Abstract
Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells.
Highlights
Carbohydrate epitopes are often used as markers for characterization of human pluripotent stem cells
Antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 ؋ 109 cells) of human embryonic stem cell lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulfglobopentaosylceramide
The sialyl-lactotetra expression was lost upon differentiation of human induced pluripotent stem cells (hiPSC) into hepatocyte-like cells, suggesting that this carbohydrate se quence is a novel marker for undifferentiated human stem cells
Summary
Carbohydrate epitopes are often used as markers for characterization of human pluripotent stem cells (hPSC). Antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 ؋ 109 cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulfglobopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. After eliminating glycan structures that were probably of mouse origin, it was concluded that, compared with human somatic cells, the human stem cells had a higher expression of the glycosphingolipids globopentaosylceramide/SSEA-3, sialyl-globopentaosylceramide/SSEA-4, fucosyl-lactotetraosylceramide/SSEA-5, di fucosyl-neolactotetraosylceramide, neolactopentaosylcerami de, neolactotetraosylceramide, globotetraosylceramide, the NeuGc-GM1 ganglioside, and gangliotriosylceramide/asialoGM2 These previous studies were done on minute amounts of hESC lines (105–106 cells). The sialyl-lactotetra expression was lost upon differentiation of hiPSC into hepatocyte-like cells, suggesting that this carbohydrate se quence is a novel marker for undifferentiated human stem cells
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