated cells that arise are distinguished by their differential expression of new glycolipid antigens, particularly ganglioside GT3 (recognized by antibody A2B5), and ganglioside 9-0-acetyl GD3 (recognized by antibody ME311). Neurons are found among the A2B5+/ME311- cells.

A pluripotent human stem-cell clone isolated from the TERA-2 teratocarcinoma line lacks antigens SSEA-3 and SSEA-4 in vitro, but expresses these antigens when grown as a xenograft tumor

Human embryonal carcinoma (EC) cells generally express the cell-surface, stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4), the epitopes of which are defined by two monoclonal antibodies that recognize different portions of an extended globoseries oligosaccharide. To examine further the relationship between these epitopes and the human EC phenotype, we investigated the properties of two newly isolated clones from the human teratocarcinoma cell line, TERA-2. One clone expresses SSEA-3 and SSEA-4; the other does not. Nevertheless, these clones otherwise resemble one another, and based upon their morphology, their expression of other cell-surface antigens, and their ability to form xenograft tumors containing a variety of cell types, we conclude that both clones are composed of pluripotent human EC cells. When exposed to retinoic acid in vitro, neither clone differentiates as extensively as other clones that we have previously derived from TERA-2. These observations indicate heterogeneity among stem cells derived from a single human teratocarcinoma, and suggest that SSEA-3 and SSEA-4 are not necessarily integral features of the human EC phenotype. On the other hand, EC cells in xenograft tumors derived from the SSEA-3- and SSEA-4-negative clone re-express these epitopes. Further, this re-expression is stable, since EC cell lines that are SSEA-3- and SSEA-4-positive grow out when the tumors are explanted in vitro. We conclude that the expression of these globoseries epitopes can be modulated by environmental influences.

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.

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.