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In the present study we have isolated and identified mesenchymal stem cells (MSCs) from the exfoliated deciduous teeth dental pulp (DP-MSCs), as plastic-adherent, spindle-shaped cells with a high proliferative potential. Immunophenotype analyses revealed that DP-MSCs were positive for mesenchymal cell markers (CD90, CD44, CD105, STRO-1, vimentin and α-SMA), and negative for hematopoietic stem cell markers (CD11b, CD33, CD34, CD45, CD235a). DPMSCs were also capable of differentiating into adipogenic, chondrogenic, myogenic and osteogenic lineages, fulfilling the functional criterion for their characterization. These results demonstrate that DP-MSCs offer a valuable, readily accessible source to obtain and store adult stem cells for future use.

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its actionin vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-γ and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additionalin vitroassessment on bone marrow highly enriched Lin− progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate thatin vivoIL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage. [ABSTRACT FROM AUTHOR]

Introduction. Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO) production both in primary human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC). Moreover, NO increases γ-globin and fetal hemoglobin levels in human erythroid progenitors. Objective. In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase γ-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. Methods. The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. Results. In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 μM up to 30 minutes). This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS), demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS) mRNA level and eNOS/Я-actin ratio in HUVEC (by twofold). In addition, bradykinin failed to increase γ-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce γ/β globin ratio in erythroid progenitors in co-cultures with HUVEC. Conclusion. Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce γ-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of γ-globin gene expression. [Projekat Ministarstva nauke Republike Srbije, br. 175053]

Mesenchymal stem cells (MSCs) were isolated from human periodontal ligament (hPDL-MSCs) and characterized by their morphology, clonogenic efficiency, proliferation and differentiation capabilities. hPDL-MSCs, derived from normal impacted third molars, possessed all of the properties of MSC, including clonogenic ability, high proliferation rate and multi-lineage (osteogenic, chondrogenic, adipogenic, myogenic) differentiation potential. Moreover, hPDL-MSCs expressed a typical MSC epitope profile, being positive for mesenchymal cell markers (CD44H, CD90, CD105, CD73, CD29, Stro-1, fibronectin, vimentin, α-SMA), and negative for hematopoietic stem cell markers (CD34, CD11b, CD45, Glycophorin-CD235a). Additionally, hPDL-MSCs, as primitive and highly multipotent cells, showed high expression of embryonic markers (Nanog, Sox2, SSEA4). The data obtained provided yet further proof that cells with mesenchymal properties can be obtained from periodontal ligament tissue. Although these cells should be further investigated to determine their clinical significance, hPDL-MSCs are believed to provide a renewable and promising cell source for new therapeutic strategies in the treatment of periodontal defects. [Projekat Ministarstva nauke Republike Srbije, br. 175062 i br. III 41011]

Introduction. 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. Objective. 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). Methods. 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. Results. 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 (Nanog, 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 α-smooth muscle actin), most appeared to have similar epitope profiles irrespective of MSC source. Conclusion. The results obtained demonstrated that both MSCs represent good alternative sources of adult MSCs that could be used in cell therapy applications. [Projekat Ministarstva nauke Republike Srbije, br. 175062]

Anaemia occurs frequently in patients with heart failure and its current treatment lacks clear targets. Emerging evidence suggests that erythroid progenitor cell expansion is an integral part of physiological response to anaemia associated with chronic stress. Understanding the underlying mechanism may provide a novel approach to anaemia management. In this study, we aimed to examine a role for nitric oxide (NO) in the regulation of bone marrow erythroid progenitor response to chronic stress. For this purpose, adult male mice were subjected to 2 h daily restraint stress for 7 or 14 consecutive days. The role of NO was assessed by subcutaneous injection with NG-nitro-L-arginine methyl ester, 30 min prior to each restraint. Chronic exposure to stress resulted in significantly increased number of bone marrow erythroid progenitors, and blockade of NO biosynthesis prior to daily stress completely prevented stress-induced erythroid progenitor cell expansion. Furthermore, chronic stress exposure led to altered expression of neural, endothelial and inducible nitric oxide synthases (NOS) in the bone marrow, both on mRNA and protein level. Decreased expression of neural and endothelial NOS, as well as reduced expression of NF-kappaB/p65 in bone marrow nuclear cell fraction, was accompanied by elevated bone marrow expression of inducible NOS in chronically stressed animals. This is the first study to demonstrate a role for NO in adaptive response of erythroid progenitors to chronic stress. Targeting NO production may be beneficial to improve bone marrow dysfunction and reduced erythroid progenitor cell expansion in chronic heart failure patients.

Macrophage migration inhibitory factor is a well-known proinflammatory cytokine that is released during systemic stress response. Although MIF can affect erythrocyte production, the role of this cytokine in stress-induced erythropoiesis is completely unknown. To extend our previous findings showing that chronic psychological stress stimulates extramedullary erythropoiesis, here we examined whether MIF is involved in the control of stress-induced erythropoietic response. Adult male C57BL/6 wild-type (WT) and MIF-KO (knock-out) mice were subjected to 2-h daily restraint stress for either 7 or 14 consecutive days. The number of erythroid progenitors and CD71/Ter119 profile of erythroid precursors were analyzed in the bone marrow and spleen. Additionally, MIF protein expression was assessed in WT mice. Our results demonstrated that chronic restraint stress enhanced the number of both erythroid progenitors and precursors in the spleen. Stress-induced increase in the number of splenic late erythroid progenitors as well as in the percentage of CD71(+)Ter119(+)-double-positive precursors was significantly more pronounced in MIF-KO mice compared to WT animals. Furthermore, repeatedly stressed WT animals demonstrated an augmented MIF expression in the spleen. Unlike the spleen, the bone marrow of chronically stressed WT mice exhibited less prominent changes in erythropoietic stress response and no significant alteration in MIF expression. In addition, MIF deficiency did not influence the bone marrow erythropoiesis in stressed animals. These findings suggest that MIF regulates extramedullary erythropoiesis by inhibiting an overexpansion of splenic immature erythroid cells during chronic stress and indicate a novel role for this cytokine under chronic stress conditions.

Background/aim: The purpose of this study was to investigate proliferation and differentiation markers in colorectal adenocarcinoma and their correlation with clinicopathological factors., Materials and Methods: Samples were collected from 38 patients with colorectal adenocarcinoma and 10 healthy controls. E-cadherin, carcinoembryonic antigen (mCEA), cyclin B1, vascular endothelial growth factor (VEGF), and erythropoietin (EPO) receptor (EPOR) were examined by immunohistochemistry; VEGF and EPO were examined by real-time PCR., Results: The tumor samples were mostly characterized by large dimension (pT3), moderate level of differentiation (G2), negative lymph node status (N0), and no metastasis. Cyclin B1 and VEGF gene and protein expressions were significantly higher in tumor tissues than in control tissues; E-cadherin expression was significantly decreased in tumor samples and in positive correlation with mCEA. EPO was almost undetectable in tumor tissues of colorectal adenocarcinoma. Significant positive correlation was detected between tumor size and cyclin B1, tumor grade, and lymph node status., Conclusion: Decreased expression of EPO, high levels of VEGF and cyclin B1 expression, predominant moderate tumor differentiation, absence of metastasis, and negative lymph node status may suggest low level of aggressiveness, better prognosis, and longer patient survival.

Interleukin 17 (IL-17) is a cytokine with pleiotropic effects associated with several inflammatory diseases. Although elevated levels of IL-17 have been described in inflammatory myopathies, its role in muscle remodeling and regeneration is still unknown. Excessive extracellular matrix degradation in skeletal muscle is an important pathological consequence of many diseases involving muscle wasting. In this study, the role of IL-17 on the expression of matrix metalloproteinase- (MMP-) 9 in myoblast cells was investigated. The expression of MMP-9 after IL-17 treatment was analyzed in mouse myoblasts C2C12 cell line. The increase in MMP-9 production by IL-17 was concomitant with its capacity to inhibit myogenic differentiation of C2C12 cells. Doxycycline (Doxy) treatment protected the myogenic capacity of myoblasts from IL-17 inhibition and, moreover, increased myotubes hypertrophy. Doxy blocked the capacity of IL-17 to stimulate MMP-9 production by regulating IL-17-induced ERK1/2 MAPK activation. Our results imply that MMP-9 mediates IL-17's capacity to inhibit myoblast differentiation during inflammatory diseases and indicate that Doxy can modulate myoblast response to inflammatory induction by IL-17., Competing Interests: The authors declare that they have no conflict of interest.

Adipose tissue is an attractive source of mesenchymal stem/stromal cells (MSCs) with potential applications in reconstructive plastic surgery and regenerative medicine. The aim of this study was to characterise human adipose tissue MSCs (ASCs) derived from healthy individuals and cancer patients and to compare their interactions with tumour cells. ASCs were isolated from adipose tissue of healthy donors, breast cancer-adjacent adipose tissue of breast cancer patients and tumour-adjacent adipose tissue of non-breast cancer patients. Their proliferation, differentiation, immunophenotype and gene expression were assessed and effects on the proliferation of human breast cancer cell line MCF-7 compared. ASCs from all sources exhibited similar morphology, proliferative and differentiation potential, showing the characteristic pattern of mesenchymal surface markers expression (CD90, CD105, CD44H, CD73) and the lack of HLA-DR and hematopoietic markers (CD11a, CD33, CD45, Glycophorin-CD235a), but uneven expression of CD34. ASCs also shared a common positive gene expression of HLA-DR, HLA-A, IL-6, TGF-β and HIF-1, but were negative for HLA-G, while the expression levels of Cox-2 and IDO-1 varied. All ASCs significantly stimulated the proliferation of MCF-7 tumour cells in direct mixed co-cultures and transwell system, although their conditioned media displayed antiproliferative activity. Data obtained showed that ASCs with similar characteristics are easily isolated from various donors and sites of origin, although ASCs could both suppress and favour tumour cells growth, emphasising the importance of cellular context within the microenvironment and pointing to the significance of safety studies to exclude any potential clinical risk of their application in regenerative medicine., (© 2013 International Federation for Cell Biology.)

Psychological stress affects different physiological processes including haematopoiesis. However, erythropoietic effects of chronic psychological stress remain largely unknown. The adult spleen contains a distinct microenvironment favourable for rapid expansion of erythroid progenitors in response to stressful stimuli, and emerging evidence suggests that inappropriate activation of stress erythropoiesis may predispose to leukaemic transformation. We used a mouse model to study the influence of chronic psychological stress on erythropoiesis in the spleen and to investigate potential mediators of observed effects. Adult mice were subjected to 2 hrs daily restraint stress for 7 or 14 consecutive days. Our results showed that chronic exposure to restraint stress decreased the concentration of haemoglobin in the blood, elevated circulating levels of erythropoietin and corticosterone, and resulted in markedly increased number of erythroid progenitors and precursors in the spleen. Western blot analysis revealed significantly decreased expression of both erythropoietin receptor and glucocorticoid receptor in the spleen of restrained mice. Furthermore, chronic stress enhanced the expression of stem cell factor receptor in the red pulp. Moreover, chronically stressed animals exhibited significantly increased expression of bone morphogenetic protein 4 (BMP4) in the red pulp as well as substantially enhanced mRNA expression levels of its receptors in the spleen. These findings demonstrate for the first time that chronic psychological stress activates BMP4-dependent extramedullary erythropoiesis and leads to the prolonged activation of stress erythropoiesis pathways. Prolonged activation of these pathways along with an excessive production of immature erythroid cells may predispose chronically stressed subjects to a higher risk of leukaemic transformation., (© 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Ahead of Print article withdrawn by publisher.

The mouse is a suitable experimental model to study the biology of mesenchymal stem cells (MSCs), as well as to be used in biocompatibility studies and tissue engineering models. However, the isolation and purification of murine MSCs is far more challenging than their counterparts from other species. In this study, we isolated, expanded and characterized mouse MSCs from bone marrow (BM-MSCs). Additionally, we analyzed the effects of two regulatory molecules, interleukin 17 (IL-17) and basic fibroblast growth factor (bFGF), on BM-MSCs growth and elucidated the signaling pathways involved. The results revealed that IL-17 increased the frequency of colony-forming units fibroblast (CFU-F) as well as the BM-MSCs proliferation in a dose-dependent manner, while bFGF supplementation had no significant effect on CFU-F frequency but induced an increase in cell proliferation. Their combined usage did not produce additive effects on BM-MSCs proliferation and even induced reduction in the number of CFU-F. Also, the involvement of both p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) signaling in proliferative activity of IL-17 and bFGF on murine BM-MSCs and, moreover, the increased co-activation of a common signaling molecule, p38 MAPK, were demonstrated. Together, the data presented highlighted the role of IL-17 and bFGF in murine BM-MSCs proliferation and pointed to the complexity and specificity of the signaling networks leading to MSCs proliferation in response to different regulatory molecules.