Human Embryonic Stem Cell Differentiation: Role of Glycosphingolipid Structure

Abstract

Glycosphingolipids (GSLs) are ubiquitous components of cell membranes that can act as mediators of cell adhesion and signal transduction and possibly be used as cell type-specific markers. A systematic survey of expression profiles of GSLs in human embryonic stem cell (hESC) lines and various differentiated derivatives was carried out using immunofluorescence, flow cytometry, and MALDI-MS and MS/MS analyses. In the undifferentiated hESCs, in addition to the well-known hESC-specific markers, SSEA-3 and SSEA-4, we identified several globosides and lacto-series GSLs, previously unrevealed in hESCs, including Gb₄Cer, Lc₄Cer, fucosyl Lc₄Cer, Globo H, and disialyl Gb₅Cer. During differentiation of hESC into embryoid body (EB) outgrowth cells, MS analyses revealed a clear-cut switch in the core structures of GSLs from globo- and lacto- to ganglio-series. To further clarify alterations is correlated with lineage-specific differentiation, we analyzed changes in GSL compositions as hESCs differentiated into neural progenitors or endodermal cells. During differentiation into neural progenitor cells, we found that the core structures of GSLs switched to mostly ganglio-series dominated by GD3. On the other hand, when hESCs differentiated into endodermal cells, patterns of GSLs were totally different from those observed in EB outgrowth or neural progenitors. The most prominent GSL identified by MALDI-MS and MS/SM analysis was Gb₄Cer, without any appreciable amount of SSEA-3 and 4 antigens, or GD3, in endodermal cells. We also demonstrated that such a switch in GSL profiling was attributable to altered expression of key glycosyltransferases (GTs) in the biosynthetic pathways, suggesting a close association of GSLs with lineage specificity and differentiation of hESCs. Therefore, these results provide new insights into the unique stage-specific transition and mechanism for alterations of GSL core structures during hESC differentiation.