1Dept of Chemical Physiology, Scripps ResearchInstitute, La Jolla, CA, USA, 2Center for iPS Cell Research andApplication (CiRA), Kyoto University, Kyoto, Japan, 3Dept ofReproductive Medicine, University of California, San Diego, La Jolla, CA, USA
Rapid and reliable methods for determining pluripotency in humanpluripotent stem cell (hPSC) populations are critically needed, not only forquality control in basic research but also for purification of differentiatedhPSCs intended for clinical use. Antibodies targeting cell surface antigens arecommonly used to identify pluripotent cells in preparations of viable cells.These pluripotency-associated antigens are often glycoproteins or glycolipids,suggesting that unique glycosylation patterns potentially recognized byspecific lectins may be a hallmark of pluripotency. Using protein microarrayscontaining 45 different lectins, we discovered unique glycosylation patternsand distinctive lectin-binding signatures that distinguish pluripotent cellsfrom non-pluripotent cells, identified by analysis of glycoproteins extractedfrom 26 hPSC and 15 differentiated cell samples. As few as 15 lectins weresufficient to accurately distinguish hPSCs from non-pluripotent cell types.These highly specific biomarkers were shared by all 12 human embryonic stemcell and 14 human induced pluripotent stem cell samples examined, regardless ofthe method of derivation, the culture condition, the cell type of origin, andthe reprogramming strategy used. We then demonstrated the utility of specificlectins as tools for identification and isolation of hPSCs from mixed cellpopulations. Lectin-mediated fluorescence staining showed extremely highconcordance with staining for validated pluripotency-associated transcriptionfactors, such as OCT4/POU5F1. In addition, we were able to separate a 1:1mixture of hPSCs:non-pluripotent cells into two cell populations that were 90%pure using lections conjugated to magnetic beads. Gene expression analysisusing cDNA microarrays suggested the differential expression offucosyltransferases and sialyltransferases may be the mechanism underlying theassociation between pluripotency and specific patterns of proteinglycosylation. Our results demonstrate that protein glycosylation differsconsiderably between pluripotent and non-pluripotent cells, and suggest thatregulation of pluripotency-associated glycoproteins may be mediated bydifferential gene expression of glycosyltransferases. Specific lectins thatrecognize the pluripotency-associated glycoproteins are potentially usefulbiomarkers that can be used to monitor pluripotency in stem cell populationsand separate pluripotent and non-pluripotent cell populations for both researchand therapeutic purposes.
