The type of stromal feeder used in limiting dilution assays influences frequency and maintenance assessment of human long-term culture initiating cells

Abstract

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-1αx + 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.