Figure 1
Figure 1. Radioprotective effect of mesenchymal stem cells genetically modified with extracellular superoxide dismutase. (A) Phenotype of mMSCs. Flow cytometric analysis was conducted on ex vivo–expanded mMSCs to determine the expression of CD11b, CD13, CD19, CD29, CD31, CD34, CD44, CD45, CD73, CD90 (Thy-1.2), CD105, CD117 (c-Kit), CD135, and Sca-1. Histograms show the relative intensity of mMSCs for various cell-surface antigens. Numbers indicate the percentage of cells in the population whose staining intensity with the specific antibody (white) was greater than that with the respective isotype control (gray). (B) Secretion of biologically active ECSOD by Ad5CMVECSOD-transduced mMSCs. mMSCs were transduced with Ad5CMVECSOD at multiplicity of infections (MOI, defined as plaque-forming units/cell) of 0, 300, or 2000 for 48 hours, the virus-containing culture medium was removed, and the cells were washed 3 times with PBS and further incubated in fresh culture medium for 48 hours. The culture supernatant was collected and analyzed for SOD activity using a SOD activity assay kit (Cayman Chemical Company, Ann Arbor, MI). Data were expressed as mean plus or minus SEM (n = 3) and analyzed statistically using a one-way analysis of variance (ANOVA) followed by post-hoc analysis with Tukey test. *P < .05 versus MOI 0; **P < .05 versus MOI 0 or 300. (C) Photomicrographs showing expression of nuclear-targeted β-galactosidase by Ad5CMVntlacZ-transduced mMSCs. mMSCs were transduced with Ad5CMVntlacZ at MOI 0, 300, or 2000 for 48 hours. The cells were X-gal stained for β-galactosidase activity and the blue nuclear-targeted β-galactosidase–positive Ad5CMVntlacZ-transduced mMSCs were identified. Original magnification ×40. (D) Intravenous administration of ECSOD gene-modified mMSCs improves survival of irradiated mice. Five-week-old female BALB/c mice were given 9 Gy total body γ irradiation from a 137Cs source (Gammacell 1000; MDS Nordion, Ottawa, ON) at a dose rate of 1.23 Gy/min. Twenty-four hours later, the animals were given a tail vein injection of 200 μL PBS, 0.5 × 106 ntlacZ gene-modified mMSCs (ntlacZ-mMSCs, MOI = 2000) in 200 μL PBS, or 0.5 × 106 ECSOD gene-modified mMSCs (ECSOD-mMSCs, MOI = 2000) in 200 μL PBS. Mouse survival was then monitored for 35 days. Kaplan-Meier survival curve was used for data analysis, and statistical significance was determined using log-rank test and one-way ANOVA followed by post-hoc analysis with Tukey test. P < .05 was considered statistically significant. The difference between the 3 groups was statistically significant by log-rank test (P = .002) and ANOVA (P < .001). Furthermore, P < .001 for ECSOD-mMSCs versus PBS, P < .001 for ECSOD-mMSCs versus ntlacZ-mMSCs, and P > .05 for ntlacZ-mMSCs versus PBS by Tukey test. In this study, 4 separate experiments were conducted and the result of each experiment was similar.

Radioprotective effect of mesenchymal stem cells genetically modified with extracellular superoxide dismutase. (A) Phenotype of mMSCs. Flow cytometric analysis was conducted on ex vivo–expanded mMSCs to determine the expression of CD11b, CD13, CD19, CD29, CD31, CD34, CD44, CD45, CD73, CD90 (Thy-1.2), CD105, CD117 (c-Kit), CD135, and Sca-1. Histograms show the relative intensity of mMSCs for various cell-surface antigens. Numbers indicate the percentage of cells in the population whose staining intensity with the specific antibody (white) was greater than that with the respective isotype control (gray). (B) Secretion of biologically active ECSOD by Ad5CMVECSOD-transduced mMSCs. mMSCs were transduced with Ad5CMVECSOD at multiplicity of infections (MOI, defined as plaque-forming units/cell) of 0, 300, or 2000 for 48 hours, the virus-containing culture medium was removed, and the cells were washed 3 times with PBS and further incubated in fresh culture medium for 48 hours. The culture supernatant was collected and analyzed for SOD activity using a SOD activity assay kit (Cayman Chemical Company, Ann Arbor, MI). Data were expressed as mean plus or minus SEM (n = 3) and analyzed statistically using a one-way analysis of variance (ANOVA) followed by post-hoc analysis with Tukey test. *P < .05 versus MOI 0; **P < .05 versus MOI 0 or 300. (C) Photomicrographs showing expression of nuclear-targeted β-galactosidase by Ad5CMVntlacZ-transduced mMSCs. mMSCs were transduced with Ad5CMVntlacZ at MOI 0, 300, or 2000 for 48 hours. The cells were X-gal stained for β-galactosidase activity and the blue nuclear-targeted β-galactosidase–positive Ad5CMVntlacZ-transduced mMSCs were identified. Original magnification ×40. (D) Intravenous administration of ECSOD gene-modified mMSCs improves survival of irradiated mice. Five-week-old female BALB/c mice were given 9 Gy total body γ irradiation from a 137Cs source (Gammacell 1000; MDS Nordion, Ottawa, ON) at a dose rate of 1.23 Gy/min. Twenty-four hours later, the animals were given a tail vein injection of 200 μL PBS, 0.5 × 106 ntlacZ gene-modified mMSCs (ntlacZ-mMSCs, MOI = 2000) in 200 μL PBS, or 0.5 × 106 ECSOD gene-modified mMSCs (ECSOD-mMSCs, MOI = 2000) in 200 μL PBS. Mouse survival was then monitored for 35 days. Kaplan-Meier survival curve was used for data analysis, and statistical significance was determined using log-rank test and one-way ANOVA followed by post-hoc analysis with Tukey test. P < .05 was considered statistically significant. The difference between the 3 groups was statistically significant by log-rank test (P = .002) and ANOVA (P < .001). Furthermore, P < .001 for ECSOD-mMSCs versus PBS, P < .001 for ECSOD-mMSCs versus ntlacZ-mMSCs, and P > .05 for ntlacZ-mMSCs versus PBS by Tukey test. In this study, 4 separate experiments were conducted and the result of each experiment was similar.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal