Arianna Scuteri

Mesenchymal Stem Cells Neuronal Differentiation Ability: A Real Perspective for Nervous System Repair?

Mesenchymal Stem Cells (MSCs) are a bone marrow-derived population present in adult tissues that possess the important property of dividing when called upon and of differentiating into specialized cells. The evidence that MSCs were able to transdifferentiate into specialized cells of tissues different from bone marrow, in particular into nervous cells, opened up the possibility of using MSCs to substitute damaged neurons, that are normally not replaced but lost, in order to repair the Nervous System. The first neuronal differentiation protocols were based on the use of a mixture of toxic drugs which induced MSCs to rapidly acquire a neuronal-like morphology with the expression of specific neuronal markers. However, many subsequent studies demonstrated that the morphological and molecular modifications of MSCs were probably due to a stress response, rather than to a real differentiation into neuronal cells, thus throwing into question the possible use of MSCs to repair the nervous system. Currently, some papers are suggesting again that it may be possible to induce neuronal differentiation of MSCs by using several differentiation protocols, and by accompanying the morphological evidence of differentiation with functional evidence, thus demonstrating that MSC-derived cells not only seem to be neurons, but that they also function like neurons. In this review, we have attempted to shed light on the capacity of MSCs to genuinely differentiate into nervous cells, and to identify the most reliable protocols for obtaining neurons from MSCs for nervous system repair.

Adult mesenchymal stem cells rescue dorsal root ganglia neurons from dying.

Mesenchymal stem cells (MSCs) can differentiate into multiple cellular lineages including neuronal cells. However, the positive effect of MSCs on repairing the nervous tissue has not yet been completely understood. In order to investigate the influence of MSCs on a neuronal population, we co-cultured MSCs, obtained by flushing the bone diaphisis from adult Sprague-Dawley rats, with DRG post-mitotic sensory neurons obtained from rat embryos at day E15. Co-cultures were maintained for 2 months. The adult rat MSCs, simply harvested in a pure culture of DRG neurons, allow the long-lasting survival and maturation of neurons otherwise committed to die. Neurons, when co-cultured with rat fibroblasts, do not survive as long as with MSCs and do not mature to the same degree. The rescue effect of MSCs on neurons is achieved only by cellular direct contact. These results provide a valid explanation for the functional improvement reported in some in vivo experiments.

Resveratrol interference with the cell cycle protects human neuroblastoma SH-SY5Y cell from paclitaxel-induced apoptosis

In previous studies we demonstrated that resveratrol acts in an antiapoptotic manner on the paclitaxel-treated human neuroblastoma (HN) SH-SY5Y cell line inhibiting the apoptotic pathways induced by the antineoplastic drug. In the present study we evaluated the antiapoptotic effect of resveratrol, studying its activity on cell cycle progression. We determined the mitotic index of cultures exposed to resveratrol and paclitaxel alone or in combination, the cell cycle distribution by flow cytometric analysis (FACS), and the modulation of some relevant cell cycle regulatory proteins. Resveratrol is able to induce S-phase cell arrest and this interference with the cell cycle is associated with an increase of cyclin E and cyclin A, a downregulation of cyclin D1, and no alteration in cyclin B1 and cdk 1 activation. The resveratrol-induced S-phase block prevents SH-SY5Y from entering into mitosis, the phase of the cell cycle in which paclitaxel exerts its activity, explaining the antiapoptotic effect of resveratrol.

NGF protects Dorsal Root Ganglion neurons from oxaliplatin by modulating JNK/Sapk and ERK1/2

The involvement of the Mitogen-Activated Protein Kinases (MAPKs) family in platinum derivative-induced peripheral neuropathy has already been demonstrated. In particular, it has been evidenced that in Dorsal Root Ganglion (DRG) neurons prolonged exposure to oxaliplatin (OHP) induces early activation of p38 and ERK1/2, which mediate neuronal apoptosis, while the neuroprotective action of JNK/Sapk is downregulated by the drug treatment. In this study, the exposure of OHP-treated neurons to a neuroprotective stimulus, represented by a high dose of NGF, counteracts OHP-induced neuronal mortality. This effect was achieved by restoring the MAPK activation existing in untreated control cells. Increased viability occurred also after the administration of retinoic acid (RA), a pro-differentiative agent able to activate both JNK/Sapk and ERK1/2. The use of specific chemical inhibitors of MAPKs confirms the importance of this class of proteins for the neuroprotective pathway, since they reverse the protective effect. In summary, our findings assess the validity of MAPKs as the target of neuroprotective therapies during chemotherapeutic treatment. Moreover they also describe a double role for ERK1/2, depending on cellular stimulation, since it mediates neuronal apoptosis after OHP exposure. However, it is also important, as is JNK/Sapk, in preserving the correct cellular differentiation that is pivotal for neuronal survival.

Emerging role of mitogen-activated protein kinases in peripheral neuropathies.

Abstract  Among the different families of intracellular molecules that can be modulated during cell damage and repair, mitogen‐activated protein kinases (MAPKs) are particularly interesting because they are involved in several intracellular pathways activated by injury and regeneration signals. Despite most of the studies have been performed in non‐neurological models, recently a causal role for MAPKs has been postulated in central nervous system disorders. However, also in some peripheral neuropathies, MAPK changes can occur and these modifications might be relevant in the pathogenesis of the damage as well as during regeneration and repair. In this review, the current knowledge on the role of MAPKs in peripheral neuropathies will be discussed.

Mesengenic differentiation: comparison of human and rat bone marrow mesenchymal stem cells.

Background and Objectives: Cellular therapies using Mesenchymal Stem Cells (MSCs) represent a promising approach for the treatment of degenerative diseases, in particular for mesengenic tissue regeneration. However, before the approval of clinical trials in humans, in vitro studies must be performed aimed at investigating MSCs’ biology and the mechanisms regulating their proliferation and differentiation abilities. Besides studies on human MSCs (hMSCs), MSCs derived from rodents have been the most used cellular type for in vitro studies. Nevertheless, the transfer of the results obtained using animal MSCs to hMSCs has been hindered by the limited knowledge regarding the similarities existing between cells of different origins. Aim of this paper is to highlight similarities and differences and to clarify the sometimes reported different results obtained using these cells. Methods and Results: We compare the differentiation ability into mesengenic lineages of rat and human MSCs cultured in their standard conditions. Our results describe in which way the source from which MSCs are derived affects their differentiation potential, depending on the mesengenic lineage considered. For osteogenic and chondrogenic lineages, the main difference between human and rat MSCs is represented by differentiation time, while for adipogenesis hMSCs have a greater differentiation potential. Conclusions: These results on the one hand suggest to carefully evaluate the transfer of results obtained with animal MSCs, on the other hand they offer a clue to better apply MSCs into clinical practice.

The fundamental role of morphology in experimental neurotoxicology: the example of chemotherapy-induced peripheral neurotoxicity.

The peripheral nervous system is a frequent target of toxic agents. The accurate identification of the sites of neurotoxic action through the morphological characterization of reliable in vivo models or in vitro systems can give fundamental clues when investigating the pathogenesis and interpreting the clinical features of drug-induced neuropathy. The morphological approach has been used to investigate almost all the anticancer drugs able to induce chemotherapy-induced peripheral neurotoxicity, i.e. platinum drugs, antitubulins and proteasome inhibitors. No models have ever been described for thalidomide. This review demonstrates that any pathogenetic study on chemotherapy-induced peripheral neurotoxicity must be based on solid morphological observations obtained in reliable animal and in vitro models. This is particularly true in this setting, since the availability of tissues of human origin is extremely limited. In fact, peripheral (generally sural) nerve biopsies are never required for diagnostic purposes in chemotherapy-treated cancer patients, and their use for a purely scientific aim, although potentially very informative, is not ethical. Moreover, several neurotoxic drugs target the dorsal root ganglia neurons, and it is very difficult to obtain high-quality specimens even from early autopsies. It is, therefore, our opinion that an extensive morphological assessment of the in vitro and in vivo effect of any potentially neurotoxic antineoplastic drugs, as well as of neuroprotectant agents, should be taken into consideration right from the earliest stages of their development.

Adult mesenchymal stem cells support cisplatin-treated dorsal root ganglion survival

Mesenchymal stem cells (MSCs) have been found to be useful in the management of different models of neurological diseases. In the present study, we tested the possible protective effect of MSCs on sensory dorsal root ganglia (DRG) explants exposed to the toxic effect of CDDP, a widely used anticancer drug. DRG explants cultured on a collagen layer and exposed to NGF for 2h (differentiating neurons) or for 5 days (fully differentiated neurons) were treated with CDDP and subsequently co-cultured with MSCs. MSCs were able to support the survival of both differentiating and fully differentiated DRG neurons up to 2 months after the drug treatment, reducing the CDDP-induced death of DRG neurons. MSCs were, however, unable to restore the correct length of DRG neurites compromised by CDDP treatment. The positive effect on neuronal survival was exerted through the contact between DRG and MSCs, and not mediated by neurotrophic factors released by the MSCs. Our observations could represent a starting point for designing a neuroprotective strategy to limit CDDP induced neuropathy without interfering with its anticancer properties.

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.