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) growth 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 for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1alpha or PF4 (LTC-IC maintenance without GAGs, 32 +/- 2%; with GAGs, 95 +/- 7%; P <.001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1alpha, 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-1alpha. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1alpha, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association 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 requires 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 interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)
Gupta
P
P
Department of Medicine, Veterans Affairs Medical Center Minneapolis, MN 55417, USA. gupta013@gold.tc.umn.edu
Oegema
T R
TR
Brazil
J J
JJ
Dudek
A Z
AZ
Slungaard
A
A
Verfaillie
C M
CM
D000328:Adult; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D004187:Disaccharides / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000494:pharmacology*; D005353:Fibronectins / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006497:Heparan Sulfate / Q000494:pharmacology*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D008222:Lymphokines / Q000494:pharmacology; D019402:Macrophage Inflammatory Proteins / Q000494:pharmacology*; D010978:Platelet Factor 4 / Q000494:pharmacology*; D017154:Stromal Cells / Q000166:cytology; D013329:Structure-Activity Relationship; D019699:Thrombospondins / Q000494:pharmacology; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D054405:Chemokine CCL3; D054407:Chemokine CCL4; D004187:Disaccharides; D016228:Endothelial Growth Factors; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D006497:Heparan Sulfate; D007377:Interleukin-3; D008222:Lymphokines; D019402:Macrophage Inflammatory Proteins; D010978:Platelet Factor 4; D019699:Thrombospondins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors