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-m 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.embryonic stem cells ͉ cerebral ischemia ͉ cell labeling S everal studies have been able to demonstrate the migrational capacity of endogenous stem or progenitor cells in rat and mouse brains during normal (1, 2) and pathophysiological conditions (3, 4). The therapeutical potential of stem cell grafting has recently been studied in various pathological conditions of the brain showing extensive cell migration after implantation. However, all investigations so far have required the invasive analysis of brain sections postmortem in various groups of animals for different survival periods. A recent investigation described the detection of labeled cells, injected into rats, but reported no specific cell migration in the in vivo MRI data (5). All other studies have investigated the potential of MRI to detect pretreated cells (5-7) by registering the MRI data ex vivo, thus permitting observation of only one time point. In the present investigation we demonstrate sufficient spatial and temporal resolution of experimental MRI at high fields to allow longitudinal studies on individual animals after stem cell implantation into the brain. We have investigated the spatial dynamics of implanted embryonic stem (ES) cells and demonstrated their high migrational potential from the implantation site in the normal brain hemisphere toward the ischemic le...

BackgroundMagnetic resonance imaging (MRI) is a promising tool for monitoring stem cell-based therapy. Conventionally, cells loaded with ironoxide nanoparticles appear hypointense on MR images. However, the contrast generated by ironoxide labeled cells is neither specific due to ambiguous background nor quantitative. A strategy to overcome these drawbacks is 19F MRI of cells labeled with perfluorocarbons. We show here for the first time that human neural stem cells (NSCs), a promising candidate for clinical translation of stem cell-based therapy of the brain, can be labeled with 19F as well as detected and quantified in vitro and after brain implantation.Methodology/Principal FindingsHuman NSCs were labeled with perfluoropolyether (PFPE). Labeling efficacy was assessed with 19F MR spectroscopy, influence of the label on cell phenotypes studied by immunocytochemistry. For in vitro MRI, NSCs were suspended in gelatin at varying densities. For in vivo experiments, labeled NSCs were implanted into the striatum of mice. A decrease of cell viability was observed directly after incubation with PFPE, which re-normalized after 7 days in culture of the replated cells. No label-related changes in the numbers of Ki67, nestin, GFAP, or βIII-tubulin+ cells were detected, both in vitro and on histological sections. We found that 1,000 NSCs were needed to accumulate in one image voxel to generate significant signal-to-noise ratio in vitro. A detection limit of ∼10,000 cells was found in vivo. The location and density of human cells (hunu+) on histological sections correlated well with observations in the 19F MR images.Conclusion/SignificanceOur results show that NSCs can be efficiently labeled with 19F with little effects on viability or proliferation and differentiation capacity. We show for the first time that 19F MRI can be utilized for tracking human NSCs in brain implantation studies, which ultimately aim for restoring loss of function after acute and neurodegenerative disorders.

Recent investigations on transient focal cerebral ischemia suggested recovery of energy metabolism during early reperfusion, but followed by secondary energy failure. As disturbances of energy metabolism are reflected by changes of the apparent diffusion coefficient (ADC) of water, the aim of the current study was to follow the dynamics of the ADC during 1 hour of middle cerebral artery occlusion (MCAO) and 10 hours of reperfusion. The right MCA was occluded in male Wistar rats inside the magnet using a remotely controlled thread occlusion model. Diffusion-, perfusion-, and T2-weighted images were performed repetitively, and ADC, perfusion, and T2 maps were calculated and normalized to the respective preischemic value. The lesion volume at each time point was defined by ADC < 80% of control. At the end of 1-hour MCAO the hemispheric lesion volume was 22.3 +/- 9.0%; it decreased to 6.4 +/- 5.7% in the first 2 hours of reperfusion (P < 0.01), but then increased again, and by the end of 10 hours of reperfusion reached 17.3 +/- 9.3%. The mean relative ADC in the end ischemic lesion volume significantly improved within 2 hours of reperfusion (from 65.7 +/- 1.2% to 90.1 +/- 6.7% of control), but later declined and decreased to 75.4 +/- 7.3% of control by the end of the experiment. Pixels with secondary deterioration of ADC showed a continuous increase of T2 value during the first 2 hours of reperfusion in spite of ADC improvement, indicating improving cytotoxic, but generation of vasogenic edema during early reperfusion. A significant decrease of the perfusion level was not observed during 10 hours of recirculation. The authors conclude that the improvement of ADC in the early phase of reperfusion may be followed by secondary deterioration that was not caused by delayed hypoperfusion.