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

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 s orted or ex vivo expanded CD34(+)/HLA-DRdim/CD2(-)/CD7(-) (34(+)/Lin(-)) ce lls. The variables examined included different stromal feeders (murine bone marrow fibroblast cell line, M2-10B4 and murine fetal liver cell line, AFT 024) and presence or absence of cytokines (MIP-1 alpha + IL-3). The absolut e LTC-IC frequency in 34(+)/Lin(-) cells measured in limiting dilution assa ys (LDA) on AFT024 (4.45 +/- 0.69%) was significantly higher than in M2-10B 4 (1.45 +/- 0.20%) LDA. Addition of MIP-1 alpha and IL-3 to AFT024 LDA incr eased 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 f or 5 weeks by replating progeny in the three LDA readout systems. The measu red 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-1 alpha and IL-3 (29.1 +/- 6.3%). Thus, the numb er of LTC-IC measured in freshly sorted 34(+) cells depends on the stromal feeder used in LDA assays. Furthermore, and most important, assessment of L TC-IC expansion or maintenance may vary significantly depending on the type of stromal feeder used to enumerate LTC-IC.