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Mesenchymal stem cells (MSCs) have been often proposed for the therapy of several neurological diseases, due to their manifold peculiar properties. In particular, since it has been previously demonstrated that these cells are able to increase the survival of untreated sensory neurons [1], in this work we evaluated their possible protective effect on sensory neurons previously exposed to toxic agents. This could be particularly relevant to design a supportive therapy to counteract the peripheral neuropathy, a very common side effect of several chemotherapeutic agents, such as platinum and taxanes compounds, which often represents their dose limiting factor [2]. Several strategies have been suggested to reduce drug neurotoxicity without affecting the antineoplastic potential, but up to now results were not encouraging [3]. Here we demonstrated that Cisplatin (CDDP) and Paclitaxel-treated sensory neurons are protected by the co-culture with MSCs, but in two different manners: through a direct contact able to block apoptosis for CDDP-treated neurons, and by the release of trophic factors (including glutathione) for Paclitaxel-treated ones. In addition, the MSCs’ effectiveness was also verified on cortical neurons, since the recent advances in targeted drug delivery allowed to drive chemotherapeutic drugs also to the central nervous system. We verified that cortical neurons are more vulnerable to the toxic action of the drugs, and overall that MSCs fail at all to protect them. All these data demonstrated that MSCs are potentially useful to limit the peripheral neuropathy onset for their protective effect on injured-sensory neurons, but they also identified for the first time a different susceptibility of cortical and sensory neurons to MSC action., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Air pollution represents a well-known environmental problem related to public health. Particulate matter (PM) is a heterogeneous mixture of chemicals, metals and soils. Its adverse effects have been correlated with particles size, being smaller particles more likely to cause a worst damage, so their study deserves more attention. Ultrafine particles (UFPs, dae < 100 nm) are short-lived particles dispersed in the environment. In Lombardy, diesel combustion and solid biomass burning are the most relevant contributors to primary UFPs emissions (15-30 nm in diameter). Toxicological studies, mainly in vitro, indicate specific effects for particles of different origin but comparative in vivo studies are scarce. PM exposure has been primarily associated to pulmonary and cardiovascular diseases through oxidative stress and inflammatory response, but recently it has been postulated that PM exposure could also be an important risk factor for neurotoxicity and could have a role in neurodegenerative diseases. In this study we analysed in BALB/c mice the effect of single and repeated intratracheal instillation of diesel (DEP) and biomass (BC) particles on respiratory, cardiovascular and central nervous systems, comparing the two different UFPs sources. The study was performed at biochemical and histopathological level. Different pro-inflammatory, cytotoxic, pro-coagulant and oxidative stress markers were measured. For the histopathological evaluation, sections of lung, heart and different parts of the central nervous system (CNS) were examined at light microscope, using standard staining tecniques and immunohistochemical methods. Inflammation was also monitored in living mice following BC or DEP intratracheal repeated administration using the FMT 1500 fluorescence tomography imaging system and the MMPSense 750 Fast probe. Our results indicate that even a single instillation of both the sources of UFPs induces a wide range of biochemical changes in the respiratory and cardiovascular systems, then confirmed by repeated instillation. In the CNS similar modifications were observed, although these were much more evident after repeated instillations. Histological examination demonstrated the presence of macrophages containing particles in the lungs after UFPs single and, more abundantly, repeated administration. However, significant changes were not observed in sections of heart and CNS. DEP was more effective in inducing oxidative stress and inflammation compared to BC., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

The difficulties encountered in the treatment of brain diseases with conventional pharmacological tools have created the need for innovative strategies. The combination of nanocarriers and alternative administration routes could represent an efficient approach to reach the brain. Intranasal administration (IN) provides a non-invasive option to deliver drugs to the brain, bypassing the BBB, reducing the first-pass effect and enhancing patient compliance. The objective of the present study was to investigate the biodistribution and bioavailability to the brain of polymeric nanoparticles (PNPs) after IN administration in healthy mice. PNPs were prepared with poly-lactide-co-glycolide polymer using nanoprecipitation method. PNPs had a polymodal distribution around 350 nm. The biodistribution of DiR-loaded PNPs was evaluated by means of 3D fluorescence tomography imaging. Our results show that 3h after a single IN administration, more than 5% of the injected dose was detectable in the brain. PNPs were quickly cleared from the thorax and the abdominal cavity, while the brain fluorescence slowly decreased ranging from 3.7% to 2.3% between 24h and 96h. Repeated IN administrations (2 administrations, 24h apart) provided a significant increment of PNPs-associated fluorescence in the brain, without affecting PNPs accumulation in other organs. These findings support nose-to-brain translocation of PNPs as a noninvasive strategy to enhance the bioavailability of therapeutics to the brain

Oxygen and glucose deprivation (OGD) due to ischemic events or trauma in the brain result in neuronal loss. The therapeutic approaches available inadequate and often the outcome is unfavorable for the patient or at least unpredictable. Stem cells could be useful for the treatment of OGD injured-neurons. Mesenchymal Stem Cells (MSCs), isolated from bone marrow as well as from various tissues, have poor immunogenicity and neuroprotective properties being able to alleviate ischemic brain injuries in animal models. The Endothelial Progenitor Cells (EPCs) are present at low frequencies both in the bone marrow and in the peripheral blood. They are thought to play a role in the recovery of cerebrovasculature integrity after stroke. In the present study we evaluated the potential neuroprotective effect of human MSCs and human EPCs on rat embryonic cortical neurons injured by OGD. OGD was induced by incubating the cortical neurons in a hypoxia chamber in a 95% N2 + 5% CO2 atmosphere at 37°C without glucose. To set up the experimental protocol, OGD was maintained for 1, 2 and 3 hours. The neurons were returned in normoxic atmosphere and after 2 and 5 days neuronal survival was evaluated by MTT assay, LDH assay and viable cellular counting. The 2 hours OGD was able to reduce neuronal viability by 50% and was chosen for the subsequent experiments. To assess MSCs and EPCs neuroprotective action, after 2 hours-long OGD the neurons were 1) co-cultured with either MSCs or EPCs seeded on a cell culture insert avoiding direct contact while sharing the same medium, or 2) cultured in a medium previously conditioned by either MSCs or EPCs. Neuronal survival was evaluated by MTT assay after 2 and 5 days. Both MSCs and EPCs increased neuronal survival after ODG. The effect was observed in absence of a direct contact between MSCs or EPCs and the injured neurons, suggesting that the release of soluble factors may be involved in their neuroprotective action. In conclusion both MSCs and EPCs could represent a potential therapeutic approach for the treatment of brain ischemic injury. Further studies are needed to identify the specific molecules and pathways that play a role in the neuroprotective effect of MSCs and EPCs., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

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.

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.

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

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.

Introduction The blood-brain barrier (BBB) plays an important role in maintaining the homeostasis of the central nervous system and in protecting the brain from potentially harmful endogenous and exogenous compounds. Nevertheless it represents also the major obstacle for the diagnosis and therapy of brain diseases. One of the most promising strategies to overcome the limited BBB penetration of drugs and contrast agents is based on nanoparticles (NP). Lipid based NP, basically liposomes and solid lipid nanoparticles (SLN), have several advantages in terms of biocompatibility, non-immunogenicity, non-toxicity; they can be used as carrier systems [1], and they have a high blood circulation residence time [2]. Moreover their surface can be easily modified with ligands which mediate a site-specific targeting. Goal of the work The objective of present investigation was to study the effect of surface characteristics of SLN covalently coupled with the monomer of ApoE-residues (141-150) (mApoE-SLN) in promoting BBB crossing and brain targeting using both in vitro and in vivo models. Methods Radiolabelled or fluorescent dye-loaded SLN, covalently coupled by DSPE-PEG(2000)-Maleimide with the monomer of ApoE-residues (141-150) [3] and functionalized with phosphatidic acid (Aβ ligands) [4], were used in the present work and produced by Nanovector s.r.l. (Torino, Italy). In vitro evaluations were performed using cultured human cerebral microvascular endothelial cells (hCMEC/D3) obtained from Institute Cochin (INSERM, Paris, France). SLN cell uptake was monitored by confocal-laser-scanning microscopy and cell-associated fluorescence was quantified by FACS analysis. Radiochemical technique was used in order to assess the ability of ApoE monomer to enhance SLN transcellular transport across the hCMEC/D3 BBB model [5]. The in vivo biodistribution of SLN, loaded with DiR (near-infrared fluorescent cyanine dye), was evaluated by means of Fluorescent Microscopy Tomography (FMT 1500, Perkin Elmer). BALB/c male mice were intravenous (IV), intratracheal (IT) or intraperitoneal (IP) administered with 50 ul of SLN formulation and tomographic data analyses were achieved using the TrueQuant software supplied (Perkin Elmer). The total amount (in picomoles) of fluorophore in the brain region was calculated by the provided software using previously generated standards of the appropriate dye [6]. Results and Discussion We demonstrated that surface functionalization of SLN with ApoE monomer plays a major role in promoting their cellular uptake within hCMEC/D3. Cell associated fluorescence was about two-fold higher in presence of SLN-mApoE compared to unfunctionalized SLN (SLN-cys) and the same trend was observed by CLSM analysis. The ability of SLN to cross the in vitro hCMEC/D3 BBB model was assessed using dual-radiolabelled formulations. With respect to SLN-cys, the presence of monomer ApoE significantly enhanced their permeability through the cell monolayer; moreover PE values obtained with the two radiotracers were equivalent for the same SLN formulation, and about 6-fold higher for SLN-mApoE (Fig. 1). These results suggest that, at least at the dose tested, SLN cross intact the cell monolayer. In vivo results confirmed the role of monomer ApoE in sustaining the delivery of SLN to the central nervous system. In particular we demonstrated that, compared to the most common routes for drug administration (IP and IV injections), IT instillation represents the best method to guarantee the biodistribution of SLN-mApoE in the brain district and to favour their retention up to 24 hours after the administration (Fig. 2).Bronchoalveolar lavage fluid (BALF) analysis does not evidence any pro-inflammatory reaction in lungs of SLN-mApoE IT-treated mice with no alteration of the alveolar-capillary barrier permeability. Conclusions The results here obtained suggest that the SLN formulation herein analysed could represent a suitable tool for sustaining drug delivery to the brain.

Transplantation of pancreatic islets has become a promising clinical option to treat patients with type 1 diabetes, alternative to the standard therapy with insulin injections. Islet transplantation is a minimally invasive therapeutic approach, and it allows a better metabolic control and a long-term insulin independence in more than 80% of patients (Ryan et al., 2002). However this therapeutic treatment has some side effects, such as 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 pancreatic islets. To avoid these side effects several strategies have been proposed and, besides the treatment with immunosuppressive drugs, promising results have been obtained with the use of Mesenchymal Stem cells (MSCs), already known in literature to be able to support the survival of many cell types (Scuteri et al., 2006). Several in vivo studies have demonstrated that the concurrent transplantation of pancreatic islets with MSCs reduces the number of islets required to achieve glycemic control in diabetic rats, but the mechanisms of these encouraging results are still unknown (Figliuzzi et al., 2009). For these reasons in this in vitro study we characterized the effect of co-culture of rat MSC on survival and functioning of rat pancreatic islets, by evaluating for 4 weeks: i) MSC adhesion to pancreatic islets; ii) viability of pancreatic islets co-cultured with MSCs; iii) the expression of insulin after co-culture; iv) the ability of co-cultured pancreatic islets to correctly adjust insulin release after variation of glucose concentration. Our results demonstrated that MSCs are able to adhere to pancreatic islets, but to increase only partly the pancreatic islet survival, which retain the ability to express and correctly release insulin after glucose variation in medium culture. Noteworthy that the insulin level in the medium of co-cultured pancreatic islets is always higher with respect to medium of pancreatic islets alone. The immunofluorescence analysis reveals that also MSCs (and not only pancreatic islets) are able to express insulin, but only in co-culture. These results, which justify the in vivo observation reported above, suggest that MSCs undergo to differentiation into a insulin-releasing phenotype after co-culture with pancreatic islets. We are now evaluating the molecular mechanisms which drive this effect, by analyzing the role of soluble factors and of proteins able to induce insulin expression. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

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.

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.

Oxygen and glucose deprivation (OGD) due to ischemic events or trauma in the brain result in neuronal loss. The therapeutic approaches available for the treatment of these conditions are not and often the outcome is unfavorable for the patient or at least unpredictable. Stem cells could be useful for the treatment of OGD injured-neurons. Mesenchymal Stem Cells (MSCs), isolated from bone marrow as well as from various tissues, have poor immunogenicity and neuroprotective properties being able to alleviate ischemic brain injuries in animal models. The Endothelial Progenitor Cells (EPCs) are present at low frequencies both in the bone marrow and in the peripheral blood and can be mobilized by the administration of drugs such as statins. They are thought to play a role in the recovery of cerebrovasculature integrity after stroke. In the present study we evaluated the potential neuroprotective effect of human MSC and human EPCs on rat embryonic cortical neurons injured by OGD. OGD was induced by incubating the cortical neurons in a hypoxia chamber in a 95% N2 + 5% CO2 atmosphere at 37°C without glucose for periods ranging from 30 minutes and 6 hours. When the neurons were returned in normoxic atmosphere they were 1) co-cultured with either MSCs or EPCs seeded on a cell culture avoiding neurons and MSCs or EPCs direct contact while sharing the same medium, or 2) cultured in a medium previously conditioned by either MSCs or EPCs. Neuronal survival was evaluated by MTT assay and viable cellular counting. Also neuronal morphology was taken into account to evaluate the potential MSCs and EPCs neuroprotective effect. Both MSCs and EPCs increased neuronal survival after ODG. This effect was observed in absence of a direct contact between MSCs or EPCs and the injured neurons, suggesting that the release of soluble factors may be the main mechanism of action. In conclusion both MSCs and EPCs could represent a potential therapeutic approach for the treatment of brain ischemic injury. Further studies are needed to identify the specific molecules involved in the neuroprotective effect of MSCs and EPCs.

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

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.

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 affecting the peripheral nervous system rather than the central one. This makes mandatory to further investigate the causes of the different response of neuronal populations to MSC treatment, in order to widen their potential use.

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.

Nowadays obesity and its related diseases represent a major health problem with an increasing worldwide prevalence. Hyperplasia and hypertrophy of adipocytes lead to an excessive fat accumulation that is not efficiently prevented by current pharmacological treatments. So the research on anti-obesity drugs with good efficacy and tolerability able both to prevent and to reduce fat accumulation is of pivotal interest. In the present study we evaluated in vitro the effects of Valproic Acid, Berberin and Resveratrol on adipogenesis. Our experimental model was represented by human Mesenchymal Stem Cells (hMSCs), physiological precursors of adipocytes that can differentiate into adipocytes also in vitro. Preliminary cytotoxicity assays were performed in order to choose non-toxic doses of the three drugs. hMSCs were induced to adipogenic differentiation and treated with Valproic Acid, Berberin and Resveratrol at the selected doses. Controls were represented by hMSCs treated for adipogenesis in absence of the drugs. At different time points intracellular lipid droplets accumulation, a typical feature of adipogenesis, was assessed by Oil Red O staining. Valproic Acid, Berberin and Resveratrol inhibited hMSCs adipogenic differentiation in a dose dependent manner as demonstrated by the reduction of the lipid droplets accumulation. To understand the molecular mechanisms of the drugs-induced adipogenesis inhibition, we focused our attention on the effects of the drugs treatment on cell cycle progression, known to be altered by many antiadipogenic drugs, and on the MAP Kinases ERK1 and ERK2, involved in the adipogenesis control. We evaluated the expression of cyclins and CDKs by immunoblotting and flow-cytometry analyses, demonstrating that Valproic Acid, Berberin and Resveratrol interfere on cell cycle progression. The expression and the phosphorylation status of the two kinases ERK1 and ERK2 were assessed by immunoblotting demonstrating an increase of ERK1 phosphorylation (i.e. activation) in hMSCs treated with Berberin and a reduction in hMSCs treated with Valproic Acid and Resveratrol compared to control cells. No changes in phosphorylation and expression of ERK2 were observed. Our study demonstrate that Valproic Acid, Berberin and Resveratrol exert an anti-adipogenic effect in our experimental model. The mechanisms of action of these drugs involve the alteration of cell cycle progression and, at least in part, ERK1/2 modulation. However other molecular pathways are likely implicated and other studies are required to identify them., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011