The AC133⁺CD38⁻ phenotype corresponds with LTC-IC and SRC function and expansion. UCB Lin⁻ cells were sorted into 4 populations (AC133⁺CD38⁻, AC133⁺CD38⁺, AC133⁻CD38⁺, and AC133⁻CD38⁻) before and after culture and then plated in LTC-IC clonogenic assays (n ≥ 3) or transplanted into NOD/SCID mice. (Ai) The AC133⁺CD38⁻ population contained significantly more LTC-ICs than the AC133⁺CD38⁺ (P = .002), AC133⁻CD38⁺ (P = .002), and AC133⁻CD38⁻ (P = .009) populations. (Aii) AC133⁺CD38⁻ cells were placed in either supportive (SCF, FMS-like tyrosine kinase 3 ligand, thrombopoietin) or differentiation (SCF, erythropoietin, interleukin-3, and GM-CSF) cultures. AC133⁺CD38⁻ expression was 31.5% ± 4.4% versus 11.9% ± 3.7% (P = .04), and LTC-IC frequency was 68.4 plus or minus 13.5 and 13.6 ± 9.4 per 2000 cells (P < .01) in the supportive and differentiation cultures, respectively. (B) Cultured cells were sorted and transplanted intravenously into NOD/SCID mice, and analyzed 7 to 8 weeks after transplantation. Because the majority of SRC activity was present in the AC133⁺CD38⁻ population, limiting dilution analysis in NOD/SCID mice (n = 52) was performed with day 0 AC133⁺CD38⁻ uncultured cells and day 8 AC133⁺CD38⁻ cultured cells. (C) Mice engrafted with AC133⁺CD38⁻ cells were secondarily transplanted into NOD/SCID mice and analyzed for the presence of human (Ci) CD45⁺HLA-ABC⁺ and (Cii) CD19⁺ and CD33⁺ cells 8 weeks after transplantation (n = 2). Statistics were calculated using the Mann-Whitney U test.