MMP-inhibitory activity of TIMP-3 is not required for expansion of hematopoietic progenitors and the recruitment of HSCs into the cell cycle. (A) MMP-inhibitory activity of TIMP-3 is not required for expansion of hematopoietic progenitors in vivo. Mutant TIMP-3 lacking MMP-inhibitory activity (TIMP-3^C1S) was over-expressed in vivo by hydrodynamic delivery. After 3 days of injection, mice were killed and examined for LT-HSCs (CD34⁻KSLFlt3⁻), ST-HSCs (CD34⁺KSLFlt3⁻), and MPPs (CD34⁺KSLFlt3⁺) by FACS, and the actual numbers of each progenitor per femur were calculated (n = 3, mean ± SD, *P < .05). Other multipotential (nmEM, nmE) or lineage-restricted (EM, E, nm) progenitors were examined by colony assay. Numbers of colonies per 1 × 10⁴ bone marrow mononuclear cells are shown (n = 3, mean ± SD). (B) Metalloproteinase-inhibitory activity is not essential for the recruitment of quiescent HSCs into the cell cycle. TIMP-3^C1S was over-expressed in vivo by hydrodynamic delivery. 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. The percentage of cycling cells (G1 + S/G2/M) was plotted on the graph (n = 3, mean ± SD). *P < .05. (C) Effects of metalloproteinase-inhibitor on the proliferation of HSCs. One hundred CD34⁻KSL cells/well were sorted into a 96-well plate and cultured as described in “Methods.” TIMP-3 (3 μg/mL), GM6001 at various concentrations (1, 5, and 25 μM), or vehicle (0.1% dimethyl sulfoxide [DMSO]) as a control were added to the culture. Cell numbers were counted at the indicated time points (n = 3, mean ± SD).