Abstract
Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.
Highlights
Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology
We focused our attention on the glycosphingolipid patterns, gangliosides, which were chemically defined by extraction, separation into the individual entities by high-performance TLC (HPTLC), and structural identification of each component by mass spectrometric procedures
Chondrogenesis was induced for 28 days in cell pellets and recognized with Alcian Blue staining (Fig. 1E). human dermal fibroblast (hDF) were used as controls for both immunophenotyping and differentiation
Summary
Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. Glycosphingolipids, gangliosides, represent an interesting class of components because: a) they are primarily exposed on the outer cell membrane in direct contact with the extracellular milieu [4]; b) they are involved in the formation of lipid rafts [5, 6]; c) they exhibit an extraordinarily large repertoire of different chemical structures [4,5,6,7]; and d) it Supplementary key words osteogenic differentiation sphingolipids stem cells characterization. We noted that in hBMSCs, ganglioside GD2, which was positive by the use of an anti-GD2 monoclonal antibody, was chemically undetectable, likely as a result of a molecular mimicry condition
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