Figure 3. In vitro expansion of LSK population.

(A) The mice with indicated genotypes were i.p. injected with tamoxifen (160 mg/kg) for three consecutive days and sacrificed at day 4. The total LSK, MP and CLP cell numbers in the femurs and tibias from each mouse were determined. Data are presented as mean ± S.D. (n.s. not significant, n = 3) (B) Whole BMN cells isolated at day 4 described in (A) were cultured in STIFA medium. At the time of plating, 2.5×10⁶ cells/well, 2×10⁶ cells/well and 1×10⁶ cells/well were plated for 3-, 6- and 9-day culture, respectively. Cells were harvested and analyzed by flow cytometry for lineage, Sca-1 and c-Kit surface markers. The absolute number of the LSK population in the harvested cells is shown as per 1×10⁶ initial BMN cells. Data are presented as mean ± S.D. (*p<0.05, n = 3) (C, D) Annexin V-binding assay of LSK (C) and lineage positive cells (D) after nine days of cell expansion in STIFA medium. Data are presented as mean ± S.D. (*p<0.05, n = 3).