Modulation of LTC-IC maintenance by differentially sulfated GAGs in the presence of multiple growth-promoting cytokines with IL-3 and MIP-1.

A total of 10 000 to 20 000 DR⁻ cells were plated in 0.4-μm transwell inserts in 6-well tissue culture clusters. Medium in the lower chambers of the wells was replaced daily by either stroma-conditioned medium (Stroma CM) or by LTBMC medium supplemented with or without a combination of cytokines (500 pg/mL G-CSF, 50 pg/mL GM-CSF, 200 pg/mL SCF, 50 pg/mL LIF, and 2 ng/nl IL-6) and with or without 5 μg/mL each of GAGs. A total of 5 ng/mL IL-3 and 10 ng/mL MIP-1α were added to all cultures, including Stroma CM. Cultures were harvested after 2 weeks (A) or 5 weeks (B). The equivalent of 500 to 1000 DR⁻ cells plated at day 0 were replated after harvesting in methylcellulose cultures for estimation of CFC generation, and the remaining cells were replated at limiting dilutions for estimation of LTC-IC frequency, as described in Methods. Numbers within the bars indicate the number of experiments. Comparison between cytokines only and other conditions: *P < .002; comparison between desulfated heparin and other conditions: §P < .001.