Long-term proliferation of CLPs in vitro, and retroviral transduction of the cCLPs. FACS-purified Lin⁻Flk2⁺IL7R⁺CD27⁺Ly6d⁻ cells (1 × 10³) were cultured on irradiated OP9 stromal cells in our modified serum-free medium (KIDMEM) or serum-containing CM with Flt3L (25 ng/mL) and different concentrations of IL-7 (0.1 and 1 ng/mL). Cells at the indicated time points were counted and FACS analyzed for CD19 and Notch1 expression. (A) Kinetics of cell proliferation during long-term culture with high (circles) or low (squares) amount of IL-7 in KIDMEM or CM. (B-C) Representative FACS profiles of cells cultured with Flt3L and 0.1 ng/mL IL-7 at early phase up to day 14 (B) or cultured with Flt3L and different concentrations of IL-7 at day 35 (C) in KIDMEM or CM. The values in each panel indicate percentages of Notch1^hiCD19⁻ and Notch1^loCD19⁺ cells. (D) Light microscopic images (20× objective) of cells at day 35 of culture with 0.1 ng/mL IL-7 in KIDMEM (left panel) or CM (right panel). (E) Limiting dilution of purified CLP cultured for 2 months under CLP conditions. (F) FACS analyses of Notch1^hiCD19⁻ CLP (shaded areas) or of Notch1^loCD19⁺ B-lineage cells (thick lines) at day 35 of culture. Isotype-matched control is shown in thin lines. (G) Retroviral transduction of cCLPs with a vector containing EGFP under the expression controls of doxycycline. Representative FACS profiles of green fluorescent protein (GFP) and CD27/CD19 expression with (bottom) or without doxycycline (top) are shown.