AC133 expression correlates with progenitor function, and the AC133⁺CD38⁻ phenotype marks a CD34^hiCD38⁻ subpopulation. (A) The expression of AC133⁺, Rho^lo, CD34⁺, and CD34⁺CD38⁻ markers was quantitatively evaluated for their progenitor function in culture by the following relationship: percentage of marker⁺ cells/number of CFU-C or LTC-ICs. Correlation between marker expression and (Ai) CFU-C or (Aii) LTC-IC function was tracked over 8 days in culture and normalized to 1 based on day 0 values. (CFU-Cs per 500 cells plated on days 0, 4, and 8 were 53.5 ± 44, 159 ± 32, and 60.5 ± 25.2, respectively. LTC-ICs per 2000 cells plated at day 0, 4, and 8 were 39 ± 12, 9 ± 1.4, and 16.3 ± 10.2, respectively.) An ideal marker would closely correlate with progenitor function resulting in a constant relative frequency during culture (dashed line). (B) Representative flow cytometry plots comparing the expression of AC133 and CD38 with CD34 and CD38 on UCB Lin⁻ cells cultured for 8 days (n = 9 independent experiments). Cells were stained with either AC133 and CD38 or CD34 and CD38. AC133⁺CD38- cells (P3) mark a CD34^hiCD38⁻ subpopulation that enriches the CD34⁺CD38⁻ population 2.5-fold (P < .001). (C) 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 CFU-C clonogenic assays (n = 5). The AC133⁺CD38⁻ subpopulation contained more CFU-GM after culture than the AC133⁺CD38⁺ (P = .005), AC133⁻CD38⁺ (P = .02), and AC133⁻CD38⁻ (P = .1) subpopulations and more CFU-mix after culture than the AC133⁺CD38⁺ (P = .05), AC133⁻CD38⁺ (P = .06), and AC133⁻CD38⁻ (P = .01) subpopulations.