Gangliosides belong to a heterogeneous family of lipids known as glycosphingolipids, which are ubiquitously expressed in vertebrate cells, and are particularly abundant in the central nervous system (1). Within mammalian cells, gangliosides are predominantly localized on the plasma membrane (2,3), where they form cell surface microdomains, including caveolae, lipid rafts, and glycolipid-enriched microdomains or cholesterol (2,4,5). Specifically, gangliosides are established to have various functions, including in cell proliferation, differentiation, immune response, adhesion, migration, apoptosis, and cell-cell and cell-substratum interactions (6). Gangliosides are classified by the presence of one or more sialic acid residues linked to different galactose and/or sialic acid residues, and are classified into asialo (o)-, a-, b- and c-series gangliosides, respectively (7).

Stem cells are widely used during research into developmental processes and offer tremendous potential in clinical applications for transplantation and tissue regeneration therapies (8). As they are undifferentiated, stem cells, specifically embryonic stem cells (ESCs), have a high potential for proliferation (self-renewal) and the capacity to differentiate into various distinct cell types (multipotency or pluripotency) (9). Induced pluripotent stem cells (iPSCs) have been generated from mouse fibroblasts via retroviral introduction of four defined transcription factors: POU domain, class 5, transcription factor 1 (Oct-4), SRY (sex determining region Y)-box 2 (Sox-2), c-Myc and Kruppel-like factor 4 (Klf-4) (10). Induction of pluripotency can be also achieved in mouse spermatogonial stem cells by self-reprogramming process (11,12). Reprogrammed pluripotent stem cells, such as iPSCs and germline-derived pluripotent stem cells, are indistinguishable from ESCs in terms of morphology, self-renewal, expression of ESC markers, and their differentiation ability (10,11,13,14). Neural stem cells (NSCs) are known to be self-renewing, multipotent cells that can differentiate into brain-forming cells, such as neurons and glial cells (astrocytes and oligodendrocytes) (15). NSCs highly express nestin, Musashi RNA-binding protein 1 (Musashi-1), Sox-2 and paired box 6 (Pax-6).

Carbohydrate-associated molecules are known to be involved in controlling cell surface interactions during development. Specifically, SSEA series were originally identified by defined carbohydrate epitopes associated with the lacto- and globo-series glycolipids, such as SSEA-1, SSEA-3 and SSEA-4 (16,17). These SSEA series were expressed in various tissues, cancer and cancer stem cells (18–23). Notably, SSEA series are present in pluripotent stem cells, such as ESCs and iPSCs. SSEA-1 (also termed CD15 and Lewis x) is present on the surface of murine embryos at the pre-implantation stage, in mouse germ cells and on the surface of teratocarcinoma stem cells (24). SSEA-1 is also produced in the thyroid, oviduct epithelium, endometrium and epididymis, and in certain areas of the brain and kidney tubules in adults (18,25). SSEA-1 production increases upon differentiation in human cells and decreases during differentiation in mice.

SSEA-3 and SSEA-4 are synthesized during oogenesis and are present in the membranes of oocytes, zygotes and early cleavage-stage embryos in human (24,26). They are present in undifferentiated primate ESCs, human embryonic germ cells, teratocarcinoma stem cells and ESCs (27).

Ganglioside biosynthesis and degradation occurs through several events: i) De novo ganglioside biosynthesis in the endoplasmic reticulum and Golgi apparatus, followed by vesicular sorting to the plasma membrane; ii) enzyme-assisted chemical modifications of molecules at the plasma membrane level; iii) internalization of gangliosides via endocytosis and recycling to the plasma membrane; iv) direct glycosylations following sorting from endosomes to the Golgi apparatus; v) degradation at the late endosomal/lysosomal level with formation of fragments of sugars (glucose, galactose, hexosamine, sialic acid) and lipids (ceramide, sphingosine, fatty acid); vi) metabolic recycling of these fragments for biosynthetic purposes (salvage pathways); and vii) further degradation of fragments to waste products (Fig. 1) (28).

Ceramides, a group of higher glycosphingolipids, are glucosylated by a glucosyl-transferase (29–31). An uncharacterized flippase enzyme caused Glc-ceramide to flip to the lumenal side of the cis-Golgi stack, where further glycosylations takes place. The first glycosylation, catalyzed by lactosyl (Lac)-ceramide synthase is galactosylation of Glc-ceramide to Lac-ceramide (Fig. 2) (32–34). Lac-ceramide is sialosylated to produce GM3, GD3 and GT3 molecules via the action of three sialyltransferases (SAT) I, II and III, each recognizing their specific acceptor substrate (35,36). GM3, GD3 and GT3 are the starting points for the ‘a-series’, ‘b-series’ and ‘c-series’ gangliosides, respectively. In each ganglioside series, N-acetyl-galactosaminyltransferase, galactosyl-transferase and SAT IV, in sequence, add an N-acetylgalactosamine, galactose and sialic acid group to the gangliosides, respectively, to produce more complex gangliosides. Further sialosylations can be performed by SAT V. From Lac-ceramide a further group of glycosphingolipids (‘O-series’) can be produced by the sequential action of N-acetyl-galactosaminyltransferase, galactosyl-transferase and sialyl-transferase IV and V, producing asialo-GM2 (GA2), asialo-GM1 (GA1), and gangliosides GM1b, GD1c and GD1α (28).

Mouse embryonic stem cells (mESCs) are derived from the inner cell mass of blastocysts (37). Established mESCs express various carbohydrate antigens, including glycolipids. Among those, SSEA-1 is the most well-established specific marker.

In E14 and Oct-4 promoter-EGFP (OG2) mESCs, small amounts of a-series gangliosides, GM3, GM1 and GD1a, were detected by high-performance thin-layer chromatography and immunocytochemistry analysis (38,39). Furthermore, in TC-1 mESCs, only glucosylceramide and lactosylceramide were present (40); however, J1 mESCs contained GM3, GM1, and GD3 (Fig. 3A) (41–43). Furthermore, 9-O acetyl GD3 was detected in 129S6/B6-F1/DsRed.T3 mESCs (44). GM3 and GD3 are known to be involved in cell adhesion and proliferation via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) 1/2 phosphorylation (Table I) (41,42,45,46). Specifically, small hairpin RNA knock-down of UDP-glucose ceramide glucosyltransferase (UGCG) to reduce glucosylceramide synthesis was demonstrated to inhibit activation of the Ras-MAPK pathway and cell proliferation (41).

OG2 mouse embryonic fibroblast (MEF) and mESCs produce GM3, GM1, and GD1a, but GM1 is not found in OG2 MEF-derived iPSCs (Fig. 3B) (39). Analysis of the cell proliferation rate in OG2 mESCs and iPSCs revealed that iPSCs have a lower proliferation than that of mESCs. GM1 is known to affect cell proliferation via the ERK 1/2-MAPK pathway and protects against apoptosis in various cell types (Table I) (39,42,47–49).

NSCs, also referred to as multipotent neural progenitor cells, can differentiate to cells of the neural linage, including neurons and glial cells (astrocytes and oligodendrocytes) (50). The NSCs highly express nestin, Musashi-1 and Sox-2; however, these marker molecules are intracellular or nuclear proteins. Several studies demonstrated that there are high levels of b-series gangliosides in NSCs (51–55). GD3 is a b-series disialoganglioside that is frequently detected in vertebrate embryos and immature proliferative cells (56,57). Particularly, GD3 and 9-O acetyl GD3 have been biochemically detected in mNSCs (44,53,58). GD3 is present in the subventricular zone of the lateral ventricle where NSCs are localized (59,60). Furthermore, GD3 expression in mouse neuroepithelial cells is enriched in NSCs, radial glia, embryonic-, postnatal-, and adult-NSCs (Fig. 3C) (53,58,61). Furthermore, NSCs differentiated from mESCs also express GD3 (unpublished data) (Fig. 3A).

The GD3 concentration is known to be high in embryonic brains, which predominantly consist of undifferentiated neural progenitor cells; however, the concentration in the brain rapidly decreases after birth (62). It has been demonstrated that NSCs derived from GD3-synthase knockout mice could not be maintained in in vitro culture (55); thus, it is speculated that GD3 has a major role in the maintenance of NSCs (51,54,63). It has been reported that when the GD3 level was reduced by an inhibitor of glucosylceramide synthesis, basic fibroblast growth factor-induced proliferation was repressed in primary mNSCs. Additionally, GD3 interacts with epidermal growth factor receptor (EGFR) (55,64). These finding imply that GD3 may induce early neural precursor cell differentiation and neurite formation (Table I) (41,43).

The pattern of ganglioside synthesis changes dramatically during nervous system development (65–67). Thus, gangliosides, including sulfatide (for the myelin sheath in the peripheral and central nerve system), galactosylceramide (for oligodendrocytes) and A2B5 (c-series gangliosides; for neural stem cells, oligodendrocytes, and astrocytes) are considered to be useful as differentiation markers of specific neural lineages (68). It was reported previously that the GD3 level is high in the brain and is involved in embryonic brain development; however, its concentration rapidly decreases during neural development, whereas other gangliosides, including GD1a, GT1b, and GQ1b, increase during aging and neural development (62). In addition, it was demonstrated that correlative changes of ganglioside composition accompany normal development in vitro and in vivo. Furthermore, b-series gangliosides, including GT1b and GQ1b, are present in mouse neuroepithelial cells (58,69).

Our previous studies demonstrated that GD3, GT1b, and GQ1b are present in cells during retinoic acid-induced neural differentiation of mESCs and embryonic carcinoma cells (Fig. 3A) (42,43). A number of approaches have been reported to determine the role of gangliosides during neural differentiation (70,71). Overproduction of gangliosides can facilitate neurite formation, which is part of the neural maturation process (43). By contrast, knock-down of Ugcg was reported to result in a decrease in the neural differentiation rate of mESCs and human dental pulp-derived mesenchymal stem cells (41,72). Therefore, these gangliosides have important regulatory roles in neural differentiation in vitro (41–43,73).

GD3 is involved in the early neural differentiation and maturation process (63,74), while GD1a induces early neural differentiation (43). By contrast with GD3, GT1b is necessary for the induction of neural differentiation and drastically enhances actin-rich dendrite generation (42,75). Furthermore, it GT1b syntheses is increased in brain synapses (74). GQ1b promotes neurite outgrowth during early neural differentiation via the ERK 1/2-MAPK pathway (Table I) (43,73,76).

Gangliosides are located on the plasma membrane and have roles in various functions of mouse stem cells. As described above, specific gangliosides are detected in mESCs, mouse iPSCs, mNSCs and differentiated neural cells. These gangliosides regulate cell proliferation and differentiation via the MAPK-ERK 1/2 pathway. Furthermore, gangliosides have been demonstrated to be useful marker molecules for detecting or sorting mouse stem cells and differentiated neural cells. Nevertheless, the functional roles of gangliosides during cellular differentiation and proliferation require further investigation. Identification of the gangliosides present in stem cells should be performed to thoroughly characterize marker gangliosides, and contribute to progression in basic stem cell research and clinical applications.