Superparamagnetic Iron Oxide Nanoparticles Function as a Long-Term, Multi-Modal Imaging Label for Non-Invasive Tracking of Implanted Progenitor Cells

<div><p>The purpose of this study was to determine the ability of superparamagnetic iron oxide (SPIO) nanoparticles to function as a long-term tracking label for multi-modal imaging of implanted engineered tissues containing muscle-derived progenitor cells using magnetic resonance imaging (MRI) and X-ray micro-computed tomography (μCT). SPIO-labeled primary myoblasts were embedded in fibrin sealant and imaged to obtain intensity data by MRI or radio-opacity information by μCT. Each imaging modality displayed a detection gradient that matched increasing SPIO concentrations. Labeled cells were then incorporated in fibrin sealant, injected into the atrioventricular groove of rat hearts, and imaged <i>in vivo</i> and <i>ex vivo</i> for up to 1 year. Transplanted cells were identified in intact animals and isolated hearts using both imaging modalities. MRI was better able to detect minuscule amounts of SPIO nanoparticles, while μCT more precisely identified the location of heavily-labeled cells. Histological analyses confirmed that iron oxide particles were confined to viable, skeletal muscle-derived cells in the implant at the expected location based on MRI and μCT. These analyses showed no evidence of phagocytosis of labeled cells by macrophages or release of nanoparticles from transplanted cells. In conclusion, we established that SPIO nanoparticles function as a sensitive and specific long-term label for MRI and μCT, respectively. Our findings will enable investigators interested in regenerative therapies to non-invasively and serially acquire complementary, high-resolution images of transplanted cells for one year using a single label.</p></div

SPIO nanoparticle-labeled cells located within the implant in three different hearts.

<p>A) Brightfield, fluorescence, and merged images showing iron deposits in progenitor cells contained within the implant (left panel), staining of filamentous actin with phalloidin (green) and DNA (blue) (middle panel) in the same section, and the merged image (right panel). Arrows indicate a few inflammatory cells that are not positive for iron. Scale bars equal 50 µm. B) A different heart showing heavily-labeled cells in the implant using the same staining as described for A). Scale bars equal 25 µm. C) Iron was also evident (brown) within a portion of the implant immediately adjacent to the right ventricular epicardium in a separate tissue section (left panel) that was immunostained for ACTN (red) and DAPI (blue) (middle panel). The right panel shows an overlay of the two images to the left. Scale bars equal 25 µm.</p

Localization of SPIO-labeled cells in whole animals by μCT.

<p>A) Arrows indicate the location of the SPIO nanoparticle-labeled muscle progenitors contained within engineered tissues that were implanted in the AV groove of an adult rat heart for 1 year. Unlike MRI images, the iron presents as a positive contrast or hyperintense signal. Coronal (A), axial (B), and sagittal (C) μCT images are shown. The panels on the left show individual μCT image slices, while the panels on the right depict the corresponding volumetric renderings from all of the acquired slices. These renderings were set to a threshold level that permitted simultaneous visualization of bone and iron-labeled cells. This processing resulted in loss of some soft tissue detail.</p

Localization of SPIO-labeled cells within the area of implants by histology.

<p>A) A photograph depicting the fibrin sealant-based implant in a heart excised one year after implantation (left panel), a low magnification image of a Masson's trichrome stained heart section (center panel), and a low magnification image from an adjacent serial section showing Prussian blue iron stain (right panel) of SPIO-labeled cells within the implant. The right atrium (RA), right ventricle (RV), and aorta are depicted along with an outline of the implant (dotted line). Scale bars equal 500 µm. B) Higher magnification brightfield images from the boxed region in A) showing an unprocessed serial section demonstrating iron (brown) can be detected in the cells of the implant and the same Masson's trichrome and Prussian blue stained sections from A). Scale bars equal 50 µm. C) The same section as the left panel from B) immunostained for the presence of macrophages (MAC387 in green), DNA (DAPI in blue), and striated muscle (ACTN in red) (left panel). Adjacent serial sections were immunostained for cardiac troponin T (cTnT in red), DNA (DAPI in blue), and striated muscle (ACTN in green) (middle panel). The right panel shows an image of ACTN, cTnT, and DAPI staining in the right ventricle. Scale bars equal 50 µm. D) Serial sections from a different heart stained for Masson's trichrome (left panel), Prussian blue with pararosaniline (middle panel), and immuno-fluorescence from secondary antibodies detecting binding of cTnT and ACTN antibodies in addition to DAPI staining of DNA. Iron is abundant in implanted cells, but is not apparent in the epicardium or myocardium of the right ventricle. The location of SPIO-labeled cells corresponds to the location of ACTN positive (red), cTnT negative (green) cells. Scale bars equal 50 µm.</p

Localization of SPIO-labeled cells in implanted rat hearts by MRI.

<p>A) Arrows indicate the location of hypointense regions resulting from the presence of SPIO nanoparticles contained in the implant. Coronal (left), axial (center), and sagittal (left) MRI slices from <i>in vivo</i> cardiac imaging acquisitions were obtained 6 months post-implantation using a 7T magnet. B) Arrows indicate the location of SPIO-labeled cells (hypointense region) in short-axis slices from <i>in vivo</i> cardiac acquisitions taken from two different rats at 6 months and 1 year after surgical implantation of engineered tissues, respectively. These images were obtained using a 4.7T magnet. C) Arrows indicate the location of iron-labeled cells within the implant in an isolated heart that had been implanted with engineered tissues for 1 year. These coronal (left) and axial (right) MRI slices correspond to the <i>in vivo</i> images shown in A).</p