Temporal changes in the carbohydrates expressed on BG01 human embryonic stem cells during differentiation as embryoid bodies

Abstract

Cell surface carbohydrates present on BG01 human embryonic stem cells after 28 days of differentiation were examined using two classes of carbohydrate binding proteins: lectins and antibodies specific for carbohydrate epitopes. Specificity of lectin staining was verified using carbohydrate ligands to block lectin interaction, glycohydrolases to cleave specific sugar residues that are receptors for these proteins, and periodate oxidation to destroy susceptible sugar residues. Specific antibodies were used to identify various tissue types and germ layers present in the 12- and 28-day differentiating embryoid bodies. Results from 12 and 28-day differentiated embryoid bodies were compared to determine changes over time. A slight increase in the sialylation of alpha-GalNAc was seen between 12 and 28 days of differentiation due to the presence of sialyl Tn and/or other sialylated alpha-GalNAc residues. Increases were also observed in GalNAc, the T antigen (Gal beta1,3 GalNAc), and difucosylated LacNAc residues during this time interval. Additionally, some distinct differences in the pattern of lectin staining between 12 and 28 days were observed. Not unexpectedly, the presence of most differentiated cell-types increased during this time period with the exception of neural progenitors, which decreased. Undifferentiated cells, which were prevalent in the 12-day EBs, were undetectable after 28 days. We conclude that several changes in glycosylation occurred during the differentiation of embryonic stem cells, and that these changes may play a role in embryonic development.

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage

“…The conservation of a specific glycan structure between different stem cell types within the same species, or conservation between different species on a certain stem cell type, may be an indication of an essential function for the glycan structure in multipotent cells [19]. At least to our knowledge, no glycosylation studies on MSCs from different species have been conducted, but we have demonstrated here similarities in stem cell glycosylation between human MSCs and both human embryonic stem cells [22][23][24] and hematopoietic stem cells [28]. It seems evident that glycan structures may be"switched on" and "off" on cell surfaces several times during cellular differentiation in developmental processes, as implied by the transition between linear and branched poly-LacNAc structures [21].…”

“…The majority of the studies performed to date have been carried out by using different glycan binding proteins, e.g. lectins or antibodies against glycan epitopes [22][23][24]. Of these binders, antibodies against SSEA-3, SSEA-4, Tra-1-60, Tra-1-81 [25,26] have been generally accepted as tools for embryonic stem cell validation [27].…”

LacdiNAc (GalNAcβ1-4GlcNAc) Contributes to Self-Renewal of Mouse Embryonic Stem Cells by Regulating Leukemia Inhibitory Factor/STAT3 Signaling

“…It has recently been demonstrated that the cell surface glycan heparan sulfate (HS) contributes to self-renewal and differentiation of mESCs by regulating BMP, Wnt, and FGF signaling [21,[27][28][29]. The expression patterns of several other cell surface glycans have now been described in undifferentiated and differentiated mESCs and hESCs [30][31][32][33][34]. However, although these glycans may be involved in the regulation of the signaling required for ESC self-renewal, with the exception of HS, their functional roles have not been demonstrated.…”

A novel role for proteomics in the discovery of cell‐surface markers on stem cells: Scratching the surface

“…Several studies on stem cell glycoproteins have been published, and because the topic is especially relevant for a discussion of cell-surface proteins, it is being included here. In two reports by Wearne et al [68,69], cell-surface glycans present on BG01 hESC that had been differentiated for 12 days as embryoid bodies (EB) and the changes in the carbohydrates that occur between 12-28 days of differentiation as EB were analyzed using fluorescence microscopy with fluorescein-labeled lectins and antibodies specific for carbohydrate epitopes. In another study, MALDI-TOF MS profiling, NMR structural analysis, and lectin-based flow cytometry were used to compare the N-glycan structures of cord blood-derived CD133 + and CD133 -cells [70].…”