Volume 15, Issue 10, Pages 1872-1880 (October 2007) A Study of the Role of Nell-1 Gene Modified Goat Bone Marrow Stromal Cells in Promoting New Bone Formation  Tara Aghaloo, Xinquan Jiang, Chia Soo, Zhiyuan Zhang, Xiuli Zhang, Jingzhou Hu, Hongya Pan, Tiffany Hsu, Benjamin Wu, Kang Ting, Xinli Zhang  Molecular Therapy  Volume 15, Issue 10, Pages 1872-1880 (October 2007) DOI: 10.1038/sj.mt.6300270 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Gene transduction and the effects on bone marrow stromal cell (BMSC) proliferation. (a) A multiplicity of infection of 50 pfu/cell achieved high transfer efficiency above 70% 3 days after AdLacZ transduction of goat BMSCs. Positive areas with X-gal staining are in blue. (b–d) Cellular morphology after transduction with AdNell-1, AdBMP-2, or untransduced control cells. Original magnification ×200. (e) Western blot probed with antibodies against Nell-1 and β-actin for confirmation of Nell-1 protein expression 3 days after gene transduction. (f) Effects of AdNell-1, AdBMP-2, and AdLacZ infection on BMSC proliferation. A growth curve represents cellular proliferation after transduction with AdNell-1, AdBMP-2, or AdLacZ. Data points represent the mean ± SD. A significant decrease in cell number after transduction with AdNell-1 compared to AdLacZ at days 3, 4, and 5; *P < 0.05. No significant difference was detected between AdBMP-2 and AdLacZ groups; P > 0.05. Molecular Therapy 2007 15, 1872-1880DOI: (10.1038/sj.mt.6300270) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 In vitro analysis of osteoblastic differentiation after bone marrow stromal cell transduction with AdNell-1, AdBMP-2, and AdLacZ. (a) alkaline phosphatase expression 12 days after gene transduction. (b) von Kossa assay comparing calcium nodules between AdNell-1-, AdBMP-2-, and AdLacZ-transduced cells 14 days after gene transfer. (c) Quantitative analysis of calcium nodules present in the three groups. A significant increase is seen in the AdNell-1 and AdBMP-2 groups when compared with the AdLacZ group; *P < 0.05, but no significant difference is seen between the AdNell-1 and AdBMP-2 groups; P > 0.05. Molecular Therapy 2007 15, 1872-1880DOI: (10.1038/sj.mt.6300270) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Histologic analysis of 2-week tissues in vivo. (a) AdNell-1-transduced bone marrow stromal cell (BMSC) injection sites showing cartilage (arrows) and osseous tissue (arrowheads) with the presence of chondroid matrix (CM) and bone marrow; hematoxylin and eosin (H&E) staining. (b) AdBMP-2 transduced BMSC injection sites showing osseous tissue intramuscularly (arrowheads) with cartilaginous tissue (arrows); H&E staining. (c) AdLacZ-transduced BMSC injection sites showing a small locus of cartilaginous (arrows) and fibroblastic (arrowheads) tissue; H&E staining. (d–f) Alcian blue staining on corresponding tissue sections of a–c to confirm the presence of cartilaginous tissue (blue) Original magnification for all figures: ×100. Molecular Therapy 2007 15, 1872-1880DOI: (10.1038/sj.mt.6300270) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Radiographic, microCT, and histologic evidence of bone formation after 4 weeks in vivo. (a) Plain radiography shows a large, defined radio-opaque mass (white arrow) with density similar to iliac bone, representing bone formation in the AdNell-1-transduced bone marrow stromal cell (BMSC) injection site on the left side as compared to the AdLacZ-treated right side. (b) An even larger radio-opaque mass with less dense bone compared to iliac bone is seen on the AdBMP-2-transduced BMSC injection site (white arrow). (c) 3D microCT analysis better demonstrates the bone mass seen in the AdNell-1 treated site (white arrow) similar to native bone, that could not be detected in AdLacZ sites. (d) 3D microCT image of a large bony nodule in the AdBMP-2-treated site (white arrow). (e) When the microCT image is bisected, a radio-dense mass is seen in the AdNell-1 treated site, (f) compared to a hollow cavity with only an outer bony shell with interspersed small bone trabeculae in the AdBMP-2-treated site. (g) Hematoxylin and eosin (H&E) histology shows the typical bony morphology in the AdNell-1 specimen compared to (h) a fatty marrow cavity with an outer bony surface and few bone trabeculae in the AdBMP-2 specimen (arrows). (i, l, o) H&E histological analysis (i) and immunostaining for Nell-1 (l) and bone morphogenetic protein-2 (BMP-2) (o) of in vivo bone formation in AdNell-1-transduced BMSC injection sites. Mature bone formation (arrows) with Haversian systems and marrow cavities with some fat cells are seen. Areas of chondroid bone matrix are also seen (CM). Nell-1 immunohistochemistry demonstrating positive brown staining in osteocytes (arrows) and some bone marrow cells (arrowheads). BMP-2 immunohistochemistry demonstrates low level staining only in new bone forming areas (arrows) and bone marrow cells (arrowheads). (j, m, p) H&E histology (j) and immunostaining for Nell-1 (m) and BMP-2 (p) of AdBMP-2-transduced BMSC injection sites demonstrates more mature but thinner trabecular bone scattered within a large amount of bone marrow in a big cavity filled with mostly fatty tissue, without obvious evidence of cartilage or chondroid matrix. Nell-1 immunohistochemistry shows no obvious staining, while BMP-2 immunohistochemistry demonstrates a strong BMP-2 expression in both the bone (arrows) and surrounding fibroblastic tissue (arrowheads). (k) Most of the AdLacZ-transduced BMSC injection sites show only fibroblastic tissue (arrows); (n) Frozen sections stained with X-gal after 4 weeks in AdLacZ-transduced BMSC injection sites (arrows). Original magnification for figure g and h: ×40; i, j, and k: ×100; l, m, n, o, and p: ×200). Molecular Therapy 2007 15, 1872-1880DOI: (10.1038/sj.mt.6300270) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Immunohistochemical analysis of intramuscular bone and cartilage formed in AdNell-1-, AdBMP-2-, or AdLacZ-transduced bone marrow stromal cell (BMSC) injection sites after 4 weeks in vivo. (a) The AdNell-1 group demonstrates significant bone matrix formation (BM) and bone marrow cavities (M) with some areas of immature bone containing large cells resembling chondrocytes (black arrows). (b) The AdBMP-2 group shows more mature bone matrix formation (BM) with marrow cavities (M) mainly filled with fatty tissue. (c) The AdLacZ group shows primarily cartilage (C) with some bone formation at the periphery (arrows and arrow heads) and marrow cavities (M). (d, e, f) Alcian blue better demonstrates the cartilage present in the corresponding tissue sections of figure a, b, and c. (g) Sox9 immunohistochemistry shows minimal staining in the AdNell-1 sites (arrows), (h) light staining in the AdBMP-2 sites, (i) but abundant positive brown staining localized in the nuclei in the AdLacZ sites (arrows). (j) Type X collagen (ColX) immunohistochemistry shows positive staining throughout the extracellular matrix and the hypertrophic chondrocytes (arrows) in the AdNell-1 sites. (k) In the AdBMP-2 sites, positive staining appears only in the extracellular matrix. (l) Isolated areas of positive staining for ColX were observed in the periphery of the cartilage where bone formation occurred in AdLacZ sites (arrows). Original magnification for all figures: ×200. Molecular Therapy 2007 15, 1872-1880DOI: (10.1038/sj.mt.6300270) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions