Rac excision from perivascular niche leads to enhanced apoptosis and decreased numbers of Nestin⁺ cells and reduced homing of WT cells into the BM of perivascular Rac-deleted mice. (A) Confirmation of deletion of Rac1 sequences in Nestin⁺ cells isolated by flow cytometry from TG Rac1 ^Δ/ΔRac3^−/− mice. The DNA size ladder is at the right. The position of floxed, deleted (Δ), and WT Rac1 bands are shown at the left. Genotypes are shown above. (B) Percentage of Nestin⁺ cells defined by expression of CD51⁺PDGFRα⁺ in the BM of the genotypes shown below each lane. Data represent mean ± standard deviation (SD); N = 5 experiments repeated four times, **P < .01. (C) Growth of cultured Nestin⁺ cells from TG WT and TG Rac1^Δ/ΔRac3^−/− mice. CD51⁺PDGFRα⁺ Nestin⁺ cells were sorted by flow cytometry from BM nucleated cells gated on live CD45/CD31/Ter119^– cells (as described in supplemental Figure 1A) and were cultured in vitro over 16 days. Data represent mean ± SD; N = 5 experiments repeated 3 times for each condition, *P < .05 (day 8) and *P < .02 (day 16). (D) Apoptosis in Nestin⁺ cells isolated from TG WT and TG Rac1^Δ/ΔRac3^−/− mice as determined by Annexin V⁺ staining using flow cytometric analysis. Data represent mean ± SD; N = 5 experiments repeated four times, ***P < .001. (E) Homing of DiD-labeled WT LDMB cells injected into recipient mice of each genotype and measured by flow cytometry 12 hours after infusion as described in Material and methods. Data are expressed as % of WT and represent analysis of the equivalent number of live cells/group and time point; mean ± SD, ***P < .001, N = 16 recipients (homing) and experiments were repeated 3 times.