ERK1 and ERK2 are involved in recruitment and maturation of human mesenchymal stem cells induced to adipogenic differentiation

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

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

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

Effects of valproic Acid, berberin and resveratrol on human mesenchymal stem cells adipogenic differentiation

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

Evaluation of neural markers expression in human mesenchymal stem cells after mesengenic differentiation

Introduction. Mesenchymal Stem Cells (MSCs) are adult multipotent cells able to differentiate in mesengenic (osteogenic, adipogenic, condrogenic) and non mesengenic lineages (e.g. neural) under appropriate culture conditions. MSCs represent a very promising therapeutic approach in different settings particularly for tissue repair and regeneration. The knowledge of human MSCs (hMSCs) biological properties is very important to optimize their clinical application. In view of MSCs application in neurodegenerative diseases, the neuronal differentiation potential of hMSCs has been also explored. Our preliminary data demonstrated that the neuronal markers beta III tubulin and NeuN were spontaneously expressed by a high percentage of undifferentiated hMSCs independently from serum presence and number of culture passages. The expression of neural markers by MSCs in absence of any differentiative agents is considered as a demonstration of MSC neural predisposition. The aim of this work was to evaluate if these markers, known to be neuronal ones, continued to be expressed also in hMSCs differentiated towards mesengenic lineages. Methods. hMSCs were obtained after patient consensus, from iliac crest bone marrow. In according to the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, the isolated hMSCs were plastic-adherent, capable of extensive proliferation when maintained in standard culture conditions, lacked of hematopoietic markers expression and presented specific surface antigens. hMSCs were differentiated toward osteogenic, adipogenic and chondrogenic lineages using specific in vitro protocols. The expression of the neuronal markers beta III tubulin and NeuN were evaluated by immunofluorescence experiments at different time points depending on the differentiation protocol used. hMSCs cultured in absence of any differentiative agent represented controls. Results. In our experiments the most of hMSCs differentiated in osteogenic and adipogenic lineages expressed the neuronal markers beta III tubulin and NeuN. Unlike, chondrogenic differentiated hMSCs didn’t express these markers. Conclusions. The finding that hMSCs differentiated into adipogenic and osteogenic lineages express neuronal markers such as beta III tubulin  and NeuN raises doubts about the reliability of these markers as indicators of neuronal differentiation and suggests that their expression could be an intrinsic property of a wide range of cellular types. Further studies are necessary to understand the specific biological role of of beta III tubulin and NeuN in hMSCs differentiated towards mesengenic lineages

Human oral squamous cell carcinoma proliferation and migration prevented by two flavonoids

Oral Cancer (OC) is one of the most frequent cancer in Head and Neck district and Oral Squamous Cell Carcinoma (OSCC) constitutes the large majority of the neoplasia arising in oral cavity. OSCC remains a hampering matters for clinics, since the overall disease free survival has not significantly increased during the last decades and invasion to surrounding tissue and to regional lymph nodes is often reported. Therefore new strategies to prevent and inhibit OSCC growth and invasion are highly desirable and new therapeutic approaches are currently tempted also with the use of natural compounds. Myricetin (MYR) and Naringenin (NAR), two naturally occurring flavonoids, widely diffused in plants, fruits and vegetable, have recently gained consideration thanks to their anti oxidant, anti inflammatory and anti tumoral properties. In this study their potential anticancer effect has been evaluated on an OSCC cell line, SCC-25 and on spontaneously immortalized non tumoral keratinocytes, HaCaT cells. MYR and NAR induce a significant cell growth inhibition in SCC-25 cells, in addition NAR selectively affected cancer cells, since it does not impair HaCaT cell growth. Furthermore an additive effect of MYR and NAR has been highlighted. The cell proliferation inhibition is not related to apoptosis induction, as demonstrated by evaluation of phosphatidyl serine membrane translocation and dapi staining. On the contrary MYR and NAR effect depends on the cell cycle progression impairment. Wound-healing and cell invasion assays, respectively performed by cell monolayer scratch and Boyden Chamber transwell test, demonstrate that the two flavonoids are able to reduce motility and invasiveness on both SCC-25 and HaCaT cells. In conclusion the results of the present study show the anticancer potential of NAR and MYR on OSCC, since both flavonoids prevent cancer cell proliferation through a cytostatic effect, by the impairment of cell cycle progression. Moreover both the flavonoids inhibit cell migration, thus highlighting their potential effect as anti metastatic agents

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

Human Mesenchymal Stem Cells and Endothelial Progenitor Cells exert a neuroprotective effect on rat cortical neurons injured by oxygen and glucose deprivation

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

Tubulin involvement in Bortezomib peripheral neurotoxicity

Axonal transport of mitochondria (Mt) controlled by specialized motor and docking proteins that distribute Mt throughout the axon where they provide energy for metabolic and synaptic activity is a vulnerable target in neuronal pathology (1). Bortezomib (BZ) is a proteasome inhibitor active in multiple myeloma (2). One of its key toxicities is painful peripheral neuropathy (BIPN), which frequently requires treatment discontinuation (3). BIPN is dose-related and predominantly sensory, resulting from axonal degeneration. Recent results indicate that BZ modifies axonal tubulin dynamic and we hypothesize that BZ alters fast axonal transport. Here we studied using time-lapse imaging the effect of different BZ concentration on axonal Mt transport in isolated dorsal root ganglion (DRG) neurons from adult male mice. We used kymograph to quantify the total number of Mt and to discriminate antero and retrogradely moving Mt from stationary Mt. Twenty-four hours of BZ treatment (0.1 to 15 µM) induced a dose-dependent reduction in Mt trafficking. Moreover, BZ had no impact on MT motion directions, but it induced a progressive reduction of both anterograde and retrograde axonal transport velocities. These events were associated with increase in tubulin polymerization and of MAP2 expression, but they occurred only after 72h of chronic BZ treatment. We have developed an in vitro model of BIPN demonstrating that transport impairment is already present before evident tubulin polymerization, suggesting that transport deficit represents an early stage of axonal dysfunction. Perpetuated transport dysfunction could impair distal organelle supply and play a critical role in advanced stages of BIPN.This work was supported by the University of Milan-Bicocca and University of Michigan research grant