This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Cell surface markers are key tools that are frequently used to characterize and separate mixed cell populations. Existing cell surface markers used to define murine embryonic stem cells (mESCs), such as stage specific embryonic antigen 1 (SSEA1), Forssman antigen (FA), alkaline phosphatase (AP) and CD9, are limiting, however, because they do not unambiguously define the pluripotent state and are not reliable indicators of differentiation commitment. To identify glycan cell surface markers that would circumvent this problem we used a panel of 18 lectins to identify epitopes specifically elevated on the surface of mESCs which, during differentiation, decrease with kinetics that precede currently used markers such as CD9, SSEA1, FA and AP. The anticipated outcome of this analysis was to identify glycans that have utility as a reliable mESC marker and as a high resolution readout for early differentiation commitment. Here we show that the lectin Dolichos biflorus agglutinin (DBA), recognizes alpha-N-acetylgalactosamine (GalNAc) cell surface epitopes on mESCs (CD9high SSEA1high APhigh DBAhigh). These glycan epitopes decline markedly in cells undergoing the first definable step of differentiation, the transition from mESCs to primitive ectoderm (CD9high SSEA1high APhigh DBAlow). Loss of GalNAc epitopes is therefore the earliest cell surface change that can be assigned to differentiating cells and the only cell surface marker known to be tightly associated with the pluripotent state. Therefore, the lectin DBA is a useful tool to characterize mESC cultures by non-destructive approaches, an indicator of differentiation commitment and a predictor of developmental potency.
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