TIMP-3 facilitates cell-cycle entry of quiescent HSCs. (A) TIMP-3 stimulates proliferation of LT-HSCs (CD34⁻KSL Flt3⁻) but not ST-HSCs (CD34⁺KSL Flt3⁻) or MPPs (CD34⁺KSL Flt3⁺). Fifty cells/well for each fraction were sorted into a 96-well plate and cultured as described in the Methods. Cell numbers were counted at the indicated time points and plotted onto the graphs (n = 3, mean ± SD). (B) Enhanced rate of the first cell division of CD34⁻KSL cells in the presence of TIMP-3. CD34⁻KSL cells were clonally sorted into a 96-well plate; the cell number for each well was counted at 48 and 68 hours after FACS sorting. Percentage of the wells containing 2 or more cells was plotted on the graph (n = 3, mean ± SD). *P < .05. (C-D) TIMP-3 recruits quiescent HSCs into the cell cycle. Mice were injected with TIMP-3 expression plasmids through tail veins by hydrodynamic delivery. (C) After 3 days of injection, BM cells were harvested and examined for the cell-cycle status of CD34⁻KSL cells, as described in the Methods (D) The percentage of cycling cells (G1 + S/G2/M) was plotted on the graph (n = 3, mean ± SD). *P < .05. (E) TIMP-3 induces DNA synthesis in HSCs. Mice were injected with TIMP-3 expression vector through tail veins by hydrodynamic delivery. After 3 days of injection, mice were injected intraperitoneally with 2 mg of BrdU. BM cells were harvested at day 4 and examined for incorporation of BrdU in CD34⁻KSL cells. Percentages of cells that have incorporated BrdU are shown (n = 3, mean ± SD). *P < .05.