Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells  Paulette A. Conget, José J. Minguell  Experimental Hematology  Volume 28, Issue 4, Pages 382-390 (April 2000) DOI: 10.1016/S0301-472X(00)00134-X

Figure 1 Flow cytometric analysis of transgene expression by MPC after Adv infection. a. Noninfected or Ad5CMVlacZ-infected MPC (MOI = 750, for 3 h). b. Noninfected or Ad5CMVGFP-infected MPC (MOI = 250, for 6 h). Cells with fluorescence intensity to the right of the vertical lines are considered as transduced cells Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 2 Efficiency of Adv-mediated gene transfer into MPC. MPC were infected at 37°C with: (a) Ad5CMVGFP at the indicated MOI for 6 hours; (b) Ad5CMVlacZ (■) or Ad5CMVGFP (•) at MOI = 250 for the indicated periods of time; (c) Ad5CMVGFP at MOI = 250 or incubated with 1 or 2 μg pGeneGrip-GFP lipofected using DOTAP for 6 hours. Forty-eight hours later, transgene expressing cells were scored by flow cytometry. Data shown represent the mean ± SD of two experiments Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 2 Efficiency of Adv-mediated gene transfer into MPC. MPC were infected at 37°C with: (a) Ad5CMVGFP at the indicated MOI for 6 hours; (b) Ad5CMVlacZ (■) or Ad5CMVGFP (•) at MOI = 250 for the indicated periods of time; (c) Ad5CMVGFP at MOI = 250 or incubated with 1 or 2 μg pGeneGrip-GFP lipofected using DOTAP for 6 hours. Forty-eight hours later, transgene expressing cells were scored by flow cytometry. Data shown represent the mean ± SD of two experiments Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 2 Efficiency of Adv-mediated gene transfer into MPC. MPC were infected at 37°C with: (a) Ad5CMVGFP at the indicated MOI for 6 hours; (b) Ad5CMVlacZ (■) or Ad5CMVGFP (•) at MOI = 250 for the indicated periods of time; (c) Ad5CMVGFP at MOI = 250 or incubated with 1 or 2 μg pGeneGrip-GFP lipofected using DOTAP for 6 hours. Forty-eight hours later, transgene expressing cells were scored by flow cytometry. Data shown represent the mean ± SD of two experiments Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 3 Adv-mediated gene transfer into MPC cultures originated from the marrow of seven individuals. MPC derived from the bone marrow of seven donors were infected with Ad5CMVGFP (MOI = 500, for 6 hours at 37°C). After 48 hours, transduced cells were scored by flow cytometry. For all cases, except PC3, PC4 and PC11, data shown represent the mean ± SD of at least three experiments Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 4 Differentiation potential of transduced MPC. Ad5CMVGFP-infected cultures (MOI = 200, for 6 hours at 37°C) were exposed either to an osteogenic (a) or an adipogenic medium (b, c). After 3 weeks, cells were examined for terminal differentiation by: (a) epifluorescent microscopic analysis after Alizarin Red S staining, photomicrography shows transduced osteoblasts (green fluorescence) embedded within mineralization nodules (red); (b) epifluorescent microscopic analysis after Nile Red staining, photomicrography shows adipocytes containing large lipid droplets (gold fluorescence) and transduced MPC (green fluorescence); (c) flow cytometric analysis of non- and Ad5CMVGFP-infected cells, where x-axis indicates autofluorescence (upper) or GFP-fluorescence (lower) and y-axis indicates cellular granularity (SSC). Cells with fluorescence intensity to the right of the vertical line are considered as transduced cells. Cells with SSC > 600 correspond to adipocytes. Notice that <0.3% of the cells were GFP-positive with a SSC > 600. Data shown are representative of three independent experiments Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 4 Differentiation potential of transduced MPC. Ad5CMVGFP-infected cultures (MOI = 200, for 6 hours at 37°C) were exposed either to an osteogenic (a) or an adipogenic medium (b, c). After 3 weeks, cells were examined for terminal differentiation by: (a) epifluorescent microscopic analysis after Alizarin Red S staining, photomicrography shows transduced osteoblasts (green fluorescence) embedded within mineralization nodules (red); (b) epifluorescent microscopic analysis after Nile Red staining, photomicrography shows adipocytes containing large lipid droplets (gold fluorescence) and transduced MPC (green fluorescence); (c) flow cytometric analysis of non- and Ad5CMVGFP-infected cells, where x-axis indicates autofluorescence (upper) or GFP-fluorescence (lower) and y-axis indicates cellular granularity (SSC). Cells with fluorescence intensity to the right of the vertical line are considered as transduced cells. Cells with SSC > 600 correspond to adipocytes. Notice that <0.3% of the cells were GFP-positive with a SSC > 600. Data shown are representative of three independent experiments Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 4 Differentiation potential of transduced MPC. Ad5CMVGFP-infected cultures (MOI = 200, for 6 hours at 37°C) were exposed either to an osteogenic (a) or an adipogenic medium (b, c). After 3 weeks, cells were examined for terminal differentiation by: (a) epifluorescent microscopic analysis after Alizarin Red S staining, photomicrography shows transduced osteoblasts (green fluorescence) embedded within mineralization nodules (red); (b) epifluorescent microscopic analysis after Nile Red staining, photomicrography shows adipocytes containing large lipid droplets (gold fluorescence) and transduced MPC (green fluorescence); (c) flow cytometric analysis of non- and Ad5CMVGFP-infected cells, where x-axis indicates autofluorescence (upper) or GFP-fluorescence (lower) and y-axis indicates cellular granularity (SSC). Cells with fluorescence intensity to the right of the vertical line are considered as transduced cells. Cells with SSC > 600 correspond to adipocytes. Notice that <0.3% of the cells were GFP-positive with a SSC > 600. Data shown are representative of three independent experiments Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 5 Expression of Adv-attachment and -internalization receptors in MPC surface. After labeling with PE-conjugated secondary antibody (control PE) or with the indicated monoclonal antibody and the secondary antibody, MPC were analyzed by flow cytometry. (a) Cytograms show FSC vs fluorescence intensity data for the whole population of MPC. (b) Histograms show fluorescence intensity data for cells with FSC < 200. In this representative experiment MFI were: control = 4, RmcB = 10, α-αvβ3 = 12, and α-αvβ5 = 28 Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 5 Expression of Adv-attachment and -internalization receptors in MPC surface. After labeling with PE-conjugated secondary antibody (control PE) or with the indicated monoclonal antibody and the secondary antibody, MPC were analyzed by flow cytometry. (a) Cytograms show FSC vs fluorescence intensity data for the whole population of MPC. (b) Histograms show fluorescence intensity data for cells with FSC < 200. In this representative experiment MFI were: control = 4, RmcB = 10, α-αvβ3 = 12, and α-αvβ5 = 28 Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 6 Adhesion of MPC to vitronectin. Suspensions of MPC, either in culture medium alone (control) or containing the indicated blocking monoclonal antibody, were loaded into VN- or BSA-coated dishes. Data shown is the relative adhesion of cells under each condition with respect to control (26% ± 3% after correction for unspecific binding [≤0.5%] to BSA), which was set to 1. Values represent the mean ± SD of two experiments Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)

Figure 7 Effect of blocking monoclonal antibodies against attachment and internalization receptors on Adv-mediated infection of MPC. MPC were infected with Ad5CMVGFP (MOI = 250, for 6 hours at 37°C) in the absence (control) or presence of the indicated monoclonal antibody. Cells were further incubated (48 hours) and the percentage of transduced cells was scored by flow cytometry. The relative number of transduced cells under each condition, was calculated with respect to control, which was set to 1. Notice that in the presence of an irrelevant antibody (pure IgG1, not shown) the relative percentage of transduced cells was similar to that obtained under control conditions. Data shown represent the mean ± SD of two experiments Experimental Hematology 2000 28, 382-390DOI: (10.1016/S0301-472X(00)00134-X)