Monitoring of implanted stem cell migration in vivo: a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat

Abstract

In vivo monitoring of stem cells after grafting is essential for a better understanding of their migrational dynamics and differentiation processes and of their regeneration potential. Migration of endogenous or grafted stem cells and neurons has been described in vertebrate brain, both under normal conditions from the subventricular zone along the rostral migratory stream and under pathophysiological conditions, such as degeneration or focal cerebral ischemia. Those studies, however, relied on invasive analysis of brain sections in combination with appropriate staining techniques. Here, we demonstrate the observation of cell migration under in vivo conditions, allowing the monitoring of the cell dynamics within individual animals, and for a prolonged time. Embryonic stem (ES) cells, constitutively expressing the GFP, were labeled by a lipofection procedure with a MRI contrast agent and implanted into rat brains. Focal cerebral ischemia had been induced 2 weeks before implantation of ES cells into the healthy, contralateral hemisphere. MRI at 78-microm isotropic spatial resolution permitted the observation of the implanted cells with high contrast against the host tissue, and was confirmed by GFP registration. During 3 weeks, cells migrated along the corpus callosum to the ventricular walls, and massively populated the borderzone of the damaged brain tissue on the hemisphere opposite to the implantation sites. Our results indicate that ES cells have high migrational dynamics, targeted to the cerebral lesion area. The imaging approach is ideally suited for the noninvasive observation of cell migration, engraftment, and morphological differentiation at high spatial and temporal resolution.

Fig 1.

Coronal multislice gradient-echo magnetic resonance images through a rat brain after stereotactical implantation of HT-22 neuronal cells into the striatum of both hemispheres. On the right hemisphere HT-22 cells were labeled with SINEREM by lipofection, whereas on the left hemisphere implanted cells remained native (see arrows). Note the strong signal decrease at the implantation location of the labeled cells on the right hemisphere. The volume of labeled cells is distinguished mainly in the two posterior contiguous image planes (mainly B and C). The hyperintensity in the upper mid-left region of the brain, just ventral of the corpus callosum, is caused by the high water content of the lateral ventricle.

Fig 2.

Coronal sections from a 2D multislice MRI experiment through a rat brain at the day of stem cell implantation (A) and 8 days (B) and 16 days (C) after implantation. Transient focal cerebral ischemia of the right hemisphere (60 min) had been induced 14 days before implantation. Note the two large circular dark tissue areas (A, arrows) demonstrating the location of the labeled stem cells, on the strongly T₂*-weighted images with the contrast produced by the USPIO-based contrast agent SINEREM in the ES cells. The cortical implantation area extends toward the subventricular zone by 8 days after implantation. By 16 days, the lesioned hemisphere, opposite to the hemisphere of implantation, shows an extended darkened area at the striatum, reflecting the massive accumulation of ES cells in the lesion periphery. In plane resolution, 50 × 50 μm²; slice thickness, 500 μm.

Fig 3.

Coronal section through a rat brain at various times after implantation of ES cells into the hemisphere contralateral to the induced transient 60-min focal ischemia. 3D data sets were recorded at the day of implantation (A) and at 6 (B) and 8 (C) days after implantation. For orientation, the necrotic tissue area is outlined on C. Note at 6 days (B) the discrete dark line (arrow in D, with higher magnification) along the corpus callosum between the cortical implantation site and the ventricular wall showing cells migrating toward the lesioned hemisphere. At 8 days (C) a dark region becomes visible in the dorsal part of the lesioned territory reflecting first arrival of USPIO-labeled cells. At higher magnification (D), the migration along the corpus callosum becomes more pronounced and is clearly visible. Taken from another animal example, the lining along the ventricular wall (E) and the accumulation of labeled stem cells on the choroid plexus (F) are also presented with high magnification.

Fig 4.

Several locations have been marked on a section from a 3D data set at 11 days after implantation (same experiment as Fig. 3). For these positions GFP immunohistochemistry is shown. Clearly, in the primary implantation sites (sites 1 and 5), in the corpus callosum (site 2), and in the periphery of the ischemic lesion (site 3) strong staining shows the presence of GFP-expressing ES cells. Note that although the cell morphology shows round shapes at the implantation sites (sites 1 and 5), ES cells become elongated already during their migration (site 2). On arrival in the lesioned hemisphere several of the cells show neuron-like shapes of cell body with long dendritic- or axon-like extensions. In the necrotic area (of the formerly ischemic core) (site 6) no GFP-expressing cells have shown up, in agreement with the lack of USPIO-induced contrast in the T₂*-weighted image. Spatial resolution of the 3D MRI experiment: 78 × 78 × 78 μm³. Microscopy of the immunohistochemically stained section used a primary magnification of ×400 for all plates. (Scale bar for sites 1–6 = 100 μm.)