Volume 12, Issue 1, Pages 33-41 (July 2005) High-Resolution Imaging of Bone Precursor Cells Within the Intact Bone Marrow Cavity of Living Mice  Philipp Mayer-Kuckuk, Terence P.F. Gade, Ian M. Buchanan, Mikhail Doubrovin, Ludmilla Ageyeva, Joseph R. Bertino, Adele L. Boskey, Ronald G. Blasberg, Jason A. Koutcher, Debabrata Banerjee  Molecular Therapy  Volume 12, Issue 1, Pages 33-41 (July 2005) DOI: 10.1016/j.ymthe.2005.02.018 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 High-efficiency accumulation of superparamagnetic iron oxide particles in bone precursor cells. (A) Micrograph of the periphery of an MSC colony 7 days postplating. (B) FACS measurement of expression of stromal cell marker CD 105 on the surface of MSCs after 14 days in culture. Blue, 97.5% of cells stained positive for CD 105. Red, MSC control lacking the CD 105 antibody. (C) Double von Kossa/ALP assay following MSC culture in osteoinductive medium. Calcified nodules stained black, while ALP expression correlates with the dark red color. The inset shows a control cultured in MSC medium. (D) Micrograph of the MC3T3-E1#4 (4) cells. (E) Micrograph of the 7F2 (7F) cells. (F) Micrograph of the MPIO–DG particles. (G–J) Composite micrograph/fluorescence images of various living BPC types labeled with DG- (green fluorescence, FITC filter) or FR- (red fluorescence, Cy5 filter) containing MPIOs. (K) Kinetics (pulse–chase) of the MPIO–DG labeling of 4 MPIO–DG (white and cross-hatched bars correspond to the percentage of positive cells and the MFI, respectively) and 7F MPIO–DG cells (black and gray columns correspond to the percentage of positive cells and the MFI, respectively). Molecular Therapy 2005 12, 33-41DOI: (10.1016/j.ymthe.2005.02.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Noninvasive magnetic resonance imaging inside the femur of living mice. (A) Photograph of the coil and holder (removable thermostat tubing is not shown). The design allows for MRI of the left mouse femur at 7 T using a gradient insert. (B) A sagittal T1-weighted spin echo image illustrating the capability to obtain high-resolution in vivo images of the mouse femur (Fe), tibia (Ti), and fibula (Fi) with an in-plane resolution of 86 × 68 μm. Molecular Therapy 2005 12, 33-41DOI: (10.1016/j.ymthe.2005.02.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Magnetic resonance imaging of bone precursor cells inside the femur of living mice. (A) A pretransplant sagittal T2*-weighted FLASH image of the mouse left hindlimb in which the femur and tibia can be clearly seen. (B) The close-up demonstrating the hyperintense marrow signal in the femur diaphysis and metaphysis. (C) The corresponding posttransplant image. (D) The close-up demonstrates the susceptibility effect mediated by injection of 1 × 105 purified MSC MPIO–DG cells and the resulting significant signal loss (red arrows indicate regions of prominent signal decrease posttransplantation). (E) Prior to transplantation, the purified MSC MPIO–DG cells were analyzed for green fluorescence by FACS (1). After MRI, bone marrow samples from transplant (2) and control (3) mice were analyzed for the presence of MSC MPIO–DG cells (green fluorescence). (F) A series of axial bone marrow images obtained pre- and posttransplantation of 1 × 105 purified MSC MPIO–DG cells. The images (50 × 50 μm in-plane resolution) represent a defined ROI (inset) within the FOV of the 3D sequence. Anatomical landmarks taken from the T1-weighted spin echo images were used to obtain almost identical ROIs at the indicated time points. Molecular Therapy 2005 12, 33-41DOI: (10.1016/j.ymthe.2005.02.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 In vivo bioluminescence imaging of reporter gene expressing bone precursor cells. (A) Fluorescence image of MSCs after transduction with the TGL virus. (B) Transduced MSCs 5 weeks post-sorting of TGL (EGFP)-positive cells (MSC TGLs). Both insets represent the corresponding micrographs. (C) In vitro luciferase assay. The luciferase activity of 1 × 105 4 TGL and 7F TGL cells was measured. To determine the influence of MPIO labeling on luciferase activity, 2 × 104 4 TGL MPIO–DG cells and 4 TGL control cells were assayed. Values are presented as means ± standard deviation of the samples. (D, G) BLI post-femoral (left side) injection of 1 × 105 4 TGL cells (control, no cell injection). The inset light signal intensity color bar ranges between 1.5 × 103 (blue) and 1 × 104 photons/cm2/s/steradian (red). (E, F and H, I) Ex vivo validation of femur BLI. (E, H) Whole femurs were prepared immediately following imaging, opened at the distal and proximal ends, and assayed for luciferase activity. (F, I) Subsequently, the bone marrow was flushed out of the bone and the samples were reanalyzed. (J, K, and L) BLI following injection of 1 × 105 MSC TGL cells into the right femur of irradiated mice. The inset light signal intensity color bar ranges between 5 × 103 (blue) and 1 × 104 photons/cm2/s/steradian (red). Molecular Therapy 2005 12, 33-41DOI: (10.1016/j.ymthe.2005.02.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Combined magnetic resonance–bioluminescence imaging. (A) Fluorescence micrograph of cytoplasmic EGFP, a part of the BLI reporter gene. Inset: Phase image. (B) High cellular accumulation of red fluorescent MPIO–FR particles (fixed cells, fluorescence overlay on differential interference contrast image). C, cytoplasm; N, nucleus. (C, D) Pretransplant sagittal T2*-weighted FLASH MR images of the left femur bone marrow space. (E, F) Corresponding posttransplant MR images of 2.5 × 105 4 TGL MPIO–DG cells. The remarkable T2* effect can be seen on the close-up (red arrows indicate regions of prominent signal decrease posttransplantation). (G, H) BLI light signal intensity color bar ranges between 1.5 × 103 (blue) and 1 × 104 photons/cm2/s/steradian (red). (I, J) Dual-color FACS confirmed the presence of cells that hold MPIO–FR paramagnetic label (red fluorescence) and express the BLI reporter gene (green fluorescence) only in 4 TGL MPIO–FR-transplanted bone marrow. Molecular Therapy 2005 12, 33-41DOI: (10.1016/j.ymthe.2005.02.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions