Marianna Monfrini

Therapeutic potential of Mesenchymal Stem Cells for the treatment of diabetic peripheral neuropathy.

ABSTRACT 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. HighlightsMSCs reduce the number of Pancreatic Islets necessary to control blood glucose level.MSCs co‐transplanted with Pancreatic Islets ameliorate diabetic neuropathy.MSCs co‐transplanted with Pancreatic Islets reduce nephrotoxicity.

Neurobasal medium toxicity on mature cortical neurons.

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.

Therapeutic Administration of Mesenchymal Stem Cells Abrogates the Relapse Phase in Chronic Relapsing-Remitting EAE

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.

Comparing the different response of PNS and CNS injured neurons to mesenchymal stem cell treatment

Abstract Mesenchymal stem cells (MSCs) are adult bone marrow‐derived stem cells actually proposed indifferently for the therapy of neurological diseases of both the Central (CNS) and the Peripheral Nervous System (PNS), as a panacea able to treat so many different diseases by their immunomodulatory ability and supportive action on neuronal survival. However, the identification of the exact mechanism of MSC action in the different diseases, although mandatory to define their real and concrete utility, is still lacking. Moreover, CNS and PNS neurons present many different biological properties, and it is still unclear if they respond in the same manner not only to MSC treatment, but also to injuries. For these reasons, in this study we compared the susceptibility of cortical and sensory neurons both to toxic drug exposure and to MSC action, in order to verify if these two neuronal populations can respond differently. Our results demonstrated that Cisplatin (CDDP), Glutamate, and Paclitaxel‐treated sensory neurons were protected by the co‐culture with MSCs, in different manners: through direct contact able to block apoptosis for CDDP‐ and Glutamate‐treated neurons, and by the release of trophic factors for Paclitaxel‐treated ones. A possible key soluble factor for MSC protection was Glutathione, spontaneously released by these cells. On the contrary, cortical neurons resulted more sensitive than sensory ones to the toxic action of the drugs, and overall MSCs failed to protect them. All these data identified for the first time a different susceptibility of cortical and sensory neurons, and demonstrated a protective action of MSCs only against drugs in peripheral neurotoxicity. HighlightsCNS neurons and PNS neurons have a different susceptibility to neurotoxic drugs.Direct contact and soluble factor release were involved in MSCs’ protective effect.MSCs protect sensory neurons but fail to protect cortical neurons from drugs toxicity.

Making connections: gap junctions are pivotal for MSCinduced long lasting survival of sensory neurons

The direct contact of Mesenchymal Stem Cells (MSCs) with Dorsal Root Ganglia sensory neurons is pivotal to prolong the neuronal survival and to support their maturation (1). Here we further investigated the mechanisms underlying this direct contact-mediated positive effect, focusing our attention on the possible interaction between MSCs and neurons, and in particular on gap junction formation. We set up direct co-cultures of MSCs and sensory neurons, and after 30 days we analyzed them. The electron microscopy analysis evidenced the presence of junctions between MSCs and neurons only in direct co-cultures. Using a diffusible dye, Calcein, we demonstrated a direct interaction among cells, with a flow of dye from MSCs to neurons. To confirm the importance of such a connection we blocked it by using a gap junction blocker, the carbenoxolone (2). The use of gap junction blocker induced a decrease of neuronal survival in co-culture, thus demonstrating the important role of gap junctions for the positive effect of MSCs. We are now investigating the possible exchanged molecules, focusing our attention on some pro-survival miRNA, such as miRNA 29b and miRNA 142-5 (3), in order to identify the molecule able to positively affect the neuronal survival.

Mesenchymal Stem Cells as New Therapeutic Approach for Diabetes and Pancreatic Disorders

Diabetes is a worldwide disease which actually includes different disorders related to glucose metabolism. According to different epidemiological studies, patients affected by diabetes present a higher risk to develop both acute and chronic pancreatitis, clinical situations which, in turn, increase the risk to develop pancreatic cancer. Current therapies are able to adjust insulin levels according to blood glucose peak, but they only partly reach the goal to abrogate the consequent inflammatory milieu responsible for diabetes-related diseases. In recent years, many studies have investigated the possible use of adult mesenchymal stem cells (MSCs) as alternative therapeutic treatment for diabetes, with promising results due to the manifold properties of these cells. In this review we will critically analyze the many different uses of MSCs for both diabetes treatment and for the reduction of diabetes-related disease development, focusing on their putative molecular mechanisms.

Synchrotron-based technique: a new high resolution imaging of nervous system

X-ray phase contrast micro-tomography (μPCI-CT) is a high resolution technique that can be used to investigate vascular and neurodegenerative disorders overcoming the limitations of the conventional imaging modalities. In fact, currently available neuroimaging techniques are based on sample-invasive imaging protocols involving dissections, staining or labeling of nervous system structures. On the other hand, μPCI-CT permits to visualize the spinal cord micro-vasculature, to detect single neuronal cells in the vertebral column and even cells infiltrating the nervous system in pathological conditions. These properties make μPCI-CT a potential powerful instrument in the study of vascular and neurodegenerative disorders as well as in the patient evaluation during medical treatment. Moreover, it would be a powerful instrument to localize in preclinical model of immune mediated diseases ectopic cells infiltrating the nervous system in a multifocal and unpredictable way. To optimize tissue fixation protocols for μPCI-CT analysis, several attempts were performed combining different protein and lipid fixation procedures and time points. The high-resolution synchrotron μPCI-CT setup allowed recognition of full-organ spinal cord anatomy of healthy rats, including anterior/posterior gray horns, the dorsal/ventral roots and ganglions, the central canal and the meninges, was clearly depicted. Superficial and deep vessels were visualized without need of any contrast-agent. At the highest resolution used, single neuronal cells perfused by surrounding vasculature were recognized allowing the detection of specific structure such as bundles of nerve fibers, single motor neurons and neuro-glial cells, cell bodies and axons as well as intra-cellular structure (i.e. cell nuclei and nucleoli). Moreover, in preclinical studies, the optimization of protocol for μPCI-CT allowed to localize ectopic infiltrating cells in nervous system organs in both mouse and rat models of Krabbe disease and Multiple Sclerosis which would allow a further accurate analysis of the areas and cell-parenchima fine interaction.

Mesenchymal stem cells protect sensory neurons, but not cortical neurons, from the chemotherapeutics-induced neurotoxicity

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.

Therapeutic potential of Mesenchymal Stem Cells for the treatment of type-1 Diabetes.

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.

Positive effect of Mesenchymal Stem Cells therapeutic administration on chronic Experimental Autoimmune Encephalomyelitis

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.