Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates:requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins

We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O -S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growt h and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell "niches" in the marrow microenvironment, We now show that long-term culture-initiating cells (LTC-IC) are maintained fo r 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and eith er MIP-1 alpha or PF4 (LTC-IC maintenance without GAGs, 32 +/- 2%; with GAG s, 95 +/- 7%; P < .001), When cultured with 5 additional cytokines, O-S-GAG s, IL-3, and MIP-1 alpha, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 /- 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1 alpha. Although O-S-GAG omission did not affect a-week expa nsion, only 20% LTC-IC were maintained for 5 weeks, When O-S-heparin was re placed by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines a lone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated hepari n, bound to MIP-1 alpha, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the associ ation and dissociation rates of heparin for PF4, were higher than those of O-S-heparin, We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month req uires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interac tions that affect presentation of proteins in a biologically active manner to progenitors. (C) 2000 by The American Society of Hematology.