In vitro modeling of human B lymphopoiesis recapitulates aberrant differentiation and expansion of early BCPs in the absence of IL-7. (A) In vitro differentiation of human BCPs with CB-derived CD34⁺ hematopoietic progenitor cells. Mononuclear cells were purified via Ficoll density centrifugation and CD34⁺ progenitor cells were isolated by positive selection using magnetic-activated cell sorting. CD34⁺ cells were stimulated with FMS-like tyrosine kinase 3 ligand (FLT3L), stem cell factor (SCF), and IL-6 after isolation and restimulated with FLT3L and SCF on day 7, with IL-7 being added to the control samples. Cells were harvested on days 14, 21, and 28 after initiation of cultures for analysis. (B-E) Absolute (B-C) and relative (D) cell counts and distribution of BCP stages (E) of in vitro differentiated BCPs on days 14, 21, and 28. Data show the median, with error bars representing the interquartile range for panels B-D and the median only of each population for panel E. Statistical analysis was performed with multiple Mann-Whitney tests and corrected for multiple comparisons with the Holm-Šídák method for data shown in panels B-D (n = 7, each with 2-5 replicates). P values are denoted as follows: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001;∗∗∗∗P < .0001. ns, not significant.