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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

Multiple Sclerosis (MS) is a neuroinflammatory and immune-mediated chronic disease of the Central Nervous System which progressively damages the axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. Most MS cases are characterized by a relapsing-remitting course, and current therapies rely only on the use of immunomodulating drugs which are, however, unable to reverse disease progression. Among the newly proposed alternative therapies, Mesenchymal Stem Cells (MSCs) are considered suitable for MS treatment due to their capacity to modulate the immune response and to modify the pattern of the released cytokines. So far, encouraging results have been obtained with the administration of MSCs before disease onset, mainly in animal models of acute Experimental Autoimmune Encephalomyelitis (EAE) in which MSCs were able to reduce inflammation, thus ameliorating also the disease’s clinical symptoms. On the contrary, only a very small number of studies have investigated the effect of MSCs on relapsing-remitting models of the disease. Here, we investigated the therapeutic potential of MSC administration, both before and after the disease’s onset, in an animal model of MS represented by Dark Agouti rats affected by chronic Relapsing-Remitting EAE. Our results demonstrated that in chronic Relapsing-Remitting EAE the administration of MSCs after the clinical disease’s appearance is able to completely abrogate the relapsing phase and to strongly reduce spinal cord demyelination. These encouraging results have demonstrated that MSCs can provide a protective and reparative strategy for MS treatment.

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a dose-limiting side effect of several antineoplastic drugs which significantly reduces patients' quality of life. Although different molecular mechanisms have been investigated, CIPN pathobiology has not been clarified yet. It has largely been recognized that Dorsal Root Ganglia are the main targets of chemotherapy and that the longest nerves are the most damaged, together with fast axonal transport. Indeed, this bidirectional cargo-specific transport has a pivotal role in neuronal function and its impairment is involved in several neurodegenerative and neurodevelopmental diseases. Literature data demonstrate that, despite different mechanisms of action, all antineoplastic agents impair the axonal trafficking to some extent and the severity of the neuropathy correlates with the degree of damage on this bidirectional transport. In this paper, we will examine the effect of the main old and new chemotherapeutic drug categories on axonal transport, with the aim of clarifying their potential mechanisms of action, and, if possible, of identifying neuroprotective strategies, based on the knowledge of the alterations induced by each drugs.

Background/Aim: Peripheral neurotoxicity is a dose-limiting factor of many chemotherapeutic agents, including cisplatin. Mesenchymal stem cells are promising for the treatment of several neurological disorders, and our aim was to verify the neuroprotective potential of human mesenchymal stem cells (hMSCs) on dorsal root ganglia (DRG) exposed to cisplatin. Materials and Methods: DRG were exposed to different cisplatin concentrations and then co-cultured with hMSCs or with hMSC-conditioned medium. Results: hMSCs showed a neuroprotective effect on cisplatininduced death of DRG, mediated by direct contact. Moreover, DRG exhibited an MSC-dependent promotion of neurite outgrowth, in particular at early time points. For this effect, the expression of Neurite Outgrowth Inhibitor (NOGO) and Myelin Associated Glycoprotein (MAG) by hMSCs was pivotal. Conclusion: hMSCs are a promising tool for reducing the neurotoxic effect of cisplatin.

Neurobasal medium (NBM) is a widely used medium for neuronal cultures, originally formulated to support survival of rat hippocampal neurons, but then optimized for several other neuronal subtypes. In the present study, the toxic effect of NBM on long-term cortical neuron cultures has been reported and investigated. A significant neuronal cell loss was observed 24 h after the total medium change performed at days in vitro 10. The neurotoxic effect was specifically because of NBM-A, a commercially derived modification of classic NBM, as neurons exposed to minimum essential medium for 24 h did not show the same mortality rate. We showed that the toxic effect was mediated by the N-methyl-d-aspartate receptor (NMDAr) as its inactivation partly prevented NBM-induced neuronal loss, and the addition of NMDAr activators, such as l-cysteine or glycine to minimum essential medium, reproduced the same toxicity rate observed in NBM. Besides the toxicity associated with NMDAr activation, the decreased antioxidative defenses also worsen (because of glutathione depletion) neuronal death, thus amplifying the effect of excitotoxic amino acids. Indeed, glutathione supplementation by the addition of its precursor N-acetyl-cysteine resulted in an increase in neuronal survival that partially prevented NBM-A toxicity. These results evidenced, on the one hand, the unsuitability of NBM-A for long-term neuronal culture, and on the other, they highlight the importance of selection of more suitable culture conditions.

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.

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.

Treatment of type-1diabetes with pancreatic islet transplantation is an intriguing therapeutic option, aimed to replace insulin administration, but very limited in clinical practice, mainly for the great number of islets necessary and for their short survival. Aim of this work is to verify the ability of Mesenchymal Stem Cells (MSCs) co-transplanted with Pancreatic Islets to improve the feasibility of this approach, by acting both on glycaemic control and on long term disease complications, such as the diabetic neuropathy. 5 groups were used (8 rats/group): a) healthy controls; b) Streptozotocin-induced diabetic rats; c) Diabetic rats transplanted with pancreatic islets (3000); d) Diabetic rats co-transplanted with pancreatic islets (2000) and MSCs (106); Diabetic rats treated with MSCs (106). Transplantations were performed after the assessment of neuropathic signs, such as the decrease of Nerve Conduction Velocity (NCV) and the 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 marked and significant glycaemia value reduction, an improvement of thermal and mechanical sensitivity, and a nearly complete restoration of NCV with respect to diabetic-untreated rats. No differences were observed between diabetic rats and diabetic rats treated with only MSCs. Co-transplantation of MSCs with Pancreatic Islets allows to reduce the successful number of pancreatic islets, to obtain a better and more physiologic glycaemic control, and to induce the regression of painful neuropathy signs, thus ameliorating diabetes complications management

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 co-transplanted 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.

There are promising studies which demonstrate that Mesenchymal Stem Cells (MSCs) are able to release neutrophic factors, to increase neuronal survival and as a consequence to repair nervous damages. For these reasons MSCs based therapeutic approach was proposed for diseases of both central and peripheral nervous system. In order to evaluate a putative positive effect of MSCs on neuronal damage recovery, two different experimental models were set up using cortical and sensory neurons. The toxic stimuli used were: Glutamate, whose toxicity is associated to Multiple Sclerosis; Cisplatin and Paclitaxel, chemotherapic drugs, which induce peripheral neuropathies. For the evaluation of the effect of MSCs, direct and indirect co-cultures were set up with rat MSCs (rMSCs); rMSCs Conditioned Medium effect was also evaluated. Our results demonstrate an important difference between cortical and sensory neurons in fact in term of susceptibility cortical neurons are much more sensible to drugs treatment. About sensory neurons we observed a protective action in direct co-cultures on Cisplatin and Glutamate treated neurons and in indirect co-cultures on Paclitaxel treated neurons; conditioned medium didn’t have any protective action. However direct and indirect co-cultures and condition medium were unable to protect cortical neurons form toxic drugs. These data could indicate that a possible MSCs based therapy would be more promising for treating peripheral nervous system diseases rather than central one.

The cell-based therapy is a promising approach to treat many degenerative diseases such as type 1 Diabetes Mellitus (T1DM). Besides the exclusive pharmacological treatment for T1DM a new approach has been recently proposed for restoring of Beta cell mass by islet transplantation. One of the principal problems of this approach is the numerical and functional loss of transplanted islets. For these reasons new strategies are studied in order to increase islets survival. In our laboratories we have already demonstrated that rat Mesenchymal Stem Cells (rMSC) are able to promote islets survival in vitro and that rMSC, if cocultured with pancreatic islets, are able to express Pdx1, a gene involved in beta cell insulin secretion. The aim of this study is to verify the effect of human Mesenchymal Stem Cells (hMSC) on the survival and function of pancreatic islets. In order to clarify which mechanism could be involved in the putative positive effect we set up different culture conditions: direct coculture, in which hMSC were in direct contact with islets; indirect coculture in which hMSC and islets shared the medium; mix cocultured in which islets were both in direct contact and shared the medium with hMSC. Preliminary results demonstrate a positive effect of hMSC on islets survival. Now we are focusing on the effect on insulin secretion regulated by hMSC in the different coculture conditions.

Besides the immunomodulatory action, Mesenchymal Stem Cells (MSCs) are able to promote neuronal and glial survival both by releasing trophic factors and through cell to cell contact. For these features MSCs are a promising tool for the treatment of inflammatory and demyelinating diseases such as Multiple Sclerosis (MS). Here we reported a pre-clinical study on Dark Agouti rats affected by a Relapsing-Remitting form of Experimental Autoimmune Encephalomyelitis (RR-EAE), one of the most suitable models for the study of RR-MS. In order to assess the possible preventive or therapeutic effect, 106 MSCs were injected i.v. (intra venous) at day 7 or at day 14 post EAE induction (p.i.) and clinical score was evaluated daily. The preventive schedule of treatment (day 7 p.i.) had no effect on EAE clinical course but the therapeutic one (day 14 p.i.) was able to hamper the relapsing phase from day 19 p.i. and till the end of the experiment (day 45 p.i.) with respect to EAE group. At day 45 p.i., histological analysis performed on spinal cord of EAE rats revealed a substantial absence of inflammatory infiltration and the presence of demyelinated plaques, assessed by Luxol fast Blue staining and by immunohistochemistry for MBP (Myelin Basic Protein). Moreover the analysis performed on serial paraffin sections revealed that the therapeutic schedule with MSCs was able to significantly reduce the extension of demyelinated areas in the spinal cord white matter with respect to EAE and EAE+MSCs day 7 p.i. groups, thus confirming clinical score evaluations. These results suggested that MSCs are able to ameliorate the clinical course of EAE animals by reducing the areas of demyelinated lesions. We are now evaluating the possible mechanism of MSCs action by investigating in vitro some putative myelinating and immunomodulating properties of MSCs. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001.

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

Transplantation of pancreatic islets is an innovative and promising clinical option to treat patients with type 1 diabetes [1]. This is a minimally invasive therapeutic approach, which allows a good metabolic control and a long-term insulin independence [2]. The therapeutic feasibility of pancreatic islets transplantation is however limited by the poor yield of pancreatic islet explants and even more the immune graft rejection, which have as a consequence the very limited lifespan of transplanted tissue [3]. To avoid these side effects besides the treatment with immunosuppressive drugs, promising results have been obtained in vivo with the use of Mesenchymal Stem cells (MSCs), already known in literature to be able to support the cellular survival through direct contact [4, 5] by the release of trophic factors [6], and by their immunomodulatory properties [7]. By means of these particular features it can be surmised that MSCs may improve the survival of pancreatic islets and, therefore, the success of the transplantation. Several in vivo studies have demonstrated the positive effect of islet-MSC co-transplantation in diabetic rats, but the mechanisms of these encouraging results are still unknown [8]. In this in vitro study we shed light on the concealed molecular mechanisms of MSC positive action, by analyzing the effect of both direct and indirect co-cultures of rat MSCs with pancreatic islets.

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.

The induction of a peripheral neuropathy is a very common side effect of many chemotherapeutic agents, including platinum compounds, and it often represents the dose limiting factor for drug clinical use. Several strategies have been suggested to reduce drug neurotoxicity without affecting the antineoplastic potential, but up to now results were not encouraging. Recently, it has been demonstrated that Mesenchymal Stem Cells (MSCs) are able to promote the survival and the maturation of untreated sensory neurons of dorsal root ganglia (DRG), which represent also the target of drug neurotoxicity. Aim of this work is to verify the neuroprotective potential of MSCs on rat DRG exposed to cisplatin (CDDP), a chemotherapeutic and neurotoxic agent. DRG post-mitotic explants from E15 rat embryos were exposed for 24 hours to different cisplatin concentrations. After 24 hours, medium was changed and DRG were directly co-cultured with human MSCs (hMSCs) or with hMSCs conditioned medium (hMSC-CM). DRG explants were photographed every day up to 1 month, and the longest neurite of each DRG was measured to evaluate neurotoxicity. DRG survival was estimated by measuring the death area percentage. The survival of CDDP-treated DRG was increased after the co-cultures with hMSCs, and both hMSCs and hMSC-CM were able to improve the neurite outgrowth of untreated and CDDP-treated DRG after 48 hours. This MSC-dependent increase of neurite length was however no longer evident at later times (1 month). This effect on neurite elongation was probably mediated by CSPG, MAG and Nogo, some proteins involved in the modulation of neurite elongation, which resulted expressed and released in the culture medium of hMSCS. Our results demonstrated a neuroprotective effect of hMSCs on CDDP toxicity and evidenced the ability of these cells to modulate neurite elongation. In this way MSCs could represent a possible mean to limit the neurotoxicity on DRG which arises after cisplatin therapy.

Mesenchymal Stem Cells (MSCs) are frequently proposed as potentially suitable for the regenerative therapeutic approach for several neurological diseases both of the central nervous system, such as Multiple Sclerosis, and at the same way also of the peripheral nervous system, thanks to their ability to increase neuronal survival and to release neurotrophic factors. Since there are great differences between neurons of the central and of the peripheral nervous system, in this study we have verified the existence of a different susceptibility of cortical and sensory neurons to the effect of MSCs after different toxic stimuli, in order to mimic the damages observed in some neurological diseases. For this aim we set up direct and indirect co-cultures of MSCs and cortical or sensory neurons previousl exposed to toxic doses of glutamate, as a paradigm of Multiple Sclerosis, or treated with two widely used chemotherapeutic drugs, cisplatin and paclitaxel, which induce peripheral neuropathies. On the same cells we evaluated also the effect of conditioned medium of MSCs, by using morphological and molecular analysis. Neuronal viability was assessed by MTT test and by count of viable cells. Our results demonstrated the protective action of MSC direct and indirect co-cultures only on sensory neurons previously exposed to the toxic agents, while conditioned medium was ineffective to rescue it. On the contrary MSCs failed at all to protect cortical neurons from the drugs used, and their conditioned medium further reduces neuronal viability. We are now investigating the putative interference of MSCs with apoptotic molecules in sensory neurons, while in cortical neurons we are evaluating the possible causes of MSC-medium toxicity by analyzing the factors released. The different effect of MSCs on cortical and sensory neurons protection observed in vitro may be not the same in vivo, where the environments differ, anyway, it suggests to address the use of MSCs against the diseases aff