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The transplantation of pancreatic islets is an innovative and intriguing therapeutic option for the long term treatment of type-1 diabetes (Remuzzi et al., 2009). Unfortunately, their clinical feasibility is limited by the great number of islets necessary to achieve glycaemic control and their short survival. A possible means to improve the performance of this technique can be represented by Mesenchymal Stem Cells (MSCs), adult stem cells alrady known to support the survival of different cellular populations (Scuteri et al., 2014). In this work the ability of Mesenchymal Stem Cells (MSCs) to improve the feasibility of this approach was verified into an in vivo model represented by Streptozotocin-induced diabetic rats. We compared 5 different groups (8 rats/group): a) healthy controls; b) Diabetic rats; c) Diabetic rats transplanted with pancreatic islets (3000); d) Diabetic rats cotransplanted with pancreatic islets (2000) and MSCs (106); Diabetic rats treated with MSCs (106). Transplantations were performed after the assessment of neuropathic signs, represented by a decreased Nerve Conduction Velocity (NCV) and an impairment of nociceptive thermal and mechanical thresholds. The same parameters were evaluated two months after the transplantation. Diabetic rats transplanted only with pancreatic islets, or co-transplanted with MSCs and a suboptimal number of pancreatic islets, showed a significant glycaemia value reduction, an improvement of thermal and mechanical sensitivity, and an improvement of NCV with respect to diabetic-untreated rats. No differences were observed between diabetic rats and diabetic rats treated with only MSCs. In conclusion, we demonstrated that co-transplantation with MSCs reduces the number of pancreatic islets needed to reach glycaemic control, and induces the regression of painful neuropathy signs, thus ameliorating diabetes complications management. Granted by MIUR – FIRB Futuro in Ricerca 2008 Prot. N° RBFR08VSVI_001., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Mesenchymal Stem Cells (MSCs) are adult multipotent cells able to increase sensory neuron survival: direct co-culture of MSCs with neurons is pivotal to observe a neuronal survival increase. Despite the identification of some mechanisms of action, little is known about how MSCs physically interact with neurons. The aim of this paper was to investigate and characterize the main mechanisms of interaction between MSCs and neurons. Morphological analysis showed the presence of gap junctions and tunneling nanotubes between MSCs and neurons only in direct co-cultures. Using a diffusible dye, we observed a flow from MSCs to neurons and further analysis demonstrated that MSCs donated mitochondria to neurons. Treatment of co-cultures with the gap junction blocker Carbenoxolone decreased neuronal survival, thus demonstrating the importance of gap junctions and, more in general, of cell communication for the MSC positive effect. We also investigated the role of extracellular vesicles; administration of direct co-cultures-derived vesicles was able to increase neuronal survival. In conclusion, our study demonstrates the presence and the importance of multiple routes of communication between MSCs and neurons. Such knowledge will allow a better understanding of the potential of MSCs and how to maximize their positive effect, with the final aim to provide the best protective treatment.

Type-1 Diabetes is generally treated with exogenous insulin administration. Despite treatment, a very common long term consequence of diabetes is the development of a disabling and painful peripheral neuropathy. The transplantation of pancreatic islets is an advanced alternative therapeutic approach, but its clinical application is still very limited, mainly because of the great number of islets required to complete the procedure and of their short-term survival. An intriguing method to improve the performance of pancreatic islets transplantation is the co-transplantation of Mesenchymal Stem Cells (MSCs), adult stem cells already known to support the survival of different cellular populations. In this proof-of-concept study, we demonstrated using an in vivo model of diabetes, the ability of allogenic MSCs to reduce the number of pancreatic islets necessary to achieve glycemic control in diabetic rats, and overall their positive effect on diabetic neuropathy, with the reduction of all the neuropathic signs showed after disease induction. The cutback of the pancreatic islet number required to control glycemia and the regression of the painful neuropathy make MSC co-transplantation a very promising tool to improve the clinical feasibility of pancreatic islet transplantation for diabetes treatment.

Neurodegenerative diseases are different and many-sided disorders affecting both the Central and the Peripheral Nervous System. Despite the very different peculiar features, all the neurodegenerative diseases are characterized by the neuronal degeneration, which may be the consequence of different processes, such as an altered protein accumulation, an axonal damage, or the exposure to toxic agents. The progressive neuronal death leads to disease progression, which is not effectively counteracted by the current symptomatic therapies. Among the newly proposed therapeutic approaches, encouraging results have been obtained with Mesenchymal Stem Cells (MSCs), adult stem cells initially proposed for their differentiation potential and for their immune-modulatory abilities. Here we first verified in vivo the protective potential of MSCs into an in vivo model of Multiple Sclerosis (MS), represented by Experimental Autoimmune Encephalomyelitis (EAE), demonstrating that intravenous administration of MSCs are able to ameliorate the clinical score and the functional skills, and to reduce demyelinated lesions. We then investigated in vitro the possible molecular mechanisms of MSC protective action, thus demonstrating that, besides immunomodulation, MSCs are able to support neuronal survival after toxic stimuli exposure by reducing the apoptosis and by inhibiting the Metalloprotease pathway, which is supposed to be involved in neurodegenerative disease progression. Moreover, MSCs are able to promote the axonal myelination through the modulation of p75 receptor. For all these abilities, MSCs can represent a promising therapeutic approach for the treatment of neurodegenerative disorders.

Multiple sclerosis (MS) is a severe chronic disease characterized by the presence of immuno-mediated demyelinating lesions and impairment of axonal transmission, which cause the reduction of nerve conduction velocity and lead to the development of neurological symptoms. Current therapy for MS is based on immunosuppressant agents, but recently Mesenchymal Stem Cells (MSCs) have been proposed as therapeutic treatment for MS, demonstrating a positive effect when administered before disease onset, mainly due to their immunomodulatory properties. Here, we investigate the therapeutic potential of MSCs into an animal model of multiple sclerosis, represented by Dark Agouti rats affected by chronic Relapsing-Remitting experimental autoimmune encephalomyelitis (EAE). In order to assess their putative effectiveness, 106 MSC were intravenously injected in EAE rats before disease onset (7 days after disease induction), to test the “preventive” schedule, or after disease onset (14 days after MSC induction), to test the “therapeutic” schedule with MSCs. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords were performed to evaluate the demyelinating lesions. Clinical score analysis demonstrated that the “preventive” schedule of treatment had no effect on EAE clinical course, while the therapeutic schedule was able to hamper relapsing phase from day 19 and till the end of the experiment (day 45) with respect to EAE group. At day 45, histological analysis performed on spinal cords of EAE rats demonstrated the presence of demyelinated plaques, assessed by Luxol fast Blue staining and by immunohystochemistry for MBP. The same lesions were present in spinal cords of rats treated with the preventive MSC administration. On the contrary the therapeutic schedule with MSCs was able to significantly reduce the extension of demyelinated areas in the spinal cords, thus confirming clinical score evaluations. These results suggested that MSCs are able to ameliorate the clinical course of EAE animals and to hamper the disease relapsing by reducing the areas of demyelinated lesions. We are now evaluating the possible mechanism of MSCs action by investigating some putative myelinating properties of MSCs.

Multiple Sclerosis (MS) is a crippling chronic disease of the Central Nervous System caused by the presence of self-antibodies which progressively damage axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. MS is characterized by a Relapsing-Remitting course, and current therapies rely only on the use of immunosuppressive drugs, which are however unable to reverse disease progression. Encouraging results have been obtained in preclinical studies with the administration of Mesenchymal Stem Cells (MSCs) before disease onset (Zappia et al., 2005). Here, we investigate the therapeutic potential of MSC administration after disease onset into an animal model of MS, represented by Dark Agouti rats affected by chronic Relapsing-Remitting Experimental Autoimmune Encephalomyelitis (EAE) (Cavaletti et al., 2004). 106 MSC were intravenously injected in EAE rats after disease onset. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords performed to evaluate the demyelinating lesions. After the first peak of disease, no further relapses were observed in EAE rats treated with MSCs, differently from what observed in EAE group. Histological analysis demonstrated the presence of demyelinated plaques in spinal cords of EAE rats, (Luxol fast Blue staining and anti-MBP immunohystochemistry). On the contrary the therapeutic schedule with MSCs significantly reduces the number and the extension of demyelinated areas in the spinal cords, confirming clinical score evaluations. These results demonstrated that MSCs ameliorate the clinical course of EAE and hamper the disease relapsing by reducing the areas of demyelinated lesions. Granted by MIUR – FIRB Futuro in Ricerca 2008 Prot. N° RBFR08VSVI_001., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Adipocytes' biology and the mechanisms that control adipogenesis have gained importance because of the need to develop therapeutic strategies to control obesity and the related pathologies. Human mesenchymal stem cells (hMSCs), undifferentiated stem cells present in the bone marrow that are physiological precursors of adipocytes, were induced to adipogenic differentiation. The molecular mechanisms on the basis of the adipogenesis were evaluated, focusing on the MAPKinases ERK1 and ERK2, which are involved in many biological and cellular processes. ERK1 and ERK2 phosphorylation was reduced with different timing and intensity for the two isoforms in treated hMSCs in comparison with control cells until day 10 and then at 14-28 days, it reached the level of untreated cultures. The total amount of ERK1 was also decreased up to day 10 and then was induced to the level of untreated cultures, whereas the expression of ERK2 was not changed following adipogenic induction. Treatment with the specific ERK1/2 inhibitor U0126 during the whole differentiation period hampered hMSCs' adipogenic differentiation, as lipid droplets appeared in very few cells and were reduced in number and size. When U0126 was administered only during the initial phase of differentiation, the number of hMSCs recruited to adipogenesis was reduced while, when it was administered later, hMSCs did not acquire a mature adipocytic phenotype. ERK1 and ERK2 are important for hMSC adipogenic differentiation since any alteration to the correct timing of their phosphorylation affects either the recruitment into the differentiation program and the extent of their maturation.

Aims In order to develop therapeutic strategies to control obesity and the related pathologies, it is very important to deepen the knowledge of adipocytes biology and the mechanisms that control adipogenesis. In the present work we have studied the molecular mechanisms at the basis of the adipocytes differentiation using the human Mesenchymal Stem Cells (hMSCs), undifferentiated stem cells present in the bone marrow that are the physiological precursors of adipocytes in the organism. In particular we have focused our attention on the MAPKinases ERK1 and ERK2, that are involved in many biological and cellular processes. Methods hMSCs, obtained from iliac crest bone marrow, were induced to adipogenic differentiation by treatment with Adipogenic Induction Medium for 10 days (determination phase), then replaced with Adipogenic Maintenance Medium until the end of treatment at day 28 (terminal differentiation phase). hMSC adipogenic differentiation was evaluated by morphological and molecular techniques. Control cells were represented by hMSCs cultured in absence of adipogenic supplements. ERK1 and ERK2 expression and phosphorylation were evaluated by immunoblotting experiments. The specific ERK inhibitor U0126 was added to the adipogenic medium at different times during the adipogenic differentiation protocol. Results hMSC treated with adipogenic differentiation protocol showed lipid droplets that increased in number and size during the whole differentiation period. In treated hMSC both ERK1 and ERK2 phosphorylation was reduced in comparison to control hMSCs, but time and intensity of these reductions were different for the two isoforms. A decrease of the total amount of ERK1 was also observed. The presence of U0126 during the whole differentiation period hampered the adipogenic differentiation of hMSCs, as very few hMSCs showed the appearance of lipid droplets that were reduced both in number and size. When U0126 was administered only during the determination phase the number of hMSCs recruited in the differentiation program was reduced, while when U0126 was administered only in the terminal differentiation phase, hMSCs did not acquired a mature adipocytic phenotype. Conclusion In this work we demonstrate that ERK1 and ERK2 are important for hMSC adipogenic differentiation. Our results suggest that ERK1 and ERK2 play a key role in determining how many cells enter into the adipogenic differentiation program and acquire the phenotipycal and molecular characteristics of mature adipocytes.