Volume 7, Issue 3, Pages 354-365 (March 2003) Efficient BMP2 gene transfer and bone formation of mesenchymal stem cells by a fiber- mutant adenoviral vector  Hajime Tsuda, Takuro Wada, Yoshinori Ito, Hiroaki Uchida, Hironari Dehari, Kiminori Nakamura, Katsunori Sasaki, Masayoshi Kobune, Toshihiko Yamashita, Hirofumi Hamada  Molecular Therapy  Volume 7, Issue 3, Pages 354-365 (March 2003) DOI: 10.1016/S1525-0016(02)00062-X Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Transduction efficiency and BMP2 expression with Adv-F/RGD into primary rat MSCs. Each assay was performed two days after infection with either AxCAZ3-F/RGD (solid circles) or AxCAZ3-F/wt (open circles) at various MOIs (particles/cell). (A) The cells were stained with X-gal, and 300 cells in three fields were counted by microscopic observation. Results are represented as the percentage of positively stained cells at each MOI. (B) The cells were lysed, and assayed for β-galactosidase activity using a commercial kit. Each experiment was repeated at least three times, the data obtained from a typical experiment are shown and represent means ± SD of triplicate determinations. (C) Representative photomicrographs of X-gal-stained MSCs transfected with either AxCAZ3-F/RGD or AxCAZ3-F/wt at an MOI of 103 particles/cell are shown (original magnification, ×100). (D) MSCs were infected with AxCAhBMP2-F/RGD (solid circles) or AxCAhBMP2-F/wt (open circles) at various MOIs (particles/cell). The culture medium was harvested 72 hours after infection. BMP2 production was measured using an ELISA kit. The data are shown as the means ± SD of quadruplicate determinations. Molecular Therapy 2003 7, 354-365DOI: (10.1016/S1525-0016(02)00062-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Expression of CAR and integrin αv in MSCs. Semi-quantitative RT-PCR was performed for CAR, integrin αv, and GAPDH. Total RNA was extracted from primary rat MSCs and from liver, spleen and kidney of Fischer rats. Reverse-transcribed cDNA was subjected to PCR using specific primers. These findings were confirmed in two experiments. Molecular Therapy 2003 7, 354-365DOI: (10.1016/S1525-0016(02)00062-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 In vitro osteogenic differentiation of MSCs. MSCs were infected with AxCAhBMP2-F/RGD (solid circles) or AxCAhBMP2-F/wt (open circles) at various MOIs (particles/cell). As markers of osteogenic differentiation, ALP activity, OC production, and Ca deposition were assayed. (A) The ALP activity of the cells was measured 7 days after infection using p-nitrophenylphosphate as the substrate. (C) The amount of osteocalcin secreted into the culture medium was measured by RIA 7 days after infection. (E) The deposited Ca was measured by decalcification of the cell layer and assay of the supernatant at 21 days after infection. ALP activity (B), OC production (D), and deposited Ca (F) of MSCs treated with rhBMP2 represent the standard in each assay. The data are shown as the mean ± SD of quadruplicate determinations. Representative macroscopic photomicrographs of Von-kossa staining are shown in (G). Molecular Therapy 2003 7, 354-365DOI: (10.1016/S1525-0016(02)00062-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Ectopic bone formation in vivo in each group implanted with MSCs alone, AxCAZ3-F/RGD-transduced MSCs, and HA alone. Representative photomicrographs show sections of MSCs alone (A, B), AxCAZ3-F/RGD-transduced MSCs (C, D), and HA alone (E, F), and the implants 4 weeks after implantation (original magnification: (A, C, E) × 40, and (B, D, F) × 100). Molecular Therapy 2003 7, 354-365DOI: (10.1016/S1525-0016(02)00062-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Ectopic bone formation in vivo in the group implanted with MSCs in the presence of rhBMP2, AxCAhBMP2-F/wt-transduced MSCs. Vascular invasion is marked by arrowheads. Representative photomicrographs show sections of MSCs in the presence of rhBMP2 (A–D). AxCAhBMP2-F/wt-transduced MSCs (E–H), the implants at 2 weeks (A, B, E, F) and 4 weeks (C, D, G, H) after implantation (original magnification: (A, C, E, G) × 40, and (B, D, F, H) × 100). Molecular Therapy 2003 7, 354-365DOI: (10.1016/S1525-0016(02)00062-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Ectopic bone formation in vivo in the group implanted with AxCAhBMP2-F/RGD-transduced MSCs. Representative photomicrographs show sections of the implants at 1 week (A, B), 2 weeks (C, D) and 4 weeks (E, F) after implantation (original magnification: (A, C, E) × 40, and (B, D, F) × 100). Molecular Therapy 2003 7, 354-365DOI: (10.1016/S1525-0016(02)00062-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Quantification of new bone formation. New bone area was calculated as a percentage of bone area per total area of each pore. An asterisk denotes the statistically significant difference between the AxCAhBMP2-F/RGD-transduced MSCs group and all other groups. (ANOVA, p < 0.05) Molecular Therapy 2003 7, 354-365DOI: (10.1016/S1525-0016(02)00062-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions