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

Expression of CD38 in human neuroblastoma SH-SY5Y cells.

Human CD38 antigen is a 42–45 kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular region. It is widely expressed in different cell types including thymocytes, activated T cells, and terminally differentiated B cells (plasma cells) and it is involved in cellular proliferation and adhesion. CD38 acts as an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPR). It has been also demonstrated that increased extracellular levels of NAD+ and cADPR are involved in inflammatory diseases and in cellular damage, such as ischemia. In the present study, we have characterized the expression of CD38 in human neuroblastoma SH-SY5Y cell line. All-trans-retinoic acid (ATRA) treatment was used to induce cell differentiation. Our results indicate that: a) even if SH-SY5Y cells have a negative phenotype express CD38 at nuclear level, ATRA treatment does not influence this pattern; b) CD38 localizing to the nucleus may co-localize with p80-coilin positive nuclear-coiled bodies; c) purified nuclei, by Western blot determinations using anti-CD38 antibodies, display a band with a molecular mass of −42 kDa; d) SH-SY5Y cells show nuclear ADP-ribosyl cyclase due to CD38 activity; e) the basal level of CD38 mRNA shows a time-dependent increase after treatment with ATRA. These results suggest that the presence of constitutive fully functional CD38 in the SH-SY5Y nucleus has some important implications for intracellular generation of cADP-ribose and subsequent nucleoplasmic calcium release

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

Embryonic rat dorsal root ganglia organotypic culture: a morphometric model to test neurotoxicology

Neurotoxicity is a common dose-limiting side-effect of several drugs (Cavaletti et al., 2008). So far a validated test method to screen drugs neurotoxicity does not exist, therefore in this interdepartment study we have analyzed the effectiveness of a morphometric neurotoxicty assessment model. Drug neurotoxicity evaluation is based on embryonic rat dorsal root ganglia (DRG) organotypic culture. DRG primary sensory neurons are the principal target of drugs neurotoxic action. In fact, primary sensory neurons lie outside the blood-nerve barrier and are supplied by capillaries with fenestrated walls. Moreover, the axons of these cells are among the longest of the entire nervous system and, therefore, are more susceptible to any agent that interferes with the energy metabolism or the structural basis of axonal transport. In particular, in this interdepartment study, the interference of the under study neurotoxic compound with NGF-induced neurite elongation is analysed. The effectiveness and reproducibility of this model, even if commonly used to test drugs, has not yet been demonstrated. In order to assess the validity of this in vitro model, antineoplastic drugs known to be in clinical use and in animal models neurotoxic (paclitaxel and oxaliplatin) or not dangerous (cyclophosphamide and 5-Fluorouracil) have been tested. DRGs explanted from E15 rat embryos have been treated for 24h with drugs concentrations comparable to those achievable in vivo. The length of the longest neurite of each DRG has been measured by ImageJ program. Experiments have been performed by three different blinded researchers in two different laboratories. Mean and standard deviation of each experiment were obtained, subsequently the mean value and standard deviation of the three independent experiments for each researcher were calculated. Data obtained by the three researchers in two different laboratories resulted statistically comparable and no significant differences were detected (One Way Anova analysis of variance and Tukey post test; p<0.05). This interdepartment in vitro study, therefore, indicates that a purely morphometric model represents a reliable tool to study drug neurotoxicity, permitting to make prediction of neurotoxic effects on humans because the concentrations tested are the same to which DRG are exposed during clinical use

Expression, distribution and glutamate uptake activity of excitatory aminoacid transporters in vitro cultures of embryonic rat dorsal root ganglia cells

Glutamate is the major mediator of excitatory signalling in the mammalian central nervous system, but it has recently been shown to play also a role in the transduction of sensory input at the periphery and in peripheral neuropathies. New advances in research have demonstrated that rat peripheral sensory terminals and dorsal root ganglia (DRG) express molecules involved in glutamate signalling, including high-affinity membrane-bound glutamate transporters (Excitatory Aminoacid Transporters, EAATs) and that alterations in their expression and/or functionality can be implicated in several models of peripheral neuropathy, neuropathic pain and hyperalgesia. Since EAATS might represent an interesting target for pharmacological intervention, the knowledge of their distribution and functionality deserves to be improved. Here we describe, through immunofluorescence assays, immunoblotting and beta-counter analysis of (H3) L-glutamate uptake, the expression, distribution and activity of the EAATs in in vitro cultures of embryonic DRG sensory neurons, sensory neurons+satellite cells and satellite cells. In this study we demonstrated that EAATs are expressed in all cultures, but that their distribution recognizes a peculiar pattern for each of them, since EAATs immunolabelling was differentially expressed in the cytoplasm of neuronal or satellite cells. This result was further confirmed by immunoblotting. Moreover, both cell types showed a strong sodium-ATP-dependent (active) glutamate uptake activity. However, the net (i.e. active transport minus passive diffusion) glutamate transport was more marked in neuronal cultures when cells were grown and maintained without satellite cells. These results, that demonstrate that functionally active EAATs can be studied in DRG cell cultures, provide further evidence for a role of glutamatergic transport in the peripheral nervous system and will be useful for testing whether any change occurs in in vitro models of peripheral nervous system damage. This work was supported in part by an unrestricted research grant from the “Fondazione Banca del Monte di Lombardia”

Biological heterogeneity of putative bladder cancer stem-like cell populations from human bladder transitional cell carcinoma samples.

Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells, the cancer stem-like cells (CSCs) or tumor initiating cells. We report on the isolation and biological characterization of putative bladder CSC populations from primary TCCs. Isolated cells were induced to proliferate in stem cell culture conditions (serum-free medium containing mitogenic growth factors). The proliferating cells formed spheroids (urospheres) and their abilities for extensive proliferation and self-renewal were assayed. Their positivity for several stem cell markers (CD133, Oct-3/4, nestin, and cytokeratins) was also assessed by immunofluorescence tests and they could have the potential to differentiate in the presence of serum. In stem cell culture conditions they gradually showed loss of proliferation, adherence to the substrate, and morphological changes, which might reflect their progressive acquisition of differentiative capacity and loss of self-renewal ability. To evaluate if effective cell selection occurred after isolation, conventional cytogenetic studies on fresh chromosome spreads immediately after isolation and after culture were carried out. In addition, a molecular cytogenetic study by UroVysion assay was carried out on paraffin-embedded tissue sections and on fresh and after culture nuclei preparations. The data collected indicated important karyotype changes and a positive selection for hypo- or near-diploid cells, losing the complexity present in fresh tumors

Antitumoral effects of Hibiscus sabdarifa on human oral squamous cell carcinoma and multiple myeloma cells

Epidemiological data consistently demonstrate a reduced cancer risk associated with a polyphenols rich diet. Hibiscus sabdarifa (HS), a polyphenols rich plant widely consumed worldwide as beverage and used in folk medicine, has recently gained interest thanks to its antioxidant, anti-inflammatory and chemopreventive properties. In the present study we investigated the antitumoral potential of HS extract in two different human tumor cell lines: Multiple Myeloma cells (RPMI 8226) and Oral Squamous Cell Carcinoma cells (SCC-25). MTT assays showed that HS extract induced a dose-dependent viability reduction in both the cells lines. For the subsequent experiments we used HS at the concentration of 5 mg/ml that was the most effective in inducing cell viability reduction after 48h of treatment. Viable cell count using trypan blue staining demonstrated that the HS extract induced decrease in cell growth of both the cell lines and this was due to a reversible cytostatic rather than a cytotoxic effect. Wound-healing and cell invasion assays, respectively performed by a scratch of cell monolayer and Boyden Chamber transwell test, demonstrated that HS extract was able to reduce motility and invasiveness in both RPMI 8226 and SCC-25 cells. The chemical inhibition of ERK1/ERK2 and PI3K, with U0126 and wortmannin respectively, reduces proliferation and migration of both SSC-25 and RPMI cells and HB extract treatment played an additive action with the inhibitors. In conclusion, our results suggest that HS extract have antitumoral properties, since it proved to inhibit tumoral cell growth and cell migration and invasiveness. It is interesting to note that HS extract is effective against two very different tumor cell lines. In fact, RPMI 8226 cells are of hematopoietic origin and grow in suspension, whereas SCC-25 cells derive from epithelium and are characterized by adherent cell growth. Therefore, although further studies are needed to clarify the molecular mechanisms involved in its action, we proposed HS as a potential chemopreventive agent

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