CD56⁺CD94^–CD117^high cells are a precursor population to CD56⁺CD94^–CD117^low/– cells. (A) The percentage of CD94^–CD117^high cells (top) and CD94⁺CD117^low/– cells (bottom) within the CD56⁺ population as a function of the percentage of CD56⁺ cells in culture. CD34⁺Lin^– cells were isolated by FACS and cultured as described in “Materials and methods.” The plots demonstrate that at early time points when the fraction of CD56⁺ cells is low the majority of CD56⁺ cells are CD94^–CD117^high. As the percentage of CD56⁺ cell increases, the fraction of CD56⁺CD94^–CD117^high cells decreases and the fraction of CD56⁺CD94⁺CD117^low/– cells increases (bottom). Results of 2 individual donors tested at days 14, 17, 19, 21, 25, and 28 are shown. (B) Proliferation rate of CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– cells by Ki67 staining. To determine whether there was a differential rate of proliferation in developing NK-cell subsets, cells at days 21 to 28 of culture were permeabilized (as described in “Materials and methods”) and stained for the nuclear antigen Ki67. The results were representative of 3 experiments. (C) CD56⁺CD94^–CD117^high cells are precursors to CD56⁺CD94⁺CD117^low/– cells. At day +21 (±3) of culture, CD56⁺CD94^–CD117^high cells were FACS sorted and cultured with cytokines and the feeder cell line (EL08.1D2). Cells were reanalyzed 1 week later for the presence of CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– populations. Shown are FACS plots after gating on the viable fraction. Results were representative of more than 3 individual experiments.