ALS is a fatal neurodegenerative disorder

ALS is a fatal neurodegenerative disorder characterized by a selective and widespread degeneration of lower and upper motor neurons (Gordon, 2013; Rowland and Shneider, 2001). The main symptomatic features of ALS are muscular atrophy, motor paralysis and difficulties in speaking, swallowing, chewing and breathing (Harms and Baloh, 2013). Respiratory failure is the most common cause of death for ALS patients, which occurs within 3–5years from the diagnosis (Al-Chalabi and Hardiman, 2013). The effect of Riluzole, the only drug approved by the Food and Drug Administration more than twenty years ago is very modest in prolonging the life of patients without ameliorating their quality of life, even lower than palliative cares (e.g. tracheostomy and invasive ventilation) (Cheah et al., 2010; Musaro, 2013). For these reasons, alternative and innovative therapeutic strategies are strongly and urgently required. Interestingly, it has been recently reported that both the ICV and the IV infusions of ematopoietic and mesenchymal stem Atglistatin induced a protective effect in two different models of ALS. Since no clear localization of stem cells within the areas of degenerating motor neurons was found, it was suggested that this was likely the response to the production of anti-inflammatory and immunomodulatory factors produced by the stem cells even far from the damaged areas (Garbuzova-Davis et al., 2008; Canzi et al., 2012; Bigini et al., 2011). Different strategies to follow the fate of transplanted stem cells in preclinical models of human disorders have been reported (Wang and Moore, 2012). In particular, the FDA approved Superparamagnetic Iron Oxide nanoparticles (SPIOn) can provide an easily transferable and non-invasive system to follow stem cells using magnetic resonance imaging. An innovative approach to further increase the reliability of stem cells tracking in different murine models of ALS by specifically labeling the cytoplasm with SPIOn and the nucleus with Hoechst-33258 has been developed by our group (Canzi et al., 2012; Bigini et al., 2012). Although this strategy enabled us to determine the interaction of various types of fetal stem cells in several models of motor neuron degeneration at different times points, perplexities were raised about the biocompatibility and the lack of cell perturbation after SPIOn internalization (Calero et al., 2014; Li et al., 2013; Reddy et al., 2012). To overcome this problem, fluorescent, biocompatible and long lasting traceable poly (methyl methacrylate) nanoparticles (PMMA-NPs) have been recently developed for the tracking of human amniotic fluid cells by ex-vivo analyses. The reliability of our approach was furthermore evaluated by in-vivo studies where the co-incubation of SPIOn and PMMA-NPs confirmed the presence of the two tracers in transplanted cells for at least three weeks after administration (Cova et al., 2013). On the basis of these results in the present study we proposed to label human umbilical cord mesenchymal stromal cells (UC-MSCs) with PMMA-NPs, that segregate into the cytoplasm, and with the nuclear dye Hoechst-33258 before to inject them ICV or intravenously in both healthy and early symptomatic SOD1G93A mice in order to track them at different time points during the disease progression. UC-MSCs were selected because they represent an innovative pool of MSCs, with a simpler, safer and cheaper collection from donors compared to the bone marrow stem cells and the cord blood mononuclear cells. In addition, mesenchymal stromal cells have shown a strong immunomodulatory and cyto-protective activity in different preclinical models of acute inflammation (Griffin et al., 2013; Stagg and Galipeau, 2013; Uccelli et al., 2008). To optimize the tracking procedures cells were labeled with PMMA-NPs, that segregate into the cytoplasm, and with the nuclear dye Hoechst-33258. Two different fluorophores, Rhodamine B (RhB) and a deep infrared dye (DIR) respectively, were conjugated to NPs in order to combine in-vivo and ex-vivo analyses. The labeling enabled us to follow the fate of systemically administered UC-MSCs for a prolonged temporal window that was hypothesized sufficient for the cells to exert a therapeutic response in SOD1G93A mice. This is the first example in preclinical ALS studies in which the tracking of the same type of stem cells is reported by using two different ways of administration and where this longitudinal tracking has been carried out in living mice by whole body scanning with optical imaging system. The results obtained by this work can be therefore considered as a robust pre-requisite to plan future experiments in which the cell-host interaction could be easily correlated with the therapeutic efficacy, in a sort of theranostic approach, independently of the pathological model or the cell-type utilized.