Role of heparan sulfate in embryoid body formation

Introduction Embryoid bodies (Ebs) are layered, ordered aggregates of cells which form when murine embryonic stem (ES) cells are grown in suspension. Ebs are made up of primitive endoderm cells overlaying an epithelium of epiblast cells, separated by a basement membrane and surrounding a central cavity. The generation of Ebs from undifferentiated ES cells has been used as a model for early development. The process of EB formation has been shown to require signalling through fibroblast growth factor (FGF) receptor 2, implicating heparan sulfate (HS) which is a necessary coreceptor for the formation of a signalling complex. Heparan sulfate proteoglycans are found both on the surface of cells and in the extracellular matrix. Within the cells, a newly synthesized HS chain is partially modified by a family of enzymes, many of which have multiple isoforms with differing substrate preferences. These modifications include the addition of sulfate groups at up to four positions on each disaccharide unit. The pattern of modifications that a HS saccharide undergoes alters its structure, affecting the ability of the HS to bind and regulate different FGF‐FGF receptor combinations, and so regulate signalling outcomes. It is hypothesized that the complement of modification enzymes that a cell expresses determines the structure of the HS that the cell produces, thereby altering the signalling response of the cell to HS‐binding factors such as FGFs. In this way, an ES cell could alter its HS to allow signalling by, for example, a particular FGF, setting in motion a chain of intracellular events leading to differentiation. Materials and methods Methods used in this study include tissue culture, RT‐PCR and immunocytochemistry. Results We have found that undifferentiated ES cells express HS as observed by cell staining with anti‐HS antibodies. They also display particular expression patterns of the different HS modification enzyme isoforms as measured by RT‐PCR. Studies on alterations in HS synthesis and structure during the process of EB formation are underway. In addition, the effect of the addition of exogenous heparin (a highly sulfated analogue of HS) and HS saccharides with variant structures on EB formation is being investigated. Discussion Alterations in the profile of modification enzymes expressed by ES cells as they differentiate may correlate with changes in the structure of the HS they make. These changes may allow the differentiating cells to control the way