Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells

Abstract

Background: Delivery and tracking of endomyocardial stem cells are limited by the inability to image transplanted cells noninvasively in the beating heart. We hypothesized that mesenchymal stem cells (MSCs) could be labeled with a iron fluorophore particle (IFP) to provide MRI contrast in vivo to assess immediate and long-term localization.

Methods and results: MSCs were isolated from swine. Short-term incubation of MSCs with IFP resulted in dose-dependent and efficient labeling. Labeled cells remained viable for multiple passages and retained in vitro proliferation and differentiation capacity. Labeled MSCs (10(4) to 10(6) cells/150 microL) were injected percutaneously into normal and freshly infarcted myocardium in swine. One, 3, and 1 animals underwent serial cardiac MRI (1.5T) for 4, 8, and 21 days, respectively. MRI contrast properties were measured both in vivo and in vitro for cells embedded in agar. Injection sites containing as few as 10(5) MSCs could be detected and contained intact IFP-bearing MSCs on histology.

Conclusions: IFP labeling of MSCs imparts useful MRI contrast, enabling ready detection in the beating heart on a conventional cardiac MR scanner after transplantation into normal and infarcted myocardium. The dual-labeled MSCs can be identified at locations corresponding to injection sites, both ex vivo using fluorescence microscopy and in vivo using susceptibility contrast on MRI. This technology may permit effective in vivo study of stem cell retention, engraftment, and migration.

Figure 1

In vitro relaxometry of IFP-labeled MSCs. A, T₁, T₂, and T₂* relaxation time constants for IFP-labeled and unlabeled MSC agar suspensions at a range of cell concentrations. Unlabeled refers to average of a range of cell concentrations from 10⁴ to 10⁶ MSCs without IFP. B, Comparison of relaxation time constants for labeled and unlabeled cell suspensions.

Figure 2

In vivo relaxometry of injected IFP-labeled MSCs. A, Typical in vivo injection of 10⁵ IFP-labeled MSCs is shown in this still frame from a cinematic SSFP magnetic resonance image immediately after injection. This cell concentration represents the minimally detected dose in vivo and was used for all subsequent relaxometry. B, T₂* parametric map of the same slice shows the lowest T₂* in the region of the injection, corresponding to the location of labeled cells. The posterobasal epicardial surface also shows typical susceptibility SSFP artifact commonly attributed to adjacent diaphragmatic surfaces. The color map corresponds to T₂* values indicated on the scale immediately to the right. C, A profile of T₂* values along the dotted white line from the middle panel. The center of the injection has a T₂* value close to the values measured in vitro (see Figure 1).

Figure 3

In vivo visualization immediately before and after IFP-MSC injection. Long-axis SSFP MRI view of left ventricle before (A) and after (B) transcatheter injection of 4×10⁶ IFP-labeled MSCs into infarct at apex (arrows) and into adjoining normal myocardium (arrowhead). Delayed hyperenhancement inversion recovery FGRE MRI highlights areas of nonviable infarcted myocardium using same view as above, before (C) and after (D) injection of IFP-labeled MSCs. MSCs appear dark against hyperenhanced infarct.

Figure 4

Serial in vivo and ex vivo MR imaging. Serial short-axis views of diastolic frames show a persistent susceptibility artifact (signal void) after injection of 10⁶ IFP-labeled MSCs imaged on days 1 (A), 4 (B), and 21 (C). D, Corresponding view of explanted heart on high-resolution 3D MRI showing signal void.

Figure 5

In vitro labeling. A, Confocal fluorescence micrograph of labeled MSCs (×100) showing green fluorescence of (Dragon Green) intracytoplasmic 0.9 μ IFP particles. DAPI nuclear counter-stain appears blue. B, Nomarski image (×100) shows outline of MSC and perinuclear accumulation of dense particles corresponding to the green fluorescence.

Figure 6

Histological confirmation of IFP-labeled cells after endomyocardial injection. A through C, Immediately after injection. A, Confocal fluorescence micrograph of round fluorescent green MSCs with DAPI nuclear counterstaining of surrounding myocardium. The cells appear intact immediately after injection with few exogenous particles in the surrounding myocardium. B, Corresponding section using differential interference microscopy. C, Overlay of images A and B. D through F, Animal shown in Figure 4, 21 days after injection. D, Endomyocardial engraftment of fluorescent green MSCs with DAPI nuclear counter-staining. MSCs appear elongated and aligned with the host myocardium. E, Corresponding section using differential interference microscopy. F, Overlay of images D and E. Cells appear intact with maintenance of DAPI nuclear staining with surrounding perinuclear IFP.