3030 – A GLYCAN BASED APPROACH TO CHARACTERIZING AND ISOLATING CELLS IN THE HEMATOPOIETIC SYSTEM

Abstract

The cell surface contains many molecules that confer identity and function. Flow cytometry and the cluster of differentiation (CD) marker system have revolutionized cell characterization and isolation. Despite this, cell populations such as hematopoietic stem and progenitors (HSPCs) remain heterogenous. Heparan sulfates (HS) are linear polysaccharides of extraordinary diversity with cell- and context-specific modification patterns. Here we utilized single-chain variable fragment antibodies (scFvs) against differentially modified HS to analyze hematopoietic cells. This approach detected different HS patterns in closely related progenitors as well as distinct lineages. HS-specific scFvs differentially bound murine HSPCs, thereby establishing unique cellular "glycotypes" and uncovering divergent and dynamic HS modification expression, specifically within the continuum of megakaryocyte and erythrocyte differentiation. Prospective HS scFv-based isolation of cellular subtypes within immunophenotypic megakaryocyte-erythrocyte progenitors (MEPs) revealed unique functional and molecular phenotypes. These discrete populations could also be purified directly by HS scFvs from a broad pool of HSPCs, as confirmed by in vitro and in vivo functional assays, as well as single-cell transcriptomics. Profiling of human HSPCs, including in differentiation assays towards megakaryocyte or erythroid cell fates revealed conserved, HS scFv binding patterns between mouse and humans, implicating HS modification patterns in lineage commitment across species. In sum, our work establishes HS glycotyping as a novel orthogonal approach to define and prospectively purify viable cell types across different hematopoietic lineages in mice and humans.