Use of lectins for probing differentiated human embryonic stem cells for carbohydrates

The carbohydrates present on the surface of differentiated human embryonic stem cells (hESCs) are not yet well established. Here, we have employed a panel of lectins and several anti-carbohydrate antibodies to determine the carbohydrates that are present at day 12 of hESC differentiation as embryoid bodies (EBs). On the basis of staining with fluorescein-labeled lectins, we have determined the presence of both terminal and internally linked alpha-d-mannopyranosyl groups, poly-N-acetyllactosaminyl chains, both alpha 2,3- and alpha 2,6-linked N-acetylneuraminic acid (Neu5Ac), alpha 1,6-linked l-fucosyl, and beta-d-galactosyl groups, and more specifically, the T, Tn, and sialyl-Tn antigens. However, no alpha 1,2-linked l-fucosyl, terminal nonreducing alpha-d-galactosyl, N-acetyl-beta-d-glucosaminyl, nor N-acetyl-alpha-d-galactosaminyl groups were found by this approach. We also established the presence of Neu5Ac alpha 2,3/2,6-Gal beta 1,4 GlcNAc-terminated chains on the surfaces of 12-day-old EBs, as indicated by the great enhancement of staining by Erythrina cristagalli agglutinin (ECA) after treatment with neuraminidase. In each case, inhibition of binding by a haptenic sugar or treatment with neuraminidase was used to eliminate the possibility of nonspecific binding of the lectins. A comparison with undifferentiated cell staining revealed an increase in alpha 2,3-linked Neu5Ac as well as a change to exclusively alpha 1,6-linked l-fucose upon differentiation.