Effect of HS oligosaccharides on LTC-IC maintenance in the presence of low-dose cytokines and MIP-1α.

CD34⁺/HLA-DR⁻ cells (9000-10 000 cells/well) were plated in 0.4-μm transwell inserts in 24-well tissue culture clusters. Medium in the lower chambers of the wells was replaced 5 days each week by LTBMC medium supplemented with a combination of low-dose recombinant cytokines (500 pg/mL granulocyte-colony-stimulating factor [G-CSF], 50 pg/mL granulocyte macrophage-colony-stimulating factor [GM-CSF], 200 pg/mL stem cell factor [SCF], 50 pg/mL leukemia inhibitory factor (LIF), and 2 ng/mL IL-6) and 200 pg/mL MIP-1α, with or without 5 μg/mL indicated HS oligosaccharides or intact (parent) HS. Cultures were harvested after 5 weeks, and cells were replated at limiting dilutions for estimations of LTC-IC frequency, as described in “Study design.” The absolute number of LTC-ICs in the starting cell population at day 0 was 2.96 ± 0.3 per 100 DR⁻ cells plated. The maintenance of LTC-IC after 5 weeks of culture in the presence of intact parent HS was 65% ± 15% of day 0 LTC-IC. n = 3 independent experiments. LTC-IC maintenance observed in the presence of the different oligosaccharides is expressed as a percentage of that seen with the intact parent HS molecule. Comparisons between No GAGs (cytokines alone) and other conditions and between intact (parent) HS and the other conditions are as indicated in Table 1. SEM bars are shown.