LTC-IC supportive activity of differentially sulfated glycosaminoglycans. A total of 10,000 to 17,000 DR⁻ cells were plated in 0.4-μm transwell inserts in 24-well tissue culture clusters. Cultures were maintained by daily replacement of the medium in the lower chambers of the wells by either stroma supernatant (Stroma SN) or by LTBMC medium supplemented with a combination of cytokines (500 pg/mL G-CSF, 50 pg/mL GM-CSF, 200 pg/mL SCF, 50 pg/mL LIF, 2 ng/mL IL-6, and 200 pg/mL MIP-1) with or without 5 μg/mL of the indicated GAGs. Cultures were harvested after 5 weeks and cells were replated at limiting dilutions for estimation of LTC-IC frequency. The absolute number of LTC-IC in the starting cell population at day 0 was 0.40 ± 0.07 per 100 DR⁻ cells plated. The absolute number of LTC-IC after 5 weeks of culture in stroma SN was 0.21 ± 0.04 per 100 DR⁻ cells initially plated at day 0 (52% of day 0 LTC-IC). Numbers within bars indicate number of experiments. (A) Comparison between cytokines only (No GAGs) and other conditions: *P < .002. Comparison between O-sulfated heparin and other conditions: §P < .02, ¶P < .002. (B) LTC-IC supportive activity of 2-O-desulfated glycosaminoglycans, prepared from unmodified heparin or from O-sulfated heparin as described in Materials and Methods. Comparison between cytokines only (No GAGs) and 2-O-desulfated O-sulfated heparin: *P < .002.