A Developmentally Regulated Heparan Sulfate Epitope Defines a Subpopulation with Increased Blood Potential During Mesodermal Differentiation

Abstract

Heparan sulfate (HS) is a mandatory coreceptor for many growth factors and morphogens involved in embryonic development; its bioactivity is dictated by complex sulfation motifs embedded within the polymer chain. Using a panel of HS-specific antibodies we have identified a unique HS epitope recognized by antibody HS4C3 that is selectively expressed during differentiation of embryonic stem (ES) cells along the mesodermal lineage to the hemangioblast stage. The appearance of this high-affinity HS4C3-binding (HS4C3(high)) epitope is transient; the epitope is specifically expressed within the emerging Brachyury(+) (Bry(+)) population and marks those cells that will become fetal liver kinase 1 (Flk1)(+). Fluorescence-activated cell sorting (FACS) separation and colony forming assays revealed that HS4C3(high)/Flk1(+) cells have a dramatically increased potential to form both blast and endothelial colonies, both of which depend upon the HS-binding growth factor vascular endothelial growth factor. Critically, expression of this HS epitope is tightly regulated, disappearing from the cell surface as the resultant hematopoietic lineages mature, in a similar manner to protein markers Bry and Flk1. In vivo studies showed a remarkable correlation with in vitro findings, with expression of HS4C3-binding epitopes restricted to newly formed mesodermal tissues during gastrulation. We believe this is the first time a defined HS epitope has been implicated in a specific developmental pathway and that this provides, in addition, a novel enrichment technique for the isolation of hemangioblasts from mixed differentiated ES cell cultures.