Expansion of CD34⁺HLA-DR⁻ CFC (A) and LTC-IC (B) in contact and noncontact cultures without cytokines and with the addition of IL-3 and MIP-1α (C and D). Twelve thousand CD34⁺HLA-DR⁻ cells were cultured for 2 to 5 weeks in LTC medium without cytokines or with the addition of IL-3 and MIP-1α (3 times per week to a final concentration of 5 ng/mL and 100 ng/mL, respectively) in contact with irradiated M2-10B4 feeders or in transwells above M2-10B4. Cells were collected after 2 and 5 weeks and 1/6 of the progeny replated in methylcellulose assay to enumerate the number of CFC (equivalent of 2,000 day 0 cells) and 5/6 of the progeny in LDA onto M2-10B4 feeders (equivalent of 10,000 day 0 cells) for 5 weeks to enumerate the number of LTC-IC. The number of CFC present at 2 and 5 weeks was divided by the number of CFC per 2,000 freshly sorted cells to calculate CFC expansion. The number of LTC-IC at 2 and 5 weeks was divided by the number of LTC-IC in freshly sorted cells to calculate LTC-IC expansion. Results represent mean ± SEM (CFC n = 10; LTC-IC n = 9). Closed bars represent contact cultures; open bars represent noncontact cultures. The number of CFC in freshly selected CD34⁺HLA-DR⁻ cells was 52 ± 15 CFC/2,000 CD34⁺HLA-DR⁻ cells. The number of LTC-IC in freshly selected CD34⁺HLA-DR⁻ cells was 46 ± 24 LTC-IC/10,000 CD34⁺HLA-DR⁻ cells. (A) Comparison between CFC expansion in contact and noncontact cultures; the number of CFC after 5 weeks in contact culture was 176 ± 53 CFC/2,000 day 0 CD34⁺HLA-DR⁻ and 218 ± 52 CFC/2,000 day 0 CD34⁺HLA-DR⁻ in noncontact culture; differences not significant. (B) Comparison between LTC-IC maintenance in contact and noncontact cultures without cytokines; the number of LTC-IC after 5 weeks in contact culture was 4.6 ± 1.6 LTC-IC/10,000 day 0 CD34⁺HLA-DR⁻ and 16.8 ± 4.7 LTC-IC/10,000 day 0 CD34⁺HLA-DR⁻ in noncontact culture; * P < .01. (C) Comparison between CFC expansion in contact and noncontact cultures supplemented with IL-3 and MIP-1α; the number of CFC after 5 weeks in contact culture supplemented with cytokines was 770 ± 152 CFC/2,000 day 0 CD34⁺HLA-DR⁻ and 981 ± 159 CFC/2,000 day 0 CD34⁺HLA-DR⁻ in cytokine supplemented noncontact culture; differences not significant. (D) Comparison between LTC-IC maintenance in contact and noncontact cultures supplemented with IL-3 and MIP-1α; the number of LTC-IC after 5 weeks in contact culture supplemented with cytokines was 7.5 ± 2.9 LTC-IC/10,000 day 0 CD34⁺HLA-DR⁻ and 42.1 ± 8.9 LTC-IC/10,000 day 0 CD34⁺HLA-DR⁻ in noncontact culture; * P < .01.