Changes of glycoconjugate expression profiles during early development

A variety of glycoconjugates, including glycosphingolipids (GSLs), expressed in mammalian tissues and cells were isolated and characterized in early biochemical studies. Later studies of virus-transformed fibroblasts demonstrated the association of GSL expression profiles with cell phenotypes. Changes of GSL expression profile were observed during mammalian embryogenesis. Cell surface molecules expressed on embryos in a stage-specific manner appeared to play key roles in regulation of cell-cell interaction and cell sorting during early development. Many mAbs showing stage-specific reactivity with mouse embryos were shown to recognize carbohydrate epitopes. Among various stage-specific embryonic antigens (SSEAs), SSEA-1 was found to react with neolacto-series GSL Le^x, while SSEA-3 and SSEA-4 reacted with globo-series Gb5 and monosialyl-Gb5, respectively. GSL expression during mouse early development was shown to shift rapidly from globo-series to neolacto/lacto-series, and then to ganglio-series. We found that multivalent Le^x caused decompaction of mouse embryos, indicating a functional role of Le^x epitope in the compaction process. Autoaggregation of mouse embryonal carcinoma (EC) F9 cells provided a useful model of the compaction process. We showed that Le^x-Le^x interaction, a novel type of molecular interavction termed carbohydrate-carbohydrate interaction (CCI), was involved in cell aggregation. Similar shifting of GSL expression profiles from globo-series and neolacto/lacto-series to ganglio-series was observed during differentiation of human EC cells and embryonic stem (ES) cells, reflecting the essential role of cell surface glycoconjugates in early development.     

Stage-specific embryonic antigen 3 as a marker of visceral extraembryonic endoderm

The distribution of the stage-specific embryonic antigen SSEA-3 was studied immunohistochemically on postimplantation mouse embryos. This carbohydrate antigen, identified as an epitope of a globo-series ganglioside isolated from human teratocarcinoma cells (Kannagi et al., 1983, J. Biol. Chem.258, 8934–8942) was originally detected on the zygote and mouse early cleavage-stage embryos. It disappears on the early blastocyst and reappears on the primitive endoderm of the implanting blastocyst (Shevinsky et al., 1982, Cell30, 697–705). We now show in the early egg cylinder (on the sixth day of pregnancy) SSEA-3 is present in the entire visceral endoderm but not in any other part of the conceptus. From Day 7 of pregnancy onward, SSEA-3 is restricted to the extraembryonic visceral endoderm and the visceral yolk sac cells. Therefore, SSEA-3 is a useful marker for this endodermal cell lineage in midgestational mouse embryos.     

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.     

Murine embryonic antigen (SSEA-1) is expressed on human cells and structurally related human blood group antigen I is expressed on mouse embryos

The stage-specific embryonic antigen (SSEA-1), present on embryonal carcinoma cells and on murine preimplantation embryos, is defined by a monoclonal antibody. The antigenic determinant of SSEA-1 is a carbohydrate structurally related to the human blood group antigen I. Since it has been suggested that the I antigen might represent a precursor or SSEA-1, we used antibodies to SSEA-1 and to I to analyze their expression on mouse preimplantation embryos. Both are expressed on mouse embryos; moreover, I is expressed on earlier embryos than SSEA-1. The I antigen is defined by its expression on human erythrocytes; accordingly, we examined expression of I and SSEA-1 on human peripheral blood elements. We find SSEA-1 to be expressed exclusively on human granulocytes while I is found only on erythrocytes. These results suggest that these closely related antigens can be independently expressed. Analysis of the expression of I and SSEA-1 was then extended to a series of mouse and human cell lines; some express both, some express only one, and some express neither of these antigens. The activation of specific glycosyltransferases and/or glycosidases during development and differentiation appears to be the biochemical mechanism regulating expression of these antigens.     

Glycolipid core structure switching from globo- to lacto- and ganglio-series during retinoic acid-induced differentiation of TERA-2-derived human embryonal carcinoma cells

We have analyzed the glycolipid markers of a recently cloned human embryonal carcinoma (EC) cell line, NTERA-2, which differentiates extensively into a variety of somatic cell types when exposed to retinoic acid. These tumor cells provide a model system that can be used to study the ontogeny of glycolipid diversity during human embryonic development. Glycolipid antigens were identified by cell surface immunofluorescence and thin-layer chromatography immunostaining using a comprehensive set of anticarbohydrate monoclonal antibodies. Undifferentiated NTERA-2 cells were found to express predominantly globo-series glycolipids, including Gb3, Gb5 (IV3GalGb4), globo-ganglioside (IV3NeuAc alpha 2----3GalGb4), globo-H (IV3Fuc alpha 1----2GalGb4), and globo-A (IV3GalNAc alpha 1----3[Fuc alpha 1----2]GalGb4). When NTERA-2 cells were induced to differentiate by culturing in the presence of 10(-5) M retinoic acid, a remarkable shift of cellular glycolipids from globo-series to lacto- and ganglio-series was observed: Globo-series structures declined, particularly during the period 7-20 days after first exposure to retinoic acid, while lacto-series structures, including fucosyl alpha 1----3 type 2 chain (Lex) and sialosyl type 2 chain, and ganglio-series structures, including GM3, GD3, 9-O-acetyl-GD3, GM2, GD2, and GT3, increased. The presence of globo-A and globo-H as the major ABH blood group antigens in undifferentiated NTERA-2 cells suggests that globo-series blood group antigens are embryonic antigens, synthesis of which switches to lacto-series during human development. Two-color immunofluorescence analysis indicated preferential expression of several ganglio- and lacto-series antigens on different subsets of differentiated cells and permitted the relationship of these subsets to the development of neurons in NTERA-2 cultures to be determined. The results suggest that glycosyltransferase, particularly those involved in controlling glycoconjugate core structure assembly, are key enzymes regulated during the differentiation of human EC cells and, by implication, during human embryogenesis.     

The beta 1,3-galactosyltransferase beta 3GalT-V is a stage-specific embryonic antigen-3 (SSEA-3) synthase

We have previously reported the molecular cloning of beta1, 3-galactosyltransferase-V (beta3GalT-V), which catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the core3 O-linked glycan GlcNAc(beta1,3)GalNAc structure. Further characterization indicated that the recombinant beta3GalT-V enzyme expressed in Sf9 insect cells also utilized the glycolipid Lc3Cer as an efficient acceptor. Surprisingly, we also found that beta3GalT-V catalyzes the transfer of Gal to the terminal GalNAc unit of the globoside Gb4, thereby synthesizing the glycolipid Gb5, also known as the stage-specific embryonic antigen-3 (SSEA-3). The SSEA-3 synthase activity of beta3GalT-V was confirmed in vivo by stable expression of the human beta3GalT-V gene in F9 mouse teratocarcinoma cells, as detected with the monoclonal antibody MC-631 by flow cytometry analysis and immunostaining of extracted glycolipids. The biological relation between SSEA-3 formation and beta3GalT-V was further documented by showing that F9 cells treated with the differentiation-inducing agent retinoic acid induced the expression of both the SSEA-3 epitope and the endogenous mouse beta3GalT-V gene. This study represents the first example of a glycosyltransferase, which utilizes two kinds of sugar acceptor substrates without requiring any additional modifier molecule.     

Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal antibodies

Three differentiation antigens of mouse teratocarcinoma stem cells are defined using a panel of ten IgM-class monoclonal antibodies raised against teratocarcinoma F9 cells. TEC-01 and four other antibodies define an antigen that corresponds to SSEA-1. TEC-02 antibody defines an antigen that is expressed on teratocarcinoma stem cells, parietal yolk sac cells PYS-2, unfertilized eggs including the zona pellucida and blastocysts. It is absent from all mouse adult tissues tested. Three other antibodies exhibit binding properties similar to TEC-02. TEC-03 antibody defines an antigen that is expressed on teratocarcinoma stem cells, PYS-2 cells and mouse blastocysts. It is absent from all mouse adult tissues except for lungs.     

Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells

A murine stage-specific embryonic antigen (SSEA3) is defined by reactivity with a monoclonal antibody prepared by immunization of a rat with 4- to 8-cell-stage mouse embryos. This antigenic determinant, present on oocytes, becomes restricted first to the inner cell mass at the blastocyst stage, and later to the primitive endoderm. Murine teratocarcinoma stem cells do not react with this antibody, whereas human teratocarcinoma stem cells are SSEA3-positive. This antigenic determinant is not expressed on a variety of other human and murine cell lines, but is found on the surface of human erythrocytes. It is a carbohydrate and is present on both cell-surface glycolipids and glycopeptides. These results demonstrate the feasibility of identifying stage-specific antigenic determinants with monoclonal antibody prepared against embryos. The need for thorough screening on a variety of cell types to establish developmentally important cross-reactivities is also emphasized.     

Monoclonal antibodies that bind to galactosylgloboside (SSEA-3 antigen)

The 3-fucosyllactosamine (3-FL) antigenic determinant, Gal(beta 1-4)[Fuc(alpha 1-3)]-GlcNAc-R, is very immunogenic in BALB/c mice. A panel of monoclonal antibodies was raised against a novel glycosphingolipid that contains a terminal 3-FL structure (structure 1): (formula: see text) Surprisingly, most of these antibodies reacted with the internal galactosylgloboside structure rather than with the terminal 3-FL epitope. The specificities of two of these antibodies, 5A3 and 8A7, were analyzed by enzyme-linked immunosorbent assay and immunostaining procedures, and compared with MC631 (anti-SSEA-3), the only other monoclonal antibody known to bind to galactosylgloboside. 5A3 and 8A7 bound equally well to structure 1 and to galactosylgloboside. These antibodies also bound to structure 1 from which the terminal galactose, or the galactose and fucose, had been removed, but not to globoside or sialylated galactosylgloboside. In contrast, MC631 did not bind to structure 1 but, as described previously, did bind to globoside and sialylated galactosylgloboside. Galactosylgloboside is a developmentally regulated antigen of mouse embryos, human teratocarcinomas, and mouse embryonic brain. Antibodies 5A3 and 8A7 will complement MC631 in the analysis of the distribution and regulation of galactosylgloboside.     

Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas

C-9-1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7-day embryos, primordial germ cells of 10∼12-day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17-day embryos. In the adult mice, C-9-1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C-9-1 antigen showed distribution similar to SSEA-1. However, C-9-1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA-1.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Glycosphingolipids of rabbit endometrium and their changes during pregnancy.

The glycolipids of nonpregnant and pregnant rabbit endometrium were characterized using a combination of biochemical and immunochemical techniques. Quantitative analyses indicated a 70% decline in acidic glycolipid (ganglioside) content during early pregnancy (day 6), and a 2.5-fold increase in neutral glycolipid content during later pregnancy (day 26). The major gangliosides of rabbit endometrium were identified by thin-layer chromatography as GM3 and GD3, with minor amounts of GM1, GD1a and GT1b. The major neutral glycolipids were identified similarly as globo-series structures Gb3 and Gb4. Monoclonal antibodies (mAbs) directed to glycolipid antigens permitted the detection of additional glycolipid species, including sialylated, sulfated and fucosylated lacto-series structures. Difucosyl Ley structure (defined by mAb AH-6) and sulfated-galactosyl structure (defined by mAb VESP 6.2) were identified by indirect immunofluorescence along the luminal surface of the endometrium during the implantation period. Rapid changes in the glycolipid composition of endometrial cells during early pregnancy may facilitate embryo adhesion and trophectoderm outgrowth during implantation.     

Sequential changes in glycolipid expression during human B cell differentiation: enzymatic bases

We have previously reported that human B cell differentiation is accompanied by sequential changes in glycosphingolipid expression. Pre-B cells contain lacto-series type II chain-based glycolipids and GM3 ganglioside; mature/activated B cells do not synthesize lacto-series compounds but express GM3 and globo-series glycolipids (Gb3 and Gb4); terminally differentiated B cells, in addition to these compounds, also contain GM2 ganglioside. At the cell surface, Gb3, Gb4 and GM2 constitute stage-specific antigens. To elucidate the biosynthetic mechanism leading to these modifications we have compared activities of the glycosyltransferases involved in the core structure assembly and the first elongation steps of neo-lacto, ganglio- and globo-series glycolipids. These glycosyltransferase activities have been measured in B cell lines and normal B lymphocytes at various stages of differentiation. We first determined the optimal requirements of the four glycosyltransferases which wynthesize Lc3, GM3, Gb4 and GM2 glycolipids in B lymphocytes and then tested these enzymes and the Gb3 synthetase in the selected B cells. The following results were obtained: ß1 → 3N-Acetylglucosaminyltransferase (Lc3 synthetase) has a high activity in pro- and pre-B cells whereas it is undetectable in more differentiated cells; α2 → 3 sialyltransferase (GM3 synthetase) is activated from the pre-B cell stage to the terminally differentiated myeloma cells; α → 4 galactosyltransferase (Gb3 synthetase) is only detected in cells representing the late stages of B cell differentiation; ß1 → 3N-Acetylgalactosaminyltransferase (Gb4 synthetase) is only found in some lymphoblastoid cell lines, representative of activated B cells whereas the ß1 → 4 N-Acetylgalactosaminyltransferase (GM2 synthetase) has a high activity in these lymphoblastoid cell lines and in terminally differentiated myeloma cells. These results suggest that the sequential shifts in the three major glycosphingolipid series observed during B cell differentiation are mostly due to sequential activations of the corresponding glycosyltransferases.     

Glycolipid glycosyltransferases in human embryonal carcinoma cells during retinoic acid induced differentiation

Retinoic acid induced differentiation of TERA-2-derived human embryonal carcinoma cells is accompanied by a dramatic reduction of extended globo-series glycolipids, including galactosyl globoside, sialylgalactosyl globoside, and globo-A antigen (each recognized by specific MoAbs). Associated with these glycolipid changes, the activities of two key enzymes, alpha 1----4 galactosyltransferase (for synthesis of globotriaosyl core structure) and beta 1----3 galactosyltransferase (for synthesis of galactosyl globoside), were found to be reduced 3- to 4-fold. The latter enzyme plays a key role in the synthesis of extended globo-series structures, and its characterization has not been reported previously. Therefore, its catalytic activity was studied in detail, including substrate specificity, detergent and phospholipid effects, pH and cation requirements, and apparent Km. During retinoic acid induced differentiation, a series of Lex glycolipid antigens (recognized by anti-SSEA-1 antibody) and their core structures (lacto-series type 2 chains) increase dramatically. In parallel with these changes in glycolipid expression, the activities of two key enzymes, beta 1----3 N-acetylglucosaminyltransferase (for extension of lacto-series type 2 chain) and alpha 1----3 fucosyltransferase (for synthesis of Lex structure), were found to increase by 4- and 2-fold, respectively. Similarly, an increase in the expression of several gangliosides (e.g., GD3 and GT3) during retinoic acid induced differentiation was mirrored by a 4-fold increase in the activity of alpha 2----3 sialyltransferase (for synthesis of ganglio core structure, GM3). The results suggest a coordinate regulation of key glycosyltransferases involved in core structure assembly and terminal chain modification.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex <Emphasis Type="Italic">N</Emphasis>-glycans or core 1-derived <Emphasis Type="Italic">O</Emphasis>-glycans are not required for the expression of stage-specific antigens SSEA-1, SSEA-3, SSEA-4, or Le<Superscript>Y</Superscript> in the preimplantation mouse embryo

The glycan epitopes termed stage-specific embryonic antigens (SSEA) occur on glycoproteins and glycolipids in mammals. However, it is not known whether these epitopes are attached to N- or O-glycans on glycoproteins and/or on glycolipids in the developing mouse embryo. In this paper the expression of the antigens SSEA-1, SSEA-3, SSEA-4 and Le^Y was examined on ovulated eggs, early embryos and blastocysts lacking either complex and hybrid N-glycans or core-1 derived O-glycans. In all cases, antigen expression determined by fluorescence microscopy of bound monoclonal antibodies to embryos at the stage of development of maximal expression was similar in mutant and control embryos. Thus, none of these developmental antigens are expressed solely on either complex N- or core 1-derived O-glycans attached to glycoproteins in the preimplantation mouse embryo. Furthermore, neither of these classes of glycan is essential for the expression of SSEA-1, SSEA-3, SSEA-4 or Le^Y on mouse embryos.     

Carbohydrate antigens expressed on stem cells and early embryonic cells

Lewis X antigen (Le(X)) is a marker of embryonic stem cells, embryonal carcinoma cells and multipotential cells of early embryos in the mouse. Le(X) is carried by branched, high-molecular weight poly-N-acetyllactosamines (embryoglycan). While embryoglycan is present in human embryonal carcinoma cells, Le(X) is not expressed in human embryonic stem cells, embryonal carcinoma cells or inner cell mass cells. Instead, these cells express SSEA-3 and SSEA-4, both of which are carried by globo-series glycolipids. Le(X) is a marker of primordial germ cells or multipotential stem cells derived from primordial germ cells both in the mouse and human. In other species of vertebrates, Le(X) is widely expressed in early embryonic cells and primordial germ cells, but the mode of expression is not completely conserved among species. Le(X) is expressed in neural stem cells from both humans and mice. Hematopoietic stem cells are not reported to express the above carbohydrate markers. A marker of these cells is CD34, a membrane-bound sialomucin. Another sialomucin, CD164 (MGC-24v) is expressed in hemotopoietic progenitor cells. As a function of Le(X) in stem cells, the promotion of integrin action is proposed, based on analyses of glycoproteins with the marker, cDNA transfection experiments and the inhibitory effects of an anti-Le(X) antibody. Most probably, Le(X) antigen as well as poly-N-acetyllactosamines play roles in the interactions on the same membrane. On the other hand, O-linked oligosaccharides on CD34 and CD164 are probably involved in the regulation of cell adhesion and proliferation via intercellular recognition.     

High-molecular-weight glycoproteins are the major carriers of the carbohydrate differentiation antigens I, i and SSEA-1 of mouse teratocarcinoma cells.

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

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

Monoclonal antibody directed to the stage-specific embryonic antigen (SSEA-1) reacts with branched glycosphingolipid similar in structure to Ii antigen

A monoclonal antibody reacting with early mouse embryos and murine embryonal carcinoma cells (F9) defines the stage-specific embryonic antigen (SSEA-1). We now report that the antigen (SSEA-1) is a complex glycolipid with the branched lacto-N-glycosyl series. Antibody to SSEA-1 reacts strongly with the branched H₄-glycosphingolipid but not with other various glycolipids so far tested. This reactivity was abolished by endo-β-galactosidase treatment. The homogeneous H₄-glycolipid not only reacted with the monoclonal antibody to SSEA-1 but also with antibody to I-(Ma), i-(Dench) and with anti-H specific lectin. Chemical analysis, including methylation, also indicates that the glycolipid antigen had a close resemblance to I-antigen.     

Expression of GD2 and GD3 gangliosides in human embryonic neural stem cells

NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.