Stroma and HDC induce NK differentiation in the CD34⁺ HPC subset that could not differentiate into NK cells with cytokines alone. (A) Semi-log plot showing the fraction of negative wells (wells containing no NK precursors) as a function of input cells per well. Three subsets of CD34⁺ cells were tested in limiting dilution culture with only cytokines (IL-15, IL-7, SCF, FLT-3L, and IL-3): CD34⁺CD38⁻NKlin⁻ (□, dashed line), CD34⁺CD38⁺NKlin⁻ (◇, dotted line), and CD34⁺NKlin⁺ (▴, solid line). No positive wells were observed for NKlin⁻ subsets cultured with cytokines alone, and the plot therefore shows a flat slope for these conditions. In contrast, CD34⁺NKlin⁺ subset showed a measurable fraction of positive wells in culture with cytokines alone, thus creating a steeper slope of the regression line. A representative donor (n = 4) is shown. (B) Calculated NKp frequencies for the 3 CD34⁺ HPC subsets cultured with the above-mentioned cytokines (n = 4 donors), expressed as number of NKp/10⁶ cells. (C) Semi-log plot of the fraction of negative wells as a function of input cells per well for the CD34⁺CD38⁺NKlin⁻ fraction cultured with cytokines alone (◇, long-dashed line), cytokines and HDC (■, gray line), cytokines and stroma (▴, black line), and cytokines and HDC and stroma (○, short-dashed line). A representative donor (n = 4) is shown. (D) Calculated NKp/10⁶ cells for CD34⁺CD38⁺NKlin⁻ fraction cultured in different conditions (n = 4 donors tested). (E) Semi-log plot of the fraction of negative wells as a function of input cells per well for the CD34⁺CD38⁻NKlin⁻ fraction cultured with cytokines alone (◇, long-dashed line), with cytokines and HDC (■, gray line), with cytokines and stroma (▴, black line), and with cytokines and HDC and stroma (○, short-dashed line). A representative donor (n = 4) is shown. (F) NKp frequency per 10⁶ cells in the CD34⁺CD38⁻NKlin⁻ population cultured in different conditions; n = 4 donors tested. Groups showing significant differences are indicated by asterisks (***P < .001, **P = .001-.01, and *P = .01-.05), 1-way analysis of variance on log-transformed values with Bonferroni posttest. (G) Fold change in mRNA levels coding for T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors and their downstream targets (WISP and CyclinD1) induced in NKlin⁻CD34⁺ HPCs after 14 days of culture of with (1) cytokines, stromal cells, and HDC versus (2) cytokines only. (H) Effect of Dickkopf-1 (DKK-1) on the generation of NK cells from CD34⁺ HPCs in the culture with cytokines, stroma, and HDC. Shown are the average number of CD56⁺ cells/well after 21 days of culture ± SEM. The difference between no addition of DKK-1 and addition at 10 ng/mL is statistically significant (P = .012). Results are the mean of 6 wells for each condition and are representative of independent 2 donors.