Volume 16, Issue 3, Pages 571-579 (March 2008) Targeted Migration of Mesenchymal Stem Cells Modified With CXCR4 Gene to Infarcted Myocardium Improves Cardiac Performance  Zhaokang Cheng, Lailiang Ou, Xin Zhou, Fei Li, Xiaohua Jia, Yinguo Zhang, Xiaolei Liu, Yuming Li, Christopher A Ward, Luis G Melo, Deling Kong  Molecular Therapy  Volume 16, Issue 3, Pages 571-579 (March 2008) DOI: 10.1038/sj.mt.6300374 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 Retroviral transduction of mesenchymal stem cells (MSCs) with the, CXCR4-eGFP fusion gene. (a) Schematic representation of the retroviral vector constructs. LTR, long terminal repeat; Ψ+, extended packaging signal; Neor, neomycin resistance gene; PPGK, murine phosphoglycerate kinase promoter. (b) Cellular localization of the CXCR4-eGFP fusion protein in retrovirally transduced MSCs without permeabilization (top) and with permeabilization (bottom). Green fluorescent protein (GFP) fluorescence is seen as green, and CXCR4 as red. Co-localization of GFP and CXCR4 appears as yellow-orange. Scale bar = 10 μm. (c) Flow cytometry analysis of eGFP (left) and surface CXCR4 (right) expression in CXCR4-MSCs. CXCR4, CXC chemokine receptor 4; eGFP, enhanced GFP. Molecular Therapy 2008 16, 571-579DOI: (10.1038/sj.mt.6300374) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 Migration and proliferation of CXCR4-MSCs. (a) Representative images of transmigrated mesenchymal stem cells (MSCs) (left) and CXCR4-MSCs (right) in response to 30 ng/ml stromal-derived factor-1 (SDF-1) in transwell assay. Scale bar = 50 μm. (b) Average number of cells migrated in transwell migration assay. Results are mean values ± SEM of five different fields from four independent experiments. *P <0.05 versus MSCs. (c) Proliferation of MSCs and CXCR4-MSCs under normal serum condition [20% fetal bovine serum (FBS)]. *P < 0.05 versus MSCs. (d) Cell proliferation and (e) viability under low serum conditions (0.5% FBS). MSCs and CXCR4-MSCs (1 × 104 cells/well) were cultured in 96-well plates with the addition of SDF-1 or anti-CXCR4 antibody. Cell number and viability were analyzed 5 days after plating. The results represent mean values ± SEM of four independent experiments. *P < 0.05 versus 20% FBS; †P < 0.05 versus 0.5% FBS; ‡P < 0.05 versus 0.5% FBS + SDF-1. CXCR4, CXC chemokine receptor 4. Molecular Therapy 2008 16, 571-579DOI: (10.1038/sj.mt.6300374) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 Homing and tissue distribution of Dil-labeled donor mesenchymal stem cells (MSCs) 3 days after systemic infusion. (a) Homing in of MSCs (left) and CXCR4-MSCs (right) toward infarcted myocardium. The presence of the cells was recognized by the red fluorescence signals presented by 1,1′-dioctadecyl-3,3,3′3′-testramethylindocarbocyanine perchlorate. Scale bar = 50 μm. (b) Relative abundance of MSCs and CXCR4-MSCs in histological sections from heart, liver, spleen, and lung. *P < 0.05 versus heart in myocardial infarction (MI) + MSCs; ‡P < 0.05 versus heart in MI + CXCR4-MSCs; ‡P < 0.05 versus spleen in MI + MSCs; §P < 0.05 versus spleen in MI + CXCR4-MSCs (MI + MSCs and normal + CXCR4-MSCs, n = 3; MI + CXCR4-MSCs, n = 4). CXCR4, CXC chemokine receptor 4; HPF, high-power field. Molecular Therapy 2008 16, 571-579DOI: (10.1038/sj.mt.6300374) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 Effect of intravenously transplanted cells on infarct size and collagen content. (a) Histomorphological appearance of the left ventricular (left) and anterior wall (right) 30 days after treatment. Significant thinning of the infarcted anterior wall and chamber dilatation are seen in the saline-treated animals, and to a lesser extent in the mesenchymal stem cell (MSC)- and CXCR4-MSC-treated animals. Significant collagen deposition was seen in the infarct region in all the animals. Under polarized light, collagen I appears red and collagen III appears green. Scale bar = 2 mm (left) and 1 mm (right), respectively. (b) Infarct size, (c) collagen content, and (d) collagen I/III ratio in animals 30 days after transplantation. Data are mean values ± SEM. *P < 0.05 versus sham-operated (sham-operated, MSCs, and CXCR4-MSCs, n = 5 each; saline, n = 6). CXCR4, CXC chemokine receptor 4; HE, hematoxylin and eosin. Molecular Therapy 2008 16, 571-579DOI: (10.1038/sj.mt.6300374) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 5 Histological examination of capillary density in isolectin-stained sections 30 days after infarction. (a) Representative micrographs of the infarct, border, and areas remote from the infarct in the four groups of animals. Scale bar = 50 μm. (b) Morphometric analysis of vessel density in the infarct, border, and areas remote from the infarct in the various groups. Data are mean values ± SEM. *P < 0.05 versus sham-operated [sham-operated, mesenchymal stem cells (MSCs), and CXCR4-MSCs, n = 5 each; saline, n = 6]. CXCR4, CXC chemokine receptor 4; HPF, high-power field. Molecular Therapy 2008 16, 571-579DOI: (10.1038/sj.mt.6300374) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 6 Immunohistochemical examination of donor cell integration and differentiation in the infarcted myocardium. (a) A cluster of enhanced green fluorescent protein (eGFP)-positive CXCR4-MSCs (black arrows) localized in the infarcted myocardium. Sections were stained with anti-GFP antibody and visualized using 3,3′-diaminobenzidine staining. Scale bar = 20 μm. (b) An eGFP-positive cell (black arrow) and corresponding DiI fluorescence (white arrow). Scale bar = 10 μm. (c) Representative sections from CXCR4-MSCs stained with an antibody that recognizes the Z-band structural protein α-actinin. Very few Dil-labeled cells in the infarct region were positive for α-actinin. Scale bar = 10 μm. (d) Sections from the same animals showed several Dil-labeled cells staining positive for von Willebrand factor (vWF) interspersed in the luminal lining of the vessel. Arrows indicate differentiated cells and arrowheads indicate undifferentiated cells. Scale bar = 10 μm. CXCR4, CXC chemokine receptor 4; MSC, mesenchymal stem cell. Molecular Therapy 2008 16, 571-579DOI: (10.1038/sj.mt.6300374) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions