Graphene oxide affects in vitro fertilization outcome by interacting with sperm membrane in an animal model

We realized the exposure of boar spermatozoa to graphene oxide (GO) at concentration of 0.5, 1, 5, 10 and 50 μg/mL in an in vitro system able to promote the capacitation, i.e. the process that allows sperm cells to became fertile. Interestingly, we found that the highest GO concentration (5, 10 and 50 μg/mL) are toxic for spermatozoa, while the lowest ones (0.5 and 1 μg/mL) seem to significantly increase the sperm cells fertilizing ability (p >.05) in an in vitro fertilization experiment. To explain this finding, we investigated the effect of GO on sperm membrane structure (atomic force microscopy) and function (confocal microscopy and flow cytometry, substrate adhesion). As a result, we found that GO is able to interact with spermatozoa membranes and, in particular, it seems to be able to extract the cholesterol, which is a key player in spermatozoa physiology, from plasma membrane of boar spermatozoa incubated under capacitation conditions. In our opinion, these results are very important because they allow identifying either a plausible mechanism of GO toxicity on spermatozoa and new strategies to manage sperm capacitation

A standardized multi-colour flow cytometry approach to characterize the many faces of peripheral circulating microparticles

Microparticles (MP) are small vescicles (<1 µm of diameter) released by several cell types and characterized by an integral plasma membrane expressing the phenotype of the cell from which they originate (Jayachandran et al., 2012). MP play a crucial role in a multitude of pathologies, Including inflammatory and autoimmune disease, diabetes, atherosclerosis, malignancies and cardiovascular disease. The role, as potential biomarker, attributed to circulating MP has been emphasized by the recent literature. In such context, multicolour flow cytometry has great potential In the MP studies (Lanuti et al., 2012). Unfortunately, consensus guidelines on MP identification and enumeration has not been reached yet, due to their small sizes. We purpose to identify, characterize and count MP from whole blood by a seven-colors flow cytometry protocol, with the aim to standardize such method and to allow the identification of both the whole compartment and different MP subpopulations (i.e. endothelial-, platelet- and leukocyte-derived MP). We optimized a novel flow cytometry protocol, using peripheral whole blood stained by a combinations of seven different antigens/probes. Different panel combinations, anticoagulants and storage conditions were evaluated to set the best protocol with the aim to obtain reliable and reproducible MP counts. MP enumeration was carried out by a single platform method by using TrueCount (BD Biosciences). Results demonstrated that the application of the newly optimized flow cytometry method here described, allows to obtain high reproducibility of MP enumeration, pointing out different MP subpopulations both in healthy donors and in different patients. This method may open new routes for the monitoring of MP numbers and phenotypes in different clinical setting

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted sub- populations of peripheral blood (PB), cord blood (CB) and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving the endothe- lial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been so far published, none of them have reached consist- ent outcomes, therefore consensus guidelines with respect to CEC and EPC identifi- cation and quantification need to be established. Here, we have carried out a deep investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/ CD146neg). This approach, combined to a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and CEC counts were consistent with previous reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies are needed to redefine EPC features in order to translate CEC and EPC characterization into clinical practice

Cryopreservation influence in the WJCs Proteome

Cryopreservation is the only mode of long-term storage of viable cells and tissues for cellular therapy, stem cell transplantation and/or tissue engineering. However the freeze-thaw process strongly contributes to cell and tissue damage with several mechanism, including oxidative stress, intracellular ice formation (IIF) dependent cell injury and altered physical cellular properties, i.e. osmotic and ion homeostasis. Our previous proteomics investigation was carried on Wharton’s jelly cells (WJCs), fibroblastlike cells with similar properties to mesenchymal stem cells, therefore a rich source of primitive cells to be potentially used in regenerative medicine. The aim of the present work was to investigate molecular changes that occur in WJCs proteome at different culture conditions (freshly and post-frozen cell preparations) and to elucidate possible mechanism involved in maintaining active proliferation and maximal cellular plasticity in order to optimize in vitro culturing procedure. To analyze changes in protein expression of WJCs we performed a comparative proteomic analysis (2DE followed by MALDI–TOF MS) between fresh and post-frozen cell culturing. WJCs postfrozen showed a qualitative and quantitative changes compared to cells from fresh preparation, expressing proteins involved in replication, cellular defense mechanism and metabolism, that ensure freeze-thaw survival. However, further investigations are needed to clarify the biological mechanisms involved in maintaining active proliferation, plasticity, multipotency cell during in vitro expansio

Reendothelization of porcine heart valve scaffolds with WJ-MSC: a new approach in the heart valve tissue engineering.

Heart valve substitution, based on biosynthetic or mechanical prosthesis replacement, is one of the most frequent surgical approach to treat heart valve diseases. Even if the prosthesis implantation gives a good life quality for patients, there are many long-term disadvantages related to the substitution, such as structural deterioration, non-structural dysfunction and re-intervention. The heart valve tissue engineering (HVTE), a novel branch of regenerative medicine, is developing innovative models and testing new methods to overcome the above reported limitations. In the present study, we investigated the possibility to reendothelize a porcine heart valve scaffold, previously decellularized, by using two cell types: Wharton’s Jelly mesenchymal stem cells (WJ-MSC) and human umbilical vein endothelial cells (HUVEC), the last used as control cells for the reendothelialization process. Both cell types showed, by fluorescence microscopy, that they were able to reconstitute a valid and functional monolayer of neo-endothelium, characterized by the surface expression of typical endothelial markers (i.e. CD144 and CD146). All together, these data suggest that both HUVEC and WJ-MSC are suitable for in vitro autologous endothelium regeneration, opening new perspectives in the field of HVTE

Proteomic insights in extracellular microvesicles from multiple sclerosis patients

To date the most important biomarkers for Multiple Sclerosis (MuS) diagnosis are the oligoclonal bands (OCBs) in CSF and Link Index. CSF is the body fluid that might better provide information about the pathological processes occurring in the CNS, because of its proximity. Anyway, it is obtained through an invasive procedure, thus tears, may represent an useful alternative source of biomarkers. Emerging evidences showed that distinct types of brain cells release high number of Extracellular Vesicles (EVs), that play important roles in the CNS, and represent a relevant source of biomarkers, relative free from confounding factors. In the present study, we analysed EVs from MuS patients obtained from tears and CSF samples. In details, 50μl of CSF or 50 μl of tears/sample were processed by a common flow cytometry no-lyse and no-wash method, in order to identify EVs. Exosomes and microvesicles (MVs) were sorted (70 μm nozzle, FACSAria III cell sorter, BD) from pooled CSF samples on the basis of their positivity to specific tetraspainins (for exosomes) or markers identifying each MV subset. Fractions were analysed by electron microscopy and Dynamic Light Scattering. Purified MV fractions undergone to FASP tryptic digestion and nanoLC-ESI-QTOF-MS/MS based shotgun proteomic approach. Identified MVs proteins were processed by Ingenuity Pathway Analysis (IPA) and PANTHER - Gene List Analysis. Our data shows the presence of subpopulations of extracellular MVs of neuronal and microglia origins in tears , indicating a cross talk between the two compartment. We also identified 55 proteins (FD

Detection and Quantification of eDNA-Associated Bacterial Membrane Vesicles by Flow Cytometry

Bacteria generate membrane vesicles, which are structures known as extracellular vesicles (EVs), reported to be involved in different pathogenic mechanisms, as it has been demonstrated that EVs participate in biofilm formation, cell-to-cell communication, bacteria–host interactions, and nutrients supply. EVs deliver nucleic acids, proteins, and polysaccharides. It has been reported that Helicobacter pylori (H. pylori) and Lactobacillus reuteri (L. reuteri), of both planktonic and biofilm phenotypes, produce EVs carrying extracellular DNA (eDNA). Here, we used polychromatic flow cytometry (PFC) to identify, enumerate, and characterize EVs as well as the eDNA-delivering EV compartment in the biofilm and planktonic phenotypes of H.pylori ATCC 43629 and L. reuteri DSM 17938. Biofilm formation was demonstrated and analyzed by fluorescence microscopy, using a classical live/dead staining protocol. The enumeration of EVs and the detection of eDNA-associated EVs were performed by PFC, analyzing both whole samples (cells plus vesicles) and EVs isolated by ultracentrifugation confirm EVs isolated by ultracentrifugation. PFC analysis was performed relying on a known-size beaded system and a mix of three different fluorescent tracers. In detail, the whole EV compartment was stained by a lipophilic cationic dye (LCD), which was combined to PKH26 and PicoGreen that selectively stain lipids and DNA, respectively. Fluorescence microscopy results displayed that both H. pylori and L. reuteri produced well-structured biofilms. PFC data highlighted that, in both detected bacterial species, biofilms produced higher EVs counts when paralleled to the related planktonic phenotypes. Furthermore, the staining with PicoGreen showed that most of the generated vesicles were associated with eDNA. These data suggest that the use of PFC, set according to the parameters here described, allows for the study of the production of eDNA-associated EVs in different microbial species in the same or several phases of growth, thus opening new perspectives in the study of microbial derived EVs in clinical samples