The transporter was successfully expressed in MSCs and

The transporter was successfully expressed in MSCs and HEK 293 cells. In both cell types, the expression of YFP-EAAT2 was confirmed by immunological staining and flow cytometry. The functionality of the transporter was determined by [3H]glutamate uptake assays, and the blood glutamate-grabbing activity was validated in healthy rats. The results demonstrated that systemic administration of CI 976 expressing EAAT2 significantly reduces blood glutamate levels. The effect on blood glutamate reduction produced by MSCs+ and HEK+ was very similar to that resulting from oxaloacetate administration. The highest dose of MSCs+ (9 × 106 cells) reduced the glutamate concentration for up to 4 h; yet, the absolute reduction was not higher than that with 3 × 106 cells or oxaloacetate. It is tempting to speculate that a higher expression level of EAAT2 or higher cell doses per animal could increase the glutamate-grabbing efficacy. Here, as well as in previous studies [26,27], the expression of EAAT2 in MSCs+ and HEK+ cells was technically optimized to gain high transporter levels per cell and simultaneously keep the cells healthy. We are aware that alternative strategies, e.g., invoking lentivirus infection, may result in even higher transduction rates, and this aspect should be addressed in forthcoming studies. Regarding the cell dose used, we established 3 × 106 cells per animal as a threshold necessary to induce a significant reduction in blood glutamate levels. The risk of cell aggregation and pulmonary embolism has been described in other studies [41]; therefore, to avoid the death of the animals, doses higher than 9 × 106 MSCs+ were not used. Owing to the lack of experience and literature on HEK cells for i.v. administration and to reduce the risk of systemic embolisms, doses higher than 3 × 106 cells were not tested. The therapeutic effect of MSCs+ and HEK+ cells was thoroughly examined in ischemic rats and compared with that of oxaloacetate treatment. In line with the results in healthy rats, the administration of transfected cells and oxaloacetate significantly reduced blood glutamate levels, which was associated with the functional recovery of the animals. However, oxaloacetate, 3 × 106 MSCs+, and surprisingly, MSCs− (3 × 106 and 9 × 106 cell doses) were the only treatments that dually caused functional recovery and a reduction in infarct volume. HEK 293 cells are widely used in in vitro cell studies because they are easy to handle, grow rapidly, are resistant to stressful conditions, and can be readily transfected [42]. Therefore, this cell line represented an excellent model to test the effect of EAAT2 cell expression on blood glutamate reduction. The functional improvement observed in ischemic animals treated with HEK+ cells confirmed that this effect was indeed mediated by the glutamate-grabbing activity associated with EAAT2 functionality. The administration of HEK+ cells and oxaloacetate in ischemic animals resulted in almost similar glutamate reductions. However, the protective effect was quite different. This suggests that, in addition to the obvious blood glutamate-grabbing effect, oxaloacetate may induce a pleiotropic mechanism protecting the animal against ischemic stroke. Oxaloacetate has been shown to serve as an antioxidant when cells are subjected to stressful stimuli, such as hydrogen peroxide, thiobarbituric acid reactive species, or excitotoxic damage [15]. MSCs are currently the most promising candidates for stem cell therapy against ischemic stroke owing to their intrinsic capability to secrete growth factors and immunomodulatory cytokines, which equip these cells with neuroprotective and neurorecovery properties [32]. Therefore, we hypothesized that the addition of a glutamate-grabbing property should increase the protective effect of MSCs against cerebral ischemia. The expression of EAAT2 in MSCs effectively conferred the expected glutamate-grabbing activity validated in healthy and ischemic animals. Unexpectedly, the protective effect observed on infarct volume reduction and functional recovery after MSC+ administration was not as pronounced as with MSCs−. This finding suggests that the transfection procedure most likely interferes with some of the intrinsic protective mechanisms of mesenchymal cells. We could exclude the effect of the transfection procedure on the release of VEGF and membrane phenotype. The quantification of IL-6 secretion, which is considered one of the most representative cytokines [34], in MSC supernatants revealed that the electroporation procedure rather than plasmid expression induced the release of this cytokine. This might explain the loss of the beneficial effect with MSCs+ that we observed with MSCs. In fact, MSCs electroporated with control plasmids (sham MSCs) were not included as an additional group in the animal study mainly because wanted to compare the behavior of MSCs+ with that of healthy MSCs.