Effects of TIMP-3 on CD34⁻KSL cells. (A) Enhanced proliferation of CD34⁻KSL cells by TIMP-3. One hundred CD34⁻KSL cells/well were sorted into a 96-well plate and cultured as described in “Methods.” (Left) Growth curve (n = 3, mean ± standard deviation [SD]). (Right) Pictures of cells cultured for 5 days (original magnification, ×100). (B) Long-term repopulating activity of CD34⁻KSL cells cultured with TIMP-3. CD34⁻KSL cells (Ly5.1) were cultured for 2 weeks as described in “Methods.” All of the cells generated from 10 CD34⁻KSL cells were transplanted into lethally irradiated congeneic hosts (Ly5.2) with competitors. Percentage of repopulation was not statistically different between control- and TIMP-3–treated groups at any time points. (C) Enhanced production of multipotential progenitor cells from HSCs cultured with TIMP-3. One hundred clonally sorted CD34⁻KSL cells were cultured in vitro for 1 week with SCF and TPO with or without TIMP-3 as described in “Methods.” Cells were then subjected to colony assays. Data are mean ± SD (n = 3, *P < .05). n, neutrophil; m, macrophage; E, erythroid cell; M, megakaryocyte. (D) TIMP-3 increases multipotential progenitors in vivo. TIMP-3 was over-expressed in vivo by hydrodynamic delivery of TIMP-3 plasmid. After 3 days of injection, mice were killed and examined for LT-HSC (CD34⁻KSLFlt3⁻), ST-HSC (CD34⁺KSLFlt3⁻), and MPP (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 BM mononuclear cells are shown (n = 3, mean ± SD).