Ganglioside expression delineates human mesenchymal stem/stromal cell populations derived from different tissue...

Prior studies have indicated that human embryonic stem cells can be distinguished from those of other mammals based on variable expression of a class of membrane glycolipids known as glycosphingolipids (GSLs), raising the question as to whether GSL display could be utilized to phenotypically define subsets of human adult stem cell populations. Adult stem cells known as "mesenchymal stem/stromal cells" (MSCs) have shown immense promise in therapeutic applications for a variety of clinical indications. Most commonly, these cells are harnessed and then culture-expanded from bone-marrow (BM-MSCs) or from adipose tissue (A-MSCs) sources. Though operational differences exist between human BM-MSCs and A-MSCs, no surface markers have been characterized to date that distinguish these as distinct subsets of culture-expanded human adult stem cells. Accordingly, we isolated GSLs from primary cultures of marrow- and adipose-derived human MSCs and an unbiased screen was performed by mass spectrometry (via matrix-assisted laser desorption/ionization (MALDI)-quadrupole ion trap (QIT)-time-of-flight (TOF), hence, via "MALDI-QIT-TOF") to analyze all component glycans. Flow cytometry was then undertaken to assess the relative levels of expression of MS-defined glycan determinants, followed by RT-qPCR to measure transcripts of genes encoding key enzymes involved in glycolipid biosynthesis. Notably, our data indicate that neither BM- nor A-MSCs display any significant level of either lacto-series or neolacto-series GSLs, but distinct differences exist in GSL species among A-MSCs and BM-MSCs: while both cell types express GSLs of the ganglio- and the globo-series, the ganglio-series GSLs GD3 and GD2 and the globo-series GSL SSEA-4 (also known as sialylGb5) are dominantly expressed only among human BM-MSCs. These structural features are shaped by divergent patterns of glycosyltransferase gene expression, with striking differences between BM- and A-MSCs in the expression of transcripts encoding GD3 synthase, GM2/GD2 synthase, and Gb5 synthase. Importantly, expression of GD3, GD2, and SSEA-4 is markedly diminished on differentiation of BM-MSCs, and co-cultures of A-MSCs and BM-MSCs show that the expression of GD3, GD2, and SSEA-4 is a cell-intrinsic feature of BM-MSCs. These data stratify the glycosignature(s) of human MSCs derived from different tissue sources, provide direct evidence that expression of these structures is cell stage-/lineage-specific, unveil the mechanistic basis of the differential expression of these glycan determinants, and draw attention to how knowledge of the MSC glycosignature can impact cytotherapeutic strategies.