Establishing a cellular microarray to visualize glycan mediated signaling in mouse embryonic stem cells

Glycosaminoglycan (GAG) mediated growth factor signaling initiates differentiation in embryonic stem cells (ESCs). Manipulation of the GAG interactome affords a novel tool to understand and direct stem cell differentiation. In this study, a high-throughput miniaturized cellular microarray platform, consisting of gelatin robotically spotted onto passivated glass slides is established to assess the effects of cellular glycan microenviroment on growth factor signaling in mouse embryonic stem cells (mESCs). Heparan sulfate (HS) acts as co-receptor for fibroblast growth factor 2 (FGF2) and the subsequent activation of the mitogen activated protein kinase (MAPK) pathway is measured by extracellular regulated kinase (ERK) phosphorylation. Using HS deficient EXT1-/-¬ ESCs, FGF2 signaling is rescued using soluble and immobilized heparin. Immobilization of glycans on cellular microarrays offer potential as a powerful tool for systematically evaluating and directing cellular differentiation.Then, using conventional culture conditions, the interactions of HS and FGF2 are screened using bis-2-methyl-4-amino-quinolyl-6-carbamide, or surfen, and compared to the commercial FGFR inhibitor PD173074. Surfen reversibly antagonizes heparan sulfate in the µm range and can HS mediated interactions can be rescued using soluble heparin, a highly sulfated HS analog