Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

BACKGROUND: Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4), to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. RESULTS: Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomato)esculetum lectin (TL), Ricinus communis agglutinin (RCA), and Concanavalin A (Con A) bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA) and Lotus tetragonolobus lectin (LTL) did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin (UEA), Phaseolus vulgaris erythro-agglutinin (PHA-E), and Maackia amurensis agglutinin (MAA) bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. CONCLUSION: Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the pluripotent state of hESCs because binding percentages and binding localization of these lectins are similar to those of SSEA-4. Non-binding lectins, DBA and LTL, may identify differentiated cell types; however, we did not find these lectins to bind to pluripotent SSEA-4 positive hESCs. This work represents a fundamental base to systematically classify pluripotent hESCs, and in future studies these lectins may be used to distinguish differentiated hESC types based on glycan presentation that accompanies differentiation

Differing Lectin Binding Profiles among Human Embryonic Stem Cells and Derivatives Aid in the Isolation of Neural Progenitor Cells

Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells

Fluorescence assisted cell sorting of hNPs using VVA lectin.

<p>hMP cells (A), hESCs (B) and hNP cells (C) stained with VVA-fluorescein lectin, analyzed for binding and used as controls. A mixed population of hESCs, hNPs and hMPs (1∶1∶1 ratio) was stained with VVA-fluorescein lectin and sorted for fluorescein positive cells (D). The sorted cells stained positive for Nestin and SOX2 (E); and were negative for OCT4 (F), and CD166 (G). Cells were stained with DAPI for nucleus. Scale bar: 10 µm.</p

Immunocytochemistry of hNP cell cultures for Nestin expression and binding to lectins.

<p>The panels in the left column show hNP cells staining for Nestin and DAPI (panels A, D, G, J, M, P, S, and V). In the same field of view as the left panel, the middle panels show binding of ConA(B), Pha-L (E), MMA (H), VVA (K), DBA (N), Pha-E (Q), LTL (T), and PNA (W) lectins in hNP cells. The merge view of lectin and Nestin staining is shown in panels in the right column. The staining amount roughly correlates with flow cytometry analysis. Scale bar: 10 µm.</p

Quantification of lectin binding in hESC, hNP and hMP cell surfaces with 8 different lectins.

<p>The percent of cells with specific carbohydrate expression was determined by flow cytometry using 8 different lectins. Each of the 3 cell types were separately stained with one of 8 lectins. The data is represented as average +/− SD of 4 independent assays of hESCs, hNP cells and hMP cells. Means with different letters are significantly different, # indicates p<0.05 compared to hESCs and * indicates p<0.05 compared to hNP cells.</p

Features of reviewed plant lectins.

<p>Features of reviewed plant lectins.</p

Flow cytometry histograms of lectin binding in hESCs, hNPs and hMPs.

<p>Percentage of cells binding to 8 different lectins was determined by flow cytometry. A representative lectin histogram plot is shown for one of 4 experimental replicates. In each panel far left grey fill peak in the histogram plot correlates with cells stained with secondary antibody only, and the shifted black tracing peak represents cells binding to a lectin. Panels in the left column show histograms of 8 different lectins binding to hESCs. Panels in the middle column are for hNP cells and panels in the right column are for hMP cells. Gating for each histogram indicates % of cells positive for the lectin-binding.</p