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Bone-marrow-derived mesenchymal stem cells (MSCs) are multipotent cells capable of selfrenewal and differentiation into multiple cell types. Accumulating preclinical and clinical evidence indicates that MSCs are good candidates to use as cell therapy in many degenerative diseases. For MSC clinical applications, an adequate number of cells are necessary so an extensive expansion is required. However, spontaneous immortalization and malignant transformation of MSCs after culture expansion have been reported in human and mouse, while very few data are present for rat MSCs (rMSCs). In this study, we monitored the chromosomal status of rMSCs at several passages in vitro, also testing the influence of four different cell culture conditions. We first used the conventional traditional cytogenetic techniques, in order to have the opportunity to observe even minor structural abnormalities and to identify low-degree mosaic conditions. Then, a more detailed genomic analysis was conducted by array comparative genomic hybridization. We demonstrated that, irrespective of culture conditions, rMSCs manifested a markedly aneuploid karyotype and a progressive chromosomal instability in all the passages we analyzed and that they are anything but stable during in vitro culture. Despite the fact that the risk of neoplastic transformation associated with this genomic instability needs to be further addressed and considering the apparent genomic stability reported for in vitro cultured human MSCs (hMSCs), our findings underline the fact that rMSCs may not in fact be a good model for effectively exploring the full clinical therapeutic potential of hMSCs.

Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA-induced neuritogenesis, we used human neuroblastoma SH-SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal-regulated kinase (ERK) 1 and 2 in RA-induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA-induced neuritogenesis (percentage of neurite-bearing cells and neurite length) but significantly reduced cell survival. In addition, we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA-induced ERK phosphorylation does not follow the classic Raf kinase-dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI 3-K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3-K inhibitor wortmannin, we observed a significant dose-dependent reduction in ERK phosphorylation. RA-induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration-dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA-induced neuritogenesis.

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The clinical usability of pancreatic islet transplantation for the treatment of type I diabetes, despite some encouraging results, is currently hampered by the short lifespan of the transplanted tissue. In vivo studies have demonstrated that co-transplantation of Mesenchymal Stem Cells (MSCs) with transplanted pancreatic islets is more effective with respect to pancreatic islets alone in ensuring glycemia control in diabetic rats, but the molecular mechanisms of this action are still unclear.The aim of this study was to elucidate the molecular mechanisms of the positive effect of MSCs on pancreatic islet functionality by setting up direct, indirect and mixed co-cultures.MSCs were both able to prolong the survival of pancreatic islets, and to directly differentiate into an “insulin-releasing” phenotype. Two distinct mechanisms mediated these effects: i) the survival increase was observed in pancreatic islets indirectly co-cultured with MSCs, probably mediated by the trophic factors released by MSCs; ii) MSCs in direct contact with pancreatic islets started to express Pdx1, a pivotal gene of insulin production, and then differentiated into insulin releasing cells. These results demonstrate that MSCs may be useful for potentiating pancreatic islets' functionality and feasibility.

The spontaneous expression of neural markers, already demonstrated in bone marrow (BM) mesenchymal stem cells (MSCs), has been considered as evidence of the MSCs' predisposition to differentiate toward neural lineages, supporting their use in stem cell-based therapy for neural repair. In this study we have evaluated, by immunocytochemistry, immunoblotting, and flow cytometry experiments, the expression of neural markers in undifferentiated MSCs from different sources: human adipose stem cells (hASCs), human skin-derived mesenchymal stem cells (hS-MSCs), human periodontal ligament stem cells (hPDLSCs,) and human dental pulp stem cells (hDPSCs). Our results demonstrate that the neuronal markers βIII-tubulin and NeuN, unlike other evaluated markers, are spontaneously expressed by a very high percentage of undifferentiated hASCs, hS-MSCs, hPDLSCs, and hDPSCs. Conversely, the neural progenitor marker nestin is expressed only by a high percentage of undifferentiated hPDLSCs and hDPSCs. Our results suggest that the expression of βIII-tubulin and NeuN could be a common feature of stem cells and not exclusive to neuronal cells. This could result in a reassessment of the use of βIII-tubulin and NeuN as the only evidence proving neuronal differentiation. Further studies will be necessary to elucidate the relevance of the spontaneous expression of these markers in stem cells.

The main dose-limiting side effect of cancer treatment with platinum compounds is peripheral neurotoxicity. To investigate the intracellular mechanisms of platinum drugs neurotoxicity we have studied the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. Both platinum compounds are toxic causing cellular death by inducing apoptosis but oxaliplatin is less neurotoxic than cisplatin. The study of the proteins involved in the intracellular transduction pathways that may cause apoptotic death, revealed a very similar pattern of changes after exposure to cisplatin or oxaliplatin. In particular, as demonstrated by densitometric analysis, after exposure to both platinum compounds the total amount of the anti-apoptotic protein Bcl-2 was significantly reduced. Conversely, the amount of the pro-apoptotic protein p53 significantly increased. Caspases 3 and 7 were activated, but their activation was a late event, indicating a secondary role in the apoptotic process. Among the mitogen activated protein kinases, only the p38 protein was activated (phosphorylated) early enough to have a possible role in inducing apoptosis, possibly through p53 stabilization. The results of the present study and the data of the literature demonstrate that the ways in which cisplatin and oxaliplatin are neurotoxic are very similar and include not only DNA damage, but also the modulation of specific molecules involved in regulating the cellular equilibrium between apoptotic death and the cell cycle.

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