The goal of this study was to evaluate if differences in culture conditions used in long-term culture assays affect enumeration of LTC-IC in freshly sorted or ex vivo expanded CD34+/HLA-DR(dim)/CD2-/CD7- (34+/Lin-) cells. The variables examined included different stromal feeders (murine bone marrow fibroblast cell line, M2-10B4 and murine fetal liver cell line, AFT024) and presence or absence of cytokines (MIP-1α + IL-3). The absolute LTC-IC frequency in 34+/Lin- cells measured in limiting dilution assays (LDA) on AFT024 (4.45 ± 0.69%) was significantly higher than in M2-10B4 (1.45 ± 0.20%) LDA. Addition of MIP-1α and IL-3 to AFT024 LDA increased the measured LTC-IC frequency to 6.8 ± 0.9%. We also determined the fraction of LTC-IC that persisted after 34+/Lin- cells were cultured for 5 weeks by replating progeny in the three LDA readout systems. The measured LTC-IC maintenance was significantly lower when M2-10B4 LDA (13.1 ± 3.5%, P < 0.05) were used compared with AFT024 LDA (36.6 ± 5.5%) or AFT024 LDA supplemented with MIP-1α and IL-3 (29.1 ± 6.3%). Thus, the number of LTC-IC measured in freshly sorted 34+ cells depends on the stromal feeder used in LDA assays. Furthermore, and most important, assessment of LTC-IC expansion or maintenance may vary significantly depending on the type of stromal feeder used to enumerate LTC-IC.
- Stromal feeder