In this study, we identified lectin biomarkers for pluripotent cells, and show that specific lectins can be used to identify, sort, and separate human pluripotent cells (hPSCs) in mixed populations with differentiated cells. Our results suggest that lectins can be integrated into strategies for purifying populations of hPSC-derived differentiated cells destined for drug screening, studies of human disease, and cell replacement therapy. It is especially critical to purge undifferentiated hPSCs from cell preparations used for clinical transplantation, because the greatest concern for using hPSC-derived cells is that residual undifferentiated cells may be tumorigenic. To our knowledge, this is the first report showing that lectins can be used to isolate and remove hPSCs from mixed cell populations. Historically, lectins have been known for their affinity for glycans on many different cell types. The idea that lectins may react with pluripotent cells was initiated in early 1980s by studies showing the ability of lectins to bind to or kill embryonal carcinoma and germ cell tumor cells 28, 29, 30, 31, 32
. However, a comprehensive, large-scale analysis of lectin binding in hPSCs was not tangible until recently 21, 22
. Consistent with our findings, hPSC-preferential binding of lectins that have high affinity to glycans containing sialic acid α2-6 and fucose α1-2 structures was observed in a study using a large quantity but relatively limited types of cell samples 21
. The presence of unique fucosylation signatures on proteins in hPSCs has also been detected and associated with the pluripotent status of the cells by proteomic analysis 17
. Although differential lectin binding to proteins extracted from pluripotent stem cells using array-based approaches has been shown by two recent reports, neither study attempted to apply this information for developing methods for isolation of pluripotent cells from mixed populations or for purging differentiated populations of residual pluripotent cells 21, 22
The data presented here show that the fucose-specific lectin UEA-I is a potential tool for isolating viable hPSCs with sustained pluripotency from heterogeneous populations (Supplementary information, Figure S3c
). Currently, separation of cell subpopulations relies in large part on antibodies to cell surface epitopes, and our research suggests that cell type-specific lectins can complement existing methods. In some cases, lectins may offer certain advantages over antibodies. They can be easily removed from cells without damaging them, by adding an oligosaccharide or monosaccharide that competes with the glycan to which the lectin binds. Also, since lectins are significantly less expensive than antibodies, they may offer advantages for procedures in which cost is an important factor, such as large-scale hPSC purifications for cell therapy and drug screening.
Cell surface antibodies are often species-specific; for example, SSEA-4 antibody, a commonly used antibody for human PSCs, does not recognize mouse PSCs, and instead binds to differentiated mouse cells. In contrast, our data suggest that the same lectins that can be used to identify human PSCs can also be used for pluripotent cells from other species, including mouse (Supplementary information, Figure S5
) and a diversity of other animals (not shown). Thus, for labs working with pluripotent cells from more than one species, the same lectin can be used for all pluripotency monitoring and isolation applications. Also, the cross-species lectin recognition of PSCs implies that certain types of glycosylation are hallmarks of the pluripotent state; this suggests that studies of specific glycosylation may reveal novel information about transitions that occur during cellular reprogramming and differentiation of PSCs.
Many of the lectins that showed preferential association with the hydrophobic proteins of hPSCs bind to fucosylated and sialylated glycans, suggesting that fucosylation and sialylation are common characteristics of PSC glycoproteins. Fucosylation and sialylation are two common types of glycosylation on the cell surface 26, 33
, and these modifications have been shown to be critical in normal embryogenesis and somatic stem cell differentiation 16, 34, 35, 36
. Our gene expression analysis suggests that the protein fucosylation and sialylation signatures in hPSCs may be due to differential expression of specific fucosyltransferases and sialyltransferases in pluripotent cells. This supports the idea that post-translational glycosylation of cell surface proteins plays a role in the regulation of transitions between pluripotent and differentiated states that occur during reprogramming of somatic cells and differentiation of pluripotent cells. It is also interesting to note that hydrophilic glycoproteins, which are primarily intracellular proteins, also differed between pluripotent and differentiated cells. In particular, we observed differences in GalNAc-containing glycoconjugates; GalNAc usually acts as a core component of O
-linked glycans, which are frequently found on serine and threonine residues in mucins and other secreted proteins 37
. This suggests that O
-linked glycosylation of serine and threonine in secreted proteins may be involved in the regulation of pluripotency, possibly by controlling autocrine or paracrine feedback loops.
Our work shows that lectin-based approaches are sensitive and reliable tools to detect pluripotency-associated protein glycosylation. The most important application of lectins to the basic and clinical applications of hPSCs is likely to be removal of residual pluripotent cells from differentiated populations, and to our knowledge, our report is the first to show the feasibility of using lectins to remove undifferentiated pluripotent cells from mixed populations. Additionally, our work suggests that glycosylation changes may also provide clues to the mechanisms underlying reprogramming, pluripotency, and differentiation. We anticipate that the use of lectins as biomarkers will become a valuable tool for stem cell research, especially for translational applications such as purifying precursor populations or purging pluripotent cells from differentiated populations.