Figure 2
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).

Effects of TIMP-3 on CD34KSL cells. (A) Enhanced proliferation of CD34KSL cells by TIMP-3. One hundred CD34KSL 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 CD34KSL cells cultured with TIMP-3. CD34KSL cells (Ly5.1) were cultured for 2 weeks as described in “Methods.” All of the cells generated from 10 CD34KSL 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 CD34KSL 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 (CD34KSLFlt3), 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).

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