![Figure 4. 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=1722430457&Signature=e7sl2cbDbOlJiNf8KdgYah5HsO9Ck9I6OO7oIb4l34k5rFVpveOibGkKnOJgLVUCgR9GsFd5178LL1ocU0Bfrc9hEWNfzpI6Ai0c2djM2bcVh8ob31kpj-JrGhUTaBYmlA8tH0x50JbYX04Ily2j2A3CEqebptW4vKVAxEmVp~EpATkl9glfgB52rr7VhMQ7q8H7Stwmse9vobvC8jrseDnUpsgfYOKqMahKsYGCpaB3FV5sQZQDl~9PPBTWTwbfJHMpAQRO4~Hxd8y-K~v-q7Cg1kSlM-jvyxKmFtacdNL~tp~1jvpB9NuIOPO7Xm2nGoikI~snhFFo01fgIW-OGQ__&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).
