Slavko Mojsilovic

In vitro expansion changes replication and differentiation capacity of mesenchymal stem cells (MSCs), increasing challenges and risks, while limiting the sufficient number of MSCs required for cytotherapy. Here, we characterized and compared proliferation, differentiation, telomere length and pluripotency marker expression in MSCs of various origins. Immunophenotyping, proliferation and differentiation assays were performed. Pluripotency marker (Nanog, Oct-4, SOX-2, SSEA-4) expression was determined by immunofluorescence. Quantitative PCR was performed for relative telomere length (RTL) analyses, while expression of relevant genes for pluripotency markers, differentiation state (Cbfa1, human placental alkaline phosphatase, peroxisome proliferator activated receptor, Sox9 and Collagen II a1), and telomerase reverse transcriptase (hTERT) was determined by semiquantitative RT-PCR. Peripheral blood MSCs (PB-MSCs) and umbilical cord MSCs (UC-MSCs) showed the highest, while periodontal ligament MSCs (PDL-MSCs) and adipose tissue MSCs (AT-MSCs) the lowest values of both the replication potential and RTL. Although MSCs from exfoliated deciduous teeth (SHEDs), PDL-MSCs and AT-MSCs showed higher mRNA expression of pluripotency markers, all MSCs expressed pluripotency marker proteins. SHEDs and PDL-MSCs showed prominent capacity for osteogenesis, PB-MSCs and UC-MSCs showed strengthened adipogenic differentiation potential, while AT-MSCs displayed similar differentiation into both lines. The MSCs populations derived from different sources, although displaying similar phenotype, exhibited high degree of variability regarding biological properties related to their self-renewal and differentiation capacity. These data indicate that for more accurate use in cell therapy, individualities of MSCs isolated from different tissues should be identified and taken into consideration when planning their use in clinical protocols.

We compared the gene expression profile of peripheral blood CD34(+) cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR-ABL oncogene. The microarray analyses have been performed in circulating CD34(+) cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR-ABL positive CML patients were in chronic phase, with a mean value of 2012±SD of CD34(+)cells/μl in peripheral blood. The gene expression profile was more prominent in CML CD34(+) cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34(+) cells (HINT1, TXN, SERBP1) and granulocytes, respectively. BCR-ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11, CDK4/6, and MYC and reduction of E2F1, KRAS, and NFKBIA gene expression in CD34(+) cells. Among genes linked to the inhibition of cellular proliferation by BCR-ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. The presence of BCR-ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34(+) cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects.

Transforming growth factor-beta (TGF-β) and oxidative stress/Reactive Oxygen Species (ROS) both have pivotal roles in health and disease. In this review we are analyzing the interplay between TGF-β and ROS in tumorigenesis and cancer progression. They have contradictory roles in cancer progression since both can have antitumor effects, through the induction of cell death, senescence and cell cycle arrest, and protumor effects by contributing to cancer cell spreading, proliferation, survival, and metastasis. TGF-β can control ROS production directly or by downregulating antioxidative systems. Meanwhile, ROS can influence TGF-β signaling and increase its expression as well as its activation from the latent complex. This way, both are building a strong interplay which can be taken as an advantage by cancer cells in order to increment their malignancy. In addition, both TGF-β and ROS are able to induce cell senescence, which in one way protects damaged cells from neoplastic transformation but also may collaborate in cancer progression. The mutual collaboration of TGF-β and ROS in tumorigenesis is highly complex, and, due to their differential roles in tumor progression, careful consideration should be taken when thinking of combinatorial targeting in cancer therapies.

Natural products have been investigated for promising new leads in pharmaceutical development. The purpose of this study was to analyze the biological effect of GE132+Natural, a novel supplement consisting of 5 compounds: Resveratrol, Ganoderma lucidum, Sulforaphane, Lycopene and Royal jelly. The antiproliferative activity of GE132+Natural was tested on 3 different human cancer cell lines: MCF7 (breast cancer cells), PC3 (prostate cancer cells), and SW480 (colon cancer cells), as well as on EA.hy 926 (normal human endothelial cell line). In addition, the cytotoxicity of GE132+- Natural on the proliferation of primary human mesenchymal stem cells isolated from dental pulp (DP=MSC), along with its in vitro impact on different peripheral blood parameters, was determined. The results revealed high antiproliferative activity of GE132+Natural on all tested cancer cell lines (PC3, MCF7 and SW480), as well as on the EA.hy 926 endothelial cell line in a dose-dependent manner. However, applie...

Interleukin-17 is Th17 cell cytokine implicated in regulation of hematopoiesis and inflammation. Besides promoting granulopoiesis, we have previously shown that IL-17 also affects erythropoiesis stimulating the development of early erythroid progenitors, BFU-E, but suppressing, at least partly via p38 MAPK, the growth of late stage erythroid progenitors, CFU-E. The aim of the present study was to investigate the involvement of other MAPKs, JNK and ERK1/2, as well as GATA transcription factors, in IL-17-mediated effects on murine bone marrow erythroid progenitors. Data obtained by use of specific MAPKs inhibitors indicated that MEK1/2-ERK1/2 MAPK signaling mediates IL-17-induced CFU-E inhibition, as well as that JNK and/or MEK1/2-ERK1/2 MAPKs activation underlies IL-17-induced stimulation of BFU-E growth. Furthermore, Western blot analyses demonstrated no effect on early hematopoiesis transcription factor, GATA-2, and enhanced expression level of erythroid-specific factor GATA-1 in m...

The interleukin (IL)-17 producing T-helper cells have been linked to pathogenesis of autoimmunity and mostly investigated in rheumatoid arthritis (RA). In this study we tested the IL-17 levels, as well as the levels of nitric oxide (NO) as possible IL-17-induced product, in patients with primary Sjögren's syndrome (pSS), an intricate and complex chronic autoimmune disorder of exocrine glands. Serum IL-17 levels and nitrite concentrations determined in patients with pSS (n=30) were compared with the values obtained in patients with RA (n=10) and healthy controls (n=15). The values obtained for IL-17 in pSS patients were also associated with the patients' clinical characteristics, particularly the rheumatoid factor (RF) and total antinuclear antibodies (tANA) levels. Serum concentrations of IL-17 were significantly (P<0.01) higher in patients with pSS (12.9 ± 28.0 pg/ml) as compared to those obtained in healthy individuals (0.2 ± 0.6 pg/ml), but not as high as the values ob...

To evaluate whether the response of hematopoietic cells to interleukin-17 (IL-17) depends on the tissue microenvironment in which hematopoiesis occurs, the influence of recombinant mouse IL-17 on spleen hematopoietic cells and cytokine release was assessed in normal mice in vitro and in vivo. In vitro, IL-17 did not significantly affect the growth of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) derived colonies. A single injection of IL-17 in vivo exhibited stimulatory effects on hematopoietic cells from both granulocytic and erythroid lineages. The increased number of metamyelocytes 48 h after treatment imply to the IL-17-induced stimulation of granulopoiesis. The number of BFU-E was increased at 24 h, while the number of CFU-E increased 6 h and 24 h after treatment. Since the same treatment in the bone marrow decreased the number of CFU-E, it may be concluded that the local microenvironment plays an important role in IL-17-mediated effects on CFU-E. IL-17 increa...

The objective of this study was to investigate the signal transduction pathways associated with the clonal development of myeloid and erythroid progenitor cells. The contribution of particular signaling molecules of protein tyrosine kinases (PTKs), mitogen-activated protein (MAP) kinase, and PI-3 kinase signaling to the growth of murine bone marrow colony forming unit-granulocyte-macrophage (CFU-GM) and erythroid (burst forming unit-erythroid [BFU-E] and colony forming unit-erythroid [CFU-E]) progenitors was examined in studies performed in the presence or absence of specific signal transduction inhibitors. The results clearly pointed to different signal transducing intermediates that are involved in cell proliferation and differentiation depending on the cell lineage, as well as on the progenitors' maturity. Lineage-specific differences were obtained when chemical inhibitors specific for receptor- or nonreceptor-PTKs, as well as for the main groups of distinctly regulated MAPK ...

Constantly growing field of biomedical applications is a new frontier that drives the development of plasma sources. The choice of the plasma system used for treatment is usually guided by the type of samples that are treated and effect these plasmas are intended to have on the samples. Some of the samples cannot undergo vacuum and due to this fact non-thermal atmospheric pressure plasmas lately have drawn considerable attention with their enormous potential for technological applications in surface modifications and biomedical applications. The necessity to use plasma for in-vivo treatments/procedures has led to several requirements for plasma sources to meet. Because of its mild plasma, low gas temperature and geometry, the plasma needle is especially convenient for medical applications. We have studied effect of the plasma needle on different living cells, from plants through bacteria such as Escherichia coli and Staphylococcus aureus to the human peripheral blood mesenchymal ste...

There are many types of plasmas that can be generated under ambient pressure and temperature conditions, the need for precise and localized treatments qualifies plasma needle to be a good match [1]. Because of its mild plasma, low gas temperature and geometry, the plasma needle is especially convenient for medical applications. This device can be used for non-contact disinfection of dental cavities and wounds, minimum-destructive precise treatment, as well as the removal of damaged tissue. Our measurements were performed on a standard size plasma needle that we originally used for the treatment of plant cells [2]. In this paper we will present results of plasma needle treatments on the human peripheral blood-derived multipotent mesenchymal stem cells (hPB-MSC) (fibroblast cells). We have treated cells in two conditions, i.e. with and without liquid environment (suspension). Results from the MTT viability and Crystal Violet adhesion tests performed with the hPB-MSC will be presented....

Mesenchymal stem cells (MSCs) are a promising tool for regenerative medicine, but due to the heterogeneity of their populations, different sources and isolation techniques, the characteristics defining MSCs are inconsistent. The aim of this study was to compare the characteristics of MSCs derived from two different human tissues: peripheral blood (PB-MSCs) and umbilical cord Wharton's Jelly (UC-MSCs). The PB-MSC and UC-MSC were isolated by adherence to plastic after gradient-density separation or an explant culture method, respectively, and compared regarding their morphology, clonogenic efficiency, proliferating rates, immunophenotype and differentiation potential. MSCs derived from both sources exhibit similar morphology, proliferation capacity and multilineage (osteogenic, chondrogenic, adipogenic and myogenic) differentiation potential. Differences were observed in the clonogenic capacity and the immunophenotype, since UC-MSCs showed higher CFU-F (colony-forming units-fibroblastic) cloning efficiency, as well as higher embryonic markers (Na-nog, Sox2, SSEA4) expression. When additional surface antigens were analyzed by flow cytometry (CD44, CD90, CD105, CD33, CD34, CD45, CD11b, CD235a) or immunofluorescent labeling (vimentin, STRO-1 and alpha-smooth muscle actin), most appeared to have similar epitope profiles irrespective of MSC source. The results obtained demonstrated that both MSCs represent good alternative sources of adult MSCs that could be used in cell therapy applications.

ABSTRACT The potential of plasma applications medicine, the connections to nanotechnologies and the results obtained by our group are reviewed. A special issue in plasma medicine is the development of the plasma sources that would achieve non-equilibrium at atmospheric pressure in an atmospheric gas mixture with no or only marginal heating of the gas, and with desired properties and mechanisms that may be controlled. Our studies have shown that control of radicals or chemically active products of the discharge, such as ROS (reactive oxygen species) and/or NO, may be used to control the growth of the seeds. Simultaneously, a specially designed plasma needle and other sources were shown to be efficient to sterilize not only colonies of bacteria but also plank-tonic samples (microorganisms protected by water) or bio films. Finally, it was shown that a plasma might induce differentiation of stem cells. Non-equilib-rium plasmas may be used in detection of different specific markers in medi-cine. For example proton transfer mass spectroscopy may be employed in the detection of volatile organic compounds without their dissociation and thus as a technique for instantaneous measurement of the presence of markers for nu-merous diseases.

ABSTRACT Here we investigate the influences of non-thermal atmospheric plasma on human mesenchymal stem cells isolated from periodontal ligament (hPDL-MSCs). A specially redesigned plasma needle was used as the source of low-temperature plasma and its effects on different hPDL-MSC functions were investigated. Cell cultures were obtained from extracted normal impacted third molars and characterized for their phenotype and multi-potential differentiation. The hPDL-MSCs possessed all the typical MSC properties, including clonogenic ability, high proliferation rate, specific phenotype and multilineage differentiation. The data regarding the interaction of plasma with hPDL-MSCs demonstrated that plasma treatment inhibited the migration of hPDL-MSCs and induced some detachment, while not affecting their viability. Additionally, plasma significantly attenuated hPDL-MSCs' proliferation, but promoted their osteogenic differentiation. The results of this study indicated that a non-thermal plasma offers specific activity with non-destructive properties that can be advantageous for future dental applications.

The effects of interleukin (IL)-17 on nitric oxide (NO) synthase (NOS) expression, as well as the participation of mitogen-activated protein kinases (MAPKs) in IL-17-mediated effects were examined in murine bone marrow cells. The results demonstrated the ability of IL-17 to upregulate the expression of mRNA for both inducible NOS and constitutive, endothelial NOS isoforms, as well as to enhance the phosphorylation of p38 MAPK. Moreover, both the NOS-inducing effect of IL-17 and the in vitro IL-17-mediated inhibition colony forming unit-erythroid (CFU-E) growth were dependent on p38 MAPK activity. The data demonstrating that the in vivo reducing effect of IL-17 on bone marrow CFU-E was prevented by co-treatment with the NOS inhibitor Nw-nitro-l-arginine methyl ester hydrochloride (L-NAME), implied that this effect is mediated through NOS activation. Besides revealing a link between the IL-17, NO, and haematopoiesis, data presented gave an insight into the mechanisms by which IL-17 exerts its modulatory effects on bone marrow cells.

Abstract Proper migration and differentiation of neural crest (NC) cells are required for normal development of craniofacial structures, heart and great vessels, sensory and autonomic nervous systems, and other organs within vertebrate embryos. Seriae-protease ...

ABSTRACT Mesenchymal stem cells (MSCs), beside regenerative potential, possess immunomodulatory properties and their use in managing immune-mediated diseases is intensively studied. We analyzed the effects of MSCs isolated from human adipose tissue (AT-MSCs), dental pulp (DP-MSCs), peripheral blood (PB-MSCs) and umbilical cord Wharton's jelly (UC-MSCs), on the proliferation of allogeneic peripheral blood mononuclear cells (PBMCs). While only AT-MSCs functioned as alloantigen presenting cells, proliferation of PBMCs in response to a phytohemagglutinin (PHA) and alloantigens in mixed lymphocytes reaction (MLR) was inhibited by all MSCs in a cell concentration-dependent manner. Conditioned medium (CM) derived from DP-MSCs, PB-MSCs and UC-MSCs, suppressed the baseline, PHA- and alloantigens-mediated proliferation of PBMC, whereas AT-MSCs-derived CM inhibited MLR, but failed to suppress the spontaneous and PHA-induced PBMCs proliferation. Differences between MSC types were observed in expression of genes related to immunomodulation, including human leukocyte antigens (HLA)-A, HLA-DR, HLA-G5, interleukin 6 (IL)-6, transforming growth factor (TGF)-beta, cyclooxygenase-2 (COX-2) and indoleamine 2,3-dioxygenase (IDO-1), under basal conditions, as well as in response to proinflammatory cytokines, interferon (IFN)-gamma and tumor necrosis factor alpha (TNF)-alpha. While AT-MSCs showed a positive constitutive expression of almost all tested genes that was augmented in response to IFN-gamma and TNF-alpha, only combined cytokine treatment increased HLA-A, COX2 and IL-6 mRNA expression in DP-MSCs and slightly stimulated the expression of HLA-G and TGF-beta in UC-MSCs. Although MSCs from different tissues showed similar potential to suppress proliferation of PBMCs, heterogeneity in the expression of genes related to immunomodulation emphasizes the importance of investigating the role of specific molecular mechanisms in the regulation of immunomodulatory activity of MSCs.

ABSTRACT Non-equilibrium plasmas proved to be able to produce large variety of chemically reactive species at a low gas temperature while maintaining uniform reaction rates over relatively large areas. The choice of the plasma system used for treatment is usually guided by the type of samples that are treated and effect these plasmas are intended to have on the samples. Some of the samples cannot undergo vacuum and due to this fact non-thermal atmospheric pressure plasmas lately have drawn considerable attention with their enormous potential for technological applications in surface modifications and biomedical applications. So far we have used several different high frequency plasma sources operating from low to atmospheric pressure for treatments of polymer, textiles [1], graphene [2], seeds [3], sterilization of bacteria and treatment of plant and stem cells [4, 5]. In order to be able to effectively use these plasma systems in treatments that demand different conditions it was necessary to make detailed diagnostics of these systems. We have used home-made derivative probes, Langmuir and catalytic probe, ICCD camera and mass-energy analyser that works both in low and atmospheric pressures to determine the optimal treatment parameters for various samples.

The study was undertaken to extend our investigation concerning both the in vivo activity of interleukin (IL)-17 and the specific role of nitric oxide (NO) in IL-17-induced effects in the process of haematopoiesis. CBA mice were simultaneously treated with IL-17 and/or nitric oxide synthase (NOS) inhibitor, l-NAME, for 5 days and changes within various haematopoietic cell lineages in bone marrow, spleen and peripheral blood were analysed. Findings showed that administration of both IL-17 and l-NAME stimulated increase in net haematopoiesis in normal mice. IL-17-enhanced myelopoiesis was characterized by stimulation of both femoral and splenic haematopoietic progenitor cells and morphologically recognizable granulocytes. Additionally, IL-17 induced alterations in the frequency of erythroid progenitor cells in both bone marrow and spleen, accompanied with their mobilization to the peripheral blood. As a consequence of these changes in the erythroid cell compartments, significant reticulocytosis was observed, which evidenced that in IL-17-treated mice effective erythropoiesis occurred. Exposure of mice to NOS inhibitor also increased the number of both granulocyte-macrophage and erythroid progenitors in bone marrow and spleens, and these alterations were followed by the mobilization of erythroid progenitors and elevated content of reticulocytes in peripheral blood. The specific role of NO in IL-17-induced haematopoiesis was demonstrated only in the IL-17-reducing effect on bone marrow late stage erythroid progenitors, CFU-E. The results demonstrated the involvement of both IL-17 and NO in the regulation of haematopoietic cell activity in various haematopoietic compartments. They further suggest that IL-17 effects are differentially mediated depending on the haematopoietic microenvironments.

Recent studies point to important roles for IL-17 and Th17 cells in sustaining chronic inflammation and articular destruction in rheumatoid arthritis (RA). We investigated the effects of TNF inhibitor on innate inflammatory and Th17 cytokines production by ex vivo lipopolysaccharide (LPS)-stimulated whole blood in patients with RA and the associations of cytokine levels in whole blood cultures with autoantibodies and markers of disease activity. Whole blood cultures from 18 healthy volunteers and 19 RA patients on etanercept therapy were stimulated with LPS and the production of IL-6, TNF-α, IL-23, IL-17A and IL-21 was measured by ELISA. After stimulation with LPS, the interleukin (IL)-17A (p = 0.020) and IL-21 (p = 0.0001) secretions were significantly higher in patients with RA than in controls, while the TNF-α (p = 0.002) was significantly lower at baseline. Etanercept significantly decreased IL-21 production (p = 0.007), while IL-6 production (p = 0.005) significantly increased after 6 months of therapy. IL-21 significantly correlated with RF (r = 0.917, p < 0.01) and antimutated citrullinated vimentin antibodies (r = 0.770, p < 0.01) at baseline. Logistic regression analysis revealed that baseline IL-21 levels (p = 0.004) were significant predictors of DAS28-ESR at 6 months follow-up. Stimulation with LPS increased production of Th17 cytokines in whole blood cultures in patients with RA. Etanercept therapy decreased IL-21 secretion, while the capacity of whole blood cells to produce IL-6 increased. IL-21 production is strongly associated with the levels of autoantibodies. Our findings suggest that IL-21 production in LPS-stimulated whole blood cultures may be predictive of clinical response to etanercept treatment in patients with RA.