![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-3C1S) 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-3C1S 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).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/116/22/10.1182_blood-2010-01-266528/6/zh89991061280004.jpeg?Expires=1721650641&Signature=k38NG2t95HFVOz477jH88TVXhDCZRAmxUU5gKH3OcvY60FuO7~491PCZ4UOq-gSe07D4tqq0ekS~klWhE3vpIm6~J1utj4Y-XxfW2HptSg8uet8Vn923yYP-Bc2p2erEdeOI8whJXRG5sjysWilGoxjyfpkTdVq0yWptHLUCS2GcH3yCMe7NpBbFkiTJEGnluCvqSQJuHSBobd3~AzLYEGOan6hnzUo3Dl9zpfD6V2tUc1M05zmhODH0tFXKDCTuNdnw5awFO4tKyszE-eEk-vobT9rIQPJyKVvC2CIVM6q0Molo2v-Hi3Jk18t~2Tuuazvfj1kAvNcux~MAClE~nA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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-3C1S) 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-3C1S 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).
