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Large hypointensities, generated by the iron particles, were clearly visible in all MRI scans of each heart observed, scattered throughout the ischemic left ventricular wall. It was remarkable that no differences could be observed between dead and living cell recipients regarding size, number, localization or distribution of the voids (Figure 2). Moreover, variation over time within each animal was absent as well: the prominent voids remained unchanged from day 5 until day 42 (Figure 2), as has been reported before by some previous studies although not given much attention 7,16_{. We} confirmed with a Prussian blue staining that the voids on the MRI images corresponded with iron particles present in the heart tissue (Figure 3). Iron was only present in intact cells with a nucleus and not free in the extracellular matrix.

It should be noted that the commonly employed reaction of horseradish peroxidase with DAB may not be used for antibody stainings in iron containing sections since iron itself reacts with the DAB substrate resulting in a false positive DAB precipitate 15_{. Although some studies acknowledged} this problem and used other labeling techniques 8,10_{, others did not notice it} 5,16-18 _{and their results} regarding the properties of the iron-positive cells need thus to be taken with caution. Regarding the findings of other studies that macrophages had phagocytozed the iron-loaded cells, only the study of Terrovitis et al 10 _{convincingly demonstrated with fluorescent double labeling that iron} colocalized with macrophages in the heart. In that study, however, immunocompetent rats were used for the transplantation experiments, leaving the question whether results are also true for immunoincompetent recipients, used by many stem cell studies.

We investigated the identity of the iron-containing cells in the immunocompromised host, using a precipitation reaction with alkaline phosphatase and red substrate which we verified not to react with iron like DAB does. In the dead-EPDC recipients, all iron-containing cells were positive for the macrophage marker CD68 (Figure 3a, d) demonstrating that the iron particles were phagocytosed by macrophages. The group that had received living EPDCs contained mainly CD68 positive iron- loaded macrophages (Figure 3b, e) but also some CD68 negative iron-loaded engrafted EPDCs (Figure 3c, f) which were, with their elongated shape, different in morphology from the round and large macrophages. This corresponds well to earlier studies where non-labeled EPDCs were found to engraft in the ischemic myocardium 12_.

We demonstrated for immunonocompromised NOD/scid mice, lacking adaptive immunity and having only altered innate immunity 19,20_{, that transplantation of iron-loaded allogeneic cells into} the infarcted hearts results in an MRI hypointensity for at least six weeks, which does not change in size or localization over time. There was no discrimination between initially engrafted living cells

Figure 2. Transplantation of living (a-h) and dead (i-p) iron-labeled EPDCs results in large hypointensities (voids, arrows) in the ischemic left ventricular wall. Number, size and localization of the voids are comparable between groups and do not change over time (day 5 until week 6). Sagittal views: a-d for living EPDCs and i-l for dead EPDCs; Short-axis views: e-h for living EPDCs and m-p for dead EPDCs.

and dead cells, the latter would be unable to integrate in the cardiac tissue. The signal was thus not dependent on engraftment of the transplanted cells, although free iron particles injected into the heart are cleared rapidly and will escape observation 3_.

In conclusion we can state that iron-labeling is not recommended for tracking of living cells over time in any of the above described transplantation models –neither for immunocompetent nor for the immunodeficient host-, as there is no discrimination between healthy, initially successfully engrafted, stem cells and dead stem cells phagocytosed by macrophages within the heart. The iron, inserted into the heart within the transplanted cells, remains present at the site of injection notwithstanding incorporation into macrophages, and was not cleared from the injection site at six weeks after transplantation as we hypothesized. Iron-labeling can, however, be very informative to evaluate whether stem cells were successfully injected into the organ of interest, to visualize the area of injection, and to pursue this site of injection over time.

Figure 3. Consecutive sections show that iron containing cells (blue) in dead-EPDC recipients (a) are positive for the macrophage marker CD68 (pink) (d), demonstrating that the macrophages had phagocytosed the iron. For living-EPDC recipients most iron containing cells (b) were positive for CD68 (e). These cells were large and round, like observed for the dead-EPDC recipients. However, in the living-EPDC recipients some differently shaped elongated iron containing cells (c) were observed which were negative for CD68 (f). Scale bars: 20 µm. Arrows indicate CD68 positive cells, arrowheads indicate Prussian blue positive but CD68 negative cells.

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General Discussion