Developmental dynamic of BM-derived CD34⁺ in B-cell differentiating condition. (A) Early B-lymphocyte development with stage-characteristic surface markers. In bold are the markers used to identify distinct populations in vitro. ImmB: immature IgM⁺ B cells; PreB: pre-B-cells; ProB: pro-B-cells. (B) Scheme of the experimental setup. Magnetically isolated CD34⁺ cells from BM aspirates were expanded in the presence of SCF, Flt3-L, and IL-6, then in presence of SCF, Flt3-L, and IL-7. From day 14 to 49, cells were cultivated in cytokine-free medium and developing common lymphoid progenitors (CLP), as well as pro-, pre-, and immature B cells were analyzed weekly by flow cytometry. (C) Distribution of B-cell subpopulations over time in culture: Live CD10⁺ cells counts and within the CD10⁺ population percentages (%) of CLP and pro-B-cells (CLP/ProB), of pre-B-cells (PreB) and of immature B-cells (ImmB) between day 14 and 49 of culture in healthy donors (HDs). Each symbol shows a different HD represented as mean and standard error of mean of 4 to 10 technical replicates at each timepoint. (D) Outcome of in vitro development of BTK-deficient (BTK1, BTK2) and IKZF1-deficient (Ikaros) CD34⁺ cells. Live CD10⁺ cell counts, and within the CD10⁺ population percentages (%) of CLP and pro-B-cells (CLP/ProB), of pre-B-cells (PreB), and of immature B-cells (ImmB) at days 14, 21, and 49. Four to 10 replicates were analyzed at each timepoint for each patient and HD. Each symbol represents a patient; mean and standard error of mean are depicted. (E) Expression of transcription factors driving B-cell specification (E2A) and commitment (PAX5) in relation to CD79a expression, evaluated by quantitative polymerase chain reaction. Mean (line) and standard error of mean (shadow) of 2 BTK patients (blue) and 7 HDs (yellow).