Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Abstract

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.

Figure 1

Glycomic profiling of hPSCs and non-pluripotent cells using lectin microarrays with validation of lectin binding to hPSCs. (A) Hydrophobic proteins isolated from the cells were labeled and analyzed by lectin microarrays. Supervised hierarchical clustering based on the array data of 13 priority lectins for the hydrophobic proteins of 26 samples of hPSCs (12 samples of hESC and 14 samples of hiPSCs, including 2 clones of hiPSCs newly reprogrammed from human primary melanocytes) and 22 samples of mammalian non-pluripotent cells (4 samples of HDFs, 4 samples of human melanoma cells, 1 sample of primary human melanocytes, 1 sample of mouse embryonic fibroblasts, 2 samples of human leukocytes, 1 sample of human adult brain tissue, 1 sample of human fetal liver tissue, 1 sample of motor neuron precursor cells differentiated from WA07 cells, 1 sample of cardiac muscle, 1 sample of adipose tissue, 2 samples of skeletal muscle, 1 sample of adrenal gland, 1 sample of lung tissue, and 1 sample of renal epithelium) was performed using the NIA Array Analysis Tool. In the dendrogram, samples acquired at the Scripps Research Institute and Kyoto University are indicated by yellow and blue solid squares, respectively. Pluripotent and non-pluripotent cells are indicated by red dots and green dots, respectively. Some of the samples are highlighted by colored lines to emphasize their relationships. Purple line: WA07 cells. Pink line: motor neuron precursor cells differentiated from WA07 cells. Black line: human primary melanocytes. Orange line: hiPSCs derived from primary melanocytes. (B) Fluorescent intensity of UEA-1, AOL, and TJA-II lectin binding with hydrophobic cell extracts based on microarray data. RFU: relative fluorescence units. (C) WA09 cells were spiked into a cell suspension of HDFs to create a mixed cell population. The cells were fixed on adhesion slides and then subjected to staining with anti-POU5F1 and SSEA-4 antibodies as well as biotinylated UEA-I lectin. WA09 cells with expression of pluripotent biomarkers POU5F1 or SSEA-4 are indicated by arrowheads and positive UEA-I staining. Insets, magnified images of the double-positive cells.

Figure 2

Lectins can be used to purge pluripotent cells from mixed populations. (A) Diagram of depletion experiments. hESCs (unlabeled) were mixed with different Calcein AM-labeled differentiated cells (green) at a ratio of 1:1. Cells were incubated with UEA-I magnetic beads. The cells captured by the magnetic beads were isolated and the unbound, eluted cells were analyzed for Calcein AM fluorescence by flow cytometry. (B) Results of depletion experiments using mixtures of WA09 and HDFs, WA09 and HEMd, and R-Olig2 hESCs and their NSC derivatives. For each sample, the unreacted mixture and the mixture reacted with beads showed about 50% Calcein AM-positive cells because half of the cells in the 1:1 mixture were labeled. After magnetic separation of the beads, nearly all of the remaining cells were Calcein AM-positive, indicating that the pluripotent cells had been removed from the mixture.

Figure 3

Lectin binding to pluripotent cells. (A) WA09 hES cells were incubated with streptavidin-AF 555 only or with streptavidin-AF 555 and biotinylated UEA-I. Human dermal fibroblasts (HDFs) were incubated with streptavidin-AF 555 and biotinylated UEA-I. Fluorescence intensity was analyzed by flow cytometry. As expected, WA09 cells incubated with streptavidin-AF 555 alone (negative controls) as well as HDFs incubated with streptavidin-AF 555 and biotinylated UEA-I both showed minimal levels of fluorescence, while WA09 cells incubated with streptavidin-AF 555 and biotinylated UEA-I showed high levels of fluorescence. (B) WA09, R-Olig2, and iPS1.HDF pluripotent cells were incubated with secondary antibody and streptavidin-AF 555 only (negative control; upper right) or SSEA-4 antibody, secondary antibody, UEA-I biotinylated lectin and streptavidin-AF 555 (treated cells, lower right), and subjected to flow cytometry. The negative control cells show minimal fluorescence, but more than 95% of the treated cells in all three tested hPSC lines show either double-positive or double-negative staining. This indicates that biotinylated UEA-I lectin can be used in flow cytometry and that it labels a similar percentage of pluripotent cells as SSEA-4, a well-recognized biomarker of human cell pluripotency.

Figure 4

Gene expression profiles of human fucosyltransferases and sialyltransferases in hPSCs and non-pluripotent cells. (A) RNA was hybridized to Illumina arrays as described in Materials and Methods. The heat-map representation of the array data indicates the relative expression levels of specific glycosyltransferases in the cells. The results of the hierarchical clustering of expression patterns in different cell types are illustrated as a dendrogram. Pluripotent and non-pluripotent cells are indicated by red and green dots, respectively. (B) The expression levels of 13 fucosyltransferases and 19 sialyltransferases in 22 samples of hPSCs (8 samples of hiPSCs and 14 samples of hESCs) and 27 samples of human non-pluripotent cells (14 samples of organ tissues and 13 samples of cultured cells). The fluorescence intensities of each gene probe were averaged, log2-transformed and plotted. Columns, mean of log2-transformed fluorescence intensities; bars, standard deviation. ^*P < 0.01, fold change of expression > 1.4 (differential expression analysis performed by GenePattern using non-transformed values of intensity).