Figure 3. A stromal-dependent self-renewing phase in pre-B cell differentiation is greatly augmented by loss of Ikaros.

a, An adherent phase in pre-B cell differentiation as revealed in stromal cultures of WT and IkE5^Δ/Δ large pre-B cells grown in the presence of IL-7 (5 ng/ml). Areas with adherent cells were marked with rectangles (left) and digitally magnified (right). Dotted circle marks the nucleus of OP9 stromal cells used as a stromal reference (scale bar, 30 μm). b, Ratio of adherent to non-adherent cells in WT and IkE5^Δ/Δ pre-B cultures at day 2 (D2) and day 3 (D3) with 5 and 0.05 ng/ml of IL-7. The mean ratio is presented ± s.d. Asterisks denote significant differences between WT and mutant pre-B cells at each culture time point (***P < 0.0001, **P < 0.01, *P < 0.05, two-tailed Student's t-test). c, Comparative expression analysis of pre-B cell differentiation genes in adherent and non-adherent pre-B cells. Hierarchical clustering of normalized gene expression values across different conditions is shown. d, Flow cytometric analysis of adherent and non-adherent cells from WT and IkE5^Δ/Δ large pre-B cell stromal cultures for CD25 and intracellular Igκ and Igκ. The percentages of positive cells relative to isotype control (grey curve) are indicated. e, Rates of propagation of WT adherent and non-adherent pre-B cell fractions grown with 5 ng/ml of IL-7. The mean number of cells generated by 5 × 10⁴ adherent (dark blue) or non-adherent (light blue) WT pre-B cells after replating on OP9 stroma for 3 days of culture is shown in the top panel. The mean number of adherent and non-adherent subsets recovered from plating either WT adherent or non-adherent pre-B cell stromal cultures is shown in the bottom panel. Error bars indicate s.d. Asterisks indicate a statistically significant difference in the growth (top panel) of WT adherent and non-adherent B cells (*P< 0.05, **P < 0.01, two-tailed Student's t-test). f, Limiting dilution colony forming assay was performed as described previously²⁹. The mean frequency of colony forming cells was calculated based on Poisson distribution and is presented in a line graph ± s.e. g, Re-association of WT and IkE5^Δ/Δ pre-B cells after replating on stroma. The mean percentage ± s.d. of stromal-adherent cells, measured 3 hrs after replating is shown. Study was performed with two independent WT and mutant pre-B cell cultures (closed and open symbols), each assayed in ten grids/well. Binding to stroma was calculated per twenty grids and averaged for each cell type (*P < 0.0001, two-tailed Student's t-test).