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Sample records for human thymic stromal

  1. Proprotein convertases generate a highly functional heterodimeric form of thymic stromal lymphopoietin in humans.

    Science.gov (United States)

    Poposki, Julie A; Klingler, Aiko I; Stevens, Whitney W; Peters, Anju T; Hulse, Kathryn E; Grammer, Leslie C; Schleimer, Robert P; Welch, Kevin C; Smith, Stephanie S; Sidle, Douglas M; Conley, David B; Tan, Bruce K; Kern, Robert C; Kato, Atsushi

    2017-05-01

    Thymic stromal lymphopoietin (TSLP) is known to be elevated and truncated in nasal polyps (NPs) of patients with chronic rhinosinusitis and might play a significant role in type 2 inflammation in this disease. However, neither the structure nor the role of the truncated products of TSLP has been studied. We sought to investigate the mechanisms of truncation of TSLP in NPs and the function of the truncated products. We incubated recombinant human TSLP with NP extracts, and determined the protein sequence of the truncated forms of TSLP using Edman protein sequencing and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. We investigated the functional activity of truncated TSLP using a PBMC-based bioassay. Edman sequencing and mass spectrometry results indicated that NP extracts generated 2 major truncated products, TSLP (residues 29-124) and TSLP (131-159). Interestingly, these 2 products remained linked with disulfide bonds and presented as a dimerized form, TSLP (29-124 + 131-159). We identified that members of the proprotein convertase were rate-limiting enzymes in the truncation of TSLP between residues 130 and 131 and generated a heterodimeric unstable metabolite TSLP (29-130 + 131-159). Carboxypeptidase N immediately digested 6 amino acids from the C terminus of the longer subunit of TSLP to generate a stable dimerized form, TSLP (29-124 + 131-159), in NPs. These truncations were homeostatic but primate-specific events. A metabolite TSLP (29-130 + 131-159) strongly activated myeloid dendritic cells and group 2 innate lymphoid cells compared with mature TSLP. Posttranslational modifications control the functional activity of TSLP in humans and overproduction of TSLP may be a key trigger for the amplification of type 2 inflammation in diseases. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  2. Directional Secretory Response of Double Stranded RNA-Induced Thymic Stromal Lymphopoetin (TSLP) and CCL11/Eotaxin-1 in Human Asthmatic Airways

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    Gustavo Nino; Shehlanoor Huseni; Perez, Geovanny F.; Krishna Pancham; Humaira Mubeen; Aleeza Abbasi; Justin Wang; Stephen Eng; Colberg-Poley, Anamaris M.; Pillai, Dinesh K; Mary C. Rose

    2014-01-01

    BACKGROUND: Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral) and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. METHODS: Primary human bronchial epithelial cells (HBEC) from c...

  3. IL-1β induces thymic stromal lymphopoietin and an atopic dermatitis-like phenotype in reconstructed healthy human epidermis.

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    Bernard, Marine; Carrasco, Cédric; Laoubi, Léo; Guiraud, Béatrice; Rozières, Aurore; Goujon, Catherine; Duplan, Hélène; Bessou-Touya, Sandrine; Nicolas, Jean-François; Vocanson, Marc; Galliano, Marie-Florence

    2017-06-01

    Atopic dermatitis (AD) is a common skin inflammatory disease characterized by the production of thymic stromal lymphopoietin (TSLP) and marked TH 2 polarization. Recent studies suggest that IL-1β contributes to the development of AD skin inflammation. Here, we have investigated the impact of IL-1β signalling on the epidermal homeostasis of both healthy subjects and AD patients [with functional filaggrin (FLG) alleles], with particular attention to TSLP production and keratinocyte differentiation. In healthy reconstructed human epidermis (RHE), IL-1β promoted (i) robust secretion of TSLP in an NF-κB-dependent manner and (ii) a significant decrease in the expression of filaggrin and other proteins of the epidermal differentiation complex. These effects were prevented by treatment of RHE with the anti-IL-1β mAb canakinumab and by the IL-1 receptor antagonist anakinra. Interestingly, RHE generated from AD donors behaved like that of healthy individuals and showed comparable responses to IL-1β signals. Collectively, our results suggest that IL-1β may be an early key mediator for the acquisition of an AD phenotype through induction of TSLP and alteration of the epidermal homeostasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  4. Human Platelets Express Functional Thymic Stromal Lymphopoietin Receptors: a Potential Role in Platelet Activation in Acute Coronary Syndrome

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    Boyuan Wang

    2013-12-01

    Full Text Available Background: Thymic stromal lymphopoietin (TSLP has been shown to be expressed in various inflammatory tissues, such as human atherosclerotic plaques. Many types of myeloid cells involved in atherosclerosis, including mast cells, lymphocytes, dendritic cells and monocytes/macrophages, present TSLP receptors (TSLPR. However, it is unknown whether platelets, which also play important roles in atherothrombosis, express TSLPR. Methods and Results: We applied flow cytometry and western blotting to show that TSLPR was expressed on the surface of human platelets. Following the addition of TSLP to platelets, the expression of CD62P, CD63, PAC-1 and p-Akt as well as aggregation and ATP release were increased significantly. A TSLPR antibody and a PI3K (phosphatidylinositol 3-kinase enzyme inhibitor (LY294002 significantly inhibited the platelet activation induced by TSLP. The expression of TSLPR, CD62P and CD63 and the increment of the expression of CD62P and CD63 induced by TSLP in the acute coronary syndrome (ACS group were markedly higher than those in the control group and the stable angina pectoris (SAP group. The expression and the increment of the expression of CD62P and CD63 induced by TSLP were positively correlated with the expression of TSLPR. Conclusion: Human platelets express functional TSLPR, which can be activated by TSLP to promote platelet activation. TSLP/TSLPR functions via activating the PI3K/AKT pathway, and this signalling pathway may be one of the mechanisms involved in thrombosis in ACS. In coronary disease patients, the determination of TSLPR in platelets may help to identify the risk of ACS.

  5. Characterization of CD34+ thymic stromal cells located in the subcapsular cortex of the human thymus

    NARCIS (Netherlands)

    Martínez-Cáceres, E.; Jaleco, A. C.; Res, P.; Noteboom, E.; Weijer, K.; Spits, H.

    1998-01-01

    In this paper we report that suspensions of human fetal thymocytes contain cells that express high levels of CD34 and Thy-1. These cells were characterized with regard to location within the thymus, phenotype, and function. Confocal laser scan analysis of frozen sections of fetal thymus with

  6. Expression of tyrosine kinase gene in mouse thymic stromal cells

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    Rinke de Wit, T. F.; Izon, D. J.; Revilla, C.; Oosterwegel, M.; Bakker, A. Q.; van Ewijk, W.; Kruisbeek, A. M.

    1996-01-01

    Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both

  7. IL-33 and Thymic Stromal Lymphopoietin in mast cell functions

    NARCIS (Netherlands)

    Saluja, Rohit; Zoltowska, Anna; Ketelaar, Maria Elizabeth; Nilsson, Gunnar

    2016-01-01

    Thymic Stromal Lymphopoietin (TSLP) and Interleukin 33 (IL-33) are two cytokines released by cells that are in proximity to our environment, e.g., keratinocytes of the skin and epithelial cells of the airways. Pathogens, allergens, chemicals and other agents induce the release of TSLP and IL-33,

  8. Characterization of human thymic exosomes.

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    Skogberg, Gabriel; Gudmundsdottir, Judith; van der Post, Sjoerd; Sandström, Kerstin; Bruhn, Sören; Benson, Mikael; Mincheva-Nilsson, Lucia; Baranov, Vladimir; Telemo, Esbjörn; Ekwall, Olov

    2013-01-01

    Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.

  9. Characterization of human thymic exosomes.

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    Gabriel Skogberg

    Full Text Available Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.

  10. Directional secretory response of double stranded RNA-induced thymic stromal lymphopoetin (TSLP) and CCL11/eotaxin-1 in human asthmatic airways.

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    Nino, Gustavo; Huseni, Shehlanoor; Perez, Geovanny F; Pancham, Krishna; Mubeen, Humaira; Abbasi, Aleeza; Wang, Justin; Eng, Stephen; Colberg-Poley, Anamaris M; Pillai, Dinesh K; Rose, Mary C

    2014-01-01

    Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral) and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. Primary human bronchial epithelial cells (HBEC) from control (n = 3) and asthmatic (n = 3) donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI) conditions and treated apically with dsRNA (viral surrogate) or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC) from normal (n = 3) and asthmatic (n = 3) donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20) vs. non-asthmatic uninfected controls (n = 20). Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay. Our data demonstrate that: 1) Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2) TSLP exposure induces unidirectional (apical) secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3) Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1. There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations.

  11. Directional secretory response of double stranded RNA-induced thymic stromal lymphopoetin (TSLP and CCL11/eotaxin-1 in human asthmatic airways.

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    Gustavo Nino

    Full Text Available Thymic stromal lymphoproetin (TSLP is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state.Primary human bronchial epithelial cells (HBEC from control (n = 3 and asthmatic (n = 3 donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI conditions and treated apically with dsRNA (viral surrogate or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC from normal (n = 3 and asthmatic (n = 3 donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20 vs. non-asthmatic uninfected controls (n = 20. Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay.Our data demonstrate that: 1 Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2 TSLP exposure induces unidirectional (apical secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3 Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1.There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations.

  12. Lipopolysaccharide attenuates induction of pro-allergic cytokines, thymic stromal lymphopoietin and interleukin 33, in respiratory epithelial cells stimulated with polyI:C and human parechovirus

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    Tsang-Hsiung Lin

    2016-10-01

    Full Text Available Epidemiological studies based on the hygiene hypothesis declare that the level of childhood exposure to environmental microbial products is inversely related to the incidence of allergic diseases in later life. Multiple types of immune cell-mediated immune regulation networks support the hygiene hypothesis. Epithelial cells are the first line of response to microbial products in the environment and bridge the innate and adaptive immune systems; however, their role in the hygiene hypothesis is unknown. To demonstrate the hygiene hypothesis in airway epithelial cells, we examined the effect of lipopolysaccharide (LPS; Toll-like receptor 4 ligand on the expression of the proallergic cytokines thymic stromal lymphopoietin (TSLP and interleukin 33 (IL33 in H292 cells (pulmonary mucoepidermoid carcinoma cells. Stimulation with the TLR ligand polyI:C and human parechovirus type 1 (HPeV1 but not LPS induced TSLP and IL33 through interferon (IFN regulatory factor 3 (IRF3 and NFB activity, which was further validated by using inhibitors (dexamethasone and Bay 11-7082 and shRNA-mediated gene knockdown. Importantly, polyI:C and HPeV1-stimulated TSLP and IL33 induction was reduced by LPS treatment by attenuating TANK-binding kinase 1, IRF3 and NFB activation. Interestingly, the basal mRNA levels of TLR signaling proteins were downregulated with long-term LPS treatment of H292 cells, which suggests that such long-term exposure modulates the expression of innate immunity signaling molecules in airway epithelial cells to mitigate the allergic response. In contrast to the effects of LPS treatment, the alarmin HMBG1 (high mobility group protein B1 acts in synergy with polyI:C to promote TSLP and IL33 expression. Our data support part of the hygiene hypothesis in airway epithelia cells in vitro. In addition to therapeutic targeting of TSLP and IL33, local application of non-pathogenic LPS may be a rational strategy to prevent allergies.

  13. Characterization of human thymic exosomes

    OpenAIRE

    Gabriel Skogberg; Judith Gudmundsdottir; Sjoerd van der Post; Kerstin Sandström; Sören Bruhn; Mikael Benson; Lucia Mincheva-Nilsson; Vladimir Baranov; Esbjörn Telemo; Olov Ekwall

    2013-01-01

    Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytom...

  14. Signal transduction around thymic stromal lymphopoietin (TSLP in atopic asthma

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    Kuepper Michael

    2008-08-01

    Full Text Available Abstract Thymic stromal lymphopoietin (TSLP, a novel interleukin-7-like cytokine, triggers dendritic cell-mediated inflammatory responses ultimately executed by T helper cells of the Th2 subtype. TSLP emerged as a central player in the development of allergic symptoms, especially in the airways, and is a prime regulatory cytokine at the interface of virus- or antigen-exposed epithelial cells and dendritic cells (DCs. DCs activated by epithelium-derived TSLP can promote naïve CD4+ T cells to adopt a Th2 phenotype, which in turn recruite eosinophilic and basophilic granulocytes as well as mast cells into the airway mucosa. These different cells secrete inflammatory cytokines and chemokines operative in inducing an allergic inflammation and atopic asthma. TSLP is, thus, involved in the control of both an innate and an adaptive immune response. Since TSLP links contact of allergen with the airway epithelium to the onset and maintainance of the asthmatic syndrome, defining the signal transduction underlying TSLP expression and function is of profound interest for a better understandimg of the disease and for the development of new therapeutics.

  15. Multiple Functions of the New Cytokine-Based Antimicrobial Peptide Thymic Stromal Lymphopoietin (TSLP

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    Louise Bjerkan

    2016-07-01

    Full Text Available Thymic stromal lymphopoietin (TSLP is a pleiotropic cytokine, hitherto mostly known to be involved in inflammatory responses and immunoregulation. The human tslp gene gives rise to two transcription and translation variants: a long form (lfTSLP that is induced by inflammation, and a short, constitutively-expressed form (sfTSLP, that appears to be downregulated by inflammation. The TSLP forms can be produced by a number of cell types, including epithelial and dendritic cells (DCs. lfTSLP can activate mast cells, DCs, and T cells through binding to the lfTSLP receptor (TSLPR and has a pro-inflammatory function. In contrast, sfTSLP inhibits cytokine secretion of DCs, but the receptor mediating this effect is unknown. Our recent studies have demonstrated that both forms of TSLP display potent antimicrobial activity, exceeding that of many other known antimicrobial peptides (AMPs, with sfTSLP having the strongest effect. The AMP activity is primarily mediated by the C-terminal region of the protein and is localized within a 34-mer peptide (MKK34 that spans the C-terminal α-helical region in TSLP. Fluorescent studies of peptide-treated bacteria, electron microscopy, and liposome leakage models showed that MKK34 exerted membrane-disrupting effects comparable to those of LL-37. Expression of TSLP in skin, oral mucosa, salivary glands, and intestine is part of the defense barrier that aids in the control of both commensal and pathogenic microbes.

  16. Inhibins Tune the Thymocyte Selection Process by Regulating Thymic Stromal Cell Differentiation

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    Ebzadrel Carbajal-Franco

    2015-01-01

    Full Text Available Inhibins and Activins are members of the TGF-β superfamily that regulate the differentiation of several cell types. These ligands were initially identified as hormones that regulate the hypothalamus-pituitary-gonadal axis; however, increasing evidence has demonstrated that they are key regulators in the immune system. We have previously demonstrated that Inhibins are the main Activin ligands expressed in the murine thymus and that they regulate thymocyte differentiation, promoting the DN3-DN4 transition and the selection of SP thymocytes. As Inhibins are mainly produced by thymic stromal cells, which also express Activin receptors and Smad proteins, we hypothesized that Inhibins might play a role in stromal cell differentiation and function. Here, we demonstrate that, in the absence of Inhibins, thymic conventional dendritic cells display reduced levels of MHC Class II (MHCII and CD86. In addition, the ratio between cTECs and mTECs was affected, indicating that mTEC differentiation was favoured and cTEC diminished in the absence of Inhibins. These changes appeared to impact thymocyte selection leading to a decreased selection of CD4SP thymocytes and increased generation of natural regulatory T cells. These findings demonstrate that Inhibins tune the T cell selection process by regulating both thymocyte and stromal cell differentiation.

  17. Current evidence of epidermal barrier dysfunction and thymic stromal lymphopoietin in the atopic march

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    Mei Li

    2014-09-01

    Full Text Available It has long been observed that the development of asthma, allergic rhinitis and food allergy are frequently preceded by atopic dermatitis, a phenomenon known as the “atopic march”. Clinical, genetic and experimental studies have supported the fact that atopic dermatitis could be the initial step of the atopic march, leading to the subsequent development of other atopic diseases. This brief review will focus on the current evidence showing that epidermal barrier dysfunction and the keratinocyte-derived cytokine thymic stromal lymphopoietin play critical roles in the onset of the atopic march.

  18. Immune recovery in acute and chronic HIV infection and the impact of thymic stromal lymphopoietin

    DEFF Research Database (Denmark)

    Gelpi, Marco; Hartling, Hans J; Thorsteinsson, Kristina

    2016-01-01

    BACKGROUND: Symptomatic primary HIV infection is associated with an adverse prognosis, and immediate initiation of combination antiretroviral therapy (cART) is recommended. However, little is known about immunological predictors of immune recovery. Thymic Stromal Lymphopoietin (TSLP) is a cytokine...... that promotes CD4+ T cells homeostatic polyclonal proliferation and regulates Th17/regulatory T-cell balance, immunological functions known to be affected during primary HIV infection. The aim of this study was to describe immune recovery in primary and chronic HIV infection and possible impact of TSLP. METHODS......: Prospective study including 100 HIV-infected individuals (primary HIV infection (N = 14), early presenters (>350 CD4+ T cells/μL, N = 42), late presenters without advanced disease (200-350 CD4+ T cells/μL, N = 24) and with advanced disease (

  19. In situ expression and serum level of thymic stromal lymphopoietin in oral lichen planus.

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    Sun, Mingxia; Tan, Wanye; Liu, Shaohua; Liu, Guixiang; Zhang, Xiaoying; Wang, Ning; Qu, Xun; Wei, Fengcai

    2014-11-01

    Oral lichen planus (OLP) is a chronic inflammatory disease of oral mucosa in which the CD8(+) T cell-mediated cytotoxicity is regarded as a major mechanism of pathogenesis. The main objective of this study is to investigate in situ expression and secretion of thymic stromal lymphopoietin (TSLP) in specimens and sera from patients with oral lichen planus. Thirty-six patients with OLP and 35 donors enrolled in specimen and serum collection. Immunohistochemical method and immunofluorescence double-staining method were used to detect the expression of thymic stromal lymphopoietin and its receptor (TSLPR) together with CD8 in OLP specimens. Enzyme-linked immunosorbent assay (ELISA) was used to detect TSLP secretion. More TSLP- or TSLPR-positive cells showed in OLP specimens than in normal controls, and TSLP-positive cells were mainly in the epithelium, while TSLPR-positive cells mainly in the lamina propria. Furthermore, the number of TSLP-positive cells in the stratum basal was associated with the amount of mononuclear cells infiltrating in the lamina propria of OLP specimens. Among infiltrating mononuclear cells in the lamina propria, some CD8-positive cells also expressed TSLPR. The TSLP serum level of patients with OLP was significantly higher than of healthy donors, but there was no statistically difference between two clinical subtypes of OLP. Our findings provided the first evidence that TSLP may enroll in the pathology of OLP and the TSLP-TSLPR interaction may play an important role in it. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. A stromal myoid cell line provokes thymic erythropoiesis between 16th to 20th weeks of intrauterine life.

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    Tamiolakis, D; Venizelos, J; Kotini, A; Karamanidis, D; Boglou, P; Papadopoulos, N

    2004-02-01

    The thymus provides an optimal cellular and humoral microenvironment for cell line committed differentiation of haematopoietic stem cells. The immigration process requires the secretion of at least one peptide called thymotaxine by cells of the reticulo-epithelial (RE) network of the thymic stromal cellular microenvironment. The thymic RE cells are functionally specialised based on their intrathymic location and this differentiation is modulated by various interaction signals of differentiating thymocytes and other non lymphatic haematopoietic stem cells. To study the role of another cell line in fetal thymic haematopoietic proliferation and differentiation in different stages of development: the stromal myoid cells. Fifteen cases of fetal thymic specimens (4th to 8th weeks: five cases 16th to 20th weeks: five cases and 28th to 32nd weeks: five cases respectively) were studied. Tissue paraffin samples were stained immunohistochemically using (i) a monoclonal antibody recognising alpha-smooth muscle actin, a contractile microfilament expressed exclusively by smooth muscle cells, myofibroblasts and related cells, (ii) a monoclonal antibody glycophorin C recognising the erythropoietic cells. Histology-Embryology Department of Democritus University of Thrace (Alexandroupolis) over ten year period (1991-2001). The number of alpha-smooth muscle actin-positive cells significantly increased during the late second and third trimester of gestation. In the above period a relevant increase in the number of glycophorin C positive cells were observed. Our data suggest that a myoid cell line is involved in the formation of an appropriate microenvironment for homing and proliferation of erythropoietic cells.

  1. The Lymphotoxin Pathway Regulates Aire-Independent Expression of Ectopic Genes and Chemokines in Thymic Stromal Cells1

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    Seach, Natalie; Ueno, Tomoo; Fletcher, Anne L.; Lowen, Tamara; Mattesich, Monika; Engwerda, Christian R.; Scott, Hamish S.; Ware, Carl F.; Chidgey, Ann P.; Gray, Daniel H. D.; Boyd, Richard L.

    2009-01-01

    Medullary thymic epithelial cells (mTEC) play an important and unique role in central tolerance, expressing tissue-restricted Ags (TRA) which delete thymocytes autoreactive to peripheral organs. Since deficiencies in this cell type or activity can lead to devastating autoimmune diseases, it is important to understand the factors which regulate mTEC differentiation and function. Lymphotoxin (LT) ligands and the LTβR have been recently shown to be important regulators of mTEC biology; however, the precise role of this pathway in the thymus is not clear. In this study, we have investigated the impact of this signaling pathway in greater detail, focusing not only on mTEC but also on other thymic stromal cell subsets. LTβR expression was found in all TEC subsets, but the highest levels were detected in MTS-15+ thymic fibroblasts. Rather than directing the expression of the autoimmune regulator Aire in mTEC, we found LTβR signals were important for TRA expression in a distinct population of mTEC characterized by low levels of MHC class II (mTEClow), as well as maintenance of MTS-15+ fibroblasts. In addition, thymic stromal cell subsets from LT-deficient mice exhibit defects in chemokine production similar to that found in peripheral lymphoid organs of Lta−/− and Ltbr−/− mice. Thus, we propose a broader role for LTα1β2-LTβR signaling in the maintenance of the thymic microenvironments, specifically by regulating TRA and chemokine expression in mTEClow for efficient induction of central tolerance. PMID:18390720

  2. Retinoid X receptor ablation in adult mouse keratinocytes generates an atopic dermatitis triggered by thymic stromal lymphopoietin

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    Li, Mei; Messaddeq, Nadia; Teletin, Marius; Pasquali, Jean-Louis; Metzger, Daniel; Chambon, Pierre

    2005-01-01

    To investigate the role of retinoid X receptors (RXRs) in epidermal homeostasis, we generated RXRαβep-/- somatic mutants in which both RXRα and RXRβ are selectively ablated in epidermal keratinocytes of adult mice. These mice develop a chronic dermatitis mimicking that observed in atopic dermatitis (AD) patients. In addition, they exhibit immunological abnormalities including elevated serum levels of IgE and IgG, associated with blood and tissue eosinophilia, indicating that keratinocyte-selective ablation of RXRs also generates a systemic syndrome similar to that found in AD patients. Furthermore, the profile of increased expression of cytokines and chemokines in skin of keratinocyte-selective RXRαβ-ablated mutants was typical of a T helper 2-type inflammation, known to be crucially involved in human AD pathogenesis. Finally, we demonstrate that thymic stromal lymphopoietin, whose expression is rapidly and strongly induced in RXRαβ-ablated keratinocytes, plays a key role in initiating the skin and systemic AD-like pathologies. PMID:16199515

  3. Thymic Epithelial Cell Development and Its Dysfunction in Human Diseases

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    Lina Sun

    2014-01-01

    Full Text Available Thymic epithelial cells (TECs are the key components in thymic microenvironment for T cells development. TECs, composed of cortical and medullary TECs, are derived from a common bipotent progenitor and undergo a stepwise development controlled by multiple levels of signals to be functionally mature for supporting thymocyte development. Tumor necrosis factor receptor (TNFR family members including the receptor activator for NFκB (RANK, CD40, and lymphotoxin β receptor (LTβR cooperatively control the thymic medullary microenvironment and self-tolerance establishment. In addition, fibroblast growth factors (FGFs, Wnt, and Notch signals are essential for establishment of functional thymic microenvironment. Transcription factors Foxn1 and autoimmune regulator (Aire are powerful modulators of TEC development, differentiation, and self-tolerance. Dysfunction in thymic microenvironment including defects of TEC and thymocyte development would cause physiological disorders such as tumor, infectious diseases, and autoimmune diseases. In the present review, we will summarize our current understanding on TEC development and the underlying molecular signals pathways and the involvement of thymus dysfunction in human diseases.

  4. Rhinovirus infection interferes with induction of tolerance to aeroantigens through OX40 ligand, thymic stromal lymphopoietin, and IL-33.

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    Mehta, Amit K; Duan, Wei; Doerner, Astrid M; Traves, Suzanne L; Broide, David H; Proud, David; Zuraw, Bruce L; Croft, Michael

    2016-01-01

    Rhinovirus infection at an early age has been associated with development of asthma, but how rhinovirus influences the immune response is not clear. Tolerance to inhaled antigen is mediated through induction of regulatory T (Treg) cells, and we examined whether rhinovirus infection of the respiratory tract can block airway tolerance by modulating Treg cells. The immune response to intranasal ovalbumin in mice was assessed with concomitant infection with RV1B, and the factors induced in vivo were compared with those made by human lung epithelial cells infected in vitro with RV16. RV1B infection of mice abrogated tolerance induced by inhalation of soluble ovalbumin, suppressing the normal generation of forkhead box protein 3-positive Treg cells while promoting TH2 cells. Furthermore, RV1B infection led to susceptibility to asthmatic lung disease when mice subsequently re-encountered aeroantigen. RV1B promoted early in vivo expression of the TNF family protein OX40 ligand on lung dendritic cells that was dependent on the innate cytokine thymic stromal lymphopoietin (TSLP) and also induced another innate cytokine, IL-33. Inhibiting each of these pathways allowed the natural development of Treg cells while minimizing TH2 differentiation and restored tolerance in the face of RV1B infection. In accordance, RV16 infection of human lung epithelial cells upregulated TSLP and IL-33 expression. These results suggest that infection of the respiratory epithelium with rhinovirus can antagonize tolerance to inhaled antigen through combined induction of TSLP, IL-33, and OX40 ligand and that this can lead to susceptibility to asthmatic lung inflammation. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  5. Thymic Stromal Lymphopoietin Is Critical for Regulation of Proinflammatory Cytokine Response and Resistance to Experimental Trypanosoma congolense Infection

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    Chukwunonso Onyilagha

    2017-07-01

    Full Text Available African trypanosomiasis (sleeping sickness poses serious threat to human and animal health in sub-Saharan Africa. Because there is currently no vaccine for preventing this disease and available drugs are not safe, understanding the mechanisms that regulate resistance and/or susceptibility to the disease could reveal novel targets for effective disease therapy and prevention. Thymic stromal lymphopoietin (TSLP plays a critical role in driving Th2 immune response. Although susceptibility to experimental Trypanosoma congolense infection in mice is associated with excessive proinflammatory responses due in part to impaired Th2 response, the role of TSLP in resistance to African trypanosomiasis has not been well studied. Here, we investigated whether TSLP is critical for maintaining Th2 environment necessary for survival of T. congolense-infected mice. We observed an increased TSLP level in mice after infection with T. congolense, suggesting a role for this cytokine in resistance to the infection. Indeed, TSLPR−/− mice were more susceptible to T. congolense infection and died significantly earlier than their wild-type (WT controls. Interestingly, serum levels of IFN-γ and TNF-α and the frequency of IFN-γ- and TNF-α-producing CD4+ T cells in the spleens and liver were significantly higher in infected TSLPR−/− mice than in the WT control mice. Susceptibility was also associated with excessive M1 macrophage activation. Treatment of TSLPR−/− mice with anti-IFN-γ mAb during infection abolished their enhanced susceptibility to T. congolense infection. Collectively, our study shows that TSLP plays a critical role in resistance to T. congolense infection by dampening the production of proinflammatory cytokines and its associated M1 macrophage activation.

  6. The human thymus microenvironment: in vivo identification of thymic nurse cells and other antigenically-distinct subpopulations of epithelial cells.

    Science.gov (United States)

    Ritter, M A; Sauvage, C A; Cotmore, S F

    1981-11-01

    We have studied the human thymus microenvironment in order to identify subsets of cells that may be responsible for the induction of different aspects of T-lymphocyte differentiation, education and MHC restriction. Using immunofluorescence on tissue sections and cell suspensions we have found MHC products (HLA-A, B, C and DR) to be present throughout the thymus epithelium whilst human T-cell antigens are absent from all non-lymphoid cells. In contrast, Thy-1 antigen (expressed on approximately 1% paediatric human thymocytes) has a differential expression amongst thymic epithelial cells, being confined to those in the subcapsular cortex and to 'thymic nurse cells' (TNC). The former represent the site to which thymocyte precursors first migrate upon entering the thymus. The latter are large epithelial cells, located within the cortex, whose plasma membrane totally enclose a number of thymus lymphocytes; these cells are therefore good candidates for the mediators of direct contact (stromal) induced thymocyte maturation.

  7. Epoc-1: a POU-domain gene expressed in murine epidermal basal cells and thymic stromal cells.

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    Yukawa, K; Yasui, T; Yamamoto, A; Shiku, H; Kishimoto, T; Kikutani, H

    1993-11-15

    POU-domain transcription factors are known as developmental regulators which control organ development and cell phenotypes. In order to clarify the roles of POU-domain transcription factors in cell differentiation, we cloned a novel POU family gene, Epoc-1, from a murine thymus cDNA library. The amino acid (aa) sequence of the POU-specific domain of Epoc-1 is almost identical to those of Oct-1 and Oct-2. However, within the POU-homeodomain, 13 out of 60 aa differ between Epoc-1 and Oct-2. Recombinant Epoc-1 products were found to bind specifically to the octamer sequence. Epoc-1 was found to be expressed in skin, thymus, stomach and testis. In situ hybridization experiments and RNase protection assays indicated that Epoc-1 is expressed in the epidermal basal cells of the skin, which contain stem cells unipotent for keratinocyte differentiation and in thymic stromal elements. These results suggest that Epoc-1 might be one of the developmental regulators which controls epidermal development and thymic organogenesis.

  8. Human thymic epithelial cells express functional HLA-DP molecules

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

    T lymphocytes, we examined whether human thymic epithelial cells (TEC) expressed HLA-DP molecules. We present evidence that TEC obtained from short time culture express low but significant levels of HLA-DP molecules. The expression of HLA-DP molecules was comparable to or higher than the expression...... of HLA-DP allospecific primed lymphocyte typing (PLT) CD4 T cell lines. IFN-gamma treatment strongly upregulated the HLA-DP allospecific PLT responses whereas other PLT responses remained largely unchanged. In conclusion, these data indicate that human thymus epithelial cells express significant levels...

  9. Human stromal (mesenchymal) stem cells

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    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self......-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number...

  10. Genetic Variants of Thymic Stromal Lymphopoietin in Nonsteroidal Anti-Inflammatory Drug-Induced Urticaria/Angioedema.

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    Plaza-Serón, María Del Carmen; Blanca-López, Natalia; Pérez-Sánchez, Natalia; Doña, Inmaculada; Acosta-Herrera, Marialbert; Pino-Yanes, María; Flores, Carlos; Cornejo-García, José Antonio; Perkins, James R; Molina, Ana; Torres, Maria José; Blanca, Miguel; Canto, María Gabriela; Ayuso, Pedro

    2016-01-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most frequent agents involved in hypersensitivity drug reactions, with NSAID-induced urticaria and/or angioedema (NIUA) being the most common entity. Mast cells are key players in NIUA and are activated by thymic stromal lymphopoietin (TSLP). This cytokine functions through recognition by its receptor, composed of IL7Rα (interleukin-7 receptor alpha) and TSLPR (TSLP receptor). These genes have been previously associated with other inflammatory diseases. We assessed the genetic association between single nucleotide polymorphisms (SNPs) in TSLP, IL7R and TSLPR and NIUA in Spanish individuals, using genotyped and imputed data. A total of 369 unrelated NIUA patients and 580 NSAID-tolerant control subjects were included, and 6 SNPs in TSLP, 6 in IL7R and 3 in TSLPR were genotyped. Further variants were imputed using Mach and the 1,000 Genomes Project (Phase 3) data. Association testing and statistical analyses were performed with Mach2dat and R. A total of 139 SNPs were tested for association following quality control. Two SNPs in TSLP (rs1816678 and rs764917) showed a nominal association (p = 0.033 and 0.024, respectively) with NIUA, although these results were not statistically significant after correcting for multiple comparisons. Although TSLP, IL7R and TSLPR are important genes involved in the development of the inflammatory response, we found no significant genetic association with NIUA in our population for common SNPs in these genes. © 2016 S. Karger AG, Basel.

  11. A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor

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    Mao, Yi; Peng, Yudong; Zeng, Qiutang; Cheng, Longxian; Wang, Boyuan; Mao, Xiaobo; Meng, Kai; Liu, Yuzhou; Lian, Yitian; Li, Dazhu

    2015-01-01

    Abnormal expression of thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) was found in patients with acute coronary syndrome. Ticagrelor, an oral platelet ADP P2Y12 receptor antagonist, is widely used in these patients. The aim of this study was to verify whether different doses of ticagrelor regulated plaque progression and platelet activity by modulating TSLP/TSLPR. Seventy-five ApoE-/- mice were randomly divided into five groups: (1) high-cholesterol diet (HCD, n = 15); (2) HCD plus ticagrelor 25 mg/kg/d (T1, n = 15); (3) HCD plus ticagrelor 50 mg/kg/d (T2, n = 15); (4) HCD plus ticagrelor 100 mg/kg/d (T3, n = 15); and (5) a normal diet group (ND, n = 15). At day 0 and at week 16, blood lipids and serum TSLP levels, expression of TSLPR, CD62, and CD63, platelet aggregation, platelet ATP release, PI3K/Akt signaling pathway, and plaque morphology were assessed. HCD increased TSLPR expression and atherosclerosis progression but high-dose ticagrelor (100 mg/kg) moderated this trend. TSLPR was positively correlated with Akt1, platelet aggregation, corrected plaque area, and vulnerability index in the T3 group (Pticagrelor only inhibited platelet activity. Besides this inhibition, high-dose ticagrelor modulated platelet activity and atherosclerosis mediated by TSLPR, potentially through the PI3K/Akt signal pathway. PMID:26517374

  12. A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor.

    Directory of Open Access Journals (Sweden)

    Yi Mao

    Full Text Available Abnormal expression of thymic stromal lymphopoietin (TSLP and its receptor (TSLPR was found in patients with acute coronary syndrome. Ticagrelor, an oral platelet ADP P2Y12 receptor antagonist, is widely used in these patients. The aim of this study was to verify whether different doses of ticagrelor regulated plaque progression and platelet activity by modulating TSLP/TSLPR. Seventy-five ApoE-/- mice were randomly divided into five groups: (1 high-cholesterol diet (HCD, n = 15; (2 HCD plus ticagrelor 25 mg/kg/d (T1, n = 15; (3 HCD plus ticagrelor 50 mg/kg/d (T2, n = 15; (4 HCD plus ticagrelor 100 mg/kg/d (T3, n = 15; and (5 a normal diet group (ND, n = 15. At day 0 and at week 16, blood lipids and serum TSLP levels, expression of TSLPR, CD62, and CD63, platelet aggregation, platelet ATP release, PI3K/Akt signaling pathway, and plaque morphology were assessed. HCD increased TSLPR expression and atherosclerosis progression but high-dose ticagrelor (100 mg/kg moderated this trend. TSLPR was positively correlated with Akt1, platelet aggregation, corrected plaque area, and vulnerability index in the T3 group (P<0.01. In conclusion, low-dose ticagrelor only inhibited platelet activity. Besides this inhibition, high-dose ticagrelor modulated platelet activity and atherosclerosis mediated by TSLPR, potentially through the PI3K/Akt signal pathway.

  13. Myeloid dendritic cells stimulated by thymic stromal lymphopoietin promote Th2 immune responses and the pathogenesis of oral lichen planus.

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    Masaki Yamauchi

    Full Text Available Oral lichen planus (OLP is a chronic inflammatory disease characterized by subepithelial T-cell infiltration. Recent studies reported that specific T helper (Th subsets, especially Th2 cells, are involved in the pathogenesis of OLP. Thymic stromal lymphopoietin (TSLP is mainly secreted by epithelial cells and potently activates myeloid dendritic cells (mDCs to induce Th2-mediated inflammation. Here, we investigated the expression of TSLP and related molecules in OLP. Buccal mucosa specimens from patients with OLP, hyperkeratosis, and ulcer were analyzed by immunohistochemistry for expression of TSLP, its receptor (TSLPR, and inflammatory cells. TSLP was detected in/around the epithelium of patients with OLP and hyperkeratosis, whereas TSLPR, CD11c (mDC, and GATA3 (Th2 were strongly expressed in the subepithelial layer only in OLP patients. Double immunofluorescence staining showed that TSLPR expression mainly co-localized with CD11c. Moreover, the number of CD11c- and GATA-3 positive cells was correlated in OLP patients. In lesions selectively extracted by laser microdissection, the mRNA expression of Th2 (IL-4, MDC, TARC, GATA3- and Th17 (IL-17, RORγt-related molecules in OLP patients was significantly higher than in other groups. These results suggest that CD11c+ mDCs expressing TSLPR contribute to aberrant Th2 immune responses and the pathogenesis of OLP via TSLP stimulation.

  14. Embryonic Trophoblasts Induce Decidual Regulatory T Cell Differentiation and Maternal–Fetal Tolerance through Thymic Stromal Lymphopoietin Instructing Dendritic Cells

    Science.gov (United States)

    Du, Mei-Rong; Guo, Pei-Fen; Piao, Hai-Lan; Wang, Song-Cun; Sun, Chan; Jin, Li-Ping; Tao, Yu; Li, Yan-Hong; Zhang, Di; Zhu, Rui; Fu, Qiang

    2014-01-01

    Physiological pregnancy requires the maternal immune system to recognize and tolerate embryonic Ags. Although multiple mechanisms have been proposed, it is not yet clear how the fetus evades the maternal immune system. In this article, we demonstrate that trophoblast-derived thymic stromal lymphopoietin (TSLP) instructs decidual CD11c+ dendritic cells (dDCs)with increased costimulatory molecules; MHC class II; and Th2/3-type, but not Th1-type, cytokines. TSLP-activated dDCs induce proliferation and differentiation of decidual CD4+CD25− T cells into CD4+CD25+FOXP3+ regulatory T cells (Tregs) through TGF-β1. TSLP-activated dDC–induced Tregs display immunosuppressive features and express Th2-type cytokines. In addition, decidual CD4+CD25+FOXP3+ Tregs promote invasiveness and HLA-G expression of trophoblasts, resulting in preferential production of Th2 cytokines and reduced cytotoxicity in decidual CD56brightCD16− NK cells. Of interest, decreased TSLP expression and reduced numbers of Tregs were observed at the maternal–fetal interface during miscarriage. Our study identifies a novel feedback loop between embryo-derived trophoblasts and maternal decidual leukocytes, which induces a tolerogenic immune response to ensure a successful pregnancy. PMID:24453244

  15. Embryonic trophoblasts induce decidual regulatory T cell differentiation and maternal-fetal tolerance through thymic stromal lymphopoietin instructing dendritic cells.

    Science.gov (United States)

    Du, Mei-Rong; Guo, Pei-Fen; Piao, Hai-Lan; Wang, Song-Cun; Sun, Chan; Jin, Li-Ping; Tao, Yu; Li, Yan-Hong; Zhang, Di; Zhu, Rui; Fu, Qiang; Li, Da-Jin

    2014-02-15

    Physiological pregnancy requires the maternal immune system to recognize and tolerate embryonic Ags. Although multiple mechanisms have been proposed, it is not yet clear how the fetus evades the maternal immune system. In this article, we demonstrate that trophoblast-derived thymic stromal lymphopoietin (TSLP) instructs decidual CD11c(+) dendritic cells (dDCs)with increased costimulatory molecules; MHC class II; and Th2/3-type, but not Th1-type, cytokines. TSLP-activated dDCs induce proliferation and differentiation of decidual CD4(+)CD25(-) T cells into CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) through TGF-β1. TSLP-activated dDC-induced Tregs display immunosuppressive features and express Th2-type cytokines. In addition, decidual CD4(+)CD25(+)FOXP3(+) Tregs promote invasiveness and HLA-G expression of trophoblasts, resulting in preferential production of Th2 cytokines and reduced cytotoxicity in decidual CD56(bright)CD16(-) NK cells. Of interest, decreased TSLP expression and reduced numbers of Tregs were observed at the maternal-fetal interface during miscarriage. Our study identifies a novel feedback loop between embryo-derived trophoblasts and maternal decidual leukocytes, which induces a tolerogenic immune response to ensure a successful pregnancy.

  16. [Effects of thymic stromal lymphopoietin (TSLP) genotypes on asthma phenotypes defined by the atopy cluster -influence of smoking habits-].

    Science.gov (United States)

    Iijima, Hiroaki; Yamada, Hideyasu; Yatagai, Yohei; Kaneko, Yoshiko; Naito, Takashi; Sakamoto, Tohru; Masuko, Hironori; Hirota, Tomomitsu; Tamari, Mayumi; Konno, Satoshi; Nishimura, Masaharu; Hizawa, Nobuyuki

    2014-02-01

    We have previously reported that a distinct sensitization pattern was associated with thymic stromal lymphopoietin (TSLP) genotype. The aim of this study is to identify the characteristics of asthma phenotypes determined by a cluster analysis of IgE responsiveness and the relationship between these phenotypes and TSLP genotypes. We studied 297 patients of adult asthma and 1571 non-asthmatic healthy adults from Ibaraki, a prefecture in central Japan and Kamishihoro, a cedar-free, birch-dominant town in northern Japan. Levels of total serum IgE and specific IgE antibodies towards 14 major inhaled allergens were measured. With the use of these measures, cluster analysis was applied to classify the phenotypes of adult asthma. We also examined the genetic effects of 2 TSLP functional single nucleotide polymorphism (SNPs) on the development of each asthma phenotype using multinomial logistic regression analysis. The cluster analysis identified four distinct clinical phenotypes of asthma, including "Dust mite dominant" (A, N=82), "Multiple pollen" (B, N=14), "Cedar dominant" (C, N=44), and "Low reactivity" (D, N=154). Asthma phenotype A consisted of younger patients with elevated IgE levels and decreased pulmonary function. Asthma phenotype B was characterized by sensitization by many pollen allergens. Asthma phenotype C was not formed in Kamishihoro. Asthma phenotype D was a group of older women who are less atopic. In current or past smokers, both TSLP SNPs (rs2289276 and rs3860933) were associated with the asthma phenotype D (odds ratio 2.11 [1.36-3.30] and 2.11 [1.34-3.33], respectively). In patients with adult asthma who are less atopic, the genetic polymorphisms of TSLP may have some important roles in the development of the disease in smokers.

  17. Flow cytometry imaging identifies rare T(H)2 cells expressing thymic stromal lymphopoietin receptor in a "proallergic" milieu.

    Science.gov (United States)

    Reefer, Amanda J; Hulse, Kathryn E; Lannigan, Josephine A; Solga, Michael D; Wright, Paul W; Kelly, Libby A; Patrie, James; Chapman, Martin D; Woodfolk, Judith A

    2010-11-01

    Thymic stromal lymphopoietin (TSLP) is expressed at sites of allergic inflammation, including eczematous skin. This cytokine has been reported to exert its T(H)2-inducing properties through dendritic cells. Expression of TSLP receptor on the surface of activated T(H)2 cells could amplify T(H)2 responses at inflamed sites through the direct actions of TSLP. To test rigorously whether T(H)2 cells induced by "proallergic" factors express TSLP receptor and characterize these cells using an experimental platform that combines flow cytometry with microscopic capabilities. CD4(+) T cells isolated from patients with atopic dermatitis or normal healthy controls were cocultured with autologous dendritic cells in the presence of T(H)2-promoting stimuli (TSLP ± allergen and staphylococcal enterotoxin B ± TSLP). Surface expression of TSLP receptor was analyzed by image-based flow cytometry, and responsiveness of purified T cells to TSLP was assessed by phosphorylation of signal transducer and activator of transcription-5 and cytokine secretion. T(H)2-promoting stimuli induced a robust population of activated T(H)2 cells (CD25(+)IL-4(+)). Regardless of the nature of the stimulus, flow cytometry imaging confirmed that T cells expressing TSLP receptor were rare, constituting a minor fraction of the IL-4(+) T cell pool; however, TSLP responsiveness was nonetheless detectable. Analysis of cell size and nuclear morphology revealed preferential expression of TSLP receptor on IL-4-expressing cells undergoing mitosis. Analysis of lesional skin in atopic dermatitis supported the view that rare IL-4(+) T cells expressing TSLP receptor are present at inflamed sites. In a "proallergic" milieu, TSLP receptor is preferentially expressed on rare actively dividing T(H)2 cells. The direct action of TSLP on T cells could amplify T(H)2 responses at sites of allergic inflammation. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  18. Phloxine O, a Cosmetic Colorant, Suppresses the Expression of Thymic Stromal Lymphopoietin and Acute Dermatitis Symptoms in Mice.

    Science.gov (United States)

    Lee, Hye Eun; Yang, Gabsik; Kim, Kyu-Bong; Lee, Byung-Mu; Lee, Joo Young

    2018-01-09

    Cosmetics are primarily applied to the skin; therefore, the association of cosmetic dyes with skin diseases or inflammation is a topic of great interest. Thymic stromal lymphopoietin (TSLP) is an interleukin 7-like cytokine that activates dendritic cells to promote Th2 inflammatory immune responses. TSLP is highly expressed in keratinocytes under inflammatory conditions, which suggests that it may play a critical role in the development of skin diseases, such as atopic dermatitis. Therefore, we investigated whether cosmetic dyes influenced the production of TSLP by keratinocytes. Phloxine O, also known as D&C Red No.27, is one of the most common red synthetic pigments and is widely used in colored cosmetics. Our results showed that Phloxine O downregulated phorbol 12-myristate 13-acetate-induced production of TSLP in a murine keratinocyte cell line (PAM212). Phloxine O also suppressed TSLP expression in KCMH-1 cells, which are mouse keratinocytes that constitutively produce high levels of TSLP. To investigate the in vivo effects of Phloxine O, we induced TSLP expression in mouse ear skin by topically applying MC903, a vitamin D3 analogue that is a well-known inducer of atopic dermatitis-like symptoms. Topical application of Phloxine O prevented MC903-induced TSLP production in mouse ear skin, attenuated the acute dermatitis-like symptoms and decreased serum IgE and histamine levels in mice. Suppression of TSLP expression by Phloxine O correlated with reduced expression of OX40 ligand and Th2 cytokines in mouse ear skin. Our results showed that Phloxine O may be beneficial to prevent dermatitis by suppressing the expression of TSLP and Th2 cytokines in skin.

  19. Interleukin-7, but not thymic stromal lymphopoietin, plays a key role in the T cell response to influenza A virus.

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    Adam W Plumb

    Full Text Available The immune response to viral infection is ideally rapid and specific, resulting in viral clearance and establishment of immune memory. Some viruses such as HIV can evade such responses leading to chronic infection, while others like Influenza A can elicit a severe inflammatory response with immune-related complications including death. Cytokines play a major role in shaping the appropriate outcomes to infection. While Interleukin-7 (IL-7 has a critical role in T and B cell development, treatment with IL-7 has recently been shown to aid the adaptive T cell response in clearance of chronic viral infection. In contrast, the IL-7-related cytokine thymic stromal lymphopoietin (TSLP has a limited role in lymphocyte development but is important in the immune response to parasitic worms and allergens. The role for these cytokines in the immune response to an acute viral infection is unclear. IL-7 and TSLP share IL-7Rα as part of their heterodimeric receptors with the gamma common chain (γc and TSLPR, respectively. We investigated the role of IL-7 and TSLP in the primary immune response to influenza A infection using hypomorphic IL-7Rα (IL-7Rα(449F and TSLPR(-/- mice. We found that IL-7, but not TSLP, plays an important role in control of influenza A virus. We also showed that IL-7 signaling was necessary for the generation of a robust influenza A-specific CD4 and CD8 T cell response and that this requirement is intrinsic to CD8 T cells. These findings demonstrate a significant role for IL-7 during acute viral infection.

  20. Andrographolide suppresses thymic stromal lymphopoietin in phorbol myristate acetate/calcium ionophore A23187-activated mast cells and 2,4-dinitrofluorobenzene-induced atopic dermatitis-like mice model

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    Li CX

    2016-02-01

    Full Text Available Chun-xiao Li,* Hua-guo Li,* Hui Zhang,* Ru-hong Cheng, Ming Li, Jian-ying Liang, Yan Gu, Bo Ling, Zhi-rong Yao, Hong Yu Department of Dermatology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Atopic dermatitis (AD is one of the most common inflammatory cutaneous diseases. Thymic stromal lymphopoietin (TSLP has been demonstrated to be an important immunologic factor in the pathogenesis of AD. The production of TSLP can be induced by a high level of intracellular calcium concentration and activation of the receptor-interacting protein 2/caspase-1/NF-κB pathway. Andrographolide (ANDRO, a natural bicyclic diterpenoid lactone, has been found to exert anti-inflammatory effects in gastrointestinal inflammatory disorders through suppressing the NF-κB pathway. Objective: To explore the effect of ANDRO on the production of TSLP in human mast cells and AD mice model. Methods: We utilized enzyme-linked immunosorbent assay, real-time reverse transcription polymerase chain reaction analysis, Western blot analysis, and immunofluorescence staining assay to investigate the effects of ANDRO on AD. Results: ANDRO ameliorated the increase in the intracellular calcium, protein, and messenger RNA levels of TSLP induced by phorbol myristate acetate/calcium ionophore A23187, through the blocking of the receptor-interacting protein 2/caspase-1/NF-κB pathway in human mast cell line 1 cells. ANDRO, via oral or local administration, also attenuated clinical symptoms in 2,4-dinitrofluorobenzene-induced AD mice model and suppressed the levels of TSLP in lesional skin. Conclusion: Taken together, ANDRO may be a potential therapeutic agent for AD through suppressing the expression of TSLP. Keywords: atopic dermatitis, thymic stromal lymphopoietin, andrographolide, human mast cell

  1. Rag defects and thymic stroma: lessons from animal models

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    Veronica eMarrella

    2014-06-01

    Full Text Available Thymocytes and thymic epithelial cells (TECs cross-talk is essential to support T-cell development and preserve thymic architecture and maturation of TECs and Foxp3+ natural regulatory T (nTreg cells. Accordingly, disruption of thymic lymphostromal cross-talk may have major implications on the thymic mechanisms that govern T cell tolerance. Several genetic defects have been described in humans that affect early stages of T cell development (leading to Severe Combined Immune Deficiency, SCID or late stages in thymocyte maturation (resulting in combined immunodeficiency. Hypomorphic mutations in SCID-causing genes may allow for generation of a limited pool of T lymphocytes with a restricted repertoire. These conditions are often associated with infiltration of peripheral tissues by activated T cells and immune dysregulation, as best exemplified by Omenn syndrome (OS. In this review, we will discuss our recent findings on abnormalities of thymic microenvironment in OS with a special focus of defective maturation of TECs, altered distribution of thymic dendritic cells (DCs and impairment of deletional and non-deletional mechanisms of central tolerance. Here, taking advantage of mouse models of OS and atypical SCID, we will discuss how modifications in stromal compartment impact and shape lymphocyte differentiation, and vice versa how inefficient T cell signalling results in defective stromal maturation. These findings are instrumental to understand the extent to which novel therapeutic strategies should act on thymic stroma to achieve full immune reconstitution.

  2. Association between larger thymic size and higher thymic output in human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy

    DEFF Research Database (Denmark)

    Kolte, Lilian; Dreves, Anne-Mette; Ersbøll, Annette K

    2002-01-01

    To examine the impact of thymic size on immune recovery in patients with human immunodeficiency virus (HIV) infection, the thymus was visualized, using computed tomographic scans, in 25 HIV-infected patients who had received highly active antiretroviral therapy (HAART) for 6-18 months and had...

  3. Thymic stromal lymphopoietin polymorphisms and allergic rhinitis risk: a systematic review and meta-analysis with 6351 cases and 11472 controls.

    Science.gov (United States)

    Sun, Qiuzhen; Liu, Yuehui; Zhang, Shaorong; Liu, Ke; Zhu, Xinhua; Liu, Jianguo; Yang, Chunping

    2015-01-01

    Previous studies suggested a close association between the thymic stromal lymphopoietin (TSLP) genetic variants and allergic diseases. Here, we explored the correlation between the TSLP polymorphisms and allergic rhinitis susceptibility using meta-analysis. We searched PubMed, Ovid, Cochrane libraries, CNKI, Wanfang, and CQVIP databases until Apr 19, 2015. Quality assessment was conducted for each article according to the Strengthening the Reporting of Genetic Association studies (STREGA). The pooled measure was calculated as the inverse variance-weighted mean of the logarithm of the OR with 95% CI to assess the strength of association between TSLP polymorphisms and the risk of allergic rhinitis. Five case-control studies with 6351 cases and 11472 controls were included in this study. TSLP rs1898671 polymorphism was significantly associated with an increased risk of allergic rhinitis (OR=1.13; 95% CI, 1.07-1.20; Prisk of allergic rhinitis in Caucasian (OR=1.14; 95% CI, 1.08-1.21; Prisk of allergic rhinitis (OR=1.13; 95% CI, 1.07-1.21; Pallergic rhinitis risk, the meta-analyses were not performed. In summary, we demonstrated that TSLP rs1898671 polymorphism was associated with a higher risk for allergic rhinitis.

  4. Targeting allergen to FcgammaRI reveals a novel T(H)2 regulatory pathway linked to thymic stromal lymphopoietin receptor.

    Science.gov (United States)

    Hulse, Kathryn E; Reefer, Amanda J; Engelhard, Victor H; Patrie, James T; Ziegler, Steven F; Chapman, Martin D; Woodfolk, Judith A

    2010-01-01

    The molecule H22-Fel d 1, which targets cat allergen to FcgammaRI on dendritic cells (DCs), has the potential to treat cat allergy because of its T-cell modulatory properties. We sought to investigate whether the T-cell response induced by H22-Fel d 1 is altered in the presence of the T(H)2-promoting cytokine thymic stromal lymphopoietin (TSLP). Studies were performed in subjects with cat allergy with and without atopic dermatitis. Monocyte-derived DCs were primed with H22-Fel d 1 in the presence or absence of TSLP, and the resulting T-cell cytokine repertoire was analyzed by flow cytometry. The capacity for H22-Fel d 1 to modulate TSLP receptor expression on DCs was examined by flow cytometry in the presence or absence of inhibitors of Fc receptor signaling molecules. Surprisingly, TSLP alone was a weak inducer of T(H)2 responses irrespective of atopic status; however, DCs coprimed with TSLP and H22-Fel d 1 selectively and synergistically amplified T(H)2 responses in highly atopic subjects. This effect was OX40 ligand independent, pointing to an unconventional TSLP-mediated pathway. Expression of TSLP receptor was upregulated on atopic DCs primed with H22-Fel d 1 through a pathway regulated by FcgammaRI-associated signaling components, including src-related tyrosine kinases and Syk, as well as the downstream molecule phosphoinositide 3-kinase. Inhibition of TSLP receptor upregulation triggered by H22-Fel d 1 blocked TSLP-mediated T(H)2 responses. Discovery of a novel T(H)2 regulatory pathway linking FcgammaRI signaling to TSLP receptor upregulation and consequent TSLP-mediated effects questions the validity of receptor-targeted allergen vaccines. Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  5. TNF-related apoptosis-inducing ligand (TRAIL) regulates midline-1, thymic stromal lymphopoietin, inflammation, and remodeling in experimental eosinophilic esophagitis.

    Science.gov (United States)

    Collison, Adam M; Sokulsky, Leon A; Sherrill, Joseph D; Nightingale, Scott; Hatchwell, Luke; Talley, Nicholas J; Walker, Marjorie M; Rothenberg, Marc E; Mattes, Joerg

    2015-10-01

    Eosinophilic esophagitis (EoE) is an inflammatory disorder of the esophagus defined by eosinophil infiltration and tissue remodeling with resulting symptoms of esophageal dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) promotes inflammation through upregulation of the E3 ubiquitin-ligase midline-1 (MID1), which binds to and deactivates the catalytic subunit of protein phosphatase 2Ac, resulting in increased nuclear factor κB activation. We sought to elucidate the role of TRAIL in EoE. We used Aspergillus fumigatus to induce EoE in TRAIL-sufficient (wild-type) and TRAIL-deficient (TRAIL(-/-)) mice and targeted MID1 in the esophagus with small interfering RNA. We also treated mice with recombinant thymic stromal lymphopoietin (TSLP) and TRAIL. TRAIL deficiency and MID1 silencing with small interfering RNA reduced esophageal eosinophil and mast cell numbers and protected against esophageal circumference enlargement, muscularis externa thickening, and collagen deposition. MID1 expression and nuclear factor κB activation were reduced in TRAIL(-/-) mice, whereas protein phosphatase 2Ac levels were increased compared with those seen in wild-type control mice. This was associated with reduced expression of CCL24, CCL11, CCL20, IL-5, IL-13, IL-25, TGFB, and TSLP. Treatment with TSLP reconstituted hallmark features of EoE in TRAIL(-/-) mice and recombinant TRAIL induced esophageal TSLP expression in vivo in the absence of allergen. Post hoc analysis of gene array data demonstrated significant upregulation of TRAIL and MID1 in a cohort of children with EoE compared with that seen in controls. TRAIL regulates MID1 and TSLP, inflammation, fibrosis, smooth muscle hypertrophy, and expression of inflammatory effector chemokines and cytokines in experimental EoE. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Epithelial cells prime the immune response to an array of gut-derived commensals towards a tolerogenic phenotype through distinct actions of thymic stromal lymphopoietin and transforming growth factor-β

    Science.gov (United States)

    Zeuthen, Louise Hjerrild; Fink, Lisbeth Nielsen; Frokiaer, Hanne

    2008-01-01

    Humans and other mammals coexist with a diverse array of microbes colonizing the intestine, termed the microflora. The relationship is symbiotic, with the microbes benefiting from a stable environment and nutrient supply, and the host gaining competitive exclusion of pathogens and continuously maintenance of the gut immune homeostasis. Here we report novel crosstalk mechanisms between the human enterocyte cell line, Caco2, and underlying human monocyte-derived DC in a transwell model where Gram-positive (G+) commensals prevent Toll-like receptor-4 (TLR4)-dependent Escherichia coli-induced semimaturation in a TLR2-dependent fashion. These findings add to our understanding of the hypo-responsiveness of the gut epithelium towards the microflora. Gut DC posses a more tolerogenic phenotype than conventional DC. Here we show that Caco2 spent medium (SM) induces tolerogenic DC with lower expression of maturation markers, interleukin (IL)-12p70, and tumour necrosis factor-α when matured with G+ and Gram-negative (G–) commensals, while IL-10 production is enhanced in DC upon encountering G+ commensals and reduced upon encountering G– bacteria. The Caco2 SM-induced tolerogenic phenotype is also seen in DC priming of naive T cells with elevated levels of transforming growth factor-β (TGF-β) and markedly reduced levels of bacteria-induced interferon-γ production. Caco2 cell production of IL-8, thymic stromal lymphopoietin (TSLP) and TGF-β increases upon microbial stimulation in a strain dependent manner. TSLP and TGF-β co-operate in inducing the tolerogenic DC phenotype but other mediators might be involved. PMID:17655740

  7. Functional Domains of Autoimmune Regulator (AIRE) Modulate INS-VNTR Transcription in Human Thymic Epithelial Cells.

    Science.gov (United States)

    Sparks, Avis E; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

    2016-05-20

    INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Functional Domains of Autoimmune Regulator (AIRE) Modulate INS-VNTR Transcription in Human Thymic Epithelial Cells*

    Science.gov (United States)

    Sparks, Avis E.; Chen, Chiachen; Breslin, Mary B.; Lan, Michael S.

    2016-01-01

    INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity. PMID:27048654

  9. Brd4 bridges the transcriptional regulators, Aire and P-TEFb, to promote elongation of peripheral-tissue antigen transcripts in thymic stromal cells.

    Science.gov (United States)

    Yoshida, Hideyuki; Bansal, Kushagra; Schaefer, Uwe; Chapman, Trevor; Rioja, Inmaculada; Proekt, Irina; Anderson, Mark S; Prinjha, Rab K; Tarakhovsky, Alexander; Benoist, Christophe; Mathis, Diane

    2015-08-11

    Aire controls immunologic tolerance by inducing a battery of thymic transcripts encoding proteins characteristic of peripheral tissues. Its unusually broad effect is achieved by releasing RNA polymerase II paused just downstream of transcriptional start sites. We explored Aire's collaboration with the bromodomain-containing protein, Brd4, uncovering an astonishing correspondence between those genes induced by Aire and those inhibited by a small-molecule bromodomain blocker. Aire:Brd4 binding depended on an orchestrated series of posttranslational modifications within Aire's caspase activation and recruitment domain. This interaction attracted P-TEFb, thereby mobilizing downstream transcriptional elongation and splicing machineries. Aire:Brd4 association was critical for tolerance induction, and its disruption could account for certain point mutations that provoke human autoimmune disease. Our findings evoke the possibility of unanticipated immunologic mechanisms subtending the potent antitumor effects of bromodomain blockers.

  10. Differential susceptibility of human thymic dendritic cell subsets to X4 and R5 HIV-1 infection.

    Science.gov (United States)

    Schmitt, Nathalie; Nugeyre, Marie-Thérèse; Scott-Algara, Daniel; Cumont, Marie-Christine; Barré-Sinoussi, Françoise; Pancino, Gianfranco; Israël, Nicole

    2006-02-28

    Human thymus can be infected by HIV-1 with potential consequences on immune regeneration and homeostasis. We previously showed that CD4 thymocytes preferentially replicate CXCR4 tropic (X4) HIV-1 dependently on interleukin (IL)-7. Here we addressed the susceptibility of thymic dendritic cells (DC) to HIV-1 infection. We investigated the replication ability of CXCR4 or CCR5 (R5) tropic HIV-1 in thymic micro-explants as well as in isolated thymic CD11clowCD14- DC, CD11chighCD14+ DC and plasmacytoid DC subsets. Thymic tissue was productively infected by both X4 and R5 viruses. However, X4 but not R5 HIV-1 replication was enhanced by IL-7 in thymic micro-explants, suggesting that R5 virus replication occurred in cells other than thymocytes. Indeed, we found that R5 HIV-1 replicated efficiently in DC isolated from thymic tissue. The replicative capacity of X4 and R5 viruses differed according to the different DC subsets. R5 but not X4 HIV-1 efficiently replicated in CD11chighCD14+ DC. In contrast, no HIV-1 replication was detected in CD11clowCD14- DC. Both X4 and R5 viruses efficiently replicated in plasmacytoid DC, which secreted interferon-alpha upon HIV-1 exposure. Productive HIV-1 infection also caused DC loss, consistent with different permissivity of each DC subset. Thymic DC sustain high levels of HIV-1 replication. DC might thus be the first target for R5 HIV-1 infection of thymus, acting as a Trojan horse for HIV-1 spread to thymocytes. Furthermore, DC death induced by HIV-1 infection may affect thymopoiesis.

  11. Stromal differentiation and architecture of the human umbilical cord.

    Science.gov (United States)

    Nanaev, A K; Kohnen, G; Milovanov, A P; Domogatsky, S P; Kaufmann, P

    1997-01-01

    In order to assess the characteristics of its stromal cells and the distribution of extracellular matrix proteins, we investigated, immunohistochemically and ultrastructurally, term, first and second trimester human umbilical cords. A differential distribution pattern of the various cytoskeletal proteins of stromal cells and extracellular matrix proteins was observed in different zones of the stroma, the subamniotic stroma, Wharton's jelly, and the vessels' adventitia. All three zones showed immunoreactivities for collagen types I, III and VI and for basement membrane molecules such as collagen type IV, laminin and heparan sulphate proteoglycan. Immunoreactivities for these extracellular matrix molecules were observed around cleft-like territories (stromal clefts) in the Wharton's jelly which were occupied by homogeneous ground substance but void of collagen fibrils and basal lamina molecules. Moreover, between the stromal clefts, slender cells were found which immunohistochemically and ultrastructurally corresponded to various stages of myofibroblastic differentiation. In earlier stages of gestation, stromal cells with a less complex expression pattern prevailed. The stromal clefts and the contractile cells together might serve as a system regulating the turgor of the cord.

  12. Mice engrafted with human fetal thymic tissue and hematopoietic stem cells develop pathology resembling chronic graft-versus-host disease.

    Science.gov (United States)

    Lockridge, Jennifer L; Zhou, Ying; Becker, Yusof A; Ma, Shidong; Kenney, Shannon C; Hematti, Peiman; Capitini, Christian M; Burlingham, William J; Gendron-Fitzpatrick, Annette; Gumperz, Jenny E

    2013-09-01

    Chronic graft-versus-host disease (cGVHD) is a significant roadblock to long-term hematopoietic stem cell (HSC) transplantation success. Effective treatments for cGVHD have been difficult to develop, in part because of a paucity of animal models that recapitulate the multiorgan pathologies observed in clinical cGVHD. Here we present an analysis of the pathology that occurs in immunodeficient mice engrafted with human fetal HSCs and implanted with fragments of human fetal thymus and liver. Starting at time points generally later than 100 days post-transplantation, the mice developed signs of illness, including multiorgan cellular infiltrates containing human T cells, B cells, and macrophages; fibrosis in sites such as lungs and liver; and thickened skin with alopecia. Experimental manipulations that delayed or reduced the efficiency of the HSC engraftment did not affect the timing or progression of disease manifestations, suggesting that pathology in this model is driven more by factors associated with the engrafted human thymic organoid. Disease progression was typically accompanied by extensive fibrosis and degradation of the thymic organoid, and there was an inverse correlation of disease severity with the frequency of FoxP3(+) thymocytes. Hence, the human thymic tissue may contribute T cells with pathogenic potential, but the generation of regulatory T cells in the thymic organoid may help to control these cells before pathology resembling cGVHD eventually develops. This model thus provides a new system to investigate disease pathophysiology relating to human thymic events and to evaluate treatment strategies to combat multiorgan fibrotic pathology produced by human immune cells. Copyright © 2013 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  13. Thymic carcinoma

    Directory of Open Access Journals (Sweden)

    Vitória Homem Machado

    2016-10-01

    Full Text Available Thymic carcinomas are a heterogeneous group of aggressive, invasive epithelial malignancies. Their incidence is rare, occurring predominantly in middle-aged men. Here we present the typical imaging findings of a thymic carcinoma. The combination of imaging characteristics with tumor location and patient age provides a roadmap for approaching the differential diagnosis. Keywords: Thymus Gland; carcinoma; mediastinal neoplasms

  14. Cloning of rat thymic stromal lymphopoietin receptor (TSLPR) and characterization of genomic structure of murine Tslpr gene

    DEFF Research Database (Denmark)

    Blagoev, Blagoy; Nielsen, Mogens M; Angrist, Misha

    2002-01-01

    , a cytokine involved in B- and T-cell function. We have cloned the TSLP receptor from rat and find that the WSXWX motif commonly found in extracellular domains of cytokine receptors is conserved as a W(T/S)XV(T/A) motif among TSLP receptors from mouse, rat and human. As in the mouse, TSLP receptor is widely...

  15. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells

    NARCIS (Netherlands)

    Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  16. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Mentink-Leusink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  17. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  18. Human mesenchymal stromal cells : biological characterization and clinical application

    NARCIS (Netherlands)

    Bernardo, Maria Ester

    2010-01-01

    This thesis focuses on the characterization of the biological and functional properties of human mesenchymal stromal cells (MSCs), isolated from different tissue sources. The differentiation capacity of MSCs from fetal and adult tissues has been tested and compared. Umbilical cord blood (UCB) has

  19. Human proT-cells generated in vitro facilitate hematopoietic stem cell-derived T-lymphopoiesis in vivo and restore thymic architecture

    National Research Council Canada - National Science Library

    Awong, Génève; Singh, Jastaranpreet; Mohtashami, Mahmood; Malm, Maria; La Motte-Mohs, Ross N; Benveniste, Patricia M; Serra, Pablo; Herer, Elaine; van den Brink, Marcel R; Zúñiga-Pflücker, Juan Carlos

    2013-01-01

    .... Here, we addressed whether in vitro-derived human progenitor T (proT)-cells could not only represent a source of thymus-seeding progenitors, but also able to influence the recovery of the thymic microenvironment...

  20. Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

    Science.gov (United States)

    Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

    2017-01-01

    Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

  1. Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

    Science.gov (United States)

    Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

    2017-05-01

    Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3(+)TCR-αβ(+) single-positive CD8(+) or CD4(+) cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

  2. Human thymic epithelial cells present superantigens to T-cell lines and thymocytes

    DEFF Research Database (Denmark)

    Jlrgensen, A; Nielsen, M; Svejgaard, A

    1996-01-01

    It is generally accepted that thymic epithelial cells (TEC) act as accessory cells in positive selection of pre-T cells. However, our knowledge of the antigen presentation and accessory cell function to human TEC is limited. Here we present results obtained by the use of serum-free cultured human...... TEC, showing that IFN-gamma-treated TEC are able to support T-cell-mediated responses to the bacterial superantigens (Sag) SEA and SEB, even at very low Sag concentrations. T-cell responses to TEC-presented Sags were dependent on the presence of the adhesion molecules ICAM-1, ICAM-2, LFA-1, and LFA-3......, but not on CD4 and CD8 molecules. There is a low but significant expression of B7 molecules on human TEC, and treatment of TEC with anti-B7.1 and anti-B7.2 antibodies before Sag pulsing leads to decreased Sag responses, indicating a significant importance of B7 molecules on TEC. Both CD4+ T-cell lines and CD4...

  3. Epigenetic Classification of Human Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Danilo Candido de Almeida

    2016-02-01

    Full Text Available Standardization of mesenchymal stromal cells (MSCs is hampered by the lack of a precise definition for these cell preparations; for example, there are no molecular markers to discern MSCs and fibroblasts. In this study, we followed the hypothesis that specific DNA methylation (DNAm patterns can assist classification of MSCs. We utilized 190 DNAm profiles to address the impact of tissue of origin, donor age, replicative senescence, and serum supplements on the epigenetic makeup. Based on this, we elaborated a simple epigenetic signature based on two CpG sites to classify MSCs and fibroblasts, referred to as the Epi-MSC-Score. Another two-CpG signature can distinguish between MSCs from bone marrow and adipose tissue, referred to as the Epi-Tissue-Score. These assays were validated by site-specific pyrosequencing analysis in 34 primary cell preparations. Furthermore, even individual subclones of MSCs were correctly classified by our epigenetic signatures. In summary, we propose an alternative concept to use DNAm patterns for molecular definition of cell preparations, and our epigenetic scores facilitate robust and cost-effective quality control of MSC cultures.

  4. Nanoparticle labeling identifies slow cycling human endometrial stromal cells

    Science.gov (United States)

    2014-01-01

    Introduction Evidence suggests that the human endometrium contains stem or progenitor cells that are responsible for its remarkable regenerative capability. A common property of somatic stem cells is their quiescent state. It remains unclear whether slow-cycling cells exist in the human endometrium. We hypothesized that the human endometrium contains a subset of slow-cycling cells with somatic stem cell properties. Here, we established an in vitro stem cell assay to isolate human endometrial-derived mesenchymal stem-like cells (eMSC). Methods Single-cell stromal cultures were initially labeled with fluorescent nanoparticles and a small population of fluorescent persistent cells (FPC) remained after culture of 21 days. Two populations of stromal cells, namely FPC and non-FPC were sorted. Results Quantitative analysis of functional assays demonstrated that the FPC had higher colony forming ability, underwent more rounds of self-renewal and had greater enrichment of phenotypically defined prospective eMSC markers: CD146+/CD140b+ and W5C5+ than the non-FPC. They also differentiate into multiple mesenchymal lineages and the expression of lineage specific markers was lower than that of non-FPC. The FPC exhibit low proliferation activities. A proliferation dynamics study revealed that more FPC had a prolonged G1 phase. Conclusions With this study we present an efficient method to label and isolate slow-proliferating cells obtained from human endometrial stromal cultures without genetic modifications. The FPC population could be easily maintained in vitro and are of interest for tissue-repair and engineering perspectives. In summary, nanoparticle labeling is a promising tool for the identification of putative somatic stem or progenitor cells when their surface markers are undefined. PMID:24996487

  5. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu....... It is concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes....

  6. A reliable and simplified sj/beta-TREC ratio quantification method for human thymic output measurement.

    Science.gov (United States)

    Ferrando-Martínez, Sara; Franco, Jaime M; Ruiz-Mateos, Ezequiel; Hernández, Ana; Ordoñez, Antonio; Gutierrez, Encarnación; Leal, Manuel

    2010-01-31

    Current techniques to peripherally assess thymic function are: the signal-joint T-cell receptor excision circle (sj-TREC) level measurement and the naive T cell and CD31+ TREC-rich subset determination. However, all of them are indirect approaches and none could be considered a direct recent thymic emigrant (RTE) marker. To overcome their limitations, Dion et al. (2004) described the sj/beta-TREC ratio that allows the peripheral quantification of the double negative to double positive intrathymic proliferation step. Nevertheless, the protocol described is expensive, sample and time-consuming, thus, limiting its usefulness. In this study, we describe a simplified protocol that reduces from 33 to 9 the amount of PCR reaction needed but maintaining the sensitivity and reproducibility of the original technique. In addition, we corroborated the effectiveness of our technique as an accurate thymic output-related marker by correlating the peripheral sj/beta-TREC ratio with a direct measurement of thymic function as the percentage of double positive thymocytes (r=0.601, p<0.001). Copyright 2009 Elsevier B.V. All rights reserved.

  7. Transcription of thymic stromal lymphopoietin via Toll-like receptor 2 in canine keratinocytes: a possible association of Staphylococcus spp. in the deterioration of allergic inflammation in canine atopic dermatitis.

    Science.gov (United States)

    Sakamoto, Mayu; Asahina, Ryota; Kamishina, Hiroaki; Maeda, Sadatoshi

    2016-06-01

    Colonization, overgrowth and subsequent infection by Staphylococcus spp. is frequently observed in canine atopic dermatitis (CAD), where it contributes to the intensity of cutaneous inflammation. The mechanisms by which staphylococci contribute to the pathogenesis of CAD are unclear. Studies suggest that thymic stromal lymphopoietin (TSLP), a cytokine induced by a cell wall component of Staphylococcus spp., may play a critical role in Th2 responses including the pathogenesis of CAD. To determine if synthetic triacylated lipopeptide (TLR1/2 ligand), a cell wall component of Staphylococcus spp., induces the transcription of TSLP via TLR2 in canine keratinocytes. Transcription of TSLP was quantified in a canine keratinocyte cell line after stimulation with synthetic triacylated lipopeptide, and again after inhibition of TLR2 by a targeted small interfering RNA. The transcription of TSLP was enhanced 6 h after stimulation with the synthetic triacylated lipopeptide; it was completely suppressed by knockdown of TLR2. The results demonstrated that a synthetic cell wall component of Staphylococcus spp. induced transcription of TSLP via TLR2 in canine keratinocytes. Additional studies will be required to investigate whether Staphylococcus spp. contributes to Th2 responses in CAD through TLR2-mediated TSLP production. © 2016 ESVD and ACVD.

  8. PPARgamma Deficiency Counteracts Thymic Senescence

    Directory of Open Access Journals (Sweden)

    David Ernszt

    2017-11-01

    Full Text Available Thymic senescence contributes to increased incidence of infection, cancer and autoimmunity at senior ages. This process manifests as adipose involution. As with other adipose tissues, thymic adipose involution is also controlled by PPARgamma. This is supported by observations reporting that systemic PPARgamma activation accelerates thymic adipose involution. Therefore, we hypothesized that decreased PPARgamma activity could prevent thymic adipose involution, although it may trigger metabolic adverse effects. We have confirmed that both human and murine thymic sections show marked staining for PPARgamma at senior ages. We have also tested the thymic lobes of PPARgamma haplo-insufficient and null mice. Supporting our working hypothesis both adult PPARgamma haplo-insufficient and null mice show delayed thymic senescence by thymus histology, thymocyte mouse T-cell recombination excision circle qPCR and peripheral blood naive T-cell ratio by flow-cytometry. Delayed senescence showed dose–response with respect to PPARgamma deficiency. Functional immune parameters were also evaluated at senior ages in PPARgamma haplo-insufficient mice (null mice do not reach senior ages due to metabolic adverse affects. As expected, sustained and elevated T-cell production conferred oral tolerance and enhanced vaccination efficiency in senior PPARgamma haplo-insufficient, but not in senior wild-type littermates according to ELISA IgG measurements. Of note, humans also show increased oral intolerance issues and decreased protection by vaccines at senior ages. Moreover, PPARgamma haplo-insufficiency also exists in human known as a rare disease (FPLD3 causing metabolic adverse effects, similar to the mouse. When compared to age- and metabolic disorder-matched other patient samples (FPLD2 not affecting PPARgamma activity, FPLD3 patients showed increased human Trec (hTrec values by qPCR (within healthy human range suggesting delayed thymic senescence, in accordance with

  9. Thymic size in uninfected infants born to HIV-positive mothers and fed with pasteurized human milk

    DEFF Research Database (Denmark)

    Jeppesen, Dorthe Lisbeth; Hasselbalch, H; Ersbøll, A K

    2003-01-01

    AIM: To examine the size of the thymus in uninfected infants born to HIV-positive mothers and to study the effects of feeding by human donor milk on the size of the thymus in these infants. METHODS: The absolute and relative thymic size was assessed by sonography as thymic index (Ti), and the Ti....../weight-ratio (Ti/w) at birth and at 4 mo of age in 12 healthy uninfected infants born to HlV-infected mothers. All infants were exclusively fed pasteurized donor milk. The results were compared with those obtained from a previous cohort of exclusively breastfed, partially breastfed and exclusively formula......-fed infants. RESULTS: At birth the Ti was reduced in infants born to HIV-infected mothers in comparison with that in control infants but this difference disappeared when their birthweights were taken into consideration (Ti/w-ratio). At 4 mo of age the geometric mean Ti of infants fed donor milk was 23...

  10. Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes

    NARCIS (Netherlands)

    S. James (Sally); J. Fox (James); F. Afsari (Farinaz); J. Lee (Jennifer); S. Clough (Sally); C. Knight (Charlotte); J. Ashmore (James); P. Ashton (Peter); O. Preham (Olivier); M.J. Hoogduijn (Martin); R.D.A.R. Ponzoni (Raquel De Almeida Rocha); Y. Hancock; M. Coles (Mark); P.G. Genever (Paul)

    2015-01-01

    textabstractBone marrow stromal cells (BMSCs, also called bone-marrow-derived mesenchymal stromal cells) provide hematopoietic support and immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. We used immortalized human BMSC clonal lines for multi-level analysis

  11. Ethanol impairs differentiation of human adipocyte stromal cells in culture.

    Science.gov (United States)

    Crabb, David W; Zeng, Yan; Liangpunsakul, Suthat; Jones, Rosemarie; Considine, Robert

    2011-09-01

    Bioinformatic resources suggest that adipose tissue expresses mRNAs for alcohol dehydrogenases (ADHs) and ALDH2, and epidemiological studies indicate that heavy alcohol use reduces adipose tissue mass. We therefore characterized the expression of alcohol metabolizing enzymes in human, rat and mouse adipose tissue, preadipocytes, and adipocytes, the ability of adipocytes to metabolize ethanol, and the effects of ethanol on differentiation of human adipose stromal cells (hASCs). Adipose tissue, preadipocytes, and adipocytes were collected from rodents or from humans undergoing bariatric surgery. hASCs were differentiated in vitro using standard methods. Gene expression and cellular differentiation were analyzed by Western blotting, RT-PCR, and microscopy. Class I ADH was expressed in human > mouse > rat adipose tissue, whereas ALDH2 was high in all samples. ADH, catalase, and ALDH2 were induced during differentiation of hASCs. The presence of 50 mM ethanol markedly reduced the differentiation of hASCs; this effect was associated with inhibition of expression of transcription factors required for differentiation, but did not depend on the ability of the cells to metabolize ethanol. Human adipose tissue expresses alcohol oxidizing enzymes. The presence of ethanol at physiologically relevant concentrations inhibits differentiation of hASCs. Ethanol could alter adipose tissue biology, inducing a form of acquired lipodystrophy, which is consistent with epidemiological studies. 2011 by the Research Society on Alcoholism.

  12. Thymic function and T cell parameters in a natural human experimental model of seasonal infectious diseases and nutritional burden

    Directory of Open Access Journals (Sweden)

    Morgan Gareth

    2011-06-01

    Full Text Available Abstract Background The study exploits a natural human experimental model of subsistence farmers experiencing chronic and seasonally modified food shortages and infectious burden. Two seasons existed, one of increased deprivation and infections (Jul-Dec, another of abundance and low infections (Jan-Jun; referred to as the hungry/high infection and harvest/low infection seasons respectively. Prior analysis showed a 10-fold excess in infectious disease associated mortality in young adults born in the hungry/high infection versus harvest/low infection season, and reduced thymic output and T cell counts in infancy. Here we report findings on the role of early life stressors as contributors to the onset of T cell immunological defects in later life. Methods We hypothesised that season of birth effects on thymic function and T cell immunity would be detectable in young adults since Kaplan-Meier survival curves indicated this to be the time of greatest mortality divergence. T cell subset analyses by flow-cytometry, sjTRECs, TCRVβ repertoire and telomere length by PCR, were performed on samples from 60 males (18-23 y selected to represent births in the hungry/high infection and harvest/low infection Results Total lymphocyte counts were normal and did not differ by birth season. CD3+ and CD4+ but not CD8+ counts were lower for those born during the hungry/high infection season. CD8+ telomere length also tended to be shorter. Overall, CD8+ TCRVβ repertoire skewing was observed with 'public' expressions and deletions seen in TCRVβ12/22 and TCRVβ24, respectively but no apparent effect of birth season. Conclusions We conclude that, although thymic function was unchanged, the CD4+ and CD3+ counts, and CD8+ telomere length results suggested that aspects of adult T cell immunity were under the influence of early life stressors. The endemicity of CMV and HBV suggested that chronic infections may modulate immunity through T cell repertoire development. The

  13. Biomechanical model of human cornea based on stromal microstructure.

    Science.gov (United States)

    Studer, H; Larrea, X; Riedwyl, H; Büchler, P

    2010-03-22

    The optical characteristics of the human cornea depends on the mechanical balance between the intra-ocular pressure and intrinsic tissue stiffness. A wide range of ophthalmic surgical procedures alter corneal biomechanics to induce local or global curvature changes for the correction of visual acuity. Due to the large number of surgical interventions performed every day, a deeper understanding of corneal biomechanics is needed to improve the safety of these procedures and medical devices. The aim of this study is to propose a biomechanical model of the human cornea, based on stromal microstructure. The constitutive mechanical law includes collagen fiber distribution based on X-ray scattering analysis, collagen cross-linking, and fiber uncrimping. Our results showed that the proposed model reproduced inflation and extensiometry experimental data [Elsheikh et al., Curr. Eye Res., 2007; Elsheikh et al., Exp. Eye Res., 2008] successfully. The mechanical properties obtained for different age groups demonstrated an increase in collagen cross-linking for older specimens. In future work such a model could be used to simulate non-symmetric interventions, and provide better surgical planning. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  14. Cryopreservation and Revival of Human Mesenchymal Stromal Cells.

    Science.gov (United States)

    Haack-Sørensen, Mandana; Ekblond, Annette; Kastrup, Jens

    2016-01-01

    Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities.The establishment of methods for large-scale expansion of clinical-grade MSCs in vitro has paved the way for their therapeutic use in clinical trials. However, the clinical application of MSCs also requires cryopreservation and banking of the cell products. To preserve autologous or allogeneic MSCs for future clinical applications, a reliable and effective cryopreservation method is required.Developing a successful cryopreservation protocol for clinical stem cell products, cryopreservation media, cryoprotectant agents (CPAs), the freezing container, the freezing temperature, and the cooling and warming rate are all aspects which should be considered.A major challenge is the selection of a suitable cryoprotectant which is able to penetrate the cells and yet has low toxicity.This chapter focuses on recent technological developments relevant for the cryopreservation of MSCs using the most commonly used cryopreservation medium containing DMSO and animal serum or human-derived products for research use and the animal protein-free cryopreservation media CryoStor (BioLife Solutions) for clinical use.

  15. Implantation of the human embryo requires Rac1-dependent endometrial stromal cell migration.

    Science.gov (United States)

    Grewal, Seema; Carver, Janet G; Ridley, Anne J; Mardon, Helen J

    2008-10-21

    Failure of the human embryo to implant into the uterine wall during the early stages of pregnancy is a major cause of infertility. Implantation involves embryo apposition and adhesion to the endometrial epithelium followed by penetration through the epithelium and invasion of the embryonic trophoblast through the endometrial stroma. Although gene-knockdown studies have highlighted several molecules that are important for implantation in the mouse, the molecular mechanisms controlling implantation in the human are unknown. Here, we demonstrate in an in vitro model for human implantation that the Rho GTPases Rac1 and RhoA in human endometrial stromal cells modulate invasion of the human embryo through the endometrial stroma. We show that knockdown of Rac1 expression in human endometrial stromal cells inhibits human embryonic trophoblast invasion into stromal cell monolayers, whereas inhibition of RhoA activity promotes embryo invasion. Furthermore, we demonstrate that Rac1 is required for human endometrial stromal cell migration and that the motility of the stromal cells increases at implantation sites. This increased motility correlates with a localized increase in Rac1 activation and a reciprocal decrease in RacGAP1 levels. These results reveal embryo-induced and localized endometrial responses that may govern implantation of the human embryo.

  16. Microarray Analysis on Gene Regulation by Estrogen, Progesterone and Tamoxifen in Human Endometrial Stromal Cells

    Directory of Open Access Journals (Sweden)

    Chun-E Ren

    2015-03-01

    Full Text Available Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies.

  17. Stromal cell contribution to human follicular lymphoma pathogenesis

    Directory of Open Access Journals (Sweden)

    Frédéric eMourcin

    2012-09-01

    Full Text Available Follicular lymphoma (FL is the prototypical model of indolent B-cell lymphoma displaying a strong dependence on a specialized cell microenvironment mimicking normal germinal center. Within malignant cell niches in invaded lymph nodes and bone marrow, external stimuli provided by infiltrating stromal cells make a pivotal contribution to disease development, progression, and drug resistance. The crosstalk between FL B cells and stromal cells is bidirectional, causing activation of both partners. In agreement, FL stromal cells exhibit specific phenotypic, transcriptomic, and functional properties. This review highlights the critical pathways involved in the direct tumor-promoting activity of stromal cells but also their role in the organization of FL cell niche through the recruitment of accessory immune cells and their polarization to a B-cell supportive phenotype. Finally, deciphering the interplay between stromal cells and FL cells provides potential new therapeutic targets with the aim to mobilize malignant cells outside their protective microenvironment and increase their sensitivity to conventional treatment.

  18. Experimental asthma persists in IL-33 receptor knockout mice because of the emergence of thymic stromal lymphopoietin-driven IL-9+and IL-13+type 2 innate lymphoid cell subpopulations.

    Science.gov (United States)

    Verma, Mukesh; Liu, Sucai; Michalec, Lidia; Sripada, Anand; Gorska, Magdalena M; Alam, Rafeul

    2017-11-10

    IL-33 plays an important role in the development of experimental asthma. We sought to study the role of the IL-33 receptor suppressor of tumorigenicity 2 (ST2) in the persistence of asthma in a mouse model. We studied allergen-induced experimental asthma in ST2 knockout (KO) and wild-type control mice. We measured airway hyperresponsiveness by using flexiVent; inflammatory indices by using ELISA, histology, and real-time PCR; and type 2 innate lymphoid cells (ILC2s) in lung single-cell preparations by using flow cytometry. Airway hyperresponsiveness was increased in allergen-treated ST2 KO mice and comparable with that in allergen-treated wild-type control mice. Peribronchial and perivascular inflammation and mucus production were largely similar in both groups. Persistence of experimental asthma in ST2 KO mice was associated with an increase in levels of thymic stromal lymphopoietin (TSLP), IL-9, and IL-13, but not IL-5, in bronchoalveolar lavage fluid. Expectedly, ST2 deletion caused a reduction in IL-13 + CD4 T cells, forkhead box P3-positive regulatory T cells, and IL-5 + ILC2s. Unexpectedly, ST2 deletion led to an overall increase in innate lymphoid cells (CD45 + lin - CD25 + cells) and IL-13 + ILC2s, emergence of a TSLP receptor-positive IL-9 + ILC2 population, and an increase in intraepithelial mast cell numbers in the lung. An anti-TSLP antibody abrogated airway hyperresponsiveness, inflammation, and mucus production in allergen-treated ST2 KO mice. It also caused a reduction in innate lymphoid cell, ILC2, and IL-9 + and IL-13 + ILC2 numbers in the lung. Genetic deletion of the IL-33 receptor paradoxically increases TSLP production, which stimulates the emergence of IL-9 + and IL-13 + ILC2s and mast cells and leads to development of chronic experimental asthma. An anti-TSLP antibody abrogates all pathologic features of asthma in this model. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  19. Low doses of IgG from atopic individuals can modulate in vitro IFN-γ production by human intra-thymic TCD4 and TCD8 cells: An IVIg comparative approach.

    Science.gov (United States)

    Sgnotto, Fábio da Ressureição; Oliveira, Marília Garcia de; Lira, Aline Aparecida de Lima; Bento-de-Souza, Luciana; Duarte, Alberto José da Silva; Victor, Jefferson Russo

    2017-07-03

    The regulatory effect of allergic responses induced by IgG antibodies on human intra-thymic cells has not been reported in the literature. The aim of this study was to evaluate the possible differential effect of purified IgG from atopic and non-atopic individuals on human intra-thymic αβT cell cytokine production. Thymic tissues were obtained from 14 patients who were less than 7 d old. Additionally, blood samples were collected from atopic and non-atopic volunteers. Thymocytes and peripheral blood mononuclear cells were cultured with purified atopic or non-atopic IgG, and intracellular cytokine production was assessed. Purified IgG did not influence the frequency or viability of human intra-thymic αβT cells. Purified non-atopic IgG induced greater IFN-γ production by intra-thymic CD4+CD8+ T cells than did the mock treatment and atopic IgG. A similar effect of purified non-atopic IgG on TCD8 cells was observed compared with the mock treatment. Atopic IgG inhibited IFN-γ and TGF-β production by intra-thymic TCD4 cells. Treatment with intravenous immunoglobulin resulted in intermediate levels of IFN-γ and TGF-β in intra-thymic TCD4 cells compared with treatment with atopic and non-atopic IgG. Peripheral TCD4 cells from non-atopic individuals produced IFN-γ only in response to atopic IgG. This report describes novel evidence revealing that IgG from atopic individuals may influence intracellular IFN-γ production by intra-thymic αβT cells in a manner that may favor allergy development.

  20. Growth and hormonal responsiveness of human endometrial stromal cells in culture.

    Science.gov (United States)

    Holinka, C F

    1988-06-01

    The present review describes and discusses published results on growth and hormonal responsiveness of human endometrial stromal cells in culture. The proliferative potential of serially subcultured cells, that is, the number of cell doublings before cells enter mitotic senescence and cease to divide, was unusually high in stromal cells from several endometrial specimens, a property that may reflect the unique proliferative capacity of human endometrium when compared to other adult tissues. Fluorescent visualization of microfilaments revealed distinct age-related changes in the distribution of cytoskeletal fibers. Addition of ovarian steroids to the culture medium of stromal cells resulted in significant morphologic changes. From comparative studies using different culture media it became evident that medium components remarkably influenced cell morphology during early culture periods in an irreversible manner. Cultured stromal cells yielded interesting results in experiments designed to define the role of polyamines in growth regulation. Proliferation was greatly inhibited when polyamine levels were reduced by specific inhibition of ornithine decarboxylase, the first and rate limiting enzyme in polyamine synthesis which produces putrescine by catalytic conversion from ornithine. The antiproliferative effects were reversed by addition of putrescine to the culture medium. These results clearly establish a causal link between polyamine depletion and growth deficiencies and reveal an essential function of polyamines in stromal cell proliferation. Hormonally regulated parameters in cultured stromal cells include aromatase activity, pregnancy-associated plasma protein-A, 51K secreted protein, prolactin and laminin. The hormonally regulated production of prolactin and laminin, both considered markers of decidualization, together with morphologic changes of stromal cells to decidual-like cells, strongly suggest that human endometrial stromal cells, when subjected to

  1. Normal Thymic Size and Low Rate of Infections in Human Donor Milk Fed HIV-Exposed Uninfected Infants from Birth to 18 Months of Age

    DEFF Research Database (Denmark)

    Jeppesen, Dorthe Lisbeth; Ersbøll, Annette Kjær; Hoppe, Tine Ursula

    2013-01-01

    fewer infections than other healthy infants. This finding along with fewer infections in exclusively breastfed infants compared to formula-fed infants supports the beneficial effect of human milk on the immune system. We suggest, when breastfeeding is not possible, that providing human donor milk......Objective. To evaluate the immune function in HIV-exposed uninfected (HIV-EU) infants fed human donor milk. Methods. Ultrasound-obtained thymic index (Ti), T-lymphocyte subsets, and the number of infections were examined from birth to 18 months of age in 18 HIV-EU infants. The infants were compared...... (P donor milk have normal growth of thymus and contract...

  2. Doxorubicin and cisplatin induce apoptosis in ovarian stromal cells obtained from cryopreserved human ovarian tissue.

    Science.gov (United States)

    Fabbri, Raffaella; Macciocca, Maria; Vicenti, Rossella; Paradisi, Roberto; Klinger, Francesca Gioia; Pasquinelli, Gianandrea; Spisni, Enzo; Seracchioli, Renato; Papi, Alessio

    2016-07-01

    To investigate mechanisms by which doxorubicin (DOX) and cisplatin (CIS) cause human ovarian stroma injury. Stromal cells from human cryopreserved ovarian tissue were cultured in the presence of 1 µM DOX and 10 µM CIS. Ovarian damage induced by treatments was evaluated by 'Live/Dead' and sulforhodamine-B assays, the expression of different apoptosis markers. Stromal cell growth was inhibited by DOX and CIS, and this effect was accompanied by apoptosis through mitochondrial pathway activation: Bax, cleaved-caspase 9, cleaved-PARP1 induction and Akt1, Bcl2, phospho-44/42-MAPK/ERK1/2 reduction were observed. DOX and CIS induced apoptosis in human ovarian stromal cells. Knowledge of mechanisms by which the drugs act is important to identify possible ways to counteract side effects of chemotherapy on ovaries.

  3. Angiogenesis Following Three-Dimensional Culture of Isolated Human Endometrial Stromal Cells

    Directory of Open Access Journals (Sweden)

    Navid Esfandiari

    2008-01-01

    Full Text Available Background: Endometriosis is the presence of endometrial tissue outside of the uterine cavity andis the most common gynecologic disorder in women of reproductive age. We have preliminaryevidence that in the presence of a 3-dimensional (3-D fibrin matrix, human endometrial glands,stroma, and neovascularization can develop in vitro, mimicking the earliest stages of endometriosis.The aim of the present study was to determine if angiogenesis can be developed in a 3-D culture ofhuman stromal cells in vitro.Materials and Methods: This was an in vitro study of human endometrial biopsies in 3-D cultureof fibrin matrix and conducted at a university affiliated infertility center. Biopsies were taken fromten normal ovulating women undergoing infertility treatment. The samples obtained from fundusof the uterine cavity were minced, stromal cells isolated and placed in a 3-D fibrin matrix culturesystem. Degree of proliferation of stromal cells, invasion of the fibrin matrix, gland formation, vesselsprouting and immunohistochemical characterization of cellular components were recorded.Results: Three-dimensional culture of human stromal cells formed sheets of cells in the fibrinmatrix. By 3-4 weeks, endothelial cell branching was observed and rudimentary capillary-likestructures formed and endothelial cells confirmed by CD31 immunostaining.Conclusion: These data show that stromal cells from endometrial explants can proliferate andinvade a fibrin matrix in vitro generating new vessels. This procedure represents a controlled,quantifiable model for the study of angiogenesis during the menstrual cycle, and in conditions suchas endometriosis and cancer.

  4. FOXN1: a master regulator gene of thymic epithelial development programme

    Directory of Open Access Journals (Sweden)

    Rosa eRomano

    2013-07-01

    Full Text Available T cell ontogeny is a sophisticated process, which takes place within the thymus through a series of well-defined discrete stages. The process requires a proper lympho-stromal interaction. In particular, cortical and medullary thymic epithelial cells (cTECs, mTECs drive T cell differentiation, education and selection processes, while the thymocyte-dependent signals allow TECs to maturate and provide an appropriate thymic microenvironment. Alterations in genes implicated in thymus organogenesis, including Tbx1, Pax1, Pax3, Pax9, Hoxa3, Eya1 and Six1, affect this well-orchestrated process, leading to disruption of thymic architecture. Of note, in both human and mice, the primordial TECs are yet unable to fully support T cell development and only after the transcriptional activation of the Forkhead-box n1 (FOXN1 gene in the thymic epithelium this essential function is acquired. FOXN1 is a master regulator in the TEC lineage specification in that it down-stream promotes transcription of genes, which, in turn, regulate TECs differentiation. In particular, FOXN1 mainly regulates TEC patterning in the fetal stage and TEC homeostasis in the postnatal thymus. An inborn null mutation in FOXN1 leads to Nude/SCID phenotype in mouse, rat and humans. In Foxn1-/- nude animals, initial formation of the primordial organ is arrested and the primordium is not colonized by hematopoietic precursors, causing a severe primary T cell immunodeficiency. In humans, the Nude/SCID phenotype is characterized by congenital alopecia of the scalp, eyebrows, and eyelashes, nail dystrophy and a severe T cell immunodeficiency, inherited as an autosomal recessive disorder. Aim of this review is to summarize all the scientific information so far available to better characterize the pivotal role of the master regulator FOXN1 transcription factor in the TEC lineage specifications and functionality.

  5. The Origin of Human Mesenchymal Stromal Cells Dictates Their Reparative Properties

    DEFF Research Database (Denmark)

    Naftali-Shani, Nili; Itzhaki-Alfia, Ayelet; Landa-Rouben, Natalie

    2013-01-01

    Human mesenchymal stromal cells (hMSCs) from adipose cardiac tissue have attracted considerable interest in regard to cell-based therapies. We aimed to test the hypothesis that hMSCs from the heart and epicardial fat would be better cells for infarct repair....

  6. Transplantation of human neonatal foreskin stromal cells in ex vivo organotypic cultures of embryonic chick femurs

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Vishnubalaji, Radhakrishnan

    2017-01-01

    We have previously reported that human neonatal foreskin stromal cells (hNSSCs) promote angiogenesis in vitro and in chick embryo chorioallantoic membrane (CAM) assay in vivo. To examine the in vivo relevance of this observation, we examined in the present study the differentiation potential of h...

  7. Mesenchymal stromal/stem cell-derived extracellular vesicles promote human cartilage regeneration in vitro

    NARCIS (Netherlands)

    Vonk, Lucienne A.; van Dooremalen, Sanne F.J.; Liv, Nalan; Klumperman, Judith; Coffer, Paul J.; Saris, Daniël B.F.; Lorenowicz, Magdalena J.

    2018-01-01

    Osteoarthritis (OA) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. Recently, allogeneic human mesenchymal stromal/stem cells (MSC) entered clinical trials as a novel therapy for OA. Increasing evidence suggests that therapeutic efficacy of MSC

  8. Comparison of cellular functionality of human mesenchymal stromal cells and PBMC

    DEFF Research Database (Denmark)

    Schmal, H; Niemeyer, P; Roesslein, M

    2007-01-01

    BACKGROUND: Human mesenchymal stromal cells (MSC) and PBMC play significant roles in repair processes following inflammation. Mechanisms of recruitment are still under investigation. METHODS AND RESULTS: MIP-1alpha induced the chemotactic migration of MSC but not of PBMC. Correlating with this, 7...

  9. A unique thymic fibroblast population revealed by the monoclonal antibody MTS-15.

    Science.gov (United States)

    Gray, Daniel H D; Tull, Dedreia; Ueno, Tomoo; Seach, Natalie; Classon, Brendan J; Chidgey, Ann; McConville, Malcolm J; Boyd, Richard L

    2007-04-15

    T cell differentiation in the thymus is dependent upon signals from thymic stromal cells. Most studies into the nature of these signals have focused only on the support provided by the thymic epithelium, but there is an emerging view that other stromal cells such as mesenchymal fibroblasts may also be involved. Study of the latter has been hindered by a lack of appropriate markers, particularly those allowing their isolation. In this study, we describe a new surface marker of thymic stroma, MTS-15, and demonstrate its specificity for fibroblasts and a subset of endothelial cells. Coculture experiments showed that the determinant could be transferred between cells. Extensive biochemical analysis demonstrated that the Ag bound by MTS-15 was the glycosphingolipid Forssman determinant, consistent with the distribution observed. Transcriptional analysis of purified MTS-15(+) thymic fibroblasts revealed a unique expression profile for a number of chemokines and growth factors important to thymocyte and epithelial cell development. In a model of cyclophosphamide-induced thymic involution and regeneration, fibroblasts were found to expand extensively and express growth factors important to epithelial proliferation and increased T cell production just before thymic regeneration. Overall, this study identifies a useful marker of thymic fibroblasts and highlights this subpopulation as a key player in thymic function by virtue of their support of both thymocytes and epithelial cells.

  10. Molecular characterisation of stromal populations derived from human embryonic stem cells

    DEFF Research Database (Denmark)

    Harkness, L.; Twine, N. A.; Abu Dawud, R.

    2015-01-01

    Human bone marrow-derived stromal (skeletal) stem cells (BM-hMSC) are being employed in an increasing number of clinical trials for tissue regeneration. A limiting factor for their clinical use is the inability to obtain sufficient cell numbers. Human embryonic stem cells (hESC) can provide...... an unlimited source of clinical grade cells for therapy. We have generated MSC-like cells from hESC (called here hESC-stromal) that exhibit surface markers and differentiate to osteoblasts and adipocytes, similar to BM-hMSC. In the present study, we used microarray analysis to compare the molecular phenotype...... of hESC-stromal and immortalised BM-hMSC cells (hMSC-TERT). Of the 7379 genes expressed above baseline, only 9.3% of genes were differentially expressed between undifferentiated hESC-stromal and BM-hMSC. Following ex vivo osteoblast induction, 665 and 695 genes exhibited >. 2-fold change (FC) in h...

  11. Immune activation is associated with decreased thymic function in ...

    African Journals Online (AJOL)

    Background: Reduced thymic function causes poor immunological reconstitution in human immunodeficiency virus (HIV)-positive patients on combined antiretroviral therapy (cART). The association between immune activation and thymic function in asymptomatic HIVpositive treatment-naive individuals has thus far not been ...

  12. A Stromal Cell Niche for Human and Mouse Type 3 Innate Lymphoid Cells.

    Science.gov (United States)

    Hoorweg, Kerim; Narang, Priyanka; Li, Zhi; Thuery, Anne; Papazian, Natalie; Withers, David R; Coles, Mark C; Cupedo, Tom

    2015-11-01

    Adaptive immunity critically depends on the functional compartmentalization of secondary lymphoid organs. Mesenchymal stromal cells create and maintain specialized niches that support survival, activation, and expansion of T and B cells, and integrated analysis of lymphocytes and their niche has been instrumental in understanding adaptive immunity. Lymphoid organs are also home to type 3 innate lymphoid cells (ILC3), innate effector cells essential for barrier immunity. However, a specialized stromal niche for ILC3 has not been identified. A novel lineage-tracing approach now identifies a subset of murine fetal lymphoid tissue organizer cells that gives rise exclusively to adult marginal reticular cells. Moreover, both cell types are conserved from mice to humans and colocalize with ILC3 in secondary lymphoid tissues throughout life. In sum, we provide evidence that fetal stromal organizers give rise to adult marginal reticular cells and form a dedicated stromal niche for innate ILC3 in adaptive lymphoid organs. Copyright © 2015 by The American Association of Immunologists, Inc.

  13. Transplantation of human bone marrow mesenchymal stromal cells reduces liver fibrosis more effectively than Wharton?s jelly?mesenchymal stromal cells

    OpenAIRE

    Rengasamy, Mathiyazhagan; Singh, Gurbind; Fakharuzi, Noor Atiqah; Siddikuzzaman,; Balasubramanian, Sudha; Swamynathan, Priyanka; Thej, Charan; Sasidharan, Gopinath; Gupta, Pawan Kumar; Das, Anjan Kumar; Rahman, Ahmad Zuhairi Abd; Fakiruddin, Kamal Shaik; Nian, Lim Moon; Zakaria, Zubaidah; Majumdar, Anish S.

    2017-01-01

    Background Mesenchymal stromal cells (MSCs) from various tissues have shown moderate therapeutic efficacy in reversing liver fibrosis in preclinical models. Here, we compared the relative therapeutic potential of pooled, adult human bone marrow (BM)- and neonatal Wharton?s jelly (WJ)-derived MSCs to treat CCl4-induced liver fibrosis in rats. Methods Sprague-Dawley rats were injected with CCl4 for 8?weeks to induce irreversible liver fibrosis. Ex-vivo expanded, pooled human MSCs obtained from ...

  14. Honokiol, a constituent of Magnolia species, inhibits adrenergic contraction of human prostate strips and induces stromal cell death

    Directory of Open Access Journals (Sweden)

    Daniel Herrmann

    2014-09-01

    Conclusions: Honokiol inhibits smooth muscle contraction in the human prostate, and induces cell death in cultured stromal cells. Because prostate smooth muscle tone and prostate growth may cause LUTS, it appears possible that honokiol improves voiding symptoms.

  15. Response of human chondrocytes and mesenchymal stromal cells to a decellularized human dermis

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    Giavaresi Gianluca

    2013-01-01

    Full Text Available Abstract Background Although progress has been made in the treatment of articular cartilage lesions, they are still a major challenge because current techniques do not provide satisfactory long-term outcomes. Tissue engineering and the use of functional biomaterials might be an alternative regenerative strategy and fulfill clinical needs. Decellularized extracellular matrices have generated interest as functional biologic scaffolds, but there are few studies on cartilage regeneration. The aim of this study was to evaluate in vitro the biological influence of a newly developed decellularized human dermal extracellular matrix on two human primary cultures. Methods Normal human articular chondrocytes (NHAC-kn and human mesenchymal stromal cells (hMSC from healthy donors were seeded in polystyrene wells as controls (CTR, and on decellularized human dermis batches (HDM_derm for 7 and 14 days. Cellular proliferation and differentiation, and anabolic and catabolic synthetic activity were quantified at each experimental time. Histology and scanning electron microscopy were used to evaluate morphology and ultrastructure. Results Both cell cultures had a similar proliferation rate that increased significantly (p p p p p p p p Conclusions The results obtained showed that in in vitro conditions HDM_derm behaves as a suitable scaffold for the growth of both well-differentiated chondrocytes and undifferentiated mesenchymal cells, thus ensuring a biocompatible and bioactive substrate. Further studies are mandatory to test the use of HDM_derm with tissue engineering to assess its therapeutic and functional effectiveness in cartilage regeneration.

  16. Isolation of Mesenchymal Stromal Cells (MSCs from Human Adenoid Tissue

    Directory of Open Access Journals (Sweden)

    Yoon Se Lee

    2013-04-01

    Full Text Available Background: Mesenchymal stromal cells (MSCs are multipotent progenitor cells that originally derived from bone marrow. Clinical use of bone marrow-derived MSC is difficult due to morbidity and low MSC abundance and isolation efficiency. Recently, MSCs have been isolated from various adult tissues. Here we report the isolation of adenoid tissue-derived MSCs (A-MSCs and their characteristics. Methods: We compared the surface markers, morphologies, and differentiation and proliferation capacities of previously established tonsil-derived MSCs (T-MSCs and bone marrow-derived MSCs (BM-MSCs with cells isolated from adenoid tissue. The immunophenotype of A-MSCs was investigated upon interferon (IFN-γ stimulation. Results: A-MSCs, T-MSCs, and BM-MSCs showed negative CD45, CD31 HLA-DR, CD34, CD14, CD19 and positive CD 90, CD44, CD73, CD105 expression. A-MSCs were fibroblast-like, spindle-shaped non-adherent cells, similar to T-MSCs and BM-MSCs. Adipogenesis was observed in A-MSCs by the formation of lipid droplets after Oil Red O staining. Osteogenesis was observed by the formation of the matrix mineralization in Alizarin Red staining. Chondrogenesis was observed by the accumulation of sulfated glycosaminoglycan-rich matrix in collagen type II staining. These data were similar to those of T-MSCs and BM-MSCs. Expression of marker genes (i.e., adipogenesis; lipoprotein lipase, proliferator-activator receptor-gamma, osteogenesis; osteocalcin, alkaline phasphatase, chondrogenesis; aggrecan, collagen type II α1 in A-MSCs were not different from those in T-MSCs and BM-MSCs. Conclusions: A-MSCs possess the characteristics of MSCs in terms of morphology, multipotent differentiation capacity, cell surface markers, and immunogeneity. Therefore, A-MSCs fulfill the definition of MSCs and represent an alternate source of MSCs.

  17. Effects of Excess Copper Ions on Decidualization of Human Endometrial Stromal Cells.

    Science.gov (United States)

    Li, Ying; Kang, Zhen-Long; Qiao, Na; Hu, Lian-Mei; Ma, Yong-Jiang; Liang, Xiao-Huan; Liu, Ji-Long; Yang, Zeng-Ming

    2017-05-01

    The aim of this study was to investigate the effects of copper ions on decidualization of human endometrial stromal cells (HESCs) cultured in vitro. Firstly, non-toxic concentrations of copper D-gluconate were screened in HESCs based on cell activity. Then, the effects of non-toxic concentrations of copper ions (0~250 μM) were examined on decidualization of human endometrial stromal cells. Our data demonstrated that the mRNA expressions of insulin-like growth factor binding protein (IGFBP-1), prolactin (PRL), Mn-SOD, and FOXO1were down-regulated during decidualization following the treatments with 100 or 250 μM copper ions. Meanwhile, the amount of malonaldehyde (MDA) in the supernatant of HESCs was increased. These results showed that in vitro decidualization of HESCs was impaired by copper treatment.

  18. CD146/MCAM defines functionality of human bone marrow stromal stem cell populations

    DEFF Research Database (Denmark)

    Harkness, Linda; Zaher, Walid; Ditzel, Nicholas

    2016-01-01

    Background Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous h......MSC population. Methods Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146+ and hMSC-CD146− cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high-content analysis...... and additionally for their ability to differentiate toward osteogenesis in vitro and form bone in vivo, and their migrational ability in vivo and in vitro was investigated. Results In vitro, the two cell populations exhibited similar growth rate and differentiation capacity to osteoblasts and adipocytes...

  19. CD146/MCAM defines functionality of human bone marrow stromal stem cell populations

    DEFF Research Database (Denmark)

    Harkness, Linda; Zaher, Walid; Ditzel, Nicholas

    2016-01-01

    BACKGROUND: Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous h...... and adipocytes on the basis of gene expression and protein production of lineage-specific markers. In vivo, hMSC-CD146(+) and hMSC-CD146(-) cells formed bone and bone marrow organ when implanted subcutaneously in immune-deficient mice. Bone was enriched in hMSC-CD146(-) cells (12.6 % versus 8.1 %) and bone......MSC population. METHODS: Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146(+) and hMSC-CD146(-) cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high...

  20. The Impact of Epithelial-Stromal Interactions on Human Breast Tumor Heterogeneity

    Science.gov (United States)

    2015-10-01

    AWARD NUMBER: W81XWH-13-1-0357 TITLE: The Impact of Epithelial-Stromal Interactions on Human Breast Tumor Heterogeneity PRINCIPAL... Heterogeneity 5b. GRANT NUMBER W81XWH-13-1-0357 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Dr. Crista Thompson 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail... Heterogeneity is a key factor underlying the variability in patient response to treatment, especially in Triple-Negative (TN) breast cancer cases. In

  1. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis

    DEFF Research Database (Denmark)

    Jafari Kermani, Abbas; Qanie, Diyako; Andersen, Thomas L

    2017-01-01

    Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells...... and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin...

  2. [Murine and human hematopoietic progenitor cultures grown on stromal layers expressing Notch ligands].

    Science.gov (United States)

    Raevskaya, A A; Savvateeva, M V; Bukhinnik, S S; Kandarakov, O F; Butylin, P A; Zhuk, S V; Demin, A M; Zaritsky, V P Krasnov A Y; Belyavsky, A V

    2017-01-01

    The ex vivo maintenance and expansion of hematopoietic stem cells and early progenitors is necessary for the successful treatment of hematopoietic and immune diseases. Multiple attempts to improve the expansion of hematopoietic stem cells (HSCs) by their cultivation in the presence of growth factor cocktails have so far failed. Novel approaches aimed at conserving the earliest precursors in their undifferentiated state are needed. These approaches should take into account local regulatory factors that are present in the HSC microenvironment and the three-dimensional architecture of their niche. In the present study, we compared the effects of two Notch ligands, i.e., Jagged1 and DLL1, on murine and human hematopoiesis in vitro. Our observations indicate that the stromal expression of Notch ligands increases the production of both the total and phenotypically early murine and human hematopoietic cells in the co-culture. On one hand, this study demonstrates the similarity of effects of stromal expression of Notch ligands on murine and human hematopoiesis in vitro. On the other hand, our study revealed a number of cell type and ligand-specific variations that are systematically described below. It seems that the effects of SCF cytokine addition on murine hematopoiesis in vitro depend on the stromal context and are oppositely directed for Jagged1 and DLL1.

  3. Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes

    Directory of Open Access Journals (Sweden)

    Sally James

    2015-06-01

    Full Text Available Bone marrow stromal cells (BMSCs, also called bone-marrow-derived mesenchymal stromal cells provide hematopoietic support and immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. We used immortalized human BMSC clonal lines for multi-level analysis of functional markers for BMSC subsets. All clones expressed typical BMSC cell-surface antigens; however, clones with trilineage differentiation capacity exhibited enhanced vascular interaction gene sets, whereas non-differentiating clones were uniquely CD317 positive with significantly enriched immunomodulatory transcriptional networks and high IL-7 production. IL-7 lineage tracing and CD317 immunolocalization confirmed the existence of a rare non-differentiating BMSC subtype, distinct from Cxcl12-DsRed+ perivascular stromal cells in vivo. Colony-forming CD317+ IL-7hi cells, identified at ∼1%–3% frequency in heterogeneous human BMSC fractions, were found to have the same biomolecular profile as non-differentiating BMSC clones using Raman spectroscopy. Distinct functional identities can be assigned to BMSC subpopulations, which are likely to have specific roles in immune control, lymphopoiesis, and bone homeostasis.

  4. The orphan nuclear receptor Nur77 regulates decidual prolactin expression in human endometrial stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Yue; Hu, Yali; Zhao, Jing; Zhen, Xin [Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008 (China); Yan, Guijun, E-mail: yanguijun33@gmail.com [Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008 (China); Sun, Haixiang, E-mail: stevensunz@163.com [Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008 (China)

    2011-01-14

    Research highlights: {yields} Decidually produced PRL plays a key role during pregnancy. {yields} Overexpression of Nur77 increased PRL mRNA expression and enhanced decidual PRL promoter activity. {yields} Knockdown of Nur77 decreased decidual PRL secretion induced by 8-Br-cAMP and MPA. {yields} Nur77 is a novel transcription factor that plays an active role in decidual prolactin expression. -- Abstract: Prolactin (PRL) is synthesized and released by several extrapituitary tissues, including decidualized stromal cells. Despite the important role of decidual PRL during pregnancy, little is understood about the factors involved in the proper regulation of decidual PRL expression. Here we present evidence that the transcription factor Nur77 plays an active role in decidual prolactin expression in human endometrial stromal cells (hESCs). Nur77 mRNA expression in hESCs was significantly increased after decidualization stimulated by 8-Br-cAMP and medroxyprogesterone acetate (MPA). Adenovirus-mediated overexpression of Nur77 in hESCs markedly increased PRL mRNA expression and enhanced decidual PRL promoter (dPRL/-332Luc) activity in a concentration-dependent manner. Furthermore, knockdown of Nur77 in hESCs significantly decreased decidual PRL promoter activation and substantially attenuated PRL mRNA expression and PRL secretion (P < 0.01) induced by 8-Br-cAMP and MPA. These results demonstrate that Nur77 is a novel transcription factor that contributes significantly to the regulation of prolactin gene expression in human endometrial stromal cells.

  5. Tumour necrosis factor-alpha (TNFα stimulates the growth of human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    F. Rougier

    1997-01-01

    Full Text Available This study reports that TNF-α is a potent mitogen for human bone marrow sternal cells in vitro (assessed by [3H]-thymidine incorporation into DNA and cell counts. In contrast, cytokines such as IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, LIF, SCF, M-CSF, G-CSF and GM-CSF had no effect. The effect of TNF-α on the growth of human bone marrow stromal cells could be of importance during inflammatory processes which take place in the marrow, for example marrow fibrosis.

  6. Good Preservation of Stromal Cells and No Apoptosis in Human Ovarian Tissue after Vitrification

    Directory of Open Access Journals (Sweden)

    Raffaella Fabbri

    2014-01-01

    Full Text Available The aim of this study was to develop a vitrification procedure for human ovarian tissue cryopreservation in order to better preserve the ovarian tissue. Large size samples of ovarian tissue retrieved from 15 female-to-male transgender subjects (18–38 years were vitrified using two solutions (containing propylene glycol, ethylene glycol, and sucrose at different concentrations in an open system. Light microscopy, transmission electron microscopy, and TUNEL assay were applied to evaluate the efficiency of the vitrification protocol. After vitrification/warming, light microscopy showed oocyte nucleus with slightly thickened chromatin and irregular shape, while granulosa and stromal cells appeared well preserved. Transmission electron microscopy showed oocytes with slightly irregular nuclear shape and finely dispersed chromatin. Clear vacuoles and alterations in cellular organelles were seen in the oocyte cytoplasm. Stromal cells had a moderately dispersed chromatin and homogeneous cytoplasm with slight vacuolization. TUNEL assay revealed the lack of apoptosis induction by vitrification in all ovarian cell types. In conclusion after vitrification/warming the stromal compartment maintained morphological and ultrastructural features similar to fresh tissue, while the oocyte cytoplasm was slightly damaged. Although these data are encouraging, further studies are necessary and essential to optimize vitrification procedure.

  7. Good preservation of stromal cells and no apoptosis in human ovarian tissue after vitrification.

    Science.gov (United States)

    Fabbri, Raffaella; Vicenti, Rossella; Macciocca, Maria; Pasquinelli, Gianandrea; Paradisi, Roberto; Battaglia, Cesare; Martino, Nicola Antonio; Venturoli, Stefano

    2014-01-01

    The aim of this study was to develop a vitrification procedure for human ovarian tissue cryopreservation in order to better preserve the ovarian tissue. Large size samples of ovarian tissue retrieved from 15 female-to-male transgender subjects (18-38 years) were vitrified using two solutions (containing propylene glycol, ethylene glycol, and sucrose at different concentrations) in an open system. Light microscopy, transmission electron microscopy, and TUNEL assay were applied to evaluate the efficiency of the vitrification protocol. After vitrification/warming, light microscopy showed oocyte nucleus with slightly thickened chromatin and irregular shape, while granulosa and stromal cells appeared well preserved. Transmission electron microscopy showed oocytes with slightly irregular nuclear shape and finely dispersed chromatin. Clear vacuoles and alterations in cellular organelles were seen in the oocyte cytoplasm. Stromal cells had a moderately dispersed chromatin and homogeneous cytoplasm with slight vacuolization. TUNEL assay revealed the lack of apoptosis induction by vitrification in all ovarian cell types. In conclusion after vitrification/warming the stromal compartment maintained morphological and ultrastructural features similar to fresh tissue, while the oocyte cytoplasm was slightly damaged. Although these data are encouraging, further studies are necessary and essential to optimize vitrification procedure.

  8. Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Stopp, Sabine; Bornhäuser, Martin; Ugarte, Fernando; Wobus, Manja; Kuhn, Matthias; Brenner, Sebastian; Thieme, Sebastian

    2013-04-01

    The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

  9. Gene Transfection of Human Turbinate Mesenchymal Stromal Cells Derived from Human Inferior Turbinate Tissues

    Directory of Open Access Journals (Sweden)

    Jin Seon Kwon

    2016-01-01

    Full Text Available Human turbinate mesenchymal stromal cells (hTMSCs are novel stem cells derived from nasal inferior turbinate tissues. They are easy to isolate from the donated tissue after turbinectomy or conchotomy. In this study, we applied hTMSCs to a nonviral gene delivery system using polyethyleneimine (PEI as a gene carrier; furthermore, the cytotoxicity and transfection efficiency of hTMSCs were evaluated to confirm their potential as resources in gene therapy. DNA-PEI nanoparticles (NPs were generated by adding the PEI solution to DNA and were characterized by a gel electrophoresis and by measuring particle size and surface charge of NPs. The hTMSCs were treated with DNA-PEI NPs for 4 h, and toxicity of NPs to hTMSCs and gene transfection efficiency were monitored using MTT assay, fluorescence images, and flow cytometry after 24 h and 48 h. At a high negative-to-positive charge ratio, DNA-PEI NPs treatment led to cytotoxicity of hTMSCs, but the transfection efficiency of DNA was increased due to the electrostatic effect between the NPs and the membranes of hTMSCs. Importantly, the results of this research verified that PEI could deliver DNA into hTMSCs with high efficiency, suggesting that hTMSCs could be considered as untapped resources for applications in gene therapy.

  10. Susceptibility of human placenta derived mesenchymal stromal/stem cells to human herpesviruses infection.

    Directory of Open Access Journals (Sweden)

    Simone Avanzi

    Full Text Available Fetal membranes (FM derived mesenchymal stromal/stem cells (MSCs are higher in number, expansion and differentiation abilities compared with those obtained from adult tissues, including bone marrow. Upon systemic administration, ex vivo expanded FM-MSCs preferentially home to damaged tissues promoting regenerative processes through their unique biological properties. These characteristics together with their immune-privileged nature and immune suppressive activity, a low infection rate and young age of placenta compared to other sources of SCs make FM-MSCs an attractive target for cell-based therapy and a valuable tool in regenerative medicine, currently being evaluated in clinical trials. In the present study we investigated the permissivity of FM-MSCs to all members of the human Herpesviridae family, an issue which is relevant to their purification, propagation, conservation and therapeutic use, as well as to their potential role in the vertical transmission of viral agents to the fetus and to their potential viral vector-mediated genetic modification. We present here evidence that FM-MSCs are fully permissive to infection with Herpes simplex virus 1 and 2 (HSV-1 and HSV-2, Varicella zoster virus (VZV, and Human Cytomegalovirus (HCMV, but not with Epstein-Barr virus (EBV, Human Herpesvirus-6, 7 and 8 (HHV-6, 7, 8 although these viruses are capable of entering FM-MSCs and transient, limited viral gene expression occurs. Our findings therefore strongly suggest that FM-MSCs should be screened for the presence of herpesviruses before xenotransplantation. In addition, they suggest that herpesviruses may be indicated as viral vectors for gene expression in MSCs both in gene therapy applications and in the selective induction of differentiation.

  11. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging

    Directory of Open Access Journals (Sweden)

    Samuel T. Mindaye

    2015-12-01

    Full Text Available Bone-marrow derived mesenchymal stromal cells (BMSCs have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5,6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results.

  12. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging.

    Science.gov (United States)

    Mindaye, Samuel T; Lo Surdo, Jessica; Bauer, Steven R; Alterman, Michail A

    2015-12-01

    Bone-marrow derived mesenchymal stromal cells (BMSCs) have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5], [6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results.

  13. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, H.; Stenvang, Jan

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators......, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3' UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central...

  14. Human Endometrial Stromal Cells Are Highly Permissive To Productive Infection by Zika Virus.

    Science.gov (United States)

    Pagani, Isabel; Ghezzi, Silvia; Ulisse, Adele; Rubio, Alicia; Turrini, Filippo; Garavaglia, Elisabetta; Candiani, Massimo; Castilletti, Concetta; Ippolito, Giuseppe; Poli, Guido; Broccoli, Vania; Panina-Bordignon, Paola; Vicenzi, Elisa

    2017-03-10

    Zika virus (ZIKV) is a recently re-emerged flavivirus transmitted to humans by mosquito bites but also from mother to fetus and by sexual intercourse. We here show that primary human endometrial stromal cells (HESC) are highly permissive to ZIKV infection and support its in vitro replication. ZIKV envelope expression was detected in the endoplasmic reticulum whereas double-stranded viral RNA colocalized with vimentin filaments to the perinuclear region. ZIKV productive infection also occurred in the human T-HESC cell line together with the induction of interferon-β (IFN-β) and of IFN-stimulated genes. Notably, in vitro decidualization of T-HESC with cyclic AMP and progesterone upregulated the cell surface expression of the ZIKV entry co-receptor AXL and boosted ZIKV replication by ca. 100-fold. Thus, endometrial stromal cells, particularly if decidualized, likely represent a crucial cell target of ZIKV reaching them, either via the uterine vasculature in the viremic phase of the infection or by sexual viral transmission, and a potential source of virus spreading to placental trophoblasts during pregnancy.

  15. Multi-layered silk film coculture system for human corneal epithelial and stromal stem cells.

    Science.gov (United States)

    Gosselin, Emily A; Torregrosa, Tess; Ghezzi, Chiara E; Mendelsohn, Alexandra C; Gomes, Rachel; Funderburgh, James L; Kaplan, David L

    2017-06-10

    With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one coculture system, to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these three-dimensional tissue systems in vitro. Coculture conditions were first optimized, including the medium, air-liquid interface culture, and surface topography and chemistry of biomaterial scaffold films based on silk protein. The silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs were studied in this in vitro system, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by quantitative reverse transcription-polymerase chain reaction. The incorporation of both cell types into the coculture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk films for corneal epithelial culture were optimized to combine a 4.0-μm-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in coculture on the optimized silk scaffolds. Copyright © 2017 John Wiley & Sons, Ltd.

  16. Expression of Siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology

    Science.gov (United States)

    Wang, Xiaoxia; Chow, Renee; Deng, Liwen; Anderson, Dan; Weidner, Noel; Godwin, Andrew K; Bewtra, Chanda; Zlotnik, Albert; Bui, Jack; Varki, Ajit; Varki, Nissi

    2011-01-01

    Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor β1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential

  17. Expression of Siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology.

    Science.gov (United States)

    Wang, Xiaoxia; Chow, Renee; Deng, Liwen; Anderson, Dan; Weidner, Noel; Godwin, Andrew K; Bewtra, Chanda; Zlotnik, Albert; Bui, Jack; Varki, Ajit; Varki, Nissi

    2011-08-01

    Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor β1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential

  18. Thymic enlargement in patients with hyperthyroidism

    Energy Technology Data Exchange (ETDEWEB)

    Myung, Jae Sung; Goo, Jin Mo; Im, Jung Gi [College of medicine and the Institute of Radiation Medicine, Seoul National University, Seoul (Korea, Republic of); Kim, Mi Young [Sejong General Hospital, Seoul (Korea, Republic of); Park, Yang Hee [National Police Hospital, Seoul (Korea, Republic of)

    2000-08-01

    To evaluate the radiologic findings and clinical feasibility of thymic enlargement in patients with hyperthyroidism. Seven patients with hyperthyroidism and anterior mediastinal bulging revealed by chest radiography were evaluated. The CT findings were analyzed with regard to the shape of the anterior mediastinal mass, surrounding infiltration, and enlargement of mediastinal lymph nodes. Whether or not tumor markers (alpha-fetoprotein, beta-human chorionic gonadotrophin, and chorionic embryonic antigen) showed increased levels was determined, and the size and thickness of the anterior mediastinal mass were measured and compared with previously described age-matched thymus data. In addition, changes in the thyroid gland were evaluated. In all seven patients, anterior mediastinal masses were bi-lobed, with no surrounding infiltration or enlarged mediastinal lymph node, and tumor marker levels showed no increase. The masses were therefore considered to be thymus. In six patients, the size of the thymus exceeded two upper standard deviations of mean value and in one patient, it was smaller than this. In three patients, PCNB (percutaneous needle biopsy) revealed normal thymic tissue and in two, follow-up chest PA demonstrated no interval change. CT showed that in three patients, the thyroid glands were diffusely enlarged. In patients with hyperthyroidism, an anterior mediastinal mass seen on chest radiographs was due to thymic enlargement. The recognition of CT findings of thymic enlargement in such patients may avoid unnecessary biopsy. (author)

  19. Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

    Science.gov (United States)

    Fazzina, R; Iudicone, P; Fioravanti, D; Bonanno, G; Totta, P; Zizzari, I G; Pierelli, L

    2016-08-25

    Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus. MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics. The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro. The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking

  20. Aged human bone marrow stromal cells maintaining bone forming capacity in vivo evaluated using an improved method of visualization

    DEFF Research Database (Denmark)

    Stenderup, Karin; Rosada, Cecilia; Justesen, J

    2004-01-01

    weeks, the implants were removed and embedded un-decalcified in methyl methacrylate (MMA). Sections were stained histochemically with Goldner's Trichrome stain and immuno-histochemically using human-specific antibodies against known osteogenic markers. Implanted human marrow stromal cells (hMSC) were...

  1. Regenerative efficacy of mesenchymal stromal cells from human placenta in sensorineural hearing loss.

    Science.gov (United States)

    Kil, Kicheol; Choi, Mi Young; Kong, Ji Sun; Kim, Woo Jin; Park, Kyoung Ho

    2016-12-01

    Hearing loss is a common chronic disorder characterized by decline of auditory function. The global population have suffered from deafness and the transplantation of stem cells is regarded as a therapeutic strategy for this disease. We collected placenta from a total of 13 samples of full term pregnant women and isolated MSCs derived from human placenta and transplanted MSCs on deaf animal model. The normal group and the sensorineural hearing loss (SNHL) group and the experimental (transplanted MSCs) group were compared and estimated hearing level using auditory brainstem response (ABR) recordings and the otoacoustic emission (OAE) test. ABR threshold value and DPOAE level showed that MSCs transplantation groups was improved than the SNHL group. And the number of spiral ganglion neurons were increased in all turn of the cochlea. And there was no evidence of acute immunological rejection and inflammation response was not observed. This study is to evaluate regenerative efficacy of hearing loss by transplanting mesenchymal stromal cells (MSCs) derived from human placenta (amnion and chorion) in deaf animal model. We identified that MSCs transplantation restored auditory impairment and promoted cell regeneration. We hope to overcome sensorineural hearing loss by transplanting stem cells such as mesenchymal stromal cells (MSCs) from easily accessible adult stem cell source in placenta. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Local anesthetic lidocaine induces apoptosis in human corneal stromal cells in vitro

    Directory of Open Access Journals (Sweden)

    Xin Zhou

    2013-12-01

    Full Text Available AIM: To demonstrate the apoptosis-inducing effect of lidocaine on human corneal stromal (HCS cells in vitro, and provide experimental basis for safety anesthetic usage in clinic of ophthalmology.METHODS: In vitro cultured HCS cells were treated with lidocaine at different doses and times, and their morphology was monitored successively with inverted phase contrast microscopy. The membrane permeability of them was detected by acridine orange/ethidium bromide (AO/EB double staining. The DNA fragmentation of them was examined by agarose gel electrophoresis, and their ultrastructure was observed by transmission electron microscopy (TEM, respectively.RESULTS: Exposure to lidocaine at doses from 0.3125g/L to 20g/L induced morphological changes of HCS cells such as cytoplasmic vacuolation, cellular shrinkage, and turning round, and elevated membrane permeability of these cells in AO/EB staining. The change of morphology and membrane permeability was dose- and time-dependent, while lidocaine at dose below 0.15625g/L could not induce these changes. Furthermore, lidocaine induced DNA fragmentation and ultrastructural changes such as cytoplasmic vacuolation, structural disorganization, chromatin condensation, and apoptotic body appearance of the cells.CONCLUSION: Lidocaine has significant cytotoxicity on human corneal stromal cells in vitro in a dose- and time-dependent manner by inducing apoptosis of these cells. The established experimental model and findings based on this model here help provide new insight into the apoptosis-inducing effect of local anesthetics in eye clinic.

  3. Characterization and angiogenic potential of human neonatal and infant thymus mesenchymal stromal cells.

    Science.gov (United States)

    Wang, Shuyun; Mundada, Lakshmi; Johnson, Sean; Wong, Joshua; Witt, Russell; Ohye, Richard G; Si, Ming-Sing

    2015-04-01

    Resident mesenchymal stromal cells (MSCs) are involved in angiogenesis during thymus regeneration. We have previously shown that MSCs can be isolated from enzymatically digested human neonatal and infant thymus tissue that is normally discarded during pediatric cardiac surgical procedures. In this paper, we demonstrate that thymus MSCs can also be isolated by explant culture of discarded thymus tissue and that these cells share many of the characteristics of bone marrow MSCs. Human neonatal thymus MSCs are clonogenic, demonstrate exponential growth in nearly 30 population doublings, have a characteristic surface marker profile, and express pluripotency genes. Furthermore, thymus MSCs have potent proangiogenic behavior in vitro with sprout formation and angiogenic growth factor production. Thymus MSCs promote neoangiogenesis and cooperate with endothelial cells to form functional human blood vessels in vivo. These characteristics make thymus MSCs a potential candidate for use as an angiogenic cell therapeutic agent and for vascularizing engineered tissues in vitro. ©AlphaMed Press.

  4. Post-thymic regulation of CD5 levels in human memory T cells is inversely associated with the strength of responsiveness to interleukin-15.

    Science.gov (United States)

    Herndler-Brandstetter, Dietmar; Brunner, Stefan; Weiskopf, Daniela; van Rijn, Ruth; Landgraf, Katja; Dejaco, Christian; Duftner, Christina; Schirmer, Michael; Kloss, Frank; Gassner, Robert; Lepperdinger, Günter; Grubeck-Loebenstein, Beatrix

    2011-08-01

    Immunologic memory is a critical feature of the adaptive immune system to fight recurrent infections. However, the mechanisms that shape the composition and function of the human memory T-cell pool remain incompletely understood. We here demonstrate that post-thymic human T-cell differentiation was associated with the downregulation, but not loss, of the inhibitory molecule CD5. The sensitivity of human CD8(+) and CD4(+) memory T cells to interleukin (IL)-15 was inversely associated with the level of CD5 expression. CD5 expression was downregulated by IL-15-mediated signaling in vitro and CD5(lo) memory T cells accumulated in the bone marrow. Persistent antigenic stimulation, as in the case of cytomegalovirus infection and rheumatoid arthritis (RA), was also associated with an increased number of CD5(lo) memory T cells. In conclusion, CD5 may be a useful marker to identify memory T-cell subsets with distinct responsiveness to the homeostatic cytokine IL-15. Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  5. NEDD8-mediated neddylation is required for human endometrial stromal proliferation and decidualization.

    Science.gov (United States)

    Liao, Yixin; Jiang, Yaling; He, Hui; Ni, Hao; Tu, Zhaowei; Zhang, Shuang; Wang, Bingyan; Lou, Jiaoying; Quan, Song; Wang, Haibin

    2015-07-01

    Does NEDD8-mediated neddylation regulate human endometrial stromal proliferation and decidualization? Neddylation inhibition by a selective NEDD8-activating enzyme inhibitor, MLN4924, significantly impairs human endometrial stromal cell (HESC) proliferation and decidualization and facilitates cell senescence, via p21 accumulation. Neddylation regulates cell proliferation and tissue remodeling during embryogenesis and tumorigenesis, while human endometrial stroma undergoes sequential proliferation, differentiation, as well as dynamic tissue remodeling during each menstrual cycle. We first analyzed the expression of NEDD8 in human endometrial tissues from 50 subjects, and then explored the consequence of neddylation inhibition by MLN4924 on HESCs proliferation, decidualization and cellular senescence. We collected 50 dated human endometrial tissues from early proliferative stage to late secretory phase of the menstrual cycle and analyzed the NEDD8 expression and cellular location in human endometrium by employing quantitative real-time PCR (qRT-PCR) and immunohistochemistry staining. Similar approaches were also used to explore the mRNA and protein expression of NEDD8 in an immortalized human endometrial stromal cell line (HESC) during proliferation and decidualization (N = 6). An MTS assay was performed to evaluate the effects of neddylation inhibition by MLN4924 on HESC proliferation. Flow cytometry and BrdU incorporation assay were conducted to determine the HESC cell cycle progression in response to MLN4924 exposure during proliferation. We also analyzed F-actin distribution by phalloidin staining and decidual marker gene expression by qRT-PCR to accesses the consequence of neddylation inhibition on HESC decidualization. Immunoblotting analysis of cullin1 and p21, and SA-β-Galactosidase staining were performed to reveal the potential molecular basis for the impaired HESC proliferation, decidualization and cellular senescence. The siRNA technique was applied to

  6. Fibulin-5 is upregulated in decidualized human endometrial stromal cells and promotes primary human extravillous trophoblast outgrowth.

    Science.gov (United States)

    Winship, Amy; Cuman, Carly; Rainczuk, Katarzyna; Dimitriadis, Evdokia

    2015-12-01

    Interactions between the highly invasive trophoblasts and the maternal uterine decidual extracellular matrix (ECM) are crucial in the determination of a successful pregnancy. Fibulin-5 (FBLN5) is a member of the fibulin family that alters cell adhesive and invasive properties and is expressed in human villous cytotrophoblasts. We aimed to determine the expression and immunolocalization of FBLN5 in human first trimester decidua and examine the effect of FBLN5 in trophoblast invasion in vitro using a first trimester placental villous outgrowth assay. We demonstrated that FBLN5 mRNA expression is upregulated in response to cAMP-mediated decidualization of primary human endometrial stromal cells, although FBLN5 itself does not enhance decidualization. We reported for the first time, FBLN5 protein production in first trimester decidual cells and also co-localization to HLAG-positive EVTs in first trimester decidua. Consequently, we investigated the effects of exogenous FBLN5 on placental villous outgrowth in vitro and demonstrated that FBLN5 promotes EVT migration/invasion. This is the first study to identify FBLN5 in decidualized human endometrial stromal cells, first trimester decidua and EVT and determine a functional role for FBLN5 in human EVTs, suggesting that decidual and or EVT-derived FBLN5 regulates EVT invasion and placentation in women. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Derivation of Stromal (Skeletal, Mesenchymal) Stem-like cells from Human Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Abdallah, Basem

    2012-01-01

    Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESC) is a pre-requisite for their use in clinical applications. However, there is no standard protocol for differentiating hESC into osteoblastic cells. The aim of this study was to identify the emergence of a human...... stromal (mesenchymal, skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESC in a feeder-free environment using serum replacement and as suspension aggregates (embryoid...... bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106 and CD166 as revealed by immunohistochemical staining and flow cytometry (FACS) analysis. Ex vivo differentiation of h...

  8. Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes.

    Science.gov (United States)

    Li, Bojun; Menzel, Ursula; Loebel, Claudia; Schmal, Hagen; Alini, Mauro; Stoddart, Martin J

    2016-05-20

    Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.

  9. miRNA Signature and Dicer Requirement during Human Endometrial Stromal Decidualization In Vitro

    Science.gov (United States)

    Estella, Carlos; Herrer, Isabel; Moreno-Moya, Juan Manuel; Quiñonero, Alicia; Martínez, Sebastián; Pellicer, Antonio; Simón, Carlos

    2012-01-01

    Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization

  10. Leukemia inhibitory factor enhances endometrial stromal cell decidualization in humans and mice.

    Directory of Open Access Journals (Sweden)

    Lorraine Lin Shuya

    Full Text Available Adequate differentiation or decidualization of endometrial stromal cells (ESC is critical for successful pregnancy in humans and rodents. Here, we investigated the role of leukemia inhibitory factor (LIF in human and murine decidualization. Ex vivo human (H ESC decidualization was induced by estrogen (E, 10(-8 M plus medroxyprogesterone acetate (MPA, 10(-7 M. Exogenous LIF (≥50 ng/ml induced STAT3 phosphorylation in non-decidualized and decidualized HESC and enhanced E+MPA-induced decidualization (measured by PRL secretion, P100 pg/mg G-CSF, IL6, IL8, and MCP1. Decidualized HESC secreted IL6, IL8, IL15 and MCP1. LIF (50 ng/ml up-regulated IL6 and IL15 (P<0.05 secretion in decidualized HESC compared to 0.5 ng/ml LIF. In murine endometrium, LIF and LIFR immunolocalized to decidualized stromal cells on day 5 of gestation (day 0 = day of plug detection. Western blotting confirmed that LIF and the LIFR were up-regulated in intra-implantation sites compared to inter-implantation sites on Day 5 of gestation. To determine the role of LIF during in vivo murine decidualization, intra-peritoneal injections of a long-acting LIF antagonist (PEGLA; 900 or 1200 µg were given just post-attachment, during the initiation of decidualization on day 4. PEGLA treatment reduced implantation site decidual area (P<0.05 and desmin staining immuno-intensity (P<0.05 compared to control on day 6 of gestation. This study demonstrated that LIF was an important regulator of decidualization in humans and mice and data provides insight into the processes underlying decidualization, which are important for understanding implantation and placentation.

  11. Leukemia Inhibitory Factor Enhances Endometrial Stromal Cell Decidualization in Humans and Mice

    Science.gov (United States)

    Yap, Joanne; Li, Priscilla; Lane, Natalie; Dimitriadis, Evdokia

    2011-01-01

    Adequate differentiation or decidualization of endometrial stromal cells (ESC) is critical for successful pregnancy in humans and rodents. Here, we investigated the role of leukemia inhibitory factor (LIF) in human and murine decidualization. Ex vivo human (H) ESC decidualization was induced by estrogen (E, 10−8 M) plus medroxyprogesterone acetate (MPA, 10−7 M). Exogenous LIF (≥50 ng/ml) induced STAT3 phosphorylation in non-decidualized and decidualized HESC and enhanced E+MPA-induced decidualization (measured by PRL secretion, P100 pg/mg G-CSF, IL6, IL8, and MCP1. Decidualized HESC secreted IL6, IL8, IL15 and MCP1. LIF (50 ng/ml) up-regulated IL6 and IL15 (P<0.05) secretion in decidualized HESC compared to 0.5 ng/ml LIF. In murine endometrium, LIF and LIFR immunolocalized to decidualized stromal cells on day 5 of gestation (day 0 = day of plug detection). Western blotting confirmed that LIF and the LIFR were up-regulated in intra-implantation sites compared to inter-implantation sites on Day 5 of gestation. To determine the role of LIF during in vivo murine decidualization, intra-peritoneal injections of a long-acting LIF antagonist (PEGLA; 900 or 1200 µg) were given just post-attachment, during the initiation of decidualization on day 4. PEGLA treatment reduced implantation site decidual area (P<0.05) and desmin staining immuno-intensity (P<0.05) compared to control on day 6 of gestation. This study demonstrated that LIF was an important regulator of decidualization in humans and mice and data provides insight into the processes underlying decidualization, which are important for understanding implantation and placentation. PMID:21966484

  12. miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

    Directory of Open Access Journals (Sweden)

    Carlos Estella

    Full Text Available Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human

  13. Expression of RAGE and HMGB1 in thymic epithelial tumors, thymic hyperplasia and regular thymic morphology.

    Directory of Open Access Journals (Sweden)

    Bernhard Moser

    Full Text Available Recently, a role of the receptor for advanced glycation endproducts (RAGE in myasthenia gravis was described. RAGE and its ligand high mobility group box 1 (HMGB1 play key roles in autoimmunity and cancer. To test whether these molecules are involved in patients with thymic abnormalities we applied immunohistochemical analysis in 33 cases of thymic epithelial tumors, comprising 27 thymomas and 6 thymic carcinomas, and 21 nonneoplastic thymuses. Both molecules were detected in neoplastic epithelial cells: RAGE staining was most intense in WHO type B2 thymomas and thymic carcinomas (pB3>thymic carcinoma (p<0.001. Conversely, HMGB1 cytoplasmic staining intensities were as follows: A and AB (none, B1 (strong, B2 (moderate, B3 and thymic carcinoma (weak; (p<0.001. Fetal thymic tissue showed a distinct expression of RAGE and HMGB1 in subcapsular cortical epithelial cells which was found in 50% of myasthenic patients. Furthermore RAGE and HMGB1 were expressed in thymocytes, macrophages, Hassall's corpuscles, thymic medulla, and germinal center cells in myasthenic patients. Immunohistochemistry results were complemented by systemic measurements (immunosorbent assay: serum levels of soluble RAGE were significantly reduced in patients with epithelial tumors (p = 0.008; and in invasive tumors (p = 0.008. Whereas RAGE was equally reduced in thymic hyperplasia and epithelial tumors (p = 0.003, HMGB1 was only elevated in malignancies (p = 0.036. Results were most pronounced in thymic carcinomas. Thus, RAGE and HMGB1 are involved in the (patho-physiology of thymus, as evidenced by differentiated thymic and systemic expression patterns that may act as diagnostic or therapeutic targets in autoimmune disease and cancer.

  14. Fine structure of the interface between the anterior limiting lamina and the anterior stromal fibrils of the human cornea.

    Science.gov (United States)

    Mathew, Jessica H; Bergmanson, Jan P G; Doughty, Michael J

    2008-09-01

    To use transmission electron microscopy (TEM) to investigate further the ultrastructural details of the collagen fibrils linking the anterior limiting lamina (ALL; Bowman's membrane) of the human cornea to the anterior stromal lamellae. Six disease-free corneas from donors aged 42 to 82 years were fixed (2% glutaraldehyde in 80 mM sodium cacodylate) and processed for TEM within 72 hours postmortem. A series of overlapping images, at 10,204x magnification, of the central corneal ALL-stroma interface were assembled. The features of the terminal ends of fibril bundles at the interface with the anterior stroma were quantitatively assessed. TEM revealed apparently terminating anterior stromal fibril bundles adjacent to the ALL. These terminating lamellae (7.8 per 100 mum) were embedded in an electron-dense material within the surrounding stromal matrix and were termed electron-dense formations (EDFs). The mean width of these stromal features was 1.6 mum. At intervals, anterior stromal lamellae approached the ALL and, in a shallow manner, inserted into the ALL. Such projections (5.4 per 100 mum) into the ALL were, on average, less than 1 mum. Numerous fibrils (29.8 per 100 mum) extended from the ALL into the stroma with a mean length of 0.8 mum. The interface the ALL forms with the anterior stroma is complex, and TEM revealed at least three different types of fibrillar arrangements, which may serve optical requirements rather than provide a structural function.

  15. SERPINB2 is a novel TGFβ-responsive lineage fate determinant of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Atteya, Muhammad

    2017-01-01

    TGF-β1, a multifunctional regulator of cell growth and differentiation, is the most abundant bone matrix growth factor. During differentiation of human bone stromal cells (hBMSCs), which constitute bone marrow osteoblast (OS) and adipocyte (AD) progenitor cells, continuous TGF-β1 (10 ng/ml) treat...

  16. Human Stromal (Mesenchymal) Stem Cells from Bone Marrow, Adipose Tissue and Skin Exhibit Differences in Molecular Phenotype and Differentiation Potential

    DEFF Research Database (Denmark)

    Al-Nbaheen, May; Vishnubalaji, Radhakrishnan; Ali, Dalia

    2013-01-01

    Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marro...

  17. Atomized human amniotic mesenchymal stromal cells for direct delivery to the airway for treatment of lung injury

    NARCIS (Netherlands)

    Kim, Sally Yunsun; Burgess, Janette K.; Wang, Yiwei; Kable, Eleanor P. W.; Weiss, Daniel J.; Chan, Hak-Kim; Chrzanowski, Wojciech

    2016-01-01

    Background: Current treatment regimens for inhalation injury are mainly supportive and rely on self-regeneration processes for recovery. Cell therapy with mesenchymal stromal cells (MSCs) is increasingly being investigated for the treatment of inhalation injury. Human amniotic MSCs (hAMSCs) were

  18. COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation

    Science.gov (United States)

    Li, Xilong; Large, Michael J.; Creighton, Chad J.; Lanz, Rainer B.; Jeong, Jae-Wook; Young, Steven L.; Lessey, Bruce A.; Palomino, Wilder A.; Tsai, Sophia Y.

    2013-01-01

    Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII; NR2F2) is an orphan nuclear receptor involved in cell-fate specification, organogenesis, angiogenesis, and metabolism. Ablation of COUP-TFII in the mouse uterus causes infertility due to defects in embryo attachment and impaired uterine stromal cell decidualization. Although the function of COUP-TFII in uterine decidualization has been described in mice, its role in the human uterus remains unknown. We observed that, as in mice, COUP-TFII is robustly expressed in the endometrial stroma of healthy women, and its expression is reduced in the ectopic lesions of women with endometriosis. To interrogate the role of COUP-TFII in human endometrial function, we used a small interfering RNA-mediated loss of function approach in primary human endometrial stromal cells. Attenuation of COUP-TFII expression did not completely block decidualization; rather it had a selective effect on gene expression. To better elucidate the role of COUP-TFII in endometrial stroma cell biology, the COUP-TFII transcriptome was defined by pairing microarray comparison with chromatin immunoprecipitation followed by deep sequencing. Gene ontology analysis demonstrates that COUP-TFII regulates a subset of genes in endometrial stroma cell decidualization such as those involved in cell adhesion, angiogenesis, and inflammation. Importantly this analysis shows that COUP-TFII plays a role in controlling the expression of inflammatory cytokines. The determination that COUP-TFII plays a role in inflammation may add insight into the role of COUP-TFII in embryo implantation and in endometrial diseases such as endometriosis. PMID:24176914

  19. Insights into the human mesenchymal stromal/stem cell identity through integrative transcriptomic profiling.

    Science.gov (United States)

    Roson-Burgo, Beatriz; Sanchez-Guijo, Fermin; Del Cañizo, Consuelo; De Las Rivas, Javier

    2016-11-21

    Mesenchymal Stromal/Stem Cells (MSCs), isolated under the criteria established by the ISCT, still have a poorly characterized phenotype that is difficult to distinguish from similar cell populations. Although the field of transcriptomics and functional genomics has quickly grown in the last decade, a deep comparative analysis of human MSCs expression profiles in a meaningful cellular context has not been yet performed. There is also a need to find a well-defined MSCs gene-signature because many recent biomedical studies show that key cellular interaction processes (i.e. inmuno-modulation, cellular cross-talk, cellular maintenance, differentiation, epithelial-mesenchymal transition) are dependent on the mesenchymal stem cells within the stromal niche. In this work we define a core mesenchymal lineage signature of 489 genes based on a deep comparative analysis of multiple transcriptomic expression data series that comprise: (i) MSCs of different tissue origins; (ii) MSCs in different states of commitment; (iii) other related non-mesenchymal human cell types. The work integrates several public datasets, as well as de-novo produced microarray and RNA-Seq datasets. The results present tissue-specific signatures for adipose tissue, chorionic placenta, and bone marrow MSCs, as well as for dermal fibroblasts; providing a better definition of the relationship between fibroblasts and MSCs. Finally, novel CD marker patterns and cytokine-receptor profiles are unravelled, especially for BM-MSCs; with MCAM (CD146) revealed as a prevalent marker in this subtype of MSCs. The improved biomolecular characterization and the released genome-wide expression signatures of human MSCs provide a comprehensive new resource that can drive further functional studies and redesigned cell therapy applications.

  20. The role of retinoic acid signaling in thymic function

    DEFF Research Database (Denmark)

    Wendland, Kerstin; Sitnik, Katarzyna; Kotarsky, Knut

    Retinoic acid (RA) is a vitamin A metabolite and member of the large family of retinoids that have been used in treatment of various forms of cancer and skin disorders. Also, vitamin A deficiency is associated with impaired ability to fight infections and RA has been shown to shape peripheral immune...... responses. However, little is known about the role of RA in the development of immune cells. We are currently investigating the role of RA signaling in thymic function. In the thymus, thymic epithelial cells (TEC) are providing the specialized microenvironment that supports T cell development and proper TEC...... maturation and homeostasis is required for the generation of a functional T cell pool. TEC development and differenti-ation is dependent on crosstalk with immune and stromal cells in the thymus and previous work of our group has suggested RA as a potential key player in this process. To study the role of RA...

  1. A histochemical comparison of human corneal stromal glycoconjugates with eight other species. Distinct species-dictated differences in binding sites of Griffonia simplicifolia I.

    Science.gov (United States)

    Panjwani, N; Baum, J

    1988-01-01

    Frozen sections of human, calf, rabbit, rat, cat, dog, goat, lamb, and hog corneas were stained with various lectins using an avidin-biotin-peroxidase complex to study glycoconjugates of stromal matrix. Staining of the stromal matrix and keratocytes with an alpha-galactose-specific lectin, Griffonia simplicifolia I (GSA-I) was species-dependent. The stromal matrices of cat, dog, and hog corneas invariably reacted intensely with this lectin, whereas those of the human, calf, rabbit, rat, and lamb did not react. A positive reaction with GSA-I could be abolished in each instance by preincubation of the sections with alpha-galactosidase. The stromal matrices and keratocytes of all nine species reacted positively with wheat germ agglutinin, concanavalin A, and Ricinus communis agglutinin but did not react with soybean agglutinin. Results of this study may help select an appropriate animal model for further investigate human corneal stromal glycoconjugates.

  2. Pleiotropic effects of cancer cells' secreted factors on human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Al-toub, Mashael; Almusa, Abdulaziz; Almajed, Mohammed

    2013-01-01

    cells' secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs. METHODS: Morphological changes were assessed using fluorescence microscopy......, but not from MCF7 and HT-29, developed an elongated, spindle-shaped morphology with bipolar processes. In association with phenotypic changes, genome-wide gene expression and bioinformatics analysis revealed an enhanced pro-inflammatory response of those MSCs. Pharmacological inhibitions of FAK and MAPKK......INTRODUCTION: Studying cancer tumors' microenvironment may reveal a novel role in driving cancer progression and metastasis. The biological interaction between stromal (mesenchymal) stem cells (MSCs) and cancer cells remains incompletely understood. Herein, we investigated the effects of tumor...

  3. GRAFTING OF HUMAN BONE MARROW STROMAL CELLS INTO SPINAL CORD INJURY: A COMPARISON OF DELIVERY METHODS

    Science.gov (United States)

    Paul, Courtney; Samdani, Amer F.; Betz, Randal R.; Fischer, Itzhak; Neuhuber, Birgit

    2011-01-01

    Study Design Three groups of 6 rats received subtotal cervical spinal cord hemisections followed with marrow stromal cell (MSC) transplants by lumbar puncture (LP), intravenous delivery (IV) or direct injection into the injury (control). Animals survived for 4 or 21 days. Objective Cell therapy is a promising strategy for the treatment of spinal cord injury (SCI). The mode of cell delivery is crucial for the translation to the clinic. Injections directly into the parenchyma may further damage already compromised tissue; therefore, less invasive methods like LP or IV delivery are preferable. Summary of Background Data Human bone marrow stromal cells (MSC) are multipotent mesenchymal adult stem cells that have a potential for autologous transplantation, obviating the need for immune suppression. While previous studies have established that MSC can be delivered to the injured spinal cord by both LP and IV, the efficacy of cell delivery has not been directly compared with respect to efficacy of delivery and effects on the host. Methods Purified MSC from a human donor were transplanted into the CSF at the lumbar region (LP), into the femoral vein (IV), or directly into the injury (control). After sacrifice, spinal cord sections were analyzed for MSC graft size, tissue sparing, host immune response, and glial scar formation, using specific antibodies as well as Nissl-myelin staining. Results LP delivery of MSC to the injured spinal cord is superior to IV delivery. Cell engraftment and tissue sparing were significantly better after LP delivery and host immune response after LP delivery was reduced compared to IV delivery. Conclusions LP is an ideal minimally-invasive technique to deliver cellular transplants to the injured spinal cord. It is superior to IV delivery and, together with the potential for autologous transplantation, lends itself for clinical application. PMID:19182705

  4. Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

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    Handgretinger Rupert

    2010-01-01

    Full Text Available Abstract Background Human multipotent mesenchymal stromal cells (MSC can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity. Results After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation. Conclusion Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

  5. Human endometrial stromal stem cells differentiate into megakaryocytes with the ability to produce functional platelets.

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    Wang, Jinju; Chen, Shuzhen; Zhang, Cheng; Stegeman, Samantha; Pfaff-Amesse, Teresa; Zhang, Ying; Zhang, Wenfeng; Amesse, Lawrence; Chen, Yanfang

    2012-01-01

    Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs). The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs) has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs). In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29) and negative hematopoietic stem cell markers (CD45 and CD34) expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO) for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1) MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation) accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2) expression; 2) The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm) and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation); 3) Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4) Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system) as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.

  6. Human endometrial stromal stem cells differentiate into megakaryocytes with the ability to produce functional platelets.

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    Jinju Wang

    Full Text Available Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs. The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs. In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29 and negative hematopoietic stem cell markers (CD45 and CD34 expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1 MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2 expression; 2 The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation; 3 Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4 Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.

  7. Non-glycanated Decorin Is a Drug Target on Human Adipose Stromal Cells

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    Alexes C. Daquinag

    2017-09-01

    Full Text Available Adipose stromal cells (ASCs have been identified as a mesenchymal cell population recruited from white adipose tissue (WAT by tumors and supporting cancer progression. We have previously reported the existence of a non-glycanated decorin isoform (ngDCN marking mouse ASCs. We identified a peptide CSWKYWFGEC that binds to ngDCN and hence can serve as a vehicle for ASC-directed therapy delivery. We used hunter-killer peptides composed of CSWKYWFGEC and a pro-apoptotic moiety to deplete ASCs and suppress growth of mouse tumors. Here, we report the discovery of the human non-glycanated decorin isoform. We show that CSWKYWFGEC can be used as a probe to identify ASCs in human WAT and tumors. We demonstrate that human ngDCN is expressed on ASC surface. Finally, we validate ngDCN as a molecular target for pharmacological depletion of human ASCs with hunter-killer peptides. We propose that ngDCN-targeting agents could be developed for obesity and cancer treatment.

  8. Adult human bone marrow stromal spheres express neuronal traits in vitro and in a rat model of Parkinson's disease

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    Suon, Sokreine; Yang, Ming; Iacovitti, Lorraine

    2006-01-01

    Adult human bone marrow stromal cells (hMSCs) grown in suspension culture gave rise to spheres of neural progenitor (NP) cells, capable of expressing both dopaminergic (DA) and GABAergic (GABA) traits. After transplantation into the Parkinsonian rat, human NPs and neurons were present at 2 weeks. Although no DA neurons appeared to survive transplantation, there were abundant GABA neurons present in the graft. By 4 weeks, however, all cells had died. Finding ways to prolong survival and promot...

  9. Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells

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    Thieme, Sebastian; Stopp, Sabine; Bornhäuser, Martin; Ugarte, Fernando; Wobus, Manja; Kuhn, Matthias; Brenner, Sebastian

    2013-01-01

    The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal str...

  10. Thymic Neuroblastoma within a Thymic Cyst in an Adult

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    Yuichiro Ueda

    2012-08-01

    Full Text Available Case Presentation: A 65-year-old female patient with no clinical manifestations was hospitalized for examination and treatment of an anterior mediastinal tumor found at the time of a regular health checkup. Enhanced computed tomography (CT and magnetic resonance imaging revealed a cystic lesion containing a solid tumor. Positron emission tomography-CT demonstrated increased uptake in the solid lesion. Tumor resection with total thymectomy was performed. A pathological diagnosis of thymic neuroblastoma within a thymic cyst was made. Micorscopic examination revealed that tumor cells of the solid component were lined with thymic epithelial cells of the inner cyst wall. Furthermore, some tumor cells of the solid component had melanin granules. These findings suggest that this tumor arose from progenitors of the thymic epithelial cells with the potential to differentiate along neural lines. Conclusions: Neuroblastoma commonly occurs in children. However, the diagnosis of neuroblastoma in adults has been reported in several case reports. We report an adult case of histogenetically informative thymic neuroblastoma within a thymic cyst. There are no standard treatment strategies and chemotherapy protocols. Complete surgical resection might be important for a better outcome.

  11. CD29 is highly expressed on epithelial, myoepithelial and mesenchymal stromal cells of human salivary glands.

    Science.gov (United States)

    Togarrati, Padma Priya; Dinglasan, Nuntana; Desai, Shivani; Ryan, William R; Muench, Marcus O

    2017-12-02

    The phenotype of the cells present in the ductal region of salivary glands have been well characterized. However, it is imperative to identify novel biomarkers that can identify different cell types present in other glandular components for the development of therapeutic strategies and diagnostics of salivary gland disorders and malignancies. Our study aimed at the characterization of the expression and distribution of various cell surface markers, especially with a focus on CD29 in human fetal as well as adult glands. Paired human midgestation fetal and adult parotid, sublingual and submandibular glands were collected. Phenotypic expression of various lineage-specific cell surface markers including CD29 was investigated in freshly collected glands. The findings were further corroborated by immunohistochemistry assay. Enriched expression of CD29 was found on acinar and ductal epithelial, mesenchymal stromal and myoepithelial cells; CD29+ cells co-expressed epithelial (CD324, CD326, NKCC1 and CD44), mesenchymal (CD73, CD90, vimentin and CD34) and myoepithelial (α-SMA) cell-specific progenitor markers in both fetal as well as adult salivary glands. CD29 is widely expressed in human salivary glands and, it could serve as a potential biomarker for devising novel cellular therapeutic and diagnostic strategies for salivary gland disorders and malignancies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  12. Cannabidiol Activates Neuronal Precursor Genes in Human Gingival Mesenchymal Stromal Cells.

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    Soundara Rajan, Thangavelu; Giacoppo, Sabrina; Scionti, Domenico; Diomede, Francesca; Grassi, Gianpaolo; Pollastro, Federica; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

    2017-06-01

    In the last years, mesenchymal stromal cells (MSCs) from oral tissues have received considerable interest in regenerative medicine since they can be obtained with minimal invasive procedure and exhibit immunomodulatory properties. This study was aimed to investigate whether in vitro pre-treatment of MSCs obtained from human gingiva (hGMSCs) with Cannabidiol (CBD), a cannabinoid component produced by the plant Cannabis sativa, may promote human gingiva derived MSCs to differentiate toward neuronal precursor cells. Specifically, we have treated the hGMSCs with CBD (5 µM) for 24 h in order to evaluate the expression of genes involved in cannabidiol signaling, cell proliferation, self-renewal and multipotency, and neural progenitor cells differentiation. Next generation sequencing (NGS) demonstrated that CBD activates genes associated with G protein coupled receptor signaling in hGMSCs. Genes involved in DNA replication, cell cycle, proliferation, and apoptosis were regulated. Moreover, genes associated with the biological process of neuronal progenitor cells (NCPs) proliferation, neuron differentiation, neurogenesis, and nervous system development were significantly modulated. From our results, we hypothesize that human gingiva-derived MSCs conditioned with CBD could represent a valid method for improving the hGMSCs phenotype and thus might be a potential therapeutic tool in the treatment of neurodegenerative diseases. J. Cell. Biochem. 118: 1531-1546, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Rac1 deletion causes thymic atrophy.

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    Lukas Hunziker

    Full Text Available The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation.

  14. Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density.

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    Cholewa, Dominik; Stiehl, Thomas; Schellenberg, Anne; Bokermann, Gudrun; Joussen, Sylvia; Koch, Carmen; Walenda, Thomas; Pallua, Norbert; Marciniak-Czochra, Anna; Suschek, Christoph V; Wagner, Wolfgang

    2011-01-01

    The composition of mesenchymal stromal cells (MSCs) changes in the course of in vitro culture expansion. Little is known how these cell preparations are influenced by culture media, plating density, or passaging. In this study, we have isolated MSCs from human adipose tissue in culture medium supplemented with either fetal calf serum (FCS) or human platelet lysate (HPL). In addition, culture expansion was simultaneously performed at plating densities of 10 or 10,000 cells/cm(2). The use of FCS resulted in larger cells, whereas HPL significantly enhanced proliferation. Notably, HPL also facilitated expansion for more population doublings than FCS (43 ± 3 vs. 22 ± 4 population doubling; p < 0.001), while plating density did not have a significant effect on long-term growth curves. To gain further insight into population dynamics, we conceived a cellular automaton model to simulate expansion of MSCS. It is based on the assumptions that the number of cell divisions is limited and that due to contact inhibition proliferation occurs only at the rim of colonies. The model predicts that low plating densities result in more heterogeneity with regard to cell division history, and favor subpopulations of higher migratory activity. In summary, HPL is a suitable serum supplement for isolation of MSC from adipose tissue and facilitates more population doublings than FCS. Cellular automaton computer simulations provided additional insights into how complex population dynamics during long-term expansion are affected by plating density and migration.

  15. Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

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    Lindolfo da Silva Meirelles

    2016-03-01

    Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747.

  16. Octanoate in Human Albumin Preparations Is Detrimental to Mesenchymal Stromal Cell Culture

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    Way-Wua Wong

    2015-01-01

    Full Text Available Cell therapies hold great promise as the next major advance in medical treatment. To enable safe, effective ex vivo culture whilst maintaining cell phenotype, growth media constituents must be carefully controlled. We have used a chemically defined mesenchymal stromal cell culture medium to investigate the influence of different preparations of human serum albumin. We examined two aspects of cell culture, growth rate as measured by population doubling time and colony forming ability which is a representative measure of the stemness of the cell population. Albumin preparations showed comparative differences in both of these criteria. Analysis of the albumin bound fatty acids also showed differences depending on the manufacturing procedure used. We demonstrated that octanoate, an additive used to stabilize albumin during pasteurization, slows growth and lowers colony forming ability during ex vivo culture. Further to this we also found the level of Na+/K+ ATPase, a membrane bound cation pump inhibited by octanoate, is increased in cells exposed to this compound. We conclude that the inclusion of human serum albumin in ex vivo growth media requires careful consideration of not only the source of albumin, but also the associated molecular cargo, for optimal cell growth and behavior.

  17. Optimizing the osteogenic differentiation of human mesenchymal stromal cells by the synergistic action of growth factors.

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    Açil, Yahya; Ghoniem, Amir-Alexander; Wiltfang, Jörg; Gierloff, Matthias

    2014-12-01

    A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p human MSCs in vitro. Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  18. Interferon γ induced compositional changes in human bone marrow derived mesenchymal stem/stromal cells.

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    Guan, Qingdong; Ezzati, Peyman; Spicer, Victor; Krokhin, Oleg; Wall, Donna; Wilkins, John A

    2017-01-01

    Mesenchymal stem/stromal cells (MSC) display a range of immunoregulatory properties which can be enhanced by the exposure to cytokines such interferon γ (IFN-γ). However the compositional changes associated with the 'licensing' of these cells have not been clearly defined. The present study was undertaken to provide a detailed comparative proteomic analysis of the compositional changes that occur in human bone marrow derived MSC following 20 h treatment with IFN-γ. 2D LC MSMS analysis of control and IFN-γ treated cells from 5 different healthy donors provided confident identification of more than 8400 proteins. In total 210 proteins were shown to be significantly altered in their expression levels (≥|2SD|) following IFN-γ treatment. The changes for several of these proteins were confirmed by flow cytometry. STRING analysis determined that approximately 30% of the altered proteins physically interacted in described interferon mediated processes. Comparison of the list of proteins that were identified as changed in the proteomic analysis with data for the same proteins in the Interferome DB indicated that ~35% of these proteins have not been reported to be IFN-γ responsive in a range of cell types. This data provides an in depth analysis of the proteome of basal and IFN-γ treated human mesenchymal stem cells and it identifies a number of novel proteins that may contribute to the immunoregulatory capacity if IFN-γ licensed cells.

  19. WISP 1 is an important survival factor in human mesenchymal stromal cells.

    Science.gov (United States)

    Schlegelmilch, Katrin; Keller, Alexander; Zehe, Viola; Hondke, Sylvia; Schilling, Tatjana; Jakob, Franz; Klein-Hitpass, Ludger; Schütze, Norbert

    2014-11-10

    WNT-induced secreted protein 1 (WISP1/CCN4), a member of the CCN protein family, acts as a downstream factor of the canonical WNT signaling pathway. Its expression is known to affect proliferation and differentiation of human mesenchymal stromal cells (hMSCs), which are fundamental for the development and maintenance of the musculoskeletal system. Whereas a dysregulated, excessive expression of WISP1 often reflects its oncogenic potential via the inhibition of apoptosis, our study emphasizes the importance of WISP1 signaling for the survival of primary human cells. We have established the efficient and specific down-regulation of endogenous WISP1 transcripts by gene silencing in hMSCs and observed cell death as a consequence of WISP1 deficiency. This was confirmed by Annexin V staining for apoptotic cells. DNA microarray analyses of WISP1 down-regulated versus control samples revealed several clusters of differentially expressed genes important for apoptosis induction such as TNF-related apoptosis-inducing ligand 1 (TRAIL) and the corresponding apoptosis-inducing receptors TRAIL-R1 and -R2. An increased expression of TRAIL and its receptors TRAIL-R1 and -R2 in WISP1-deficient hMSCs was confirmed by immunocytofluorescence. Accordingly, WISP1 deficiency is likely to cause TRAIL-induced apoptosis. This is an important novel finding, which suggests that WISP1 is indispensable for the protection of healthy hMSCs against TRAIL-induced apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Expression of CD44 standard form and variant isoforms in human bone marrow stromal cells

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    Xiaofeng Wang

    2017-05-01

    Full Text Available Human Bone Marrow Stromal Cells (hBMSCs can migrate from bone marrow to injured tissues, where they may differentiate into different types of new cells for replacement of dysfunctional cells. CD44 plays an important role in stem cell movement. The expression distribution of CD44 standard form (CD44S and CD44 variants (CD44V is closely related to cell movement and tissue migration. The aim of this study was to evaluate the expressions of CD44S and CD44V in hBMSCs. The hBMSCs from four human subjects were cultured in vitro. Phenotypic properties were analyzed by flow cytometry, and adipocyte and osteoblast differentiations were evaluated at passage 4. The expressions of CD44S and CD44V were examined using quantitative real-time polymerase chain reaction (q-PCR. Results showed that hBMSCs were successfully cultured, with positive expressions of markers of mesenchymal cells (CD90, CD73, CD105, and negative expressions of markers of hematopoietic cells (CD34, CD45. The cultured hBMSCs can be induced to differentiate into adipocytes and osteoblasts. Q-PCR results showed that the expression of CD44S was significantly higher than the expressions of different CD44V isoforms in different samples. These results revealed significant differences in the distributions of CD44S and CD44V gene expressions, demonstrating a dominant CD44S expression in hBMCSs.

  1. Kisspeptin-10 inhibits stromal-derived factor 1-induced invasion of human endometrial cancer cells.

    Science.gov (United States)

    Schmidt, Elena; Haase, Maike; Ziegler, Elke; Emons, Günter; Gründker, Carsten

    2014-02-01

    The cross talk between metastatic cancer cells and target sites is critical for the development and progression of metastases. Disruption of this interaction will allow to design mechanism-based effective and specific therapeutic interventions for metastases. We have established a coculture system of cells derived from different tumor entities and MG63 human osteoblastlike cells to analyze tumor cell invasion. Recently, we have shown that breast cancer cell invasion was dramatically increased when cocultured with MG63 cells.Using this model, we have now analyzed whether stromal-derived factor 1 (SDF-1) is responsible for human endometrial cancer cell invasion and whether kisspeptin-10 (KP-10) treatment affects SDF-1-induced invasion of endometrial cancer cells in vitro. Invasion was quantified by assessment of endometrial cancer cell migration rate through an artificial basement membrane in a modified Boyden chamber during coculture with MG63 cells or after treatment with SDF-1α, SDF-1β, or the combination of both SDF-1 isoforms. In addition, the role of SDF-1 in invasion of endometrial cancer cells was analyzed by blocking SDF-1 secretion during coculture with MG64 cells. Furthermore, the effects of KP-10 treatment on MG63 coculture-driven and SDF-1-induced invasion were analyzed. Endometrial cancer cell invasion was significantly increased when cocultured with MG63 cells. Treatment with KP-10 reduced the ability to invade a reconstituted basement membrane and to migrate in response to the cellular stimulus. This effect was significant in a dose window of 10(-13) to 10(-11) mol/L. During coculture, SDF-1 protein expression of MG63 cells was significantly increased. The MG63 coculture-induced increase of endometrial cancer cell invasion could be blocked by anti-SDF-1 antibodies. Treatment of endometrial cancer cells in monoculture (without MG63) with SDF-1α, SDF-1β, or the combination of both isoforms resulted in a significant increase of endometrial cancer

  2. Human corneal fibrillogenesis. Collagen V structural analysis and fibrillar assembly by stromal fibroblasts in culture.

    Science.gov (United States)

    Ruggiero, F; Burillon, C; Garrone, R

    1996-08-01

    The stroma of the developing cornea is a highly organized extracellular matrix formed essentially by uniform, small-diameter collagen fibrils with constant interfibrillar spacing. Unlike the fibrillogenesis of chicken cornea, the assembly and maturation of human corneal fibrils have been poorly investigated. In the current study, the authors aimed to ascertain the heterotypic organization (collagens I and V) of the human corneal fibrils at the supramolecular level. To gain more insight into the molecular structure of collagen V, its cellular source, and its role in fibrillogenesis, the authors used cultured human corneal fibroblasts. The structure of human corneal stroma after brief homogenization of the tissue was analyzed by immunogold labeling using specific polyclonal antibodies and rotary shadowing. Biochemical, electron microscopic, and immunolabeling approaches were used to investigate the collagen fibril formation and the extracellular matrix synthesis using human corneal fibroblasts grown in culture as a model system. The authors showed that in human corneal stroma, collagen I is distributed uniformly along the striated fibrils, in contrast to collagen V, which could be identified only at sites at which the fibrils partially were disrupted. Rotary shadowing observations of the homogenate revealed that collagen VI, a major component of the human cornea, was associated closely with the collagen fibril surface. Corneal fibroblasts synthesize and deposit a collagenous matrix with fibrils resembling those of the human cornea in appearance and collagen composition. Biochemical data indicate that a high concentration (20% to 30%) of collagen V is synthesized by stromal fibroblasts and that collagen V molecules are processed similarly to matrix forms in which the extension peptides are retained on the molecules. The heterotypic nature (collagens I and V) of human corneal fibrils was determined. Results indicate that human corneal fibroblasts synthesize the major

  3. Derivation of Insulin Producing Cells From Human Endometrial Stromal Stem Cells and Use in the Treatment of Murine Diabetes

    OpenAIRE

    Santamaria, Xavier; Massasa, Efi E; Feng, Yuzhe; Wolff, Erin; Taylor, Hugh S

    2011-01-01

    Pancreatic islet cell transplantation is an effective approach to treat type 1 diabetes, however the shortage of cadaveric donors and limitations due to rejection require alternative solutions. Multipotent cells derived from the uterine endometrium have the ability to differentiate into mesodermal and ectodermal cellular lineages, suggesting the existence of mesenchymal stem cells in this tissue. We differentiated human endometrial stromal stem cells (ESSC) into insulin secreting cells using ...

  4. Leukemia inhibitory factor increases the proliferation of human endometrial stromal cells and expression of genes related to pluripotency

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    Mojdeh Salehnia

    2017-08-01

    Full Text Available Background: Concerning the low population of human endometrial mesenchymal cells within the tissue and their potential application in the clinic and tissue engineering, some researches have been focused on their in vitro expansion. Objective: The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF as a proliferative factor on the expansion and proliferation of human endometrial stromal cells. Materials and Methods: In this experimental study, the isolated and cultured human endometrial stromal cells from women at ovulatory phase aged 20-35 years, after fourth passage were divided into control and LIF-treated groups. In the experimental group, the endometrial cells were treated by 10 ng/ml LIF in culture media and the cultured cells without adding LIF considered as control group. Both groups were evaluated and compared for proliferation rate using MTT assay, for CD90 marker by flow cytometric analysis and for the expression of Oct4, Nanog, PCNA and LIFr genes using real-time RT-PCR. Results: The proliferation rate of control and LIF-treated groups were 1.17±0.17 and 1.61±0.06 respectively and there was a significant increase in endometrial stromal cell proliferation following in vitro treatment by LIF compared to control group (p=0.049. The rate of CD90 positive cells was significantly increased in LIFtreated group (98.96±0.37% compared to control group (94.26±0.08% (p=0.0498. Also, the expression ratio of all studied genes was significantly increased in the LIFtreated group compared to control group (p=0.0479. Conclusion: The present study showed that LIF has a great impact on proliferation, survival, and maintenance of pluripotency of human endometrial stromal cells and it could be applicable in cell therapies.

  5. Leukemia inhibitory factor increases the proliferation of human endometrial stromal cells and expression of genes related to pluripotency.

    Science.gov (United States)

    Salehnia, Mojdeh; Fayazi, Mehri; Ehsani, Shokreya

    2017-04-01

    Concerning the low population of human endometrial mesenchymal cells within the tissue and their potential application in the clinic and tissue engineering, some researches have been focused on their in vitro expansion. The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) as a proliferative factor on the expansion and proliferation of human endometrial stromal cells. In this experimental study, the isolated and cultured human endometrial stromal cells from women at ovulatory phase aged 20-35 years, after fourth passage were divided into control and LIF-treated groups. In the experimental group, the endometrial cells were treated by 10 ng/ml LIF in culture media and the cultured cells without adding LIF considered as control group. Both groups were evaluated and compared for proliferation rate using MTT assay, for CD90 marker by flow cytometric analysis and for the expression of Oct4, Nanog, PCNA and LIFr genes using real-time RT-PCR. The proliferation rate of control and LIF-treated groups were 1.17±0.17 and 1.61±0.06 respectively and there was a significant increase in endometrial stromal cell proliferation following in vitro treatment by LIF compared to control group (p=0.049). The rate of CD90 positive cells was significantly increased in LIF-treated group (98.96±0.37%) compared to control group (94.26±0.08%) (p=0.0498). Also, the expression ratio of all studied genes was significantly increased in the LIF-treated group compared to control group (p=0.0479). The present study showed that LIF has a great impact on proliferation, survival, and maintenance of pluripotency of human endometrial stromal cells and it could be applicable in cell therapies.

  6. Mesenchymal Stromal/stem Cell-derived Extracellular Vesicles Promote Human Cartilage RegenerationIn Vitro.

    Science.gov (United States)

    Vonk, Lucienne A; van Dooremalen, Sanne F J; Liv, Nalan; Klumperman, Judith; Coffer, Paul J; Saris, Daniël B F; Lorenowicz, Magdalena J

    2018-01-01

    Osteoarthritis (OA) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. Recently, allogeneic human mesenchymal stromal/stem cells (MSC) entered clinical trials as a novel therapy for OA. Increasing evidence suggests that therapeutic efficacy of MSC depends on paracrine signalling. Here we investigated the role of extracellular vesicles (EVs) secreted by human bone marrow derived MSC (BMMSC) in human OA cartilage repair. To test the effect of BMMSC-EVs on OA cartilage inflammation, TNF-alpha-stimulated OA chondrocyte monolayer cultures were treated with BMMSC-EVs and pro-inflammatory gene expression was measured by qRT-PCR after 48 h. To assess the impact of BMMSC-EVs on cartilage regeneration, BMMSC-EVs were added to the regeneration cultures of human OA chondrocytes, which were analyzed after 4 weeks for glycosaminoglycan content by 1,9-dimethylmethylene blue (DMMB) assay. Furthermore, paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-O) and type II collagen (immunostaining). We show that BMMSC-EVs inhibit the adverse effects of inflammatory mediators on cartilage homeostasis. When co-cultured with OA chondrocytes, BMMSC-EVs abrogated the TNF-alpha-mediated upregulation of COX2 and pro-inflammatory interleukins and inhibited TNF-alpha-induced collagenase activity. BMMSC-EVs also promoted cartilage regeneration in vitro . Addition of BMMSC-EVs to cultures of chondrocytes isolated from OA patients stimulated production of proteoglycans and type II collagen by these cells. Our data demonstrate that BMMSC-EVs can be important mediators of cartilage repair and hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis.

  7. Mechanical properties of cross-linked collagen meshes after human adipose derived stromal cells seeding.

    Science.gov (United States)

    Ochoa, Ignacio; Peña, Estefania; Andreu, Enrique J; Pérez-Ilzarbe, Maitane; Robles, Jose E; Alcaine, Clara; López, Tania; Prósper, Felipe; Doblaré, Manuel

    2011-02-01

    The main goal of this study was to evaluate the potential of collagen meshes derived from porcine dermis as scaffolds for repairing pelvic organ prolapses. Mechanical properties of collagen meshes with different cross-linking percentages before and after Adipose Derived Stromal Cells (ADSC) seeding were studied as well as the cell-scaffold interaction. Uniaxial tensile tests of the collagen meshes with three different cross-linking percentages (full-, partial-, and noncross-linked) were carried out along orthogonal directions. Their mechanical properties were studied with the same tests before and after seeding with human derived adipose stem cells (ADSC) after 1 and 7 days. Histological analyses were performed to determine adhesion and proliferation of ADSC. Significant differences in mechanical properties of the unseeded meshes were observed between each orthogonal direction independently of the cross-linking percentage. A better cell adhesion rate was observed in the cross-linked meshes. An increase in the mechanical properties after cell seeding was observed with a direct relation with the degree of cross-linking. All meshes analyzed showed a marked anisotropy that should be taken into account during the surgical procedure. The cross-linking treatment increased cell adhesion and the mechanical properties of the collagen meshes after seeding. These results suggest that the mechanical properties of this type of collagen mesh could be useful as scaffolds for repair of pelvic organ prolapse. 2010 Wiley Periodicals, Inc.

  8. Stromal interaction molecule 1 regulates growth, cell cycle, and apoptosis of human tongue squamous carcinoma cells.

    Science.gov (United States)

    Cui, Xiaobo; Song, Laixiao; Bai, Yunfei; Wang, Yaping; Wang, Boqian; Wang, Wei

    2017-04-30

    Oral tongue squamous cell carcinoma (OTSCC) is the most common type of oral carcinomas. However, the molecular mechanism by which OTSCC developed is not fully identified. Stromal interaction molecule 1 (STIM1) is a transmembrane protein, mainly located in the endoplasmic reticulum (ER). STIM1 is involved in several types of cancers. Here, we report that STIM1 contributes to the development of human OTSCC. We knocked down STIM1 in OTSCC cell line Tca-8113 with lentivirus-mediated shRNA and found that STIM1 knockdown repressed the proliferation of Tca-8113 cells. In addition, we also showed that STIM1 deficiency reduced colony number of Tca-8113 cells. Knockdown of STIM1 repressed cells to enter M phase of cell cycle and induced cellular apoptosis. Furthermore, we performed microarray and bioinformatics analysis and found that STIM1 was associated with p53 and MAPK pathways, which may contribute to the effects of STIM1 on cell growth, cell cycle, and apoptosis. Finally, we confirmed that STIM1 controlled the expression of MDM2, cyclin-dependent kinase 4 (CDK4), and growth arrest and DNA damage inducible α (GADD45A) in OTSCC cells. In conclusion, we provide evidence that STIM1 contributes to the development of OTSCC partially through regulating p53 and MAPK pathways to promote cell cycle and survival. © 2017 The Author(s).

  9. Xeno-Free Strategies for Safe Human Mesenchymal Stem/Stromal Cell Expansion: Supplements and Coatings

    Directory of Open Access Journals (Sweden)

    M. Cimino

    2017-01-01

    Full Text Available Human mesenchymal stem/stromal cells (hMSCs have generated great interest in regenerative medicine mainly due to their multidifferentiation potential and immunomodulatory role. Although hMSC can be obtained from different tissues, the number of available cells is always low for clinical applications, thus requiring in vitro expansion. Most of the current protocols for hMSC expansion make use of fetal bovine serum (FBS as a nutrient-rich supplement. However, regulatory guidelines encourage novel xeno-free alternatives to define safer and standardized protocols for hMSC expansion that preserve their intrinsic therapeutic potential. Since hMSCs are adherent cells, the attachment surface and cell-adhesive components also play a crucial role on their successful expansion. This review focuses on the advantages/disadvantages of FBS-free media and surfaces/coatings that avoid the use of animal serum, overcoming ethical issues and improving the expansion of hMSC for clinical applications in a safe and reproducible way.

  10. Characterization of Human Knee and Chin Adipose-Derived Stromal Cells

    Directory of Open Access Journals (Sweden)

    Magali Kouidhi

    2015-01-01

    Full Text Available Animal study findings have revealed that individual fat depots are not functionally equivalent and have different embryonic origins depending on the anatomic location. Mouse bone regeneration studies have also shown that it is essential to match the Hox code of transplanted cells and host tissues to achieve correct repair. However, subcutaneous fat depots from any donor site are often used in autologous fat grafting. Our study was thus carried out to determine the embryonic origins of human facial (chin and limb (knee fat depots and whether they had similar features and molecular matching patterns. Paired chin and knee fat depots were harvested from 11 subjects and gene expression profiles were determined by DNA microarray analyses. Adipose-derived stromal cells (ASCs from both sites were isolated and analyzed for their capacity to proliferate, form clones, and differentiate. Chin and knee fat depots expressed a different HOX code and could have different embryonic origins. ASCs displayed a different phenotype, with chin-ASCs having the potential to differentiate into brown-like adipocytes, whereas knee-ASCs differentiated into white adipocytes. These results highlighted different features for these two fat sites and indicated that donor site selection might be an important factor to be considered when applying adipose tissue in cell-based therapies.

  11. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis.

    Science.gov (United States)

    Jafari, Abbas; Qanie, Diyako; Andersen, Thomas L; Zhang, Yuxi; Chen, Li; Postert, Benno; Parsons, Stuart; Ditzel, Nicholas; Khosla, Sundeep; Johansen, Harald Thidemann; Kjærsgaard-Andersen, Per; Delaisse, Jean-Marie; Abdallah, Basem M; Hesselson, Daniel; Solberg, Rigmor; Kassem, Moustapha

    2017-02-14

    Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. miR-141-3p inhibits human stromal (mesenchymal) stem cell proliferation and differentiation

    DEFF Research Database (Denmark)

    Qiu, Weimin; Kassem, Moustapha

    2014-01-01

    Wnt signaling determines human stromal (mesenchymal) stem cell (hMSC) differentiation fate into the osteoblast or adipocyte lineage. microRNAs (miRNAs) are small RNA molecules of 21-25 nucleotides that regulate many aspects of osteoblast biology. Thus, we examined miRNAs regulated by Wnt signaling...... activity, gene expression and in vitro mineralized matrix formation. Bioinformatic studies, Western blot analysis and 3'UTR reporter assay demonstrated that cell division cycle 25A (CDC25A) is a direct target of miR-141-3p. siRNA-mediated knock-down of CDC25A inhibited hMSC proliferation and osteoblast...... in hMSC. We identified miRNA (miR)-141-3p as a Wnt target which in turn inhibited Wnt signaling. Moreover, miR-141-3p inhibited hMSC proliferation by arresting cells at the G1 phase of the cell cycle. miR-141-3p inhibited osteoblast differentiation of hMSC as evidenced by reduced alkaline phosphatase...

  13. Gene methylation of human ovarian carcinoma stromal progenitor cells promotes tumorigenesis.

    Science.gov (United States)

    Ho, Chih-Ming; Shih, Daniel Tzu-Bi; Hsiao, Chih-Chiang; Huang, Shih-Hung; Chang, Shwu-Fen; Cheng, Wen-Fang

    2015-11-23

    This study aimed to investigate whether the DNA methylation of human ovarian carcinoma stromal progenitor cells (OCSPCs) could promote the tumorigenesis of ovarian carcinoma. OCSPCs were first isolated from fresh tumor tissues and ascites of ovarian cancer patients. In vivo and in vitro experiments on the effect of the OCSPCs on tumorigenesis and the effects of DNA demethylation on the OCSPCs were then performed. The OCSPCs possessed self-renewal and multipotent differentiation capacity with elevated expressions of OCT4, NANOG, BMP2, BMP4, Rex-1, AC133 and TGF-β. The OCSPCs, when combined with tumor cells in vivo could promote tumor growth. The methylation profiles of tumor suppressor genes (TSGs) were significantly higher in the OCSPCs than in ovarian cancer cells (p cells. The expression levels of TSGs were re-expressed by 5-aza-2-dC to inhibit the self-renewal and growth of OCSPCs. OCSPCs with decreased TSG expressions in the ovarian tumor microenvironment were able to promote tumorigenesis which could be reversed by DNA demethylation. DNA demethylation reversing the expression of TSGs in OCSPCs may represent a potential therapeutic target for ovarian cancer.

  14. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    Science.gov (United States)

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  15. Deciphering the stromal and hematopoietic cell network of the adventitia from non-aneurysmal and aneurysmal human aorta.

    Directory of Open Access Journals (Sweden)

    Charles-Antoine Dutertre

    Full Text Available Aneurysm is associated to a complex remodeling of arteries that affects all their layers. Although events taking place in the intima and the media have received a particular attention, molecular and cellular events taking place in the adventitia have started to be deciphered only recently. In this study, we have precisely described the composition and distribution of stromal and hematopoietic cells in human arterial adventitia, both at steady state and in the setting of aortic aneurysm. Using polychromatic immunofluorescent and flow cytometry analyses, we observed that unlike the medial layer (which comprises mostly macrophages and T cells among leukocytes, the adventitia comprises a much greater variety of leukocytes. We observed an altered balance in macrophages subsets in favor of M2-like macrophages, an increased proliferation of macrophages, a greater number of all stromal cells in aneurysmal aortas. We also confirmed that in this pathological setting, adventitia comprised blood vessels and arterial tertiary lymphoid organs (ATLOs, which contained also M-DC8(+ dendritic cells (slanDCs that could participate in the induction of T-cell responses. Finally, we showed that lymphatic vessels can be detected in aneurysmal adventitia, the functionality of which will have to be evaluated in future studies. All together, these observations provide an integrative outlook of the stromal and hematopoietic cell network of the human adventitia both at steady state and in the context of aneurysm.

  16. Adiponectin Effect on The Viability of Human Endometrial Stromal Cells and mRNA Expression of Adiponectin Receptors

    Directory of Open Access Journals (Sweden)

    Somayeh Bohlouli

    2013-01-01

    Full Text Available Background: Adiponectin is one of the most important adipokines secreted from fattytissue that has a direct inhibitory effect on the development of cancer cells. Adiponectinplays an important role in human reproduction system and fertility of women. Adiponectinconcentration decreases in women with endometriosis and endometrial cancer.The aim of the present study was to investigate the effect of adiponectin on humanendometrial stromal cell (HESC viability as well as mRNA expression of Adipo R1and Adipo R2 receptors.Materials and Methods: In this experimental study, eight endometrial biopsies weretaken and stromal cells were separated by enzymatic digestion and cell filtrations. Stromalcells of each biopsy were divided into four groups: control, 10, 100, and 200 ng/mladiponectin concentrations. The effect of adiponectin on viability of the normal HESCswas studied by trypan blue staining and the relative expression levels of Adipo R1 andR2 were analyzed by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR. Data were analyzed by one way ANOVA and unpaired student’s t test andp<0.05 was considered significant.Results: Adiponectin decreased viability of normal human endometrial stromal cells ina dose and time dependent manner. Expression of Adipo R1 and Adipo R2 receptors didnot change in the presence of adiponectin.Conclusion: Adiponectin can directly influence the viability of HESCs and decreasetheir viability, but it didn’t change expression of adiponectin receptors.

  17. Expansion in microcarrier-spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells.

    Science.gov (United States)

    Lin, Youshan Melissa; Lim, Jessica Fang Yan; Lee, Jialing; Choolani, Mahesh; Chan, Jerry Kok Yen; Reuveny, Shaul; Oh, Steve Kah Weng

    2016-06-01

    Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. heMSC expanded on either collagen-coated Cytodex 3 microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100β, MMP13 and ALPL, were investigated and compared in both conditions. BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100β) but not of hypertrophic genes (e.g., MMP13 and ALPL). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights

  18. Amelioration of streptozotocin-induced diabetes in mice with cells derived from human marrow stromal cells.

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    Min Zhao

    Full Text Available BACKGROUND: Pluri-potent bone marrow stromal cells (MSCs provide an attractive opportunity to generate unlimited glucose-responsive insulin-producing cells for the treatment of diabetes. We explored the potential for human MSCs (hMSCs to be differentiated into glucose-responsive cells through a non-viral genetic reprogramming approach. METHODS AND FINDINGS: Two HMSC lines were transfected with three genes: PDX-1, NeuroD1 and Ngn3 without subsequent selection, followed by differentiation induction in vitro and transplantation into diabetic mice. Human MSCs expressed mRNAs of the archetypal stem cell markers: Sox2, Oct4, Nanog and CD34, and the endocrine cell markers: PDX-1, NeuroD1, Ngn3, and Nkx6.1. Following gene transfection and differentiation induction, hMSCs expressed insulin in vitro, but were not glucose regulated. After transplantation, hMSCs differentiated further and approximately 12.5% of the grafted cells expressed insulin. The graft bearing kidneys contained mRNA of insulin and other key genes required for the functions of beta cells. Mice transplanted with manipulated hMSCs showed reduced blood glucose levels (from 18.9+/-0.75 to 7.63+/-1.63 mM. 13 of the 16 mice became normoglycaemic (6.9+/-0.64 mM, despite the failure to detect the expression of SUR1, a K(+-ATP channel component required for regulation of insulin secretion. CONCLUSIONS: Our data confirm that hMSCs can be induced to express insulin sufficient to reduce blood glucose in a diabetic mouse model. Our triple gene approach has created cells that seem less glucose responsive in vitro but which become more efficient after transplantation. The maturation process requires further study, particularly the in vivo factors influencing the differentiation, in order to scale up for clinical purposes.

  19. FOXN1GFP/w Reporter hESCs Enable Identification of Integrin-β4, HLA-DR, and EpCAM as Markers of Human PSC-Derived FOXN1+ Thymic Epithelial Progenitors

    Directory of Open Access Journals (Sweden)

    Chew-Li Soh

    2014-06-01

    Full Text Available Thymic epithelial cells (TECs play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1GFP/w line as a readout, we developed a reproducible protocol for generating FOXN1-GFP+ thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-β4 (ITGB4, CD104 and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1+ TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1+ TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.

  20. Epithelial cells prime the immune response to an array of gut-derived commensals towards a tolerogenic phenotype through distinct actions of thymic stromal lymphopoietin and transforming growth factor-beta

    DEFF Research Database (Denmark)

    Zeuthen, Louise Hjerrild; Fink, Lisbeth Nielsen; Frøkiær, Hanne

    2007-01-01

    Humans and other mammals coexist with a diverse array of microbes colonizing the intestine, termed the microflora. The relationship is symbiotic, with the microbes benefiting from a stable environment and nutrient supply, and the host gaining competitive exclusion of pathogens and continuously......-induced semimaturation in a TLR2-dependent fashion. These findings add to our understanding of the hypo-responsiveness of the gut epithelium towards the microflora. Gut DC posses a more tolerogenic phenotype than conventional DC. Here we show that Caco2 spent medium (SM) induces tolerogenic DC with lower expression...

  1. Flotillin-1 protein is upregulated in human endometrial cancer and localization shifts from epithelial to stromal with increasing tumor grade.

    Science.gov (United States)

    Winship, Amy Louise; Rainczuk, Kate; Dimitriadis, Evdokia

    2016-01-01

    Endometrial cancer is the most common invasive gynecological malignancy. Flotillin-1 is an integral membrane protein and estrogen responsive gene. Flotillin-1 expression and localization in human endometrial cancers grades 1-3 was investigated using real-time RT-PCR and immunohistochemistry. Flotillin-1 mRNA levels were unchanged in endometrial cancer versus benign endometrium. Flotillin-1 protein was significantly reduced in the epithelial compartment with increasing tumor grade, although levels increased in the tumor stroma across grades. We have identified a novel factor in human endometrial cancer and observed a shift in epithelial to stromal localization with increasing tumor grade in women.

  2. Adult human bone marrow stromal spheres express neuronal traits in vitro and in a rat model of Parkinson's disease.

    Science.gov (United States)

    Suon, Sokreine; Yang, Ming; Iacovitti, Lorraine

    2006-08-23

    Adult human bone marrow stromal cells (hMSCs) grown in suspension culture gave rise to spheres of neural progenitor (NP) cells, capable of expressing both dopaminergic (DA) and GABAergic (GABA) traits. After transplantation into the Parkinsonian rat, human NPs and neurons were present at 2 weeks. Although no DA neurons appeared to survive transplantation, there were abundant GABA neurons present in the graft. By 4 weeks, however, all cells had died. Finding ways to prolong survival and promote the appropriate neurotransmitter phenotype is essential if hMSCs are to be clinically useful.

  3. Thymic hyperplasia in Graves′ disease

    Directory of Open Access Journals (Sweden)

    Narendra Kotwal

    2013-01-01

    Full Text Available Graves′ disease is an autoimmune thyroid condition characterized by the production of autoantibodies against the thyrotropin receptor. It is known to be associated with autoimmune conditions such as myasthenia gravis, Addison′s disease, type 1 diabetes mellitus, and vitiligo. We present a case of rare autoimmune association of Graves′ disease with thymic hyperplasia which regressed after treatment with antithyroid drugs. Exact pathophysiology of thymic hyperplasia in Graves′ is not well understood; it is likely to be the result of rather than the cause of Graves′ disease.

  4. Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Wen-Hui [Department of Orthopaedics, The Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001 (China); Yan, Ba-Yi; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Medicine, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Department of Anatomy, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Yang, Wu-Lin, E-mail: wulinyoung@163.com [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium (SBIC), Agency for Science, Technology and Research - A*STAR (Singapore)

    2014-01-24

    Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

  5. Human platelet lysate permits scale-up of dental pulp stromal cells for clinical applications.

    Science.gov (United States)

    Govindasamy, Vijayendran; Ronald, Veronica Sainik; Abdullah, Aimi Naim Binti; Ganesan Nathan, Kavitha R; Aziz, Zeti Adura Che Abdul; Abdullah, Mariam; Zain, Rosnah Binti; Kasim, Noor Hayaty Abu; Musa, Sabri; Bhonde, Ramesh R

    2011-11-01

    BACKGROUND AIMS. Dental pulp stromal cells (DPSC) are considered to be a promising source of stem cells in the field of regenerative therapy. However, the usage of DPSC in transplantation requires large-scale expansion to cater for the need for clinical quantity without compromising current good manufacturing practice (cGMP). Existing protocols for cell culturing make use of fetal bovine serum (FBS) as a nutritional supplement. Unfortunately, FBS is an undesirable additive to cells because it carries the risk of transmitting viral and prion diseases. Therefore, the present study was undertaken to examine the efficacy of human platelet lysate (HPL) as a substitute for FBS in a large-scale set-up. METHODS. We expanded the DPSC in Dulbecco's modified Eagle's medium-knock-out (DMEM-KO) with either 10% FBS or 10% HPL, and studied the characteristics of DPSC at pre- (T25 culture flask) and post- (5-STACK chamber) large-scale expansion in terms of their identity, quality, functionality, molecular signatures and cytogenetic stability. RESULTS. In both pre- and post-large-scale expansion, DPSC expanded in HPL showed extensive proliferation of cells (c. 2-fold) compared with FBS; the purity, immune phenotype, colony-forming unit potential and differentiation were comparable. Furthermore, to understand the gene expression profiling, the transcriptomes and cytogenetics of DPSC expanded under HPL and FBS were compared, revealing similar expression profiles. CONCLUSIONS. We present a highly economized expansion of DPSC in HPL, yielding double the amount of cells while retaining their basic characteristics during a shorter time period under cGMP conditions, making it suitable for therapeutic applications.

  6. Expansion strategies for human mesenchymal stromal cells culture under xeno-free conditions.

    Science.gov (United States)

    Tozetti, Patrícia Aparecida; Caruso, Samia Rigotto; Mizukami, Amanda; Fernandes, Taisa Risque; da Silva, Fernanda Borges; Traina, Fabiola; Covas, Dimas Tadeu; Orellana, Maristela Delgado; Swiech, Kamilla

    2017-09-01

    Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost-effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno-free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix-derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno-free bioprocess. UCM MSC were cultured in a scalable planar (compact 10-layer flasks and roller bottles) and 3-D microcarrier-based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 104 and 1.4 ± 0.3 × 104  cells/cm2 . UCM MSC-based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5-23.0 × 104  cells/cm2 ) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno-free expansion processes represents an important step toward a GMP compliant large-scale production platform for MSC-based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358-1367, 2017. © 2017 American Institute of Chemical Engineers.

  7. Genetically engineered mesenchymal stromal cells producing TNFα have tumour suppressing effect on human melanoma xenograft.

    Science.gov (United States)

    Tyciakova, Silvia; Matuskova, Miroslava; Bohovic, Roman; Polakova, Katarina; Toro, Lenka; Skolekova, Svetlana; Kucerova, Lucia

    2015-01-01

    Mesenchymal stromal cells (MSC) are a promising tool for targeted cancer therapy due to their tumour-homing ability. Intrinsic resistance enables the MSC to longer tolerate therapeutic factors, such as prodrug converting enzymes, cytokines and pro-apoptotic proteins. Tumour necrosis factor alpha (TNFα) is known to be cytotoxic to a variety of cancer cells and exert a tumour-destructive capacity. MSC were retrovirally transduced to stable express an exogenous gene encoding the desired therapeutic agent hTNFα. The effect of a TNFα-producing adipose tissue-derived MSC (AT-MSC/hTNFα) was tested on the tumour cell lines of different origins: melanoma (A375), breast carcinoma (SKBR3, MDA-MB-231), colon carcinoma (HT29), ovarian carcinoma (SKOV3) and glioblastoma (U87-MG) cells. The tumour suppressing effect of AT-MSC/hTNFα on A375 melanoma xenografts was monitored in an immunodeficient mouse model in vivo. Engineered AT-MSC are able to constitutively secrete human TNFα protein, induce apoptosis of tumour cell lines via caspase 3/7 activation and inhibit the tumour cell proliferation in vitro. Melanoma A375 and breast carcinoma SKBR3 cells were the most sensitive, and their proliferation in vitro was reduced by conditioned media produced by AT-MSC/hTNFα to 60% and 40%, respectively. The previously reported tumour supportive effect of AT-MSC on subcutaneous A375 melanoma xenograft growth was neutralised and suppressed by engineered AT-MSC stably producing hTNFα. When AT-MSC/hTNFα were coinjected with A375 melanoma cells, the tumour mass inhibition was up to 97.5%. The results of the present study demonstrate that tumour cells respond to hTNFα-based treatment mediated by genetically engineered AT-MSC/hTNFα both in vitro and in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

  8. The NLRP3 Inflammasome Promotes Age-Related Thymic Demise and Immunosenescence

    Directory of Open Access Journals (Sweden)

    Yun-Hee Youm

    2012-01-01

    Full Text Available The collapse of thymic stromal cell microenvironment with age and resultant inability of the thymus to produce naive T cells contributes to lower immune-surveillance in the elderly. Here we show that age-related increase in ‘lipotoxic danger signals’ such as free cholesterol (FC and ceramides, leads to thymic caspase-1 activation via the Nlrp3 inflammasome. Elimination of Nlrp3 and Asc, a critical adaptor required for inflammasome assembly, reduces age-related thymic atrophy and results in an increase in cortical thymic epithelial cells, T cell progenitors and maintenance of T cell repertoire diversity. Using a mouse model of irradiation and hematopoietic stem cell transplantation (HSCT, we show that deletion of the Nlrp3 inflammasome accelerates T cell reconstitution and immune recovery in middle-aged animals. Collectively, these data demonstrate that lowering inflammasome-dependent caspase-1 activation increases thymic lymphopoiesis and suggest that Nlrp3 inflammasome inhibitors may aid the re-establishment of a diverse T cell repertoire in middle-aged or elderly patients undergoing HSCT.

  9. Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

    Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

  10. Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

    Directory of Open Access Journals (Sweden)

    Montzka Katrin

    2009-03-01

    Full Text Available Abstract Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as

  11. Genetically engineered mesenchymal stromal cells produce IL-3 and TPO to further improve human scaffold-based xenograft models.

    Science.gov (United States)

    Carretta, Marco; de Boer, Bauke; Jaques, Jenny; Antonelli, Antonella; Horton, Sarah J; Yuan, Huipin; de Bruijn, Joost D; Groen, Richard W J; Vellenga, Edo; Schuringa, Jan Jacob

    2017-07-01

    Recently, NOD-SCID IL2Rγ -/- (NSG) mice were implanted with human mesenchymal stromal cells (MSCs) in the presence of ceramic scaffolds or Matrigel to mimic the human bone marrow (BM) microenvironment. This approach allowed the engraftment of leukemic samples that failed to engraft in NSG mice without humanized niches and resulted in a better preservation of leukemic stem cell self-renewal properties. To further improve our humanized niche scaffold model, we genetically engineered human MSCs to secrete human interleukin-3 (IL-3) and thrombopoietin (TPO). In vitro, these IL-3- and TPO-producing MSCs were superior in expanding human cord blood (CB) CD34 + hematopoietic stem/progenitor cells. MLL-AF9-transduced CB CD34 + cells could be transformed efficiently along myeloid or lymphoid lineages on IL-3- and TPO-producing MSCs. In vivo, these genetically engineered MSCs maintained their ability to differentiate into bone, adipocytes, and other stromal components. Upon transplantation of MLL-AF9-transduced CB CD34 + cells, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) developed in engineered scaffolds, in which a significantly higher percentage of myeloid clones was observed in the mouse compartments compared with previous models. Engraftment of primary AML, B-cell ALL, and biphenotypic acute leukemia (BAL) patient samples was also evaluated, and all patient samples could engraft efficiently; the myeloid compartment of the BAL samples was better preserved in the human cytokine scaffold model. In conclusion, we show that we can genetically engineer the ectopic human BM microenvironment in a humanized scaffold xenograft model. This approach will be useful for functional study of the importance of niche factors in normal and malignant human hematopoiesis. Copyright © 2017 ISEH - International Society for Experimental Hematology. All rights reserved.

  12. Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors.

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    Maren Schwenke

    Full Text Available Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF triggered chemotaxis (directed locomotion, whereas platelet-derived growth factor (PDGF-BB elicited both chemotaxis and chemokinesis (non-directed locomotion of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating

  13. Human mesenchymal stromal cells as cellular drug-delivery vectors for glioblastoma therapy: a good deal?

    Science.gov (United States)

    Clavreul, Anne; Pourbaghi-Masouleh, Milad; Roger, Emilie; Lautram, Nolwenn; Montero-Menei, Claudia N; Menei, Philippe

    2017-09-29

    Glioblastoma (GB) is the most malignant brain tumor in adults. It is characterized by angiogenesis and a high proliferative and invasive capacity. Standard therapy (surgery, radiotherapy and chemotherapy with temozolomide) is of limited efficacy. Innovative anticancer drugs targeting both tumor cells and angiogenesis are urgently required, together with effective systems for their delivery to the brain. We assessed the ability of human mesenchymal stromal cells (MSCs) to uptake the multikinase inhibitor, sorafenib (SFN), and to carry this drug to a brain tumor following intranasal administration. MSCs were primed with SFN and drug content and release were quantified by analytical chemistry techniques. The ability of SFN-primed MSCs to inhibit the survival of the human U87MG GB cell line and endothelial cells was assessed in in vitro assays. These cells were then administered intranasally to nude mice bearing intracerebral U87MG xenografts. Their effect on tumor growth and angiogenesis was evaluated by magnetic resonance imaging and immunofluorescence analyses, and was compared with the intranasal administration of unprimed MSCs or SFN alone. MSCs took up about 9 pg SFN per cell, with no effect on viability, and were able to release 60% of the primed drug. The cytostatic activity of the released SFN was entirely conserved, resulting in a significant inhibition of U87MG and endothelial cell survival in vitro. Two intranasal administrations of SFN-primed MSCs in U87MG-bearing mice resulted in lower levels of tumor angiogenesis than the injection of unprimed MSCs or SFN alone, but had no effect on tumor volume. We also observed an increase in the proportion of small intratumoral vessels in animals treated with unprimed MSCs; this effect being abolished if the MSCs were primed with SFN. We show the potential of MSCs to carry SFN to brain tumors following an intranasal administration. However, the therapeutic effect is modest probably due to the pro

  14. Wnt5a Regulates the Assembly of Human Adipose Derived Stromal Vascular Fraction-Derived Microvasculatures.

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    Venkat M Ramakrishnan

    Full Text Available Human adipose-derived stromal vascular fraction (hSVF cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1 hSVF because of the rapid UEA1+ endothelium (EC loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%. To determine which Wnt isoform(s and receptor(s may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0-150 ng/ml was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that

  15. Association between in vivo bone formation and ex vivo migratory capacity of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Andersen, Rikke K; Zaher, Walid; Larsen, Kenneth H

    2015-01-01

    INTRODUCTION: There is a clinical need for developing systemic transplantation protocols for use of human skeletal stem cells (also known bone marrow stromal stem cells) (hBMSC) in tissue regeneration. In systemic transplantation studies, only a limited number of hBMSC home to injured tissues...... vivo transwell migration and following intravenous injection, better in vivo homing ability to bone fracture when compared to LBF clones. Comparative microarray analysis of HBF versus LBF clones identified enrichment of gene categories of chemo-attraction, adhesion and migration associated genes. Among...

  16. a stromal myoid cell line provokes thymic erythropoiesis between

    African Journals Online (AJOL)

    hi-tech

    2004-02-02

    Feb 2, 2004 ... in different stages of development obtained after pregnancy failure due to chorioamnionitis (five samples), to implantation of the foetus in the region of the internal os resulting in placenta praevia (six samples), and to endometriosisadenomyosis (four samples). Selection Criteria: All cases were chosen from.

  17. A stromal myoid cell line provokes thymic erythropoiesis between ...

    African Journals Online (AJOL)

    Alexandroupolis) over ten year period (1991-2001). Results: The number of alpha-smooth muscle actin - positive cells significantly increased during the late second and third trimester of gestation. In the above period a relevant increase in the ...

  18. The Effect of Copper on Endometrial Receptivity and Induction of Apoptosis on Decidualized Human Endometrial Stromal Cells.

    Science.gov (United States)

    Carrascosa, Jose P; Cotán, David; Jurado, Inmaculada; Oropesa-Ávila, Manuel; Sánchez-Martín, Pascual; Savaris, Ricardo F; Tan, Justin; Sánchez-Alcázar, Jose A; Tan, Seang L; Horcajadas, José Antonio

    2017-01-01

    Intrauterine devices (IUDs) have been widely used to prevent pregnancies with great efficacy during decades. It has been demonstrated that IUD alters the endometrial gene expression, but there is no scientific data about how copper, a metal commonly used in these devices, by itself, is able to influence the processes of endometrial receptivity and apoptosis in decidualized human endometrial stromal cells. Five endometrial samples were obtained from fertile women and processed by a standard protocol to obtain human endometrial stromal cells for in vitro studies. Stromal cells were cultured in vitro and decidualized for 8 days. At day 6, copper was added to the treatment group or camptothecin as positive control for apoptosis until day 8. Five endometrial samples were used in each group. The aim of this study was to analyze the effect of copper in apoptosis and necrosis by flow cytometry, to visualize the apoptotic microtubule network during apoptosis by immunofluorescence, and finally to determine the gene expression profile of a panel of 192 genes related to endometrial receptivity and immune system by quantitative reverse transcription PCR (RT-qPCR). Copper, compared to the decidualized group, induced changes in the gene expression by an order of magnitude in 49 genes (42 up- and 9 downregulated). This alteration in the decidualization gene signature by copper includes 19 genes involved in the endometriosis pathology and others related to other gynecological disorders such as preeclampsia and infertility. Our results indicate that copper does not increase the apoptosis level induced by the decidualization treatment. However, copper alters the gene expression of some biomarkers of endometrial receptivity and immune response.

  19. In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

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    Vishnubalaji Radhakrishnan

    2012-01-01

    Full Text Available Abstract Background Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs and adult dermal skin (hADSSCs using explants cultures and were compared with bone marrow (hMSC-TERT and adipose tissue-derived mesenchymal stem cells (hADMSCs for their potential differentiation into osteoblasts, adipocytes, and endothelial cells. Results Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC, with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues. Conclusions Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs

  20. Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle.

    Science.gov (United States)

    De Clercq, Katrien; Held, Katharina; Van Bree, Rieta; Meuleman, Christel; Peeraer, Karen; Tomassetti, Carla; Voets, Thomas; D'Hooghe, Thomas; Vriens, Joris

    2015-06-01

    Are members of the transient receptor potential (TRP) channel superfamily functionally expressed in the human endometrial stroma? The Ca(2+)-permeable ion channels TRPV2, TRPV4, TRPC6 and TRPM7 are functionally expressed in primary endometrial stromal cells. Intercellular communication between epithelial and stromal endometrial cells is required to initiate decidualization, a prerequisite for successful implantation. TRP channels are possible candidates as signal transducers involved in cell-cell communication, but no fingerprint is available of the functional distribution of TRP channels in the human endometrium during the luteal phase of the menstrual cycle. Endometrial biopsy samples (previously frozen) from patients of reproductive age with regular menstrual cycles, who were undergoing diagnostic laparoscopic surgery for pain and/or infertility, were analysed. Samples were obtained from the menstrual (Days 1-5, n = 3), follicular (Days 6-14, n = 6), early luteal (Days 15-20, n = 5) and late luteal (Days 21-28, n = 5) phases. In addition, a total of 13 patient samples taken during the luteal phase were used to set up primary cell cultures for further experiments. Quantitative real-time PCR (qRT-PCR), immunocytochemistry, Fura2-based Ca(2+)-microfluorimetry and whole-cell patch clamp experiments were performed to study the functional expression pattern of TRP channels. Specific pharmacological agents, such as Δ(9)-tetrahydrocannabinol, GSK1016790A and 1-oleoyl-2-acetyl-glycerol, were used to functionally assess the expression of TRPV2, TRPV4 and TRPC6, respectively. Expression of TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4 and TRPM7 was detected at the mRNA level in endometrial biopsies (n = 19) and in primary endometrial stromal cell cultures obtained from patients during the luteal phase (n = 5) of the menstrual cycle. Messenger RNA levels of TRPV2, TRPC4 and TRPC6 were significantly increased (P endometrial stromal cells. Ca(2+)-microfluorimetry revealed

  1. Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

    Science.gov (United States)

    Hanga, Mariana P.; Heathman, Thomas R. J.; Coopman, Karen; Nienow, Alvin W.; Williams, David J.; Hewitt, Christopher J.

    2017-01-01

    ABSTRACT Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high‐throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum‐based medium was applied to a serum‐free process in the ambr15, resulting in >250% increase in yield compared to the serum‐based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum‐free and automated processing improved the reproducibility more than 10‐fold compared to the serum‐based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination

  2. In vitro epigenetic reprogramming of human cardiac mesenchymal stromal cells into functionally competent cardiovascular precursors.

    Directory of Open Access Journals (Sweden)

    Matteo Vecellio

    Full Text Available Adult human cardiac mesenchymal-like stromal cells (CStC represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS in the presence of 5 µM all-trans Retinoic Acid (ATRA, 5 µM Phenyl Butyrate (PB, and 200 µM diethylenetriamine/nitric oxide (DETA/NO, to create a novel epigenetically active cocktail (EpiC. Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker I(f current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors.

  3. Distribution of mediastinal ectopic thymic tissue in patients without thymic disease

    Directory of Open Access Journals (Sweden)

    Behnam Sanei

    2015-01-01

    Conclusion: Because of high incidence of ectopic thymic tissue in mediastinum in patients without thymic disease, we recommend wide excision of thymus gland and all of adipose tissue in patients with myasthenia gravis.

  4. Mechanical properties of cross-linked collagen meshes after human adipose derived stromal cells seeding

    OpenAIRE

    Ochoa, I. (Ignacio); Peña, E. (Estefanía); Andreu, E.J. (Enrique José); Perez-Ilzarbe, M. (Maitane); Robles, J.E. (José Enrique); Alcaine, C. (C.); Lopez, T. (Tania); Prosper, F. (Felipe); Doblare, M. (M.)

    2010-01-01

    The main goal of this study was to evaluate the potential of collagen meshes derived from porcine dermis as scaffolds for repairing pelvic organ prolapses. Mechanical properties of collagen meshes with different cross-linking percentages before and after Adipose Derived Stromal Cells (ADSC) seeding were studied as well as the cell-scaffold interaction. Uniaxial tensile tests of the collagen meshes with three different cross-linking percentages (full-, partial-, and non...

  5. High-throughput assay for the identification of compounds regulating osteogenic differentiation of human mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Hugo Alves

    Full Text Available Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. They present multilineage differentiation potential and trophic and immunosuppressive abilities, making them the best candidate for clinical applications. Several molecules have been described to increase bone formation and were mainly discovered by candidate approaches towards known signaling pathways controlling osteogenesis. However, their bone forming potential is still limited, making the search for novel molecules a necessity. High-throughput screening (HTS not only allows the screening of a large number of diverse chemical compounds, but also allows the discovery of unexpected signaling pathways and molecular mechanisms for a certain application, even without the prior knowledge of the full molecular pathway. Typically HTS is performed in cell lines, however, in this manuscript we have performed a phenotypical screen on more clinically relevant human mesenchymal stromal cells, as a proof of principle that HTS can be performed in those cells and can be used to find small molecules that impact stem cell fate. From a library of pharmacologically active small molecules, we were able to identify novel compounds with increased osteogenic activity. These compounds allowed achieving levels of bone-specific alkaline phosphatase higher than any other combination previously known. By combining biochemical techniques, we were able to demonstrate that a medium to high-throughput phenotypic assay can be performed in academic research laboratories allowing the discovery of novel molecules able to enhance stem cell differentiation.

  6. Survival of human mesenchymal stromal cells from bone marrow and adipose tissue after xenogenic transplantation in immunocompetent mice

    DEFF Research Database (Denmark)

    Niemeyer, P; Vohrer, J; Schmal, H

    2008-01-01

    INTRODUCTION: Mesenchymal stromal cells (MSC) represent an attractive cell population for tissue engineering purposes. As MSC are described as immunoprivileged, non-autologous applications seem possible. A basic requirement is the survival of MSC after transplantation in the host. The purpose...... of the current paper was to evaluate the survival of undifferentiated and osteogenically induced human MSC from different origins after transplantation in immunocompetent mice. METHODS: Human MSC were isolated from bone marrow (BMSC) and adipose tissue (ASC). After cultivation on mineralized collagen, MSC were...... transplanted subcutaneously into immunocompetent mice (n=12). Undifferentiated MSC (group A) were compared with osteogenic-induced MSC (group B). Human-specific in situ hybridization and anti-vimentin staining was used to follow MSC after transplantation. Quantitative evaluation of lymphocytes and macrophages...

  7. Wound-healing potential of human umbilical cord blood-derived mesenchymal stromal cells in vitro--a pilot study.

    Science.gov (United States)

    You, Hi-Jin; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2015-11-01

    Our previous studies demonstrated that human bone marrow-derived mesenchymal stromal cells have great potential for wound healing. However, it is difficult to clinically utilize cultured stem cells. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialized for cartilage repair as a first cell therapy product that uses allogeneic stem cells. Should hUCB-MSCs have a superior effect on wound healing as compared with fibroblasts, which are the main cell source in current cell therapy products for wound healing, they may possibly replace fibroblasts. The purpose of this in vitro study was to compare the wound-healing activity of hUCB-MSCs with that of fibroblasts. This study was particularly designed to compare the effect of hUCB-MSCs on diabetic wound healing with those of allogeneic and autologous fibroblasts. Healthy (n = 5) and diabetic (n = 5) fibroblasts were used as the representatives of allogeneic and autologous fibroblasts for diabetic patients in the control group. Human UCB-MSCs (n = 5) were used in the experimental group. Cell proliferation, collagen synthesis and growth factor (basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor-β) production were compared among the three cell groups. Human UCB-MSCs produced significantly higher amounts of vascular endothelial growth factor and basic fibroblast growth factor when compared with both fibroblast groups. Human UCB-MSCs were superior to diabetic fibroblasts but not to healthy fibroblasts in collagen synthesis. There were no significant differences in cell proliferation and transforming growth factor-β production. Human UCB-MSCs may have greater capacity for diabetic wound healing than allogeneic or autologous fibroblasts, especially in angiogenesis. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR and NKG2D

    Directory of Open Access Journals (Sweden)

    Alessandro Poggi

    2006-01-01

    Full Text Available Human natural killer (NK lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis. Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity.

  9. Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

    Science.gov (United States)

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2017-02-01

    Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

  10. Adipose tissue-derived stromal cells inhibit TGF-β1-induced differentiation of human dermal fibroblasts and keloid scar-derived fibroblasts in a paracrine fashion.

    Science.gov (United States)

    Spiekman, Maroesjka; Przybyt, Ewa; Plantinga, Josée A; Gibbs, Susan; van der Lei, Berend; Harmsen, Martin C

    2014-10-01

    Adipose tissue-derived stromal cells augment wound healing and skin regeneration. It is unknown whether and how they can also influence dermal scarring. The authors hypothesized that adipose tissue-derived stromal cells inhibit adverse differentiation of dermal fibroblasts induced by the pivotal factor in scarring, namely, transforming growth factor (TGF)-β. TGF-β1-treated adult human dermal fibroblasts and keloid scar-derived fibroblasts were incubated with adipose tissue-derived stromal cell-conditioned medium and assessed for proliferation and differentiation, particularly the production of collagen, expression of SM22α, and development of hypertrophy and contractility. TGF-β1-induced proliferation of adult human dermal fibroblasts was abolished by adipose tissue-derived stromal cell-conditioned medium. Simultaneously, the medium reduced SM22α gene and protein expression of TGF-β1-treated adult human dermal fibroblasts, and their contractility was reduced also. Furthermore, the medium strongly reduced transcription of collagen I and III genes and their corresponding proteins. In contrast, it tipped the balance of matrix turnover to degradation through stimulating gene expression of matrix metalloproteinase (MMP)-1, MMP-2, and MMP-14, whereas MMP-2 activity was up-regulated also. Even in end-stage myofibroblasts (i.e., keloid scar-derived fibroblasts), adipose tissue-derived stromal cell-conditioned medium suppressed TGF-β1-induced myofibroblast contraction and collagen III gene expression. The authors show that adipose tissue-derived stromal cells inhibit TGF-β1-induced adverse differentiation and function of adult human dermal fibroblasts and TGF-β1-induced contraction in keloid scar-derived fibroblasts, in a paracrine fashion.

  11. Multipotent mesenchymal stromal cells enhance insulin secretion from human islets via N-cadherin interaction and prolong function of transplanted encapsulated islets in mice

    OpenAIRE

    Montanari, Elisa; Meier, Raphael P. H.; Mahou, Redouan; Seebach, Jörg D.; Wandrey, Christine; Gerber-Lemaire, Sandrine; Buhler, Leo H.; Gonelle-Gispert, Carmen

    2017-01-01

    Background Multipotent mesenchymal stromal cells (MSC) enhance viability and function of islets of Langerhans. We aimed to examine the interactions between human MSC and human islets of Langerhans that influence the function of islets. Methods Human MSC and human islets (or pseudoislets, obtained after digestion and reaggregation of islet cells) were cocultured with or without cellular contact and glucose-stimulated insulin secretion assays were performed to assess cell function. The expressi...

  12. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Simonsen, Janne Lytoft; Rosada, Cecilia; Serakinci, Nedime

    2002-01-01

    . The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing......, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.......Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells...

  13. Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

    Science.gov (United States)

    Tomlinson, Matthew J; Dennis, Caitriona; Yang, Xuebin B; Kirkham, Jennifer

    2015-08-01

    The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

  14. In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Henk-Jan Prins

    2014-03-01

    Full Text Available One of the applications of bone marrow stromal cells (BMSCs that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing “bulk-cultured” BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

  15. Adult stromal cells derived from human adipose tissue provoke pancreatic cancer cell death both in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Beatrice Cousin

    Full Text Available BACKGROUND: Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC on pancreatic tumor cell proliferation. PRINCIPAL FINDINGS: Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate. ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth. CONCLUSION: These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.

  16. Effects of a hyperandrogenaemic state on the proliferation and decidualization potential in human endometrial stromal cells.

    Science.gov (United States)

    Freis, Alexander; Renke, Tobias; Kämmerer, Ulrike; Jauckus, Julia; Strowitzki, Thomas; Germeyer, Ariane

    2017-04-01

    Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women, involving hyperandrogenaemia and insulin resistance. Treatment options include dexamethasone, as well as the off-label use of metformin. To evaluate the impact of those drugs on cyclic changes in endometrial development, we tested possible effects of metformin and dexamethasone on endometrial stromal cells decidualisation, proliferation, and gene regulation in a hyperandrogenaemic microenvironment in vitro. Ten endometrial biopsies (of which five were decidualized in vitro) were used from regularly cycling women. Cells were treated with testosterone, dexamethasone, and metformin in different concentrations. Thereafter, cells were assessed for proliferation and decidualization capacity, as well as mTor and MMP-2 gene regulation. Metformin showed a dose-dependent negative effect on prolactin secretion, a known decidualization marker. This effect was stronger in a hyperandrogenaemic condition and could not be compensated by dexamethasone. Testosterone had a dose dependent negative effect on proliferation in decidualized endometrial stromal cells. Dexamethasone slightly compensated the negative proliferative effect only in low-dose testosterone. High-dose metformin also showed a dose-dependent reduction in endometrial stromal cell proliferation without a major impact by testosterone or dexamethasone in decidualized and non-decidualized cells. High-dose metformin significantly reduced the expression of matrix metalloproteinase-2 (MMP-2) and mechanistic Target of Rapamycin (mTor), regardless of the concentration of dexamethasone and testosterone. The strongest effect could be observed for the combination with high-dose dexamethasone. When therapies, such as metformin and dexamethasone, are used to normalize peripheral androgen levels in patients with PCOS, their effect on the endometrial microenvironment should be taken into consideration as well, especially metformin has to be used with

  17. Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

    Science.gov (United States)

    Charbord, P; Tippens, D; Wight, T S; Gown, A M; Singer, J W

    1987-01-01

    This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

  18. The Impact of Epithelial Stromal Interactions on Human Breast Tumor Heterogeneity

    Science.gov (United States)

    2016-12-01

    Dr. Crista Thompson CONTRACTING ORGANIZATION: Royal Institution for the Advancement of Learning Montreal QC H3A 2T5 REPORT DATE: December 2016...Institution for the Advancement of Learning 845 Sherbrooke St. W. suite 531 Montreal QC H3A 2T5 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES...epithelial  and  stromal  LCM  isolates  and  subjected  to  microarray‐based  gene  expression  profiling  via  Agilent  SurePrint G3 8x60K chips using

  19. Human mesenchymal stromal cell lysates as a novel strategy to recover liver function.

    Science.gov (United States)

    Herencia, Carmen; Almadén, Yolanda; Martínez-Moreno, Julio M; Espejo, Isabel; Herrera, Concha; Pérez-Sánchez, Carlos; Guerrero, Fátima; Ciria, Rubén; Briceño, Francisco Javier; Ferrín, Gustavo; de la Mata, Manuel; Muñoz-Castañeda, Juan R

    2015-01-01

    It is unknown if the beneficial effects of mesenchymal stromal cells (MSC) transplantation into the liver are dependent on their anchorage and differentiation into hepatocytes or rather the result of the release of stem cell intracellular content with hepatoprotector properties. The effects of intact MSC transplantation were compared with the infusion of MSC lysates in an experimental rat model of acute liver failure. A more powerful hepatoprotective and antiapoptotic effect was obtained after infusion of MSC lysates than intact MSC. Changes in IL-6 levels and miRNAs might explain the beneficial effects of MSC lysates. Infusion of MSC lysates show a better hepatoprotective effect than the transplantation of intact MSC.

  20. Bacillus Calmette-Guérin vaccination, thymic size, and thymic output in healthy newborns

    DEFF Research Database (Denmark)

    Birk, Nina Marie; Nissen, Thomas Nørrelykke; Zingmark, Vera

    2017-01-01

    Background: The Bacillus Calmette-Guérin vaccine (BCG) has been associated with beneficial nonspecific effects on infant health. We aimed to examine the effect of BCG at birth on thymic size and the associations between thymic output, circulating lymphocytes, risk of infection, and thymic size....... Methods: In infants randomized to BCG or no BCG, thymic index (TI), and thymic/weight index (TWI) were measured by ultrasound at birth and at the age of 3 mo. T cell subpopulations including CD4+ T cells, CD8+ T cells, and recent thymic emigrants (RTEs) were assessed by flow cytometry. Infections up...... to age 3 mo were parent-reported. Results: BCG vaccination did not affect thymic size at age 3 mo, measured as TI. At birth, the number of lymphocytes, CD4+ T cells, CD8+ T cells, and RTEs were positively associated with TI and TWI. Furthermore, a reduced risk of infections up to age 3 mo was associated...

  1. Bacillus Calmette-Guérin vaccination, thymic size, and thymic output in healthy newborns

    DEFF Research Database (Denmark)

    Birk, Nina Marie; Nissen, Thomas Nørrelykke; Zingmark, Vera

    2017-01-01

    BACKGROUND: The Bacillus Calmette-Guérin vaccine (BCG) has been associated with beneficial nonspecific effects on infant health. We aimed to examine the effect of BCG at birth on thymic size and the associations between thymic output, circulating lymphocytes, risk of infection, and thymic size....... METHODS: In infants randomized to BCG or no BCG, thymic index (TI), and thymic/weight index (TWI) were measured by ultrasound at birth and at the age of 3 mo. T cell subpopulations including CD4+ T cells, CD8+ T cells, and recent thymic emigrants (RTEs) were assessed by flow cytometry. Infections up...... to age 3 mo were parent-reported. RESULTS: BCG vaccination did not affect thymic size at age 3 mo, measured as TI. At birth, the number of lymphocytes, CD4+ T cells, CD8+ T cells, and RTEs were positively associated with TI and TWI. Furthermore, a reduced risk of infections up to age 3 mo was associated...

  2. A proteome study of secreted prostatic factors affecting osteoblastic activity: galectin-1 is involved in differentiation of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Andersen, H; Jensen, Ole N; Moiseeva, Elena P

    2003-01-01

    /ionization time of flight mass spectrometry (MALDI-TOF MS). One of these spots was identified as galectin-1. We examined whether PC3 CM, recombinant galectin-1 alone, or combined with insulin-like growth factor-I (IGF-I) had any effects on the proliferation or differentiation of human bone marrow stromal (h...

  3. Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells

    DEFF Research Database (Denmark)

    Burns, Jorge S.; Harkness, Linda; Aldahmash, Abdullah

    2017-01-01

    Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as "Limited" or "Significant". Previously reported stepwise...

  4. Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue–Derived Human Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Galeano-Garces, Catalina; Camilleri, Emily T.; Riester, Scott M.; Dudakovic, Amel; Larson, Dirk R.; Qu, Wenchun; Smith, Jay; Dietz, Allan B.; Im, Hee-Jeong; Krych, Aaron J.; Larson, A. Noelle; Karperien, Marcel; van Wijnen, Andre J.

    2017-01-01

    Objective: To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue–derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems. Design: Both

  5. Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells

    DEFF Research Database (Denmark)

    Burns, Jorge S.; Harkness, Linda; Aldahmash, Abdullah

    2017-01-01

    Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as "Limited" or "Significant". Previously reported stepwise acquisi...

  6. Interferon-gamma and tumor necrosis factor-alpha sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis

    DEFF Research Database (Denmark)

    Fluhr, Herbert; Krenzer, Stefanie; Stein, Gerburg M

    2007-01-01

    The subtle interaction between the implanting embryo and the maternal endometrium plays a pivotal role during the process of implantation. Human endometrial stromal cells (ESCs) express Fas and the implanting trophoblast cells secrete Fas ligand (FASLG, FasL), suggesting a possible role for Fas...

  7. Enhanced cyclooxygenase-2 expression levels and metalloproteinase 2 and 9 activation by Hexachlorobenzene in human endometrial stromal cells.

    Science.gov (United States)

    Chiappini, Florencia; Bastón, Juan Ignacio; Vaccarezza, Agustina; Singla, José Javier; Pontillo, Carolina; Miret, Noelia; Farina, Mariana; Meresman, Gabriela; Randi, Andrea

    2016-06-01

    Hexachlorobenzene (HCB) is an organochlorine pesticide that induces toxic reproductive effects in laboratory animals. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). Endometriosis is characterized by the presence of functional endometrial tissues outside the uterine cavity. Experimental studies indicate that exposure to organochlorines can interfere with both hormonal regulation and immune function to promote endometriosis. Altered expression of metalloproteinases (MMPs) in patients with endometriosis, suggests that MMPs may play a critical role. In the endometriotic lesions, prostaglandin E2 (PGE2) produced by cyclooxygenase-2 (COX-2), binds to its EP4 receptor (EP4), and via c-Src kinase induces MMPs activation, promoting endometriosis. We examined the HCB action on MMP-2 and MMP-9 activities and expression, COX-2 levels, PGE2 signaling, and the AhR involvement in HCB-induced effects. We have used different in vitro models: (1) human endometrial stromal cell line T-HESC, (2) primary cultures of Human Uterine Fibroblast (HUF), and (3) primary cultures of endometrial stromal cells from eutopic endometrium of control (CESC) and subjects with endometriosis (EESC). Our results show that HCB enhances MMP-2 and MMP-9 activities in T-HESC, HUF and ESC cells. The MMP-9 levels were elevated in all models, while the MMP-2 expression only increased in ESC cells. HCB enhanced COX-2 and EP4 expression, PGE2 secretion and the c-Src kinase activation in T-HESC. Besides, we observed that AhR is implicated in these HCB-induced effects. In conclusion, our results show that HCB exposure could contribute to endometriosis development, affecting inflammation and invasion parameters of human endometrial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. PICTORIAL ESSAY Thymic masses: A radiological review

    African Journals Online (AJOL)

    emission tomography-computed tomography (PET-CT) is seen with increasing age.[2] Various disorders can affect the thymus, e.g. epithelial, lymphoid and germ cell neoplasms; thymic cysts; hyperplasia; and hamartomatous lesions. On chest X-ray, thymic abnormalities typically manifest as focal or diffuse thickening of the ...

  9. MicroRNA-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting LRP3

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Alajez, Nehad M

    2017-01-01

    3 (LRP3) in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during......Understanding the regulatory networks underlying lineage differentiation and fate determination of human bone marrow stromal cells (hBMSC) is a prerequisite for their therapeutic use. The goal of the current study was to unravel the novel role of the low-density lipoprotein receptor-related protein...... the osteogenic induction of the imCL1 clone. Data from functional and gene expression assays demonstrated the role of LRP3 as a molecular switch promoting hBMSC lineage differentiation into osteoblasts and inhibiting differentiation into adipocytes. Interestingly, microRNA (miRNA) expression profiling identified...

  10. Manufacturing of Human Umbilical Cord Mesenchymal Stromal Cells on Microcarriers in a Dynamic System for Clinical Use

    Directory of Open Access Journals (Sweden)

    Florian Petry

    2016-01-01

    Full Text Available The great properties of human mesenchymal stromal cells (hMSCs make these cells an important tool in regenerative medicine. Because of the limitations of hMSCs derived from the bone marrow during isolation and expansion, hMSCs derived from the umbilical cord stroma are a great alternative to overcome these issues. For a large expansion of these cells, we performed a process transfer from static culture to a dynamic system. For this reason, a microcarrier selection out of five microcarrier types was made to achieve a suitable growth surface for the cells. The growth characteristics and metabolite consumption and production were used to compare the cells growth in 12-well plate and spinner flask. The goal to determine relevant process parameters to transfer the expansion process into a stirred tank bioreactor was achieved.

  11. Stem cells from human exfoliated deciduous tooth exhibit stromal-derived inducing activity and lead to generation of neural crest cells from human embryonic stem cells.

    Science.gov (United States)

    Karbalaie, Khadijeh; Tanhaei, Somayyeh; Rabiei, Farzaneh; Kiani-Esfahani, Abbas; Masoudi, Najmeh Sadat; Nasr-Esfahani, Mohammad Hossein; Baharvand, Hossein

    2015-01-01

    The neural crest is a transient structure of early vertebrate embryos that generates neural crest cells (NCCs). These cells can migrate throughout the body and produce a diverse array of mature tissue types. Due to the ethical and technical problems surrounding the isolation of these early human embryo cells, researchers have focused on in vitro studies to produce NCCs and increase their knowledge of neural crest development. In this experimental study, we cultured human embryonic stem cells (hESCs) on stromal stem cells from human exfoliated deciduous teeth (SHED) for a two-week period. We used different approaches to characterize these differentiated cells as neural precursor cells (NPCs) and NCCs. In the first co-culture week, hESCs appeared as crater-like structures with marginal rosettes. NPCs derived from these structures expressed the early neural crest marker p75 in addition to numerous other genes associated with neural crest induction such as SNAIL, SLUG, PTX3 and SOX9. Flow cytometry analysis showed 70% of the cells were AP2/P75 positive. Moreover, the cells were able to self-renew, sustain multipotent differentiation potential, and readily form neurospheres in suspension culture. SHED, as an adult stem cell with a neural crest origin, has stromal-derived inducing activity (SDIA) and can be used as an NCC inducer from hESCs. These cells provide an invaluable resource to study neural crest differentiation in both normal and disordered human neural crest development.

  12. Human pathological basis of blood vessels and stromal tissue for nanotechnology.

    Science.gov (United States)

    Nishihara, Hiroshi

    2014-07-01

    The recent development of nanotechnology has already produced clinically applicable "nanodrugs," which are largely dependent on a novel concept for the drug delivery system. Thus the elucidation of local pharmacokinetics of nanodrugs is indispensable for the further development of nanomedicine; however, the detailed pathophysiology associated with nano-sized materials especially in pathologic lesions has not been well-described. In this review article, the microscopic appearance of vascular pericytes in addition to endothelial cells is discussed in the normal state and also in several pathological conditions which could be the major targets for nanomedicine. Moreover, the role of stromal tissue including myofibroblasts is also focused on, as well as inflammatory cells. Finally, the significance of disease-specific tissue structure in the establishment of personalized nanomedicine is discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Human T cells upregulate CD69 after coculture with xenogeneic genetically-modified pig mesenchymal stromal cells.

    Science.gov (United States)

    Li, Jiang; Andreyev, Oleg; Chen, Man; Marco, Michael; Iwase, Hayato; Long, Cassandra; Ayares, David; Shen, Zhongyang; Cooper, David K C; Ezzelarab, Mohamed B

    2013-01-01

    Mesenchymal stromal cells (MSC) obtained from α1,3-galactosyltransferase gene knock-out pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46 pMSC) suppress in vitro human anti-pig cellular responses as efficiently as allogeneic human MSC. We investigated the immunoregulatory effects of GTKO/CD46 pMSC on human CD4(+) and CD8(+) T cell proliferation in response to pig aortic endothelial cells (pAEC). pMSC efficiently suppressed T cell proliferation, which was associated with downregulation of granzyme B expression. No induction of CD4(+)CD25(+)Foxp3(hi) regulatory T cells or T cell apoptosis was documented. In correlation with T cell proliferation, CD25 expression was upregulated on T cells in response to pAEC but not to pMSC. In contrast, CD69 expression was upregulated on T cells in response to both pMSC and pAEC, which was associated with a significant increase in the phosphorylation of STAT5. GTKO/CD46 pMSC possibly regulate human T cell responses through modulation of CD69 expression and STAT5 signaling. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Osteogenic Differentiation of Human and Ovine Bone Marrow Stromal Cells in response to β-Glycerophosphate and Monosodium Phosphate.

    Science.gov (United States)

    Bottagisio, Marta; Lovati, Arianna B; Lopa, Silvia; Moretti, Matteo

    2015-08-01

    Bone defects are severe burdens in clinics, and thus cell therapy offers an alternative strategy exploiting the features of bone marrow stromal cells (BMSCs). Sheep are a suitable orthopedic preclinical model for similarities with humans. This study compares the influence of two phosphate sources combined with bone morphogenetic protein-2 (BMP-2) on the osteogenic potential of human and ovine BMSCs. β-Glycerophosphate (β-GlyP) and monosodium phosphate (NaH2PO4) were used as organic and inorganic phosphate sources. Osteogenic differentiation of the BMSCs was assessed by calcified matrix, alkaline phosphatase (ALP) activity, and gene expression analysis. A higher calcified matrix deposition was detected in BMSCs cultured with NaH2PO4. Although no significant differences were detected among media for human BMSCs, β-GlyP with or without BMP-2 determined a positive trend in ALP levels compared to NaH2PO4. In contrast, NaH2PO4 had a positive effect on ALP levels in ovine BMSCs. β-GlyP better supported the expression of COL1A1 in human BMSCs, whereas all media enhanced RUNX2 and SPARC expression. Ovine BMSCs responded poorly to any media for RUNX2, COL1A1, and SPARC expression. NaH2PO4 improved calcified matrix deposition without upregulating the transcriptional expression of osteogenic markers. A further optimization of differentiation protocols needs to be performed to translate the procedures from preclinical to clinical models.

  15. Expression of adrenomedullin in human ovaries, ovarian sex cord-stromal tumors and cultured granulosa-luteal cells.

    Science.gov (United States)

    Liu, Jianqi; Bützow, Ralf; Hydén-Granskog, Christel; Voutilainen, Raimo

    2009-02-01

    The aim of the present study was to characterise the expression pattern of the multifunctional vasoactive peptide adrenomedullin (ADM) in human ovarian tumors, and to find hormonal regulators of ADM expression in human ovaries. The expression of ADM messenger RNA (mRNA) was higher in granulosa cell tumors than in fibrothecomas and normal ovaries, as analysed by Northern blots. In normal ovaries, ADM immunoreactivity was localised in both granulosa and thecal cells. Eight of the 90 granulosa cell tumors (9%) showed moderate and 53 (59%) weak ADM immunoreactivity, whereas 27% (11/41) of the fibrothecomas displayed weak ADM staining. FSH, protein kinase A activator (Bu)(2)cAMP, prostaglandin E(2) (PGE(2)), activin A and the broad protein kinase regulator staurosporine decreased ADM mRNA accumulation in cultured granulosa-luteal cells time- and dose-dependently. FSH, (Bu)(2)cAMP and PGE(2) increased progesterone secretion and the accumulation of the steroidogenic acute regulatory protein mRNA in these cells. In conclusion, ADM is expressed in normal human ovaries and sex cord-stromal tumors, particularly in those of granulosa cell origin. FSH, PGE(2,) (Bu)(2)cAMP and activin A suppress ADM gene expression in granulosa-luteal cells. Expression of ADM in human ovaries and its hormonal regulation in granulosa cells suggests a paracrine role for ADM in ovarian function.

  16. Reconstruction of hematopoietic inductive microenvironment after transplantation of VCAM-1-modified human umbilical cord blood stromal cells.

    Directory of Open Access Journals (Sweden)

    Yao Liu

    Full Text Available The hematopoietic inductive microenvironment (HIM is where hematopoietic stem/progenitor cells grow and develop. Hematopoietic stromal cells were the key components of the HIM. In our previous study, we had successfully cultured and isolated human cord blood-derived stromal cells (HUCBSCs and demonstrated that they could secret hemopoietic growth factors such as GM-CSF, TPO, and SCF. However, it is still controversial whether HUCBSCs can be used for reconstruction of HIM. In this study, we first established a co-culture system of HUCBSCs and cord blood CD34(+ cells and then determined that using HUCBSCs as the adherent layer had significantly more newly formed colonies of each hematopoietic lineage than the control group, indicating that HUCBSCs had the ability to promote the proliferation of hematopoietic stem cells/progenitor cells. Furthermore, the number of colonies was significantly higher in vascular cell adhesion molecule-1 (VCAM-1-modified HUCBSCs, suggesting that the ability of HUCBSCs in promoting the proliferation of hematopoietic stem cells/progenitor cells was further enhanced after having been modified with VCAM-1. Next, HUCBSCs were infused into a radiation-damaged animal model, in which the recovery of hematopoiesis was observed. The results demonstrate that the transplanted HUCBSCs were "homed in" to bone marrow and played roles in promoting the recovery of irradiation-induced hematopoietic damage and repairing HIM. Compared with the control group, the HUCBSC group had significantly superior effectiveness in terms of the recovery time for hemogram and myelogram, CFU-F, CFU-GM, BFU-E, and CFU-Meg. Such differences were even more significant in VCAM-1-modified HUCBSCs group. We suggest that HUCBSCs are able to restore the functions of HIM and promote the recovery of radiation-induced hematopoietic damage. VCAM-1 plays an important role in supporting the repair of HIM damage.

  17. Laminin production by human endometrial stromal cells relates to the cyclic and pathologic state of the endometrium

    DEFF Research Database (Denmark)

    Faber, M; Wewer, U M; Berthelsen, J G

    1986-01-01

    The cyclic changes in the presence of the basement membrane glycoprotein laminin in endometrial stromal cells was studied by immunohistochemistry. The interstitial matrix around the stromal cells of the proliferative phase of the normal menstrual cycle was unreactive with antibodies to laminin. H...

  18. In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells

    NARCIS (Netherlands)

    Prins, H.J.; Braat, A.K.; Gawlitta, D.; Dhert, W.J.A.; Egan, D.A.; Tijssen-Slump, E.; Yuan, Huipin; Coffer, P.J.; Rozemuller, H.; Martens, A.C.

    2014-01-01

    One of the applications of bone marrow stromal cells (BMSCs) that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that

  19. In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells.

    NARCIS (Netherlands)

    Prins, H.J.; Braat, A.K.; Gawlitta, D.; Dhert, W.J.A.|info:eu-repo/dai/nl/10261847X; Egan, D.A.; Tijssen-Slump, E.; Yuan, H.; Coffer, P.J; Rozemuller, H.; Martens, A.C.M.

    2014-01-01

    One of the applications of bone marrow stromal cells (BMSCs) that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that

  20. Encapsulating Non-Human Primate Multipotent Stromal Cells in Alginate via High Voltage for Cell-Based Therapies and Cryopreservation

    Science.gov (United States)

    Gryshkov, Oleksandr; Pogozhykh, Denys; Hofmann, Nicola; Pogozhykh, Olena; Mueller, Thomas; Glasmacher, Birgit

    2014-01-01

    Alginate cell-based therapy requires further development focused on clinical application. To assess engraftment, risk of mutations and therapeutic benefit studies should be performed in an appropriate non-human primate model, such as the common marmoset (Callithrix jacchus). In this work we encapsulated amnion derived multipotent stromal cells (MSCs) from Callithrix jacchus in defined size alginate beads using a high voltage technique. Our results indicate that i) alginate-cell mixing procedure and cell concentration do not affect the diameter of alginate beads, ii) encapsulation of high cell numbers (up to 10×106 cells/ml) can be performed in alginate beads utilizing high voltage and iii) high voltage (15–30 kV) does not alter the viability, proliferation and differentiation capacity of MSCs post-encapsulation compared with alginate encapsulated cells produced by the traditional air-flow method. The consistent results were obtained over the period of 7 days of encapsulated MSCs culture and after cryopreservation utilizing a slow cooling procedure (1 K/min). The results of this work show that high voltage encapsulation can further be maximized to develop cell-based therapies with alginate beads in a non-human primate model towards human application. PMID:25259731

  1. Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering.

    Science.gov (United States)

    Zaky, S H; Ottonello, A; Strada, P; Cancedda, R; Mastrogiacomo, M

    2008-12-01

    The heterogeneous population of non-haematopoietic cells residing in the bone marrow (bone marrow stromal cells, BMSCs) and the different fractions and components obtained from platelet-rich plasma provide an invaluable source of autologous cells and growth factors for bone and other connective tissue reconstruction. In this study, we investigated the effect of an allogenic platelet lysate on human BMSCs proliferation and differentiation. Cell proliferation and number of performed cell doublings were enhanced in cultures supplemented with the platelet-derived growth factors (platelet lysate, PL), either with or without the concomitant addition of fetal bovine serum (FBS), compared to cultures performed in the presence of FBS and FGF2. Both in vitro and in vivo osteogenic differentiation were unaltered in cells maintained in medium supplemented with PL and not FBS (Only PL) and in cells maintained in medium containing FBS and FGF2. Interestingly, the in vitro cartilage formation was more effective in the pellet of BMSCs expanded in the Only PL medium. In particular, a chondrogenic differentiation was observed in pellets of some in vitro-expanded BMSCs in the Only PL medium, whereas pellets from parallel cell cultures in medium containing FBS did not respond to the chondrogenic induction. We conclude that the platelet lysate from human source is an effective and even more beneficial substitute for fetal bovine serum to support the in vitro expansion of human BMSCs for subsequent tissue-engineering applications. (c) 2008 John Wiley & Sons, Ltd.

  2. Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells

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    Agneta Månsson-Broberg

    2016-04-01

    Full Text Available The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

  3. Distribution of mediastinal ectopic thymic tissue in patients without thymic disease.

    Science.gov (United States)

    Sanei, Behnam; Tabatabie, Seid Abbas; Bigdelian, Hamid; Hashemi, Seid Mozafar; Davarpanah, Amir Hossein; Jafari, Hamid Reza; Sanei, Mohammad Hossein

    2015-01-01

    Different approaches to treat myasthenia gravis showed variable outcomes probably because of remained ectopic thymic tissue. The aim of this study is to determine incidence of thymic tissue in cases without any thymic disease. Seventy-two patients scheduled for open heart surgery during 2000 and 2007 without thymic disease that were enrolled in the study at Chamran Hospital. Intraoperative biopsies were taken form aortopulmonary window, aortocaval groove and left and right pericardiophrenic regions. Finally, the distribution of ectopic thymic tissue was evaluated in above regions. Thymic tissue was found overall in 70.85% of patients. The most common sites for thymic tissue were left pericardiophrenic (50%) and right pericardiophrenic (31.9%) regions. Frequencies of ectopic thymus in aortopulmonary window and aortocaval groove were 19.4% and 12.5%, respectively. Because of high incidence of ectopic thymic tissue in mediastinum in patients without thymic disease, we recommend wide excision of thymus gland and all of adipose tissue in patients with myasthenia gravis.

  4. Recurrent Herpetic Stromal Keratitis in Mice: A Model for Studying Human HSK

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    Patrick M. Stuart

    2012-01-01

    Full Text Available Herpes simplex virus 1 (HSV-1 infection of the cornea leads to a potentially blinding disease, termed herpetic stromal keratitis (HSK that is characterized by lesions of an immunoinflammatory nature. In spite of the fact that HSK typically presents as a recurrent disease due to reactivation of virus which latently infects the trigeminal ganglia, most murine studies of HSK have employed a primary and not recurrent model of the disease. This report documents the several recurrent models of HSK that have been developed and how data generated from these models differs in some important aspects from data generated following primary infection of the cornea. Chief among these differences is the fact that recurrent HSK takes place in the context of an animal that has a preexisting anti-HSV immune response, while primary HSK occurs in an animal that is developing such a response. We will document both differences and similarities that derive from this fundamental difference in these models with an eye towards possible vaccines and therapies that demonstrate promise in treating HSK.

  5. Recurrent Herpetic Stromal Keratitis in Mice: A Model for Studying Human HSK

    Science.gov (United States)

    Stuart, Patrick M.; Keadle, Tammie L.

    2012-01-01

    Herpes simplex virus 1 (HSV-1) infection of the cornea leads to a potentially blinding disease, termed herpetic stromal keratitis (HSK) that is characterized by lesions of an immunoinflammatory nature. In spite of the fact that HSK typically presents as a recurrent disease due to reactivation of virus which latently infects the trigeminal ganglia, most murine studies of HSK have employed a primary and not recurrent model of the disease. This report documents the several recurrent models of HSK that have been developed and how data generated from these models differs in some important aspects from data generated following primary infection of the cornea. Chief among these differences is the fact that recurrent HSK takes place in the context of an animal that has a preexisting anti-HSV immune response, while primary HSK occurs in an animal that is developing such a response. We will document both differences and similarities that derive from this fundamental difference in these models with an eye towards possible vaccines and therapies that demonstrate promise in treating HSK. PMID:22593769

  6. DHP-derivative and low oxygen tension effectively induces human adipose stromal cell reprogramming.

    Directory of Open Access Journals (Sweden)

    Min Ki Jee

    Full Text Available BACKGROUND AND METHODS: In this study, we utilized a combination of low oxygen tension and a novel anti-oxidant, 4-(3,4-dihydroxy-phenyl-derivative (DHP-d to directly induce adipose tissue stromal cells (ATSC to de-differentiate into more primitive stem cells. De-differentiated ATSCs was overexpress stemness genes, Rex-1, Oct-4, Sox-2, and Nanog. Additionally, demethylation of the regulatory regions of Rex-1, stemnesses, and HIF1alpha and scavenging of reactive oxygen species were finally resulted in an improved stem cell behavior of de-differentiate ATSC (de-ATSC. Proliferation activity of ATSCs after dedifferentiation was induced by REX1, Oct4, and JAK/STAT3 directly or indirectly. De-ATSCs showed increased migration activity that mediated by P38/JUNK and ERK phosphorylation. Moreover, regenerative efficacy of de-ATSC engrafted spinal cord-injured rats and chemical-induced diabetes animals were significantly restored their functions. CONCLUSIONS/SIGNIFICANCE: Our stem cell remodeling system may provide a good model which would provide insight into the molecular mechanisms underlying ATSC proliferation and transdifferentiation. Also, these multipotent stem cells can be harvested may provide us with a valuable reservoir of primitive and autologous stem cells for use in a broad spectrum of regenerative cell-based disease therapy.

  7. Late Adherent Human Bone Marrow Stromal Cells Form Bone and Restore the Hematopoietic Microenvironment In Vivo

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    Verônica Fernandes Vianna

    2013-01-01

    Full Text Available Bone marrow stromal cells (BMSCs are a valuable resource for skeletal regenerative medicine because of their osteogenic potential. In spite of the very general term “stem cell,” this population of cells is far from homogeneous, and different BMSCs clones have greatly different phenotypic properties and, therefore, potentially different therapeutic potential. Adherence to a culture flask surface is a primary defining characteristic of BMSCs. We hypothesized that based on the adherence time we could obtain an enriched population of cells with a greater therapeutic potential. We characterized two populations of bone marrow-derived cells, those that adhered by three days (R-cells and those that did not adhere by three days but did by six days (L-cells. Clones derived from L-cells could be induced into adipogenic, chondrogenic, and osteogenic differentiation in vitro. L-cells appeared to have greater proliferative capacity, as manifested by larger colony diameter and clones with higher CD146 expression. Only clones from L-cells developed bone marrow stroma in vivo. We conclude that the use of late adherence of BMSCs is one parameter that can be used to enrich for cells that will constitute a superior final product for cell therapy in orthopedics.

  8. Human Adenomyosis Endometrium Stromal Cells Secreting More Nerve Growth Factor: Impact and Effect.

    Science.gov (United States)

    Li, Yan; Zou, Shien; Xia, Xian; Zhang, Shaofen

    2015-09-01

    Abnormal expression of nerve growth factor (NGF) was found in adenomyosis (AM). We collected AM foci from patients and eutopic endometrium from non-AM controls. Endometrium stromal cells (ESCs) were cultured. Different levels of 17β-estradiol, tumor necrosis factor (TNF), CoCl2, and H2O2 were added to the culture system separately, then the expression level of NGF in ESCs was detected. After adding different levels of NGF, the proliferation and apoptosis of ESCs and aromatase expression were detected. We found that 17β-estradiol promoted NGF production in AM ESCs but not in control ESCs; TNF promoted NGF production in both AM and control ESCs; and CoCl2 inhibited NGF production in control ESCs, but had no effect in AM ESCs. Nerve growth factor promoted the proliferation and synthesis of aromatase in AM ESCs. In conclusion, locally increased estrogen levels and inflammation may cause increased NGF production in the uterus of patients with AM. Nerve growth factor stimulated the proliferation and increased aromatase expression of ESCs from AM foci, suggesting NGF might contribute to the pathology and etiology of AM. © The Author(s) 2014.

  9. Improvement of interleukin 2 production, clonogenic capability and restoration of stromal cell function in human immunodeficiency virus-type-1 patients after highly active antiretroviral therapy.

    Science.gov (United States)

    Isgrò, Antonella; Aiuti, Alessandro; Mezzaroma, Ivano; Addesso, Maria; Riva, Elisabetta; Giovannetti, Antonello; Mazzetta, Francesca; Alario, Cecilia; Mazzone, Annamaria; Ruco, Luigi; Aiuti, Fernando

    2002-09-01

    Haematological abnormalities frequently occur in patients infected by human immunodeficiency virus-type 1 (HIV-1). Increasing evidence indicates that bone marrow suppression (BM) results from viral infection of accessory cells, with impaired stromal function and alteration of haematopoietic growth factor network. We have investigated the effects of antiretroviral therapy on cytokine and chemokine production by BM cells and stromal cells in a group of HIV-1-infected subjects before and during treatment. Compared with uninfected controls, an altered cytokine and chemokine production by BM cells was observed before treatment, characterized by decreased interleukin 2 (IL-2) and elevated tumour necrosis factor-alpha, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell-expressed and secreted) levels, along with a defective BM clonogenic activity. Antiretroviral therapy showed increased BM clonogenic capability, associated with normalization of IL-2 production and chemokine receptors expression on CD34+ cells. Pre-therapy, BM accessory cells were represented by macrophage-like cells, in some cases positive for HIV-1 DNA, suggesting that these cells are the main target of HIV-1 infection. During therapy, the stromal cells became predominantly fibroblastoid-like, as observed in normal controls, and were negative for HIV-1 DNA. Controlling HIV-1 replication may produce amelioration of stem cell activity, and restoration of stromal cell pattern and functions, with increased IL-2 production at BM level.

  10. An in vivo model to assess magnesium alloys and their biological effect on human bone marrow stromal cells.

    Science.gov (United States)

    Yoshizawa, Sayuri; Chaya, Amy; Verdelis, Kostas; Bilodeau, Elizabeth A; Sfeir, Charles

    2015-12-01

    Magnesium (Mg) alloys have many unique qualities which make them ideal candidates for bone fixation devices, including biocompatibility and degradation in vivo. Despite a rise in Mg alloy production and research, there remains no standardized system to assess their degradation or biological effect on human stem cells in vivo. In this study, we developed a novel in vivo model to assess Mg alloys for craniofacial and orthopedic applications. Our model consists of a collagen sponge seeded with human bone marrow stromal cells (hBMSCs) around a central Mg alloy rod. These scaffolds were implanted subcutaneously in mice and analyzed after eight weeks. Alloy degradation and biological effect were determined by microcomputed tomography (microCT), histological staining, and immunohistochemistry (IHC). MicroCT showed greater volume loss for pure Mg compared to AZ31 after eight weeks in vivo. Histological analysis showed that hBMSCs were retained around the Mg implants after 8 weeks. Furthermore, immunohistochemistry showed the expression of dentin matrix protein 1 and osteopontin around both pure Mg and AZ31 with implanted hBMSCs. In addition, histological sections showed a thin mineral layer around all degrading alloys at the alloy-tissue interface. In conclusion, our data show that degrading pure Mg and AZ31 implants are cytocompatible and do not inhibit the osteogenic property of hBMSCs in vivo. These results demonstrate that this model can be used to efficiently assess the biological effect of corroding Mg alloys in vivo. Importantly, this model may be modified to accommodate additional cell types and clinical applications. Magnesium (Mg) alloys have been investigated as ideal candidates for bone fixation devices due to high biocompatibility and degradation in vivo, and there is a growing need of establishing an efficient in vivo material screening system. In this study, we assessed degradation rate and biological effect of Mg alloys by transplanting Mg alloy rod with

  11. Thymic Carcinoma Management Patterns among International Thymic Malignancy Interest Group (ITMIG) Physicians with Consensus from the Thymic Carcinoma Working Group.

    Science.gov (United States)

    Shepherd, Annemarie; Riely, Gregory; Detterbeck, Frank; Simone, Charles B; Ahmad, Usman; Huang, James; Korst, Robert; Rajan, Arun; Rimner, Andreas

    2017-04-01

    Thymic carcinomas are rare epithelial malignancies with limited data to guide management. To identify areas of agreement and variability in current clinical practice, a 16-question electronic survey was given to members of the International Thymic Malignancy Interest Group (ITMIG). Areas of controversy were discussed with the Thymic Carcinoma Working Group and consensus was achieved, as described. A total of 100 ITMIG members responded. There was general agreement regarding the role for multimodality therapy with definitive surgical resection in physically fit patients with advanced but resectable disease. Areas of controversy included the need for histologic confirmation before surgery, the role of adjuvant therapy, the optimal first-line chemotherapy regimen, and the recommended treatment course for marginally resectable disease with invasion into the great vessels, pericardium, and lungs. The results of the questionnaire provide a description of the management of thymic carcinoma by 100 ITMIG members with a specific interest or expertise in thymic malignancies. Although there was agreement in some areas, clinical practice appears to vary significantly. There is a great need for collaborative research to identify optimal evaluation and treatment strategies. Given the need for multimodality therapy in many cases, a multidisciplinary discussion of the management of patients with thymic carcinoma is critical. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

  12. The effect of age on thymic function

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    Donald B. Palmer

    2013-10-01

    Full Text Available Age-related regression of the thymus is associated with a decline in naïve T cell output. This is thought to contribute to the reduction in T cell diversity seen in older individuals and linked with increased susceptibility to infection, autoimmune disease and cancer. Thymic involution is one of the most dramatic and ubiquitous changes seen in the ageing immune system, but the mechanisms which underlying this process are poorly understood. However, a picture is emerging, implicating the involvement of both extrinsic and intrinsic factors. In this review we assess the role of the thymic microenvironment as a potential target that regulates thymic involution, question whether thymocyte development in the aged thymus is functionally impaired and explore the kinetics of thymic involution.

  13. The temporal expression of estrogen receptor alpha-36 and runx2 in human bone marrow derived stromal cells during osteogenesis

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    Francis, W.R., E-mail: w.francis@swansea.ac.uk [Institute of Life Science, College of Medicine, Swansea University (United Kingdom); Owens, S.E.; Wilde, C. [Institute of Life Science, College of Medicine, Swansea University (United Kingdom); Pallister, I. [Institute of Life Science, College of Medicine, Swansea University (United Kingdom); Trauma and Orthopaedics, Morriston Hospital, Swansea (United Kingdom); Kanamarlapudi, V. [Institute of Life Science, College of Medicine, Swansea University (United Kingdom); Zou, W., E-mail: weizou60@hotmail.com [College of Life Sciences, Liaoning Normal University, Dalian 116081 (China); Liaoning Key Laboratories of Biotechnology and Molecular Drug Research and Development, Dalian 116081 (China); Xia, Z. [Institute of Life Science, College of Medicine, Swansea University (United Kingdom)

    2014-10-24

    Highlights: • ERα36 is the predominant ERα isoform involved in bone regulation in human BMSC. • ERα36 mRNA is significantly upregulated during the process of osteogenesis. • The pattern of ERα36 and runx2 mRNA expression is similar during osteogenesis. • ERα36 appears to be co-localised with runx2 during osteogenesis. - Abstract: During bone maintenance in vivo, estrogen signals through estrogen receptor (ER)-α. The objectives of this study were to investigate the temporal expression of ERα36 and ascertain its functional relevance during osteogenesis in human bone marrow derived stromal cells (BMSC). This was assessed in relation to runt-related transcription factor-2 (runx2), a main modulatory protein involved in bone formation. ERα36 and runx2 subcellular localisation was assessed using immunocytochemistry, and their mRNA expression levels by real time PCR throughout the process of osteogenesis. The osteogenically induced BMSCs demonstrated a rise in ERα36 mRNA during proliferation followed by a decline in expression at day 10, which represents a change in dynamics within the culture between the proliferative stage and the differentiative stage. The mRNA expression profile of runx2 mirrored that of ERα36 and showed a degree subcellular co-localisation with ERα36. This study suggests that ERα36 is involved in the process of osteogenesis in BMSCs, which has implications in estrogen deficient environments.

  14. Fetal Bone Marrow-Derived Mesenchymal Stem/Stromal Cells Enhance Humanization and Bone Formation of BMP7 Loaded Scaffolds.

    Science.gov (United States)

    Shafiee, Abbas; Baldwin, Jeremy G; Patel, Jatin; Holzapfel, Boris M; Fisk, Nicholas M; Khosrotehrani, Kiarash; Hutmacher, Dietmar W

    2017-09-01

    Tissue engineered constructs built with human cells capable of generating a bone-like organ within the mouse have attracted considerable interest over the past decade. Here, we aimed to compare the utility of human mesenchymal stem/stromal cells (MSC) isolated from fetal term placenta (fPL-MSC) and fetal first trimester bone marrow (fBM-MSC) in a polycaprolactone scaffold/BMP7-based model in nude mice. Furthermore, fPL-MSC were co-seeded with fetal placenta-derived endothelial colony forming cells (ECFC) to assess the impact of ECFC on fPL-MSC osteogenesis. X-ray radiography and micro computed tomography analyses showed enhanced bone formation in all BMP7 groups; however there was no difference after 2 months in bone formation between scaffolds seeded with fPL-MSC alone or combination of ECFC and fPL-MSC. Of interest, fBM-MSC showed the highest level of bone formation. Additionally, endochondral ossification contributed in generation of bone in fBM-MSC. Histological analysis showed the primary role of BMP in generation of cortical and trabecular bone, and the recruitment of hematopoietic cells to the scaffolds. Current in vivo engineered bone organs can potentially be used for drug screening or as models to study bone tissue development in combination with haematopoiesis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Fetal mesenchymal stromal cells differentiating towards chondrocytes acquire a gene expression profile resembling human growth plate cartilage.

    Directory of Open Access Journals (Sweden)

    Sandy A van Gool

    Full Text Available We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs differentiating towards chondrocytes as an alternative model for the human growth plate (GP. Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

  16. Fetal mesenchymal stromal cells differentiating towards chondrocytes acquire a gene expression profile resembling human growth plate cartilage.

    Science.gov (United States)

    van Gool, Sandy A; Emons, Joyce A M; Leijten, Jeroen C H; Decker, Eva; Sticht, Carsten; van Houwelingen, Johannes C; Goeman, Jelle J; Kleijburg, Carin; Scherjon, Sicco A; Gretz, Norbert; Wit, Jan Maarten; Rappold, Gudrun; Post, Janine N; Karperien, Marcel

    2012-01-01

    We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

  17. Gateway-compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells.

    Science.gov (United States)

    Petrakis, Spyros; Raskó, Tamas; Mátés, Lajos; Ivics, Zoltan; Izsvák, Zsuzsanna; Kouzi-Koliakou, Kokkona; Koliakos, George

    2012-07-01

    The Gateway technology cloning system and transposon technology represent state-of-the-art laboratory techniques. Combination of these molecular tools allows rapid cloning of target genes into expression vectors. Here, we describe a novel Gateway technology-compatible transposon plasmid that combines the advantages of Gateway recombination cloning with the Sleeping Beauty (SB) transposon-mediated transgene integrations. In our system the transposition is catalyzed by the novel hyperactive SB100x transposase, and provides highly efficient and precise transgene integrations into the host genome. A Gateway-compatible transposon plasmid was generated in which the potential target gene can be fused with a yellow fluorescent protein (YFP) tag at the N-terminal. The vector utilizes the CAGGS promoter to control fusion protein expression. The transposon expression vector encoding the YFP-interferon-β protein (IFNB1) fusion protein together with the hyperactive SB100x transposase was used to generate stable cell lines in human embryonic kidney (HEK293) and rat adipose-derived stromal cells (ASC). ASCs and HEK293 cells stably expressed and secreted the human IFNB1 for up to 4 weeks after transfection. The generated Gateway-compatible transposon plasmid can be utilized for numerous experimental approaches, such as gene therapy or high-throughput screening methods in primary cells, representing a valuable molecular tool for laboratory applications. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Secreted galectin-3 as a possible biomarker for the immunomodulatory potential of human umbilical cord mesenchymal stromal cells.

    Science.gov (United States)

    Liu, Guang-Yang; Xu, Yi; Li, Yan; Wang, Li-Hua; Liu, Yong-Jun; Zhu, Delin

    2013-10-01

    Human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) possess broad and potent immunomodulatory activities and have shown great potential in anti-inflammatory therapies. However, a biomarker that can be used to assess quickly and efficiently the immunomodulatory function of UC-MSCs has not been identified. Several studies have revealed that galectin-3 (Gal-3), a member of the human galectin family, is involved in the immunosuppressive function of MSCs. Gal-3 gene expression in UC-MSCs was analyzed using quantitative reverse transcriptase polymerase chain reaction and Western blotting. Blocking of Gal-3 expression in UC-MSCs with small interfering RNA was employed to analyze whether the immunosuppressive function of UC-MSCs was affected. We found that UC-MSCs expressed Gal-3 both on the cell surface and in secreted form, and the expression levels of Gal-3 did not show significant variation after cell passaging. We further showed that Gal-3 expression correlated with the immunosuppressive function of UC-MSCs because knock-down of Gal-3 expression with small interfering RNA significantly abrogated the inhibitory effects of UC-MSCs on mitogen-stimulated and alloantigen-stimulated proliferation of human peripheral blood mononuclear cells; meanwhile, the inhibitory effect of UC-MSCs was reversed by adding back recombinant Gal-3 to the co-culture systems. The inhibitory activities of human UC-MSCs were not reduced even when they were separated from human peripheral blood mononuclear cells in a transwell co-culture system, indicating that the soluble form of Gal-3 was the major effector. The Gal-3 protein secreted by UC-MSCs shows good correlation with immunosuppressive potential and may serve as a possible biomarker for the potency test of UC-MSCs. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  19. Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells.

    Science.gov (United States)

    Ito, Sawa; Barrett, A John; Dutra, Amalia; Pak, Evgenia; Miner, Samantha; Keyvanfar, Keyvan; Hensel, Nancy F; Rezvani, Katayoun; Muranski, Pawel; Liu, Paul; Larochelle, Andre; Melenhorst, J Joseph

    2015-01-01

    Mesenchymal stromal cells (MSCs) support the growth and differentiation of normal hematopoietic stem cells (HSCs). Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs) in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML) were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6weeks. Four samples showed CD34(+)CD38(-) predominance, and four were predominantly CD34(+)CD38(+). CD34(+) CD38(-) predominant leukemia cells maintained the CD34(+) CD38(-) phenotype and were viable for 6weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34(+) CD38(+) predominant leukemic cells maintained the CD34(+)CD38(+) phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34(+) blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest that MSCs provide pro-survival benefits to leukemic blasts through cell-cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs. Published by Elsevier B.V.

  20. Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery.

    Science.gov (United States)

    Lankford, Lee; Chen, Y Julia; Saenz, Zoe; Kumar, Priyadarsini; Long, Connor; Farmer, Diana; Wang, Aijun

    2017-06-01

    In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3-5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities. Copyright © 2017 International Society for Cellular Therapy. Published

  1. Thymic measurements in pathologically proven normal thymus and thymic hyperplasia: intraobserver and interobserver variabilities.

    Science.gov (United States)

    Araki, Tetsuro; Sholl, Lynette M; Gerbaudo, Victor H; Hatabu, Hiroto; Nishino, Mizuki

    2014-06-01

    To determine the intraobserver and interobserver variabilities of thymic measurements on computed tomography (CT) in patients with pathologic diagnosis of thymic hyperplasia or normal thymus. Thirty-three patients with pathologic diagnosis of thymic hyperplasia (n = 25) or normal thymus (n = 8) who had identifiable thymus gland on CT were retrospectively studied. Two radiologists independently measured thymic size and CT attenuation. Concordance correlation coefficients (CCCs) and Bland-Altman plots were used to assess intraobserver and interobserver agreements. The intraobserver and interobserver agreements of thymic diameters and the lobe length were moderate, with CCCs ranging from 0.73 to 0.89 and from 0.72 to 0.81, respectively. Higher agreement was noted among patients whose measurements were performed on the same CT image in two independent measurements, with intraobserver CCC ≥ 0.95 for diameters and length. After providing readers with an instruction for consistent selection of CT image for measurements, the intraobserver and interobserver agreements improved, resulting in CCCs ranging from 0.81 to 0.92 and from 0.77 to 0.85 for diameters and length, respectively. Thymic lobe thickness had the least agreement. CT attenuation measurements were highly reproducible, with CCCs ranging from 0.88 to 0.97. In patients with thymic CT attenuation >30 HU (Hounsfield unit), the attenuation measurements were more reproducible with narrower 95% limits of agreement. Thymic size measurements had moderate-to-high intraobserver and interobserver agreements, when the instruction for consistent selection of images was provided to the readers. CT attenuation was highly reproducible, with higher reproducibility for thymic glands with >30 HU. Awareness of thymic measurement variability is necessary when interpreting measured values of normal thymus and thymic pathology on CT. Copyright © 2014 AUR. Published by Elsevier Inc. All rights reserved.

  2. Thymic measurements in pathologically proven normal thymus and thymic hyperplasia: Intra- and interobserver variability

    Science.gov (United States)

    Araki, Tetsuro; Sholl, Lynette M.; Gerbaudo, Victor H.; Hatabu, Hiroto; Nishino, Mizuki

    2014-01-01

    Objective: Determine the intra- and interobserver variability of thymic measurements on computed tomography (CT) in patients with pathological diagnosis of thymic hyperplasia or normal thymus. Materials and Methods: Thirty-three patients with pathological diagnosis of thymic hyperplasia (n=25) or normal thymus (n=8) who had identifiable thymus gland on CT were retrospectively studied. Two radiologists independently measured thymic size and CT attenuation. Concordance correlation coefficients (CCCs) and Bland-Altman plots were used to assess intra- and interobserver agreements. Results: The intra- and interobserver agreements of thymic diameters and the lobe length were moderate, with CCCs ranging 0.73-0.89 and 0.72-0.81, respectively. Higher agreement was noted among patients whose measurements were performed on the same CT image in two independent measurements, with intraobserver CCC ≥0.95 for diameters and length. After providing readers with an instruction for consistent selection of CT image for measurements, the intra- and interobserver agreement improved, resulting in CCCs ranging 0.81-0.92 and 0.77-0.85 for diameters and length, respectively. Thymic lobe thickness had the least agreement. CT attenuation measurements were highly reproducible, with CCCs ranging 0.88-0.97. In patients with thymic CT attenuation >30HU, the attenuation measurements were more reproducible with narrower 95% limits of agreement. Conclusion: Thymic size measurements had moderate to high intra- and interobserver agreement, when the instruction for consistent selection of images were provided to the readers. CT attenuation was highly reproducible, with higher reproducibility for thymic glands with >30HU. Awareness of thymic measurement variability is necessary when interpreting measured values of normal thymus and thymic pathology on CT. PMID:24809315

  3. Human Serum is as Efficient as Fetal Bovine Serum in Supporting Proliferation and Differentiation of Human Multipotent Stromal (Mesenchymal) Stem Cells In Vitro and In Vivo

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Haack-Sørensen, Mandana; Al-Nbaheen, May

    2011-01-01

    BACKGROUND: Human multipotent stromal (skeletal, mesenchymal) stem cells (hMSC) are employed in an increasing number of clinical trials for tissue regeneration of age-related degenerative diseases. However, routine use of fetal bovine sera (FBS) for their in vitro expansion is not optimal and may...... pose a health risk for patients. METHODS: We carried out a side-by-side comparison of the effects of allogenic pooled human serum (HuS) versus FBS on hMSC proliferation and differentiation in vitro and in vivo. As a model for hMSC, we employed telomerase-immortalized hMSC; hMSC-TERT cell line. RESULTS....... FBS. hMSC-TERT or primary bone marrow derived hMSC induced to osteoblastic or adipocytic differentiation in the presence of HuS or FBS showed comparable levels of gene expression and protein production of osteoblastic markers (CBFA1/Runx2, alkaline phosphastase, collagen type I and osteocalcin...

  4. The monitoring of gene functions on a cell-defined siRNA microarray in human bone marrow stromal and U2OS cells

    Directory of Open Access Journals (Sweden)

    Hi Chul Kim

    2016-06-01

    Full Text Available Here, we developed a cell defined siRNA microarray (CDSM for human bone marrow stromal cells (hBMSCs designed to control the culture of cells inside the spot area without reducing the efficiency of siRNA silencing, “Development of a cell-defined siRNA microarray for analysis of gene functionin human bone marrow stromal cells” (Kim et al., 2016 [1]. First, we confirmed that p65 protein inhibition efficiency was maintained when hBMSCs were culture for 7 days on the siRNA spot, and siRNA spot activity remained in spite of long term storage (10 days and 2 months. Additionally, we confirmed p65 protein inhibition in U2OS cells after 48 h reverse transfection.

  5. Quantitative proteomics and transcriptomics reveals metabolic differences in attracting and non-attracting human-in-mouse glioma stem cell xenografts and stromal cells

    Directory of Open Access Journals (Sweden)

    Norelle C. Wildburger

    2015-09-01

    Full Text Available Bone marrow-derived human mesenchymal stem cells (BM-hMSCs show promise as cell-based delivery vehicles for anti-glioma therapeutics, due to innate tropism for gliomas. However, in clinically relevant human-in-mouse glioma stem cell xenograft models, BM-hMSCs tropism is variable. We compared the proteomic profile of cancer and stromal cells in GSCXs that attract BM-hMSCs (“attractors” with those to do not (“non-attractors” to identify pathways that may modulate BM-hMSC homing, followed by targeted transcriptomics. The results provide the first link between fatty acid metabolism, glucose metabolism, ROS, and N-glycosylation patterns in attractors. Reciprocal expression of these pathways in the stromal cells suggests microenvironmental cross-talk.

  6. Multi-lineage differentiation of human mesenchymal stromal cells on the biophysical microenvironment of cell-derived matrix.

    Science.gov (United States)

    Choi, Dong Hoon; Suhaeri, Muhammad; Hwang, Mintai P; Kim, Ik Hwan; Han, Dong Keun; Park, Kwideok

    2014-09-01

    We obtained fibroblast- (FDM) and preosteoblast- (PDM) derived matrices in vitro from their respective cells. Our hypothesis was that these naturally occurring cell-derived matrices (CDMs) would provide a better microenvironment for the multi-lineage differentiation of human mesenchymal stromal cells (hMSCs) than those based on traditional single-protein-based platforms. Cells cultured for 5-6 days were decellularized with detergents and enzymes. The resulting matrices showed a fibrillar surface texture. Under osteogenic conditions, human bone-marrow-derived stromal cells (HS-5) exhibited higher amounts of both mineralized nodule formation and alkaline phosphatase (ALP) expression than those cultured on plastic or gelatin. Osteogenic markers (Col I, osteopontin, and cbfa1) and ALP activity from cells cultured on PDM were notably upregulated at 4 weeks. The use of FDM significantly improved the cellular expression of chondrogenic markers (Sox 9 and Col II), while downregulating that of Col I at 4 weeks. Both CDMs were more effective in inducing cellular synthesis of glycosaminoglycan content than control substrates. We also investigated the effect of matrix surface texture on hMSC (PT-2501) differentiation; soluble matrix (S-matrix)-coated substrates exhibited a localized fibronectin (FN) alignment, whereas natural matrix (N-matrix)-coated substrates preserved the naturally formed FN fibrillar alignment. hMSCs cultured for 4 weeks on N-matrices under osteogenic or chondrogenic conditions deposited a greater amount of calcium and proteoglycan than those cultured on S-matrices as assessed by von Kossa and Safranin O staining. In contrast to the expression levels of lineage-specific markers for cells cultured on gelatin, FN, or S-matrices, those cultured on N-matrices yielded highly upregulated levels. This study demonstrates not only the capacity of CDM for being an effective inductive template for the multi-lineage differentiation of hMSCs, but also the critical

  7. A xenogeneic-free bioreactor system for the clinical-scale expansion of human mesenchymal stem/stromal cells.

    Science.gov (United States)

    Dos Santos, Francisco; Campbell, Andrew; Fernandes-Platzgummer, Ana; Andrade, Pedro Z; Gimble, Jeffrey M; Wen, Yuan; Boucher, Shayne; Vemuri, Mohan C; da Silva, Cláudia L; Cabral, Joaquim M S

    2014-06-01

    The large cell doses (>1 × 10(6)  cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC

  8. Apatite formation on bioactive calcium-silicate cements for dentistry affects surface topography and human marrow stromal cells proliferation.

    Science.gov (United States)

    Gandolfi, Maria Giovanna; Ciapetti, Gabriela; Taddei, Paola; Perut, Francesca; Tinti, Anna; Cardoso, Marcio Vivan; Van Meerbeek, Bart; Prati, Carlo

    2010-10-01

    The effect of ageing in phosphate-containing solution of bioactive calcium-silicate cements on the chemistry, morphology and topography of the surface, as well as on in vitro human marrow stromal cells viability and proliferation was investigated. A calcium-silicate cement (wTC) mainly based on dicalcium-silicate and tricalcium-silicate was prepared. Alpha-TCP was added to wTC to obtain wTC-TCP. Bismuth oxide was inserted in wTC to prepare a radiopaque cement (wTC-Bi). A commercial calcium-silicate cement (ProRoot MTA) was tested as control. Cement disks were aged in DPBS for 5 h ('fresh samples'), 14 and 28 days, and analyzed by ESEM/EDX, SEM/EDX, ATR-FTIR, micro-Raman techniques and scanning white-light interferometry. Proliferation, LDH release, ALP activity and collagen production of human marrow stromal cells (MSC) seeded for 1-28 days on the cements were evaluated. Fresh samples exposed a surface mainly composed of calcium-silicate hydrates CSH (from the hydration of belite and alite), calcium hydroxide, calcium carbonate, and ettringite. Apatite nano-spherulites rapidly precipitated on cement surfaces within 5 h. On wTC-TCP the Ca-P deposits appeared thicker than on the other cements. Aged cements showed an irregular porous calcium-phosphate (Ca-P) coating, formed by aggregated apatite spherulites with interspersed calcite crystals. All the experimental cements exerted no acute toxicity in the cell assay system and allowed cell growth. Using biochemical results, the scores were: fresh cements>aged cements for cell proliferation and ALP activity (except for wTC-Bi), whereas fresh cementsbismuth on cell proliferation was reduced by the progressive increase of the biocoating thickness on aged cement. In conclusion, the experimental cements have adequate biological properties to be used as root-end/root repair filling materials or pulp capping materials. The alfa-TCP doped cement represents a new potential bioactive material for expanded applications in

  9. Effect of water-soluble P-chitosan and S-chitosan on human primary osteoblasts and giant cell tumor of bone stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Tang, T; Zhang, G; PY Lau, Carol; Zheng, L Z; Xie, X H; Wang, X L; Patrick, Y; Qin, L; Kumta, Shekhar M [Department of Orthopaedics and Traumatology, Chinese University of Hong Kong (Hong Kong); Wang, X H; He, K, E-mail: kumta@cuhk.edu.hk [Department of Mechanical Engineering, Institute of Bio-manufacturing Engineering, Tsinghua University, Beijing (China)

    2011-02-15

    Water-soluble phosphorylated chitosan (P-chitosan) and disodium (1 {yields} 4)-2-deoxy-2-sulfoamino-{beta}-D-glucopyranuronan (S-chitosan) are two chemically modified chitosans. In this study, we found that P-chitosan significantly promotes cell proliferation of both human primary osteoblasts (OBs) and the OB like stromal cell component of the giant cell tumor of bone (GCTB) cells at the concentration from 125 to 1000 {mu}g ml{sup -1} at all time points of 1, 3, 5 and 7 days after treatment. Further investigation of the osteogenic effect of the P-chitosan suggested that it regulates the levels of osteoclastogenic factors, receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression. An interesting finding is that S-chitosan at lower concentration (100 {mu}g ml{sup -1}) stimulates cell proliferation while a higher dose (1000 {mu}g ml{sup -1}) of S-chitosan inhibits it. The inhibitory effect of S-chitosan on human primary GCT stromal cells was greater than that of OBs (p < 0.05). Taken together, our findings elucidated the osteogenic effect of P-chitosan and the varying effects of S-chitosan on the proliferation of human primary OBs and GCT stromal cells and provided us the rationale for the construction of novel bone repair biomaterials with the dual properties of bone induction and bone tumor inhibition.

  10. Cancer cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts.

    Science.gov (United States)

    Lau, Tat-San; Chung, Tony Kwok-Hung; Cheung, Tak-Hong; Chan, Loucia Kit-Ying; Cheung, Leonard Wing-Hong; Yim, So-Fan; Siu, Nelson Shing-Shun; Lo, Kwok-Wai; Yu, May Mei-Yung; Kulbe, Hagen; Balkwill, Frances R; Kwong, Joseph

    2014-01-01

    We have investigated the role of cytokine lymphotoxin in tumour-stromal interactions in human ovarian cancer. We found that lymphotoxin overexpression is commonly shared by the cancer cells of various ovarian cancer subtypes, and lymphotoxin-beta receptor (LTBR) is expressed ubiquitously in both the cancer cells and cancer-associated fibroblasts (CAFs). In monoculture, we showed that ovarian cancer cells are not the major lymphotoxin-responsive cells. On the other hand, our co-culture studies demonstrated that the cancer cell-derived lymphotoxin induces chemokine expression in stromal fibroblasts through LTBR-NF-κB signalling. Amongst the chemokines being produced, we found that fibroblast-secreted CXCL11 promotes proliferation and migration of ovarian cancer cells via the chemokine receptor CXCR3. CXCL11 is highly expressed in CAFs in ovarian cancer biopsies, while CXCR3 is found in malignant cells in primary ovarian tumours. Additionally, the overexpression of CXCR3 is significantly associated with the tumour grade and lymph node metastasis of ovarian cancer, further supporting the role of CXCR3, which interacts with CXCL11, in promoting growth and metastasis of human ovarian cancer. Taken together, these results demonstrated that cancer-cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts. Our findings suggest that lymphotoxin-LTBR and CXCL11-CXCR3 signalling represent therapeutic targets in ovarian cancer. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  11. CD146 (MCAM in human cs-DLK1−/cs-CD34+ adipose stromal/progenitor cells

    Directory of Open Access Journals (Sweden)

    Susanne Hörl

    2017-07-01

    Full Text Available To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs we sorted the stromal vascular faction (SVF of human abdominal subcutaneous white adipose tissue (sWAT according to cell surface (cs expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1−/cs-CD34+/cs-CD146− ASCs, ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146+ and ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146− cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146+ endothelial cells (CD31+/CD34+/CD146+ and pericytes (CD31−/CD34−/CD146+ ASCs. The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34+ and contained low amounts of intracellular CD146 protein (CD31−/CD34+/CD146+. Additionally, we detected wavy CD34−/CD146+ and CD34dim/CD146+ cells. CD34dim/CD146+ cells were slightly more bulky than CD34−/CD146+ cells. Both CD34−/CD146+ and CD34dim/CD146+ cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34+/CD146+ and CD34−/CD146+ cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1−/cs-CD34+/cs-CD146− ASCs.

  12. Tumor-associated Endo180 requires stromal-derived LOX to promote metastatic prostate cancer cell migration on human ECM surfaces.

    Science.gov (United States)

    Caley, Matthew P; King, Helen; Shah, Neel; Wang, Kai; Rodriguez-Teja, Mercedes; Gronau, Julian H; Waxman, Jonathan; Sturge, Justin

    2016-02-01

    The diverse composition and structure of extracellular matrix (ECM) interfaces encountered by tumor cells at secondary tissue sites can influence metastatic progression. Extensive in vitro and in vivo data has confirmed that metastasizing tumor cells can adopt different migratory modes in response to their microenvironment. Here we present a model that uses human stromal cell-derived matrices to demonstrate that plasticity in tumor cell movement is controlled by the tumor-associated collagen receptor Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) and the crosslinking of collagen fibers by stromal-derived lysyl oxidase (LOX). Human osteoblast-derived and fibroblast-derived ECM supported a rounded 'amoeboid-like' mode of cell migration and enhanced Endo180 expression in three prostate cancer cell lines (PC3, VCaP, DU145). Genetic silencing of Endo180 reverted PC3 cells from their rounded mode of migration towards a bipolar 'mesenchymal-like' mode of migration and blocked their translocation on human fibroblast-derived and osteoblast-derived matrices. The concomitant decrease in PC3 cell migration and increase in Endo180 expression induced by stromal LOX inhibition indicates that the Endo180-dependent rounded mode of prostate cancer cell migration requires ECM crosslinking. In conclusion, this study introduces a realistic in vitro model for the study of metastatic prostate cancer cell plasticity and pinpoints the cooperation between tumor-associated Endo180 and the stiff microenvironment imposed by stromal-derived LOX as a potential target for limiting metastatic progression in prostate cancer.

  13. Optimization of Human Corneal Endothelial Cells for Culture: The Removal of Corneal Stromal Fibroblast Contamination Using Magnetic Cell Separation

    Directory of Open Access Journals (Sweden)

    Gary S. L. Peh

    2012-01-01

    Full Text Available The culture of human corneal endothelial cells (CECs is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated “labeled” and “flow-through” cell fractions were collected separately, cultured, and morphologically assessed. Cells from the “flow-through” fraction displayed compact polygonal morphology and expressed Na+/K+ATPase indicative of corneal endothelial cells, whilst cells from the “labeled” fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs.

  14. Direct Reprogramming of Human Bone Marrow Stromal Cells into Functional Renal Cells Using Cell-free Extracts

    Directory of Open Access Journals (Sweden)

    Evangelia Papadimou

    2015-04-01

    Full Text Available The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs, also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes—formation of “domes” and tubule-like structures—and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.

  15. Human platelet lysate gel provides a novel three dimensional-matrix for enhanced culture expansion of mesenchymal stromal cells.

    Science.gov (United States)

    Walenda, Gudrun; Hemeda, Hatim; Schneider, Rebekka K; Merkel, Rudolf; Hoffmann, Bernd; Wagner, Wolfgang

    2012-12-01

    Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that-apart from heparin-consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and reseeding procedure by pipetting cells together with their HPL-matrix-this method does not require washing steps and can easily be automated. The immunophenotype and in vitro differentiation potential toward adipogenic, osteogenic, and chondrogenic lineage were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities, and nonenzymatic passaging procedures for culture expansion of MSCs.

  16. Long-Term Safety of Transplanting Human Bone Marrow Stromal Cells into the Extravascular Spaces of the Choroid of Rabbits

    Directory of Open Access Journals (Sweden)

    Adi Tzameret

    2017-01-01

    Full Text Available Incurable neuroretinal degeneration diseases cause severe vision loss and blindness in millions of patients worldwide. In previous studies, we demonstrated that transplanting human bone marrow stromal cells (hBMSCs in the extravascular spaces of the choroid (EVSC of the Royal College of Surgeon rats ameliorated retinal degeneration for up to 5 months. Assessing the safety of hBMSC treatment and graft survival in a large animal is a crucial step before initiating clinical trials. Here, we transplanted hBMSCs into the EVSC compartment of New Zealand White rabbits. No immunosuppressants were used. Transplanted cells were spread across the EVSC covering over 80 percent of the subretinal surface. No cells were detected in the sclera. Cells were retained in the EVSC compartment 10 weeks following transplantation. Spectral domain optical coherence tomography (SD-OCT and histopathology analysis demonstrated no choroidal hemorrhages, retinal detachment, inflammation, or any untoward pathological reactions in any of transplanted eyes or in the control noninjected contralateral eyes. No reduction in retinal function was recorded by electroretinogram up to 10 weeks following transplantation. This study demonstrates the feasibility and safety of transplanting hBMSCs in the EVSC compartment in a large eye model of rabbits.

  17. Evaluation of gene delivery strategies to efficiently overexpress functional HLA-G on human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Joana S Boura

    2014-01-01

    Full Text Available Mesenchymal stromal cells (MSC constitutively express low levels of human leukocyte antigen-G (HLA-G, which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immunomodulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1 and a viral system (Lv-HLA-G1 using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK cell–mediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR9 expression is the mechanism responsible for the abrogation of HLA-G1's immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSC's immunomodulatory properties.

  18. Route of delivery influences biodistribution of human bone marrow-derived mesenchymal stromal cells following experimental bone marrow transplantation

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    Wang FJ

    2015-12-01

    Full Text Available Mesenchymal stromal cells (MSCs have shown promise as treatment for graft-versus-host disease (GvHD following allogeneic bone marrow transplantation (alloBMT. Mechanisms mediating in vivo effects of MSCs remain largely unknown, including their biodistribution following infusion. To this end, human bone-marrow derived MSCs (hMSCs were injected via carotid artery (IA or tail vein (TV into allogeneic and syngeneic BMT recipient mice. Following xenogeneic transplantation, MSC biodistribution was measured by bioluminescence imaging (BLI using hMSCs transduced with a reporter gene system containing luciferase and by scintigraphic imaging using hMSCs labeled with [99mTc]-HMPAO. Although hMSCs initially accumulated in the lungs in both transplant groups, more cells migrated to organs in alloBMT recipient as measured by in vivo BLI and scintigraphy and confirmed by ex vivo BLI imaging, immunohistochemistry and quantitative RT-PCR. IA injection resulted in persistent whole–body hMSC distribution in alloBMT recipients, while hMSCs were rapidly cleared in the syngeneic animals within one week. In contrast, TV-injected hMSCs were mainly seen in the lungs with fewer cells traveling to other organs. Summarily, these results demonstrate the potential use of IA injection to alter hMSC biodistribution in order to more effectively deliver hMSCs to targeted tissues and microenvironments.

  19. Hyaluronan-CD44 interaction promotes growth of decidual stromal cells in human first-trimester pregnancy.

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    Rui Zhu

    Full Text Available Hyaluronan (HA and its receptor CD44 are expressed at the maternal-fetal interface, but its role in early pregnancy remains unclear. Here, we found that primary decidual stromal cells (DSCs continuously secreted HA and expressed its receptor CD44. Pregnancy-associated hormones up-regulated HA synthetase (HAS 2 transcription and HA release from DSCs. High molecular weight-HA (HMW-HA, but not medium molecular weight (MMW-HA or low molecular weight (LMW-HA, promoted proliferation and inhibited apoptosis of DSCs in a CD44-dependent manner. The in-cell Western analysis revealed HMW-HA activated PI3K/AKT and mitogen-activated protein kinase (MAPK/ERK1/2 signaling pathways time-dependently. Blocking these pathways by specific inhibitor LY294002 or U0126 abrogated HMW-HA-regulated DSc proliferation and apoptosis. Finally, we have found that HA content, HA molecular weight, HAS2 mRNA level, and CD44 expression were significantly decreased in DSCs from unexplained miscarriage compared with the normal pregnancy. Collectively, our results indicate that higher level and greater molecular mass of HA at maternal-fetal interface contributes to DSc growth and maintenance of DSCs in human early pregnancy.

  20. Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers.

    Science.gov (United States)

    Liu, Shutong; de Castro, Luis F; Jin, Ping; Civini, Sara; Ren, Jiaqiang; Reems, Jo-Anna; Cancelas, Jose; Nayak, Ramesh; Shaw, Georgina; O'Brien, Timothy; McKenna, David H; Armant, Myriam; Silberstein, Leslie; Gee, Adrian P; Hei, Derek J; Hematti, Peiman; Kuznetsov, Sergei A; Robey, Pamela G; Stroncek, David F

    2017-04-27

    Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.

  1. Hydrocortisone regulates types I and III collagen gene expression and collagen synthesis in human marrow stromal cells.

    Science.gov (United States)

    Fernández, M; Minguell, J J

    1997-01-01

    Hematopoiesis is the resultant of the orderly molecular and cellular interactions between progenitor cells and stroma. In vitro studies (Dexter-type cultures) have shown that initiation of hematopoiesis only occurs after establishment of a hydrocortisone-dependent layer of stromal cells. Although the molecular basis for the requirement of hydrocortisone are not well understood, data have shown that synthesis/assembly of extracellular matrix molecules (proteoglycans and fibronectin) is regulated by hydrocortisone. Since interstitial collagens are abundantly expressed in the marrow stroma, we investigated whether hydrocortisone may also modulate the expression of collagen types I and III. For these studies, human bone marrow fibroblast cultures were grown in standard culture medium either in the absence or presence of 10(-7) M hydrocortisone. Under both conditions, bone marrow fibroblasts synthesized collagen types I and III, and expressed the respective genes. However, hydrocortisone produced a decrease in the synthesis of interstitial collagens and also in the relative abundance of pro-alpha 1(I) and pro-alpha 1(III) mRNAs. The results of this study are consistent with the assumption that glucocorticoids regulate the expression of several extracellular matrix molecules in the marrow stroma and thus permit in vitro hematopoiesis to occur.

  2. Inhibition of histone deacetylase activity in human endometrial stromal cells promotes extracellular matrix remodelling and limits embryo invasion.

    Directory of Open Access Journals (Sweden)

    Carlos Estella

    Full Text Available Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs to degrade extracellular matrix (ECM proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs. The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion.

  3. Inhibition of Histone Deacetylase Activity in Human Endometrial Stromal Cells Promotes Extracellular Matrix Remodelling and Limits Embryo Invasion

    Science.gov (United States)

    Atkinson, Stuart P.; Quiñonero, Alicia; Martínez, Sebastián; Pellicer, Antonio; Simón, Carlos

    2012-01-01

    Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs) to degrade extracellular matrix (ECM) proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs). The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs) that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs) related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA) increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion. PMID:22291969

  4. MicroRNA-34a inhibits osteoblast differentiation and in vivo bone formation of human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Holmstrøm, Kim; Qiu, Weimin

    2014-01-01

    Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, microRNAs (miRNAs) were identified as novel key regulators of human stromal (skeletal, mesenchymal) stem cells (hMSC) differentiation. Here, we identified miRNA......-34a (miR-34a) and its target protein networks as modulator of osteoblastic (OB) differentiation of hMSC. miRNA array profiling and further validation by quantitative RT-PCR revealed that miR-34a was upregulated during OB differentiation of hMSC, and in situ hybridization confirmed its OB expression...... in vivo. Overexpression of miR-34a inhibited early commitment and late OB differentiation of hMSC in vitro, whereas inhibition of miR-34a by anti-miR-34a enhanced these processes. Target prediction analysis and experimental validation confirmed Jagged1 (JAG1), a ligand for Notch 1, as a bona fide target...

  5. Induction of mitochondrial apoptotic pathway by raloxifene and estrogen in human endometrial stromal ThESC cell line.

    Science.gov (United States)

    Nikolic, Ivana; Andjelkovic, Marija; Zaric, Milan; Zelen, Ivanka; Canovic, Petar; Milosavljevic, Zoran; Mitrovic, Marina

    2017-03-01

    Endometrial hyperplasia is a condition that occurs as a result of hormonal imbalance between estrogen and progesterone. Morphological disturbance of endometrial cells occurs consequently leading towards endometrial cancer. In therapy of endometrial hyperplasia SERMs are used to supress effects of locally high estrogen level in uterus. There is strong evidence suggesting that estrogen could be involved in cell death - apoptosis. There are no experimental data demstrating the direct apoptotic effect of both raloxifene and estrogen on the ThESC cell line. The aim of our study wa sto investigate both cytotoxic and apototic mechanism of raloxifene and estrogen - induced death in the ThESC cell line. In order to determine their cytotoxic and apoptotic effects, various doses of raloxifene and estrogen were applied to the ThESC cell line for 24 h. After the treatment MTT assay, FACS analysis and immunofluoroscence method were conducted. The results of this study for the first time demonstrated the cytotoxic and apoptotic effects of raloxifene and estrogen on human endometrial stromal cell line suggesting the involvement of the inner, mitochondrial apoptotic pathway. Our results demonstrated apoptotic effects of investigated drugs in the ThESC cell line through increasing the Bax/Bcl-2 ratio and activation of caspase 3.

  6. Dexamethasone Regulates EphA5, a Potential Inhibitory Factor with Osteogenic Capability of Human Bone Marrow Stromal Cells

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Yamada

    2016-01-01

    Full Text Available We previously demonstrated the importance of quality management procedures for the handling of human bone marrow stromal cells (hBMSCs and provided evidence for the existence of osteogenic inhibitor molecules in BMSCs. One candidate inhibitor is the ephrin type-A receptor 5 (EphA5, which is expressed in hBMSCs and upregulated during long-term culture. In this study, forced expression of EphA5 diminished the expression of osteoblast phenotypic markers. Downregulation of endogenous EphA5 by dexamethasone treatment promoted osteoblast marker expression. EphA5 could be involved in the normal growth regulation of BMSCs and could be a potential marker for replicative senescence. Although Eph forward signaling stimulated by ephrin-B-Fc promoted the expression of ALP mRNA in BMSCs, exogenous addition of EphA5-Fc did not affect the ALP level. The mechanism underlying the silencing of EphA5 in early cultures remains unclear. EphA5 promoter was barely methylated in hBMSCs while histone deacetylation could partially suppress EphA5 expression in early-passage cultures. In repeatedly passaged cultures, the upregulation of EphA5 independent of methylation could competitively inhibit osteogenic signal transduction pathways such as EphB forward signaling. Elucidation of the potential inhibitory function of EphA5 in hBMSCs may provide an alternative approach for lineage differentiation in cell therapy strategies and regenerative medicine.

  7. Proliferation and osteoblastic differentiation of human bone marrow stromal cells on hydroxyapatite/bacterial cellulose nanocomposite scaffolds.

    Science.gov (United States)

    Fang, Bo; Wan, Yi-Zao; Tang, Ting-Tang; Gao, Chuan; Dai, Ke-Rong

    2009-05-01

    In this study, we prepared hydroxyapatite/bacterial cellulose (HAp/BC) nanocomposite scaffolds utilizing the biomimetic technique, and investigated the proliferation and osteoblastic differentiation of stromal cells derived from human bone marrow (hBMSC) on them. Scanning electron microscopy proved that cells could adhere and spread on scaffolds. The hBMSC seeded on the nanocomposites exhibited better adhesion and activity than those seeded upon the pure BC. After 6 days of culture on scaffolds, the cells proliferated faster on the nanocomposites than on the pure BC, as assessed by Alamar Blue assay. Real-time reverse transcription PCR results showed that the alkaline phosphatase (ALP) activity of hBMSC and the expression of osteopontin, osteocalcin, bone sialoprotein, and ALP mRNA were all higher for up to 7 days for hBMSC cultured on the nanocomposites than for those cultured upon the pure BC with and without the presence of osteogenic supplements (L-ascorbic acid, glycerophosphate, and dexamethasone, pproliferation, and differentiation in cultured hBMSC can be modulated by the HAp/BC nanocomposite scaffold properties. In summary, we have developed a scaffold that displays in vitro biocompatibility, which may have potential use for bone tissue engineering.

  8. Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics.

    Science.gov (United States)

    Sun, Hongli; Wu, Chengtie; Dai, Kerong; Chang, Jiang; Tang, Tingting

    2006-11-01

    In the present study, the effects of a calcium magnesium silicate bioactive ceramic (akermanite) on proliferation and osteoblastic differentiation of human bone marrow stromal cells (hBMSC) have been investigated and compared with the classical ceramic (beta-tricalcium phosphate, beta-TCP). Akermanite and beta-TCP disks were seeded with hBMSC and kept in growth medium or osteogenic medium for 10 days. Proliferation and osteoblastic differentiation were evaluated on day 1, 4, 7 and 10. The data from the Alamar Blue assay and lactic acid production assay showed that hBMSC proliferated more significantly on akermanite than on beta-TCP. The analysis of osteoblast-related genes, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), indicated that akermanite ceramics enhanced the expression of osteoblast-related genes, but type I collagen (COL I) showed no noticeable difference among akermanite and beta-TCP ceramics. Furthermore, this stimulatory effect was observed not only in osteogenic medium, but also in normal growth medium without osteogenic reagents such as l-ascorbic acid, glycerophosphate and dexamethasone. This result suggests that akermanite can promote osteoblastic differentiation of hBMSC in vitro even without osteogenic reagents, and may be used as a bioactive material for bone regeneration and tissue engineering applications.

  9. Graves' Patient with Thymic Expression of Thyrotropin Receptors and Dynamic Changes in Thymic Hyperplasia Proportional to Graves' Disease Activity.

    Science.gov (United States)

    Song, Young Shin; Won, Jae-Kyung; Kim, Mi Jeong; Lee, Ji Hyun; Kim, Dong-Wan; Chung, June-Key; Park, Do Joon; Park, Young Joo

    2016-05-01

    Thymic hyperplasia is frequently observed in Graves' disease. However, detectable massive enlargement of the thymus is rare, and the mechanism of its formation has remained elusive. This case showed dynamic changes in thymic hyperplasia on serial computed tomography images consistent with changes in serum thyrotropin receptor (TSH-R) antibodies and thyroid hormone levels. Furthermore, the patient's thymic tissues underwent immunohistochemical staining for TSH-R, which demonstrated the presence of thymic TSH-R. The correlation between serum TSH-R antibody levels and thymic hyperplasia sizes and the presence of TSH-R in her thymus suggest that TSH-R antibodies could have a pathogenic role in thymic hyperplasia.

  10. Decellularized adipose tissue microcarriers as a dynamic culture platform for human adipose-derived stem/stromal cell expansion.

    Science.gov (United States)

    Yu, Claire; Kornmuller, Anna; Brown, Cody; Hoare, Todd; Flynn, Lauren E

    2017-03-01

    With the goal of designing a clinically-relevant expansion strategy for human adipose-derived stem/stromal cells (ASCs), methods were developed to synthesize porous microcarriers derived purely from human decellularized adipose tissue (DAT). An electrospraying approach was applied to generate spherical DAT microcarriers with an average diameter of 428 ± 41 μm, which were soft, compliant, and stable in long-term culture without chemical crosslinking. Human ASCs demonstrated enhanced proliferation on the DAT microcarriers relative to commercially-sourced Cultispher-S microcarriers within a spinner culture system over 1 month. ASC immunophenotype was maintained post expansion, with a trend for reduced expression of the cell adhesion receptors CD73, CD105, and CD29 under dynamic conditions. Upregulation of the early lineage-specific genes PPARγ, LPL, and COMP was observed in the ASCs expanded on the DAT microcarriers, but the cells retained their multilineage differentiation capacity. Comparison of adipogenic and osteogenic differentiation in 2-D cultures prepared with ASCs pre-expanded on the DAT microcarriers or Cultispher-S microcarriers revealed similar adipogenic and enhanced osteogenic marker expression in the DAT microcarrier group, which had undergone a higher population fold change. Further, histological staining results suggested a more homogeneous differentiation response in the ASCs expanded on the DAT microcarriers as compared to either Cultispher-S microcarriers or tissue culture polystyrene. A pilot chondrogenesis study revealed higher levels of chondrogenic gene and protein expression in the ASCs expanded on the DAT microcarriers relative to all other groups, including the baseline controls. Overall, this study demonstrates the promise of applying dynamic culture with tissue-specific DAT microcarriers as a means of deriving regenerative cell populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

    Science.gov (United States)

    Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

    2013-12-01

    Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  12. Quantitative analysis of expression level of estrogen and progesterone receptors and VEGF genes in human endometrial stromal cells after treatment with nicotine.

    Science.gov (United States)

    Totonchi, Hamidreza; Miladpour, Behnoosh; Mostafavi-Pour, Zohreh; Khademi, Fatemeh; Kasraeian, Maryam; Zal, Fatemeh

    2016-10-01

    Cigarette smoke is a complex mixture of toxic chemicals, including nicotine, carbon monoxide, and several recognized carcinogens and mutagens. Nicotine has a direct disturbing influence on steroid hormones (estrogen and progesterone), which are essential components of the female reproductive system, but the effect of nicotine on the hormone receptors is not yet clear. The aim of this study was to elucidate the effect of nicotine on the expression of estrogen receptor (ER), progesterone receptor (PR), and vascular endothelial growth factor (VEGF) in endometrial stromal cells. Expression levels of PR, ER, and VEGF in human endometrial stromal primary cells treated with nicotine (0, 10 -11 , 10 -8 , and 10 -6  μM) for 24 h were measured by quantitative real-time PCR. MTT assay demonstrated that nicotine decreased cell viability in a dose-dependent manner. Real-time PCR data showed that despite decrease in ER expression in the nicotine-treated groups compared with the control, nicotine exerted an increased inhibitory effect on PR expression compared to that on ER expression. VEGF mRNA expression in nicotine-treated endometrial stromal cells was increased. The results from this study provide novel evidence for inhibitory effects of nicotine on steroid hormones receptor expression in human primary endometrial cells. Also, our data suggest that nicotine might have angiogenesis effects on these cells.

  13. Curcumin attenuates TNF-α-induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and proinflammatory cytokines in human endometriotic stromal cells.

    Science.gov (United States)

    Kim, Ki-Hyung; Lee, Eun Na; Park, Jin Kyeong; Lee, Ja-Rang; Kim, Ji-Hyun; Choi, Hak-Jong; Kim, Bong-Seon; Lee, Hee-Woo; Lee, Kyu-Sup; Yoon, Sik

    2012-07-01

    Curcumin, a naturally occurring polyphenolic compound from Curcuma longa, has long been used in folk medicine as an antiinflammatory remedy in Asian countries. Endometriosis is a chronic gynecological inflammatory disorder in which immune system deregulation may play a role in its initiation and progression. A number of mediators, including cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1); proinflammatory cytokines such as tumour necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6 and IL-8; and chemokines such as monocyte chemotactic protein-1 (MCP-1), play key roles in the pathogenesis of endometriosis. The aim of our study was to explore the effect of curcumin on the expression of these critical molecules in human ectopic endometriotic stromal cells isolated from women with endometriosis. Endometriotic stromal cells treated with curcumin showed marked suppression of TNF-α-induced mRNA expression of ICAM-1 and VCAM-1. Curcumin treatment also significantly decreased the TNF-α-induced cell surface and total protein expression of ICAM-1 and VCAM-1 in a dose-dependent manner. In addition, treatment of endometriotic stromal cells with curcumin markedly inhibited TNF-α-induced secretion of IL-6, IL-8 and MCP-1. Furthermore, curcumin inhibited the activation of transcription factor NF-κB, a key regulator of inflammation, in human endometriotic stromal cells. These findings suggest that curcumin may have potential therapeutic uses in the prevention and treatment of endometriosis. Copyright © 2011 John Wiley & Sons, Ltd.

  14. The oil-resin of the tropical rainforest tree Copaifera langsdorffii reduces cell viability, changes cell morphology and induces cell death in human endometriotic stromal cultures.

    Science.gov (United States)

    Henriques da Silva, Julianna; Borges, Vinicius Raphael de Almeida; Pereira, Leonardo da Cunha Boldrini; Ferrari, Renato; de Mattos, Rômulo Medina; Barros, Eliane Gouveia de Oliveira; Palmero, Celia Yelimar; Fernandes, Patricia Dias; de Carvalho, Patricia Ribeiro; Pereira de Sousa, Valeria; Cabral, Lucio Mendes; Nasciutti, Luiz Eurico

    2015-12-01

    The hormonal treatment for endometriosis frequently fails to completely eradicate endometriotic implants. A new therapeutic treatment is needed. This study investigates the in-vitro effect of Copaifera langsdorffii oil-resin on human eutopic and ectopic endometrium stromal cell cultures (EuESCs and EctESCs). A nanocomposite system containing the copaiba oil-resin (NanoCOR) was developed and acute toxicity test was performed. Endometrial stromal cells (ESCs) from non-endometriotics controls (CESCs), EuESCs and EctESCs were isolated and treated with different concentrations of NanoCOR, at different time intervals to evaluate its effect on cell morphology, proliferation, viability, necrosis and apoptosis induction. When treated with 50 μg/ml of NanoCOR, the morphology of EctESCs changed, as the actin microfilaments were disorganized, disassembled or disrupted. Moreover, at 24 h of treatment with NanoCOR, the EctESCs viability was inhibited, and a significant number of these cells underwent apoptosis. In EuESCs, these effects were observed only at 48 h. Finally, the treatment of EctESCs with NanoCOR increased the lactate dehydrogenase release into the extracellular medium more than in EuESCs. Our data indicate that NanoCOR has a greater impact on the behaviour of human endometriotic stromal cells than on the eutopic endometrium stromal cells, supporting the idea that NanoCOR should be further investigated as a novel and valuable alternative to treat endometriosis. © 2015 Royal Pharmaceutical Society.

  15. Chemokine CCL28 induces apoptosis of decidual stromal cells via binding CCR3/CCR10 in human spontaneous abortion.

    Science.gov (United States)

    Sun, Chan; Zhang, Yuan-Yuan; Tang, Chuan-Ling; Wang, Song-Cun; Piao, Hai-Lan; Tao, Yu; Zhu, Rui; Du, Mei-Rong; Li, Da-Jin

    2013-10-01

    Spontaneous abortion is the most common complication of pregnancy. Immune activation and the subsequent inflammation-induced tissue injury are often observed at the maternal-fetal interface as the final pathological assault in recurrent spontaneous abortion. However, the precise mechanisms responsible for spontaneous abortion involving inflammation are not fully understood. Chemokine CCL28 and its receptors CCR3 and CCR10 are important regulators in inflammatory process. Here, we examined the expression of CCL28 and its receptors in decidual stromal cells (DSCs) by immunochemistry and flow cytometry (FCM), and compared their expression level in DSCs from normal pregnancy versus spontaneous abortion, and their relationship to inflammatory cytokines production by DSCs. We further analyzed regulation of the pro-inflammatory cytokines on CCL28 expression in DSCs by real-time polymerase chain reaction, In-cell Western and FCM. The effects of CCL28-CCR3/CCR10 interaction on DSC apoptosis was investigated by Annexin V staining and FCM analysis or DAPI staining and nuclear morphology. Higher levels of the inflammatory cytokines interleukin (IL)-1β, IL-17A and tumor necrosis factor-α, and increased CCR3/CCR10 expression were observed in DSCs from spontaneous abortion compared with normal pregnancy. Treatment with inflammatory cytokines differently affected CCL28 and CCR3/CCR10 expression in DSCs. Human recombinant CCL28 promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10 and CCL28. However, CCL28 did not affect DSC growth. These results suggest that the inflammation-promoted up-regulation of CCL28 and its receptors interaction in DSCs is involved in human spontaneous abortion via inducing DSC apoptosis.

  16. A Good Manufacturing Practice-grade standard protocol for exclusively human mesenchymal stromal cell-derived extracellular vesicles.

    Science.gov (United States)

    Pachler, Karin; Lener, Thomas; Streif, Doris; Dunai, Zsuzsanna A; Desgeorges, Alexandre; Feichtner, Martina; Öller, Michaela; Schallmoser, Katharina; Rohde, Eva; Gimona, Mario

    2017-04-01

    Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs. Depletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit. Bone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV. The 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human MSC-derived EVs. With this Good Manufacturing Practice-grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  17. Increased migration of human mesenchymal stromal cells by autocrine motility factor (AMF resulted in enhanced recruitment towards hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Juan Bayo

    Full Text Available BACKGROUND AND AIMS: Several reports described the migration of human mesenchymal stromal cells (MSCs towards tumor-released factors. Autocrine motility factor (AMF is produced by several tumors including hepatocellular carcinoma (HCC. The aim of this study was to analyze AMF involvement on MSC migration towards human HCC. METHODS: Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2 activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated. RESULTS: AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro. CONCLUSION: AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation.

  18. Increased migration of human mesenchymal stromal cells by autocrine motility factor (AMF) resulted in enhanced recruitment towards hepatocellular carcinoma.

    Science.gov (United States)

    Bayo, Juan; Fiore, Esteban; Aquino, Jorge B; Malvicini, Mariana; Rizzo, Manglio; Peixoto, Estanislao; Andriani, Oscar; Alaniz, Laura; Piccioni, Flavia; Bolontrade, Marcela; Podhajcer, Osvaldo; Garcia, Mariana G; Mazzolini, Guillermo

    2014-01-01

    Several reports described the migration of human mesenchymal stromal cells (MSCs) towards tumor-released factors. Autocrine motility factor (AMF) is produced by several tumors including hepatocellular carcinoma (HCC). The aim of this study was to analyze AMF involvement on MSC migration towards human HCC. Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord) were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2) activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated. AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF) also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro. AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation.

  19. Fluorescent Immortalized Human Adipose Derived Stromal Cells (hASCs-TS/GFP+) for Studying Cell Drug Delivery Mediated by Microvesicles.

    Science.gov (United States)

    Cocce, Valentina; Balducci, Luigi; Falchetti, Maria L; Pascucci, Luisa; Ciusani, Emilio; Brini, Anna T; Sisto, Francesca; Piovani, Giovanna; Alessandri, Giulio; Parati, Eugenio; Cabeza, Laura; Pessina, Augusto

    2017-11-24

    A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASCs) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs). We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigated for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells. The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells. Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would use less resource and time and it could possibly contribute to simplification of GMP procedures. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Hypoxia-controlled EphA3 marks a human endometrium-derived multipotent mesenchymal stromal cell that supports vascular growth.

    Directory of Open Access Journals (Sweden)

    Catherine To

    Full Text Available Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs, but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.

  1. ADAM17 deletion in thymic epithelial cells alters aire expression without affecting T cell developmental progression.

    Directory of Open Access Journals (Sweden)

    David M Gravano

    2010-10-01

    Full Text Available Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach.We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted.In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and the mechanisms by which it regulates intrathymic T cell development

  2. Comparable change in stromal refractive index of cat and human corneas following blue-IRIS

    Science.gov (United States)

    Wozniak, Kaitlin T.; Gearhart, Sara M.; Savage, Daniel E.; Ellis, Jonathan D.; Knox, Wayne H.; Huxlin, Krystel R.

    2017-05-01

    Blue intratissue refractive index shaping (blue-IRIS) is a method with potential to correct ocular refraction noninvasively in humans. To date, blue-IRIS has only ever been applied to cat corneas and hydrogels. To test the comparability of refractive index change achievable in cat and human tissues, we used blue-IRIS to write identical phase gratings in ex vivo feline and human corneas. Femtosecond pulses (400 nm) were focused ˜300 μm below the epithelial surface of excised cat and human corneas and scanned to write phase gratings with lines ˜1 μm wide, spaced 5 μm apart, using a scan speed of 5 mm/s. Additional cat corneas were used to test writing at 3 and 7 mm/s in order to document speed dependence of the refractive index change magnitude. The first-order diffraction efficiency was immediately measured and used to calculate the refractive index change attained. Our data show that blue-IRIS induces comparable refractive index changes in feline and human corneas, an essential requirement for further developing its use as a clinical vision correction technique.

  3. Immunologic competence in adults following thymic irradiation in infancy

    Energy Technology Data Exchange (ETDEWEB)

    Ammann, A.J.; Wara, W.M.; Wara, D.W.; Phillips, T.L.

    1977-07-01

    Removal of, or irradiation to, the thymus during the neonatal period in man has resulted in no reported adverse effects on cellular immunity, although thymectomy in neonatal experimental animals is known to produce profound immunological disturbances. Adverse effects in humans may not be recognized until several decades have passed. The immunological capabilities of 7 adults with histories of thymic irradiation as infants were evaluated; normal tests results indicated intact immune systems in all cases. The 3 women tested, however, had abnormal clinical histories, including 2 with multiple tumors and 1 with chronic mucocutaneous candidiasis.

  4. Low oxygen tension reveals distinct HOX codes in human cord blood-derived stromal cells associated with specific endochondral ossification capacities in vitro and in vivo.

    Science.gov (United States)

    Liedtke, Stefanie; Sacchetti, Benedetto; Laitinen, Anita; Donsante, Samantha; Klöckers, Robert; Laitinen, Saara; Riminucci, Mara; Kogler, Gesine

    2017-10-01

    Effects of oxygen tension on the generation, expansion, proliferation and differentiation of stromal cell types is widely described in the literature. However, data on the internal heterogeneity of applied cell populations at different O 2 levels and possible impacts on differentiation potentials are controversial. Here, the expression of 39 human HOX genes was determined in neonatal cord blood stromal cells and linked to differentiation-associated signatures. In cord blood, unrestricted somatic stromal cells (USSCs), lacking HOX gene expression, and cord blood-derived multipotent stromal cells (CB-MSCs), expressing about 20 HOX genes, are distinguished by their specific HOX code. Interestingly, 74% of the clones generated at 21% O 2 were HOX-negative USSCs, whereas 73% of upcoming clones at 3% O 2 were HOX-positive CB-MSCs. In order to better categorize distinct cell lines generated at 3% O 2 , the expression of all 39 HOX genes within HOX clusters A, B, C and D were tested and new subtypes defined: cells negative in all four HOX clusters (USSCs); cells positive in all four clusters (CB-MSCs ABCD ); and subpopulations missing a single cluster (CB-MSCs ACD and CB-MSCs BCD ). Comprehensive qPCR analyses of established chondro-osteomarkers revealed subtype-specific signatures verifiably associated with in vitro and in vivo differentiation capacity. The data presented here underline the necessity of better characterizing distinct cell populations at a clonal level, taking advantage of the inherent specific HOX code as a distinguishing feature between individual subtypes. Moreover, the correlation of subtype-specific molecular signatures with in vitro and in vivo bone formation is discussed. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Improved performance of aging human mesenchymal stromal cells for bone tissue engineering

    NARCIS (Netherlands)

    Alves, H.A.D.C.R.

    2011-01-01

    In the last decades, an improvement in general life conditions has led to an increace in human life span but, concomitantly, also led to an increasing incidence of bone related disorders. A substancial effort has been placed in the development of new techologies to overcome current limitations of

  6. Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

    DEFF Research Database (Denmark)

    Haack-Sorensen, M.; Friis, T.; Bindslev, L.

    2008-01-01

    conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly...

  7. Enhanced cellular responses of human bone marrow stromal cells cultured on pretreated surface with allogenic platelet-rich plasma.

    Science.gov (United States)

    Shin, Seung Han; Yoo, Jeong Joon; Kim, Ha Na; Nam, Jinwoo; Kim, Hee Joong

    2012-01-01

    The principal objective of this study was to evaluate the effects of surface pretreatment with platelet-rich plasma (PRP) on the cellular functions of human bone marrow stromal cells (hBMSCs). The surfaces of tissue culture plates (TCPs) were pretreated by adding PRP followed by centrifugation to bring platelets closer to the surface, followed by incubation for 60 min at 37°C. Then, hBMSCs were seeded onto TCP and TCP pretreated with PRP (TCP-PRP), followed by culture in osteogenic medium. Cell attachment, proliferation, and osteogenic differentiation were evaluated. Field emission scanning electron microscope (FE-SEM; JSM-7401F, JEOL Ltd., Japan) observations were conducted. The attachment of hBMSCs was significantly lower on TCP-PRP than on TCP. However, when the cell numbers were normalized with those observed on day 1 of culture, cellular proliferation on 5 days was significantly higher on TCP-PRP. Alkaline phosphatase activity, an index of early phase of osteoblastic differentiation, was significantly higher on TCP-PRP on day 14. Calcium deposition amount, an index of terminal osteoblastic differentiation, was also significantly higher on TCP-PRP on days 14 and 21. The results of von Kossa staining confirmed that, on day 21, the area of mineralized nodules was significantly larger on TCP-PRP. FE-SEM observation demonstrated that activated platelets and fibrin network covered the surface after PRP treatment. An increase in the number of hBMSCs and their cellular products was evident on the FE-SEM observation, and the fibrin network remained on day 21. Our results demonstrate that a PRP-treated surface enhanced early proliferation and late osteogenic differentiation of hBMSCs.

  8. MicroRNA Levels as Prognostic Markers for the Differentiation Potential of Human Mesenchymal Stromal Cell Donors.

    Science.gov (United States)

    Georgi, Nicole; Taipaleenmaki, Hanna; Raiss, Christian C; Groen, Nathalie; Portalska, Karolina Janaeczek; van Blitterswijk, Clemens; de Boer, Jan; Post, Janine N; van Wijnen, Andre J; Karperien, Marcel

    2015-08-15

    The ability of human mesenchymal stromal/stem cells (hMSCs) to differentiate into various mesenchymal cell lineages makes them a promising cell source for the use in tissue repair strategies. Since the differentiation potential of hMSCs differs between donors, it is necessary to establish biomarkers for the identification of donors with high differentiation potential. In this study, we show that microRNA (miRNA) expression levels are effective for distinguishing donors with high differentiation potential from low differentiation potential. Twenty hMSC donors were initially tested for marker expression and differentiation potential. In particular, the chondrogenic differentiation potential was evaluated on the basis of histological matrix formation, mRNA expression levels of chondrogenic marker genes, and quantitative glycosaminoglycan deposition. Three donors out of twenty were identified as donors with high chondrogenic potential, whereas nine showed moderate and eight showed low chondrogenic potential. Expression profiles of miRNAs involved in chondrogenesis and cartilage homeostasis were used for the distinction between high-performance hMSCs and low-performance hMSCs. Global mRNA expression profiles of the donors before the onset of chondrogenic differentiation revealed minor differences in gene expression between low and high chondrogenic performers. However, analysis of miRNA expression during a 7-day differentiation period identified miR-210 and miR-630 as positive regulators of chondrogenesis. In contrast, miR-181 and miR-34a, which are negative regulators of chondrogenesis, were upregulated during differentiation in low-performing donors. In conclusion, profiling of hMSC donors for a specific panel of miRNAs may have a prognostic value for selecting donors with high differentiation potential to improve hMSC-based strategies for tissue regeneration.

  9. Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors: assessment of immunophenotype and differentiation capacity in vitro.

    Science.gov (United States)

    van Vollenstee, Fiona A; Jackson, Carlo; Hoffmann, Danie; Potgieter, Marnie; Durandt, Chrisna; Pepper, Michael S

    2016-10-01

    Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.

  10. Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro.

    Directory of Open Access Journals (Sweden)

    Andreas Matthäus Bader

    Full Text Available Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, "post-ischemic" cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic

  11. Human Placenta-Derived Mesenchymal Stromal-Like Cells Enhance Angiogenesis via T Cell-Dependent Reprogramming of Macrophage Differentiation.

    Science.gov (United States)

    He, Shuyang; Gleason, Joseph; Fik-Rymarkiewicz, Ewa; DiFiglia, Andrea; Bharathan, Mini; Morschauser, Andrew; Djuretic, Ivana; Xu, Yan; Krakovsky, Michael; Jankovic, Vladimir; Buensuceso, Charito; Edinger, James; Herzberg, Uri; Hofgartner, Wolfgang; Hariri, Robert

    2017-06-01

    Peripheral arterial disease (PAD) is a leading cause of limb loss and mortality worldwide with limited treatment options. Mesenchymal stromal cell (MSC) therapy has demonstrated positive effects on angiogenesis in preclinical models and promising therapeutic efficacy signals in early stage clinical studies; however, the mechanisms underlying MSC-mediated angiogenesis remain largely undefined. Here, we investigated the mechanism of action of human placenta-derived MSC-like cells (PDA-002) in inducing angiogenesis using mice hind limb ischemia model. We showed that PDA-002 improved blood flow and promoted collateral vessel formation in the injured limb. Histological analysis demonstrated that PDA-002 increased M2-like macrophages in ischemic tissue. Analysis of the changes in functional T cell phenotype in the draining lymph nodes revealed that PDA-002 treatment was associated with the induction of cytokine and gene expression signatures of Th2 response. Angiogenic effect of PDA-002 was markedly reduced in Balb/c nude mice compared with wild type. This reduction in efficacy was reversed by T cell reconstitution, suggesting T cells are essential for PDA-002-mediated angiogenesis. Furthermore, effect of PDA-002 on macrophage differentiation was also T cell-dependent as a PDA-002-mediated M2-like macrophage skewing was only observed in wild type and T cell reconstituted nude mice, but not in nude mice. Finally, we showed that PDA-002-treated animals had enhanced angiogenic recovery in response to the second injury when PDA-002 no longer persisted in vivo. These results suggest that PDA-002 enhances angiogenesis through an immunomodulatory mechanism involving T cell-dependent reprogramming of macrophage differentiation toward M2-like phenotype. Stem Cells 2017;35:1603-1613. © 2017 AlphaMed Press.

  12. Inhibition of chronic prostate inflammation by hyaluronic acid through an immortalized human prostate stromal cell line model.

    Directory of Open Access Journals (Sweden)

    Ming-Che Liu

    Full Text Available Benign prostatic hyperplasia (BPH is the most common urologic disease among elderly men. A well-established in vitro cell model is required to determine the therapeutic mechanism of BPH inflammation. In this study, we attempted to establish an immortalized human prostate stromal cell line by transfecting with HPV-16 E6/E7 and designated as ihPSC. No significant difference was found in fibroblast-like morphology between primary hPSC and ihPSC. The ihPSC possessed a significantly higher cell proliferation rate than primary hPSC. The prostate-specific markers and proteins including cytoskeleton (α-SMA and vimentin and smooth muscle (calponin, especially the androgen receptor (AR were also examined in ihPSC, almost identical to the primary hPSC. To create an in vitro model featuring chronic prostatic inflammation, ihPSC was stimulated with IFN-γ+IL-17 and then treated with the high molecular weight hyaluronic acid hylan G-F 20 as an alternative strategy for inhibiting BPH inflammation. Hylan G-F 20 could dose-dependently diminish the inflammation-induced proliferation in ihPSC. The enhanced expressions of inflammatory molecules including IL-1β, IL-6, IL-8, cyclooxygenase 2 (COX2, inducible nitrogen oxide synthase (iNOS, and Toll-like receptor 4 (TLR4 were all abolished by hylan G-F 20. For inflammatory signaling, hylan G-F 20 can also diminish the IFN-γ+IL-17-increased expression of iNOS and p65 in ihPSC. These findings suggest that ihPSC could provide a mechanism-based platform for investigating prostate inflammation. The hylan G-F 20 showed strong anti-inflammatory effects by decreasing inflammatory cytokines and signalings in the ihPSC, indicating its therapeutic potentials in BPH treatment in the future.

  13. Poly(γ-Glutamic Acid) as an Exogenous Promoter of Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.

    Science.gov (United States)

    Antunes, Joana C; Tsaryk, Roman; Gonçalves, Raquel M; Pereira, Catarina Leite; Landes, Constantin; Brochhausen, Christoph; Ghanaati, Shahram; Barbosa, Mário A; Kirkpatrick, C James

    2015-06-01

    Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(γ-glutamic acid) (γ-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, γ-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-γ-PGA-supplemented medium (CM+γ-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding γ-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in γ-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, γ-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that γ-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration.

  14. Interleukin-1β stimulates stromal-derived factor-1α expression in human subacromial bursa.

    Science.gov (United States)

    Blaine, Theodore A; Cote, Mindy A; Proto, Al; Mulcahey, Mary; Lee, Francis Y; Bigliani, Louis U

    2011-11-01

    Chemokines produced by synoviocytes of the subacromial bursa are up-regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF-1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL-1β and IL-6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti-human antibodies to IL-1, IL-6, and SDF-1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL-1β and IL-6) and SDF-1α expression was measured by ELISA and RT-PCR. SDF-1α, IL-1β, and IL-6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose-dependent increase in SDF-1α production in the supernatants of cells treated with IL-1β. SDF-1α mRNA expression was also increased in bursal cells treated with IL-1β. IL-6 caused a minimal but not statistically significant increase in SDF-1α expression. SDF-1α, IL-1β, and IL-6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF-1α gene expression and protein production are stimulated by IL-1β. IL-1β produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease. Copyright © 2011 Orthopaedic Research Society.

  15. Good Preservation of Stromal Cells and No Apoptosis in Human Ovarian Tissue after Vitrification

    OpenAIRE

    Raffaella Fabbri; Rossella Vicenti; Maria Macciocca; Gianandrea Pasquinelli; Roberto Paradisi; Cesare Battaglia; Nicola Antonio Martino; Stefano Venturoli

    2014-01-01

    The aim of this study was to develop a vitrification procedure for human ovarian tissue cryopreservation in order to better preserve the ovarian tissue. Large size samples of ovarian tissue retrieved from 15 female-to-male transgender subjects (18–38 years) were vitrified using two solutions (containing propylene glycol, ethylene glycol, and sucrose at different concentrations) in an open system. Light microscopy, transmission electron microscopy, and TUNEL assay were applied to evaluate the ...

  16. Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

    Directory of Open Access Journals (Sweden)

    Justyna Czapla

    Full Text Available The aim of the present study was to isolate mesenchymal stromal cells (MSC with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI. The study aimed also to investigate the feasibility of xenogeneic MSCs implantation.MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO. Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb. MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed.In two murine models (hindlimb ischemia and MI we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.

  17. Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

    Science.gov (United States)

    Czapla, Justyna; Matuszczak, Sybilla; Wiśniewska, Ewa; Jarosz-Biej, Magdalena; Smolarczyk, Ryszard; Cichoń, Tomasz; Głowala-Kosińska, Magdalena; Śliwka, Joanna; Garbacz, Marcin; Szczypior, Mateusz; Jaźwiec, Tomasz; Langrzyk, Agnieszka; Zembala, Michał; Szala, Stanisław

    2016-01-01

    The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.

  18. Engineering new bone via a minimally invasive route using human bone marrow-derived stromal cell aggregates, microceramic particles, and human platelet-rich plasma gel.

    Science.gov (United States)

    Chatterjea, Anindita; Yuan, Huipin; Fennema, Eelco; Burer, Ruben; Chatterjea, Supriyo; Garritsen, Henk; Renard, Auke; van Blitterswijk, Clemens A; de Boer, Jan

    2013-02-01

    There is a rise in the popularity of arthroscopic procedures in orthopedics. However, the majority of cell-based bone tissue-engineered constructs (TECs) rely on solid preformed scaffolding materials, which require large incisions and extensive dissections for placement at the defect site. Thus, they are not suitable for minimally invasive techniques. The aim of this study was to develop a clinically relevant, easily moldable, bone TEC, amenable to minimally invasive techniques, using human mesenchymal stromal cells (hMSCs) and calcium phosphate microparticles in combination with an in situ forming platelet-rich plasma gel obtained from human platelets. Most conventional TECs rely on seeding and culturing single-cell suspensions of hMSCs on scaffolds. However, for generating TECs amenable to the minimally invasive approach, it was essential to aggregate the hMSCs in vitro before seeding them on the scaffolds as unaggregated MSCs did not generate any bone. Twenty four hours of in vitro aggregation was determined to be optimal for maintaining cell viability in vitro and bone formation in vivo. Moreover, no statistically significant difference was observed in the amount of bone formed when the TECs were implanted via an open approach or a minimally invasive route. TECs generated using MSCs from three different human donors generated new bone through the minimally invasive route in a reproducible manner, suggesting that these TECs could be a viable alternative to preformed scaffolds employed through an open surgery for treating bone defects.

  19. Interferon-γ differentially modulates the impact of tumor necrosis factor-α on human endometrial stromal cells.

    Science.gov (United States)

    Spratte, Julia; Oemus, Anne; Zygmunt, Marek; Fluhr, Herbert

    2015-09-01

    The pro-inflammatory T helper (Th)-1 cytokines, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), are immunological factors relevant at the feto-maternal interface and involved in the pathophysiology of implantation disorders. The synergistic action of the two cytokines has been described with regard to apoptotic cell death and inflammatory responses in different cell types, but little is known regarding the human endometrium. Therefore, we examined the interaction of TNF-α and IFN-γ in human endometrial stromal cells (ESCs). ESCs were isolated from specimens obtained during hysterectomy and decidualized in vitro. Cells were incubated with TNF-α, IFN-γ or signaling-inhibitor. Insulin-like growth factor binding protein (IGFBP)-1, prolactin (PRL), leukemia inhibitory factor (LIF), interleukin (IL)-6, IL-8, regulated on activation normal T-cell expressed and secreted protein (RANTES) and monocyte chemotactic protein (MCP)-1 were measured using ELISA and real-time RT-PCR. Nuclear factor of transcription (NF)-κB and its inhibitor (IκBα) were analyzed by in-cell western assay and transcription factor assay. TNF-α inhibited and IFN-γ did not affect the decidualization of ESCs. In contrast, IFN-gamma differentially modulated the stimulating effect of TNF-alpha on cytokines by enhancing IL-6, RANTES and MCP-1 and attenuating LIF mRNA expression. These effects were time- and dose-dependent. IFN-γ had no impact on the initial activation of NF-κB signaling. Histone-deacetylase activity was involved in the modulating effect of IFN-γ on RANTES secretion. These observations showed a distinct pattern of interaction of the Th-1 cytokines, TNF-α and IFN-γ in the human endometrium, which could play an important role in the pathophysiology of implantation disorders. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z

  20. Recombinant human collagen-based microspheres mitigate cardiac conduction slowing induced by adipose tissue-derived stromal cells.

    Directory of Open Access Journals (Sweden)

    Nicoline W Smit

    Full Text Available Stem cell therapy to improve cardiac function after myocardial infarction is hampered by poor cell retention, while it may also increase the risk of arrhythmias by providing an arrhythmogenic substrate. We previously showed that porcine adipose tissue-derived-stromal cells (pASC induce conduction slowing through paracrine actions, whereas rat ASC (rASC and human ASC (hASC induce conduction slowing by direct coupling. We postulate that biomaterial microspheres mitigate the conduction slowing influence of pASC by interacting with paracrine signaling.To investigate the modulation of ASC-loaded recombinant human collagen-based microspheres, on the electrophysiological behavior of neonatal rat ventricular myocytes (NRVM.Unipolar extracellular electrograms, derived from microelectrode arrays (8x8 electrodes containing NRVM, co-cultured with ASC or ASC loaded microspheres, were used to determine conduction velocity (CV and conduction heterogeneity. Conditioned medium (Cme of (cocultures was used to assess paracrine mechanisms.Microspheres did not affect CV in control (NRVM monolayers. In co-cultures of NRVM and rASC, hASC or pASC, CV was lower than in controls (14.4±1.0, 13.0±0.6 and 9.0± 1.0 vs. 19.5±0.5 cm/s respectively, p<0.001. Microspheres loaded with either rASC or hASC still induced conduction slowing compared to controls (13.5±0.4 and 12.6±0.5 cm/s respectively, p<0.001. However, pASC loaded microspheres increased CV of NRVM compared to pASC and NRMV co-cultures (16.3±1.3 cm/s, p< 0.001 and did not differ from controls (p = NS. Cme of pASC reduced CV in control monolayers of NRVM (10.3±1.1 cm/s, p<0.001, similar to Cme derived from pASC-loaded microspheres (11.1±1.7 cm/s, p = 1.0. The presence of microspheres in monolayers of NRVM abolished the CV slowing influence of Cme pASC (15.9±1.0 cm/s, p = NS vs. control.The application of recombinant human collagen-based microspheres mitigates indirect paracrine conduction slowing through

  1. Human adipose-derived stromal cells in a clinically applicable injectable alginate hydrogel

    DEFF Research Database (Denmark)

    Larsen, Bjarke Follin; Juhl, Morten; Cohen, Smadar

    2015-01-01

    is to inject the cells in an in situ cross-linked alginate hydrogel. METHODS: ASCs from abdominal human tissue were embedded in alginate hydrogel and alginate hydrogel modified with Arg-Gly-Asp motifs (RGD-alginate) and cultured for 1 week. Cell viability, phenotype, immunogenicity and paracrine activity were...... determined by confocal microscopy, dendritic cell co-culture, flow cytometry, reverse transcriptase quantitative polymerase chain reaction, Luminex multiplex, and lymphocyte proliferation experiments. RESULTS: ASCs performed equally well in alginate and RGD-alginate. After 1 week of alginate culture, cell...... viability was >93%. Mesenchymal markers CD90 and CD29 were reduced compared with International Society for Cellular Therapy criteria. Cells sedimented from the alginates during cultivation regained the typical level of these markers, and trilineage differentiation was performed by standard protocols...

  2. Notch2 controls prolactin and insulin-like growth factor binding protein-1 expression in decidualizing human stromal cells of early pregnancy.

    Directory of Open Access Journals (Sweden)

    Gerlinde R Otti

    Full Text Available Decidualization, the transformation of the human uterine mucosa into the endometrium of pregnancy, is critical for successful implantation and embryonic development. However, key regulatory factors controlling differentiation of uterine stromal cells into hormone-secreting decidual cells have not been fully elucidated. Hence, we herein investigated the role of the Notch signaling pathway in human decidual stromal cells (HDSC isolated from early pregnancy samples. Immunofluorescence of first trimester decidual tissues revealed expression of Notch2 receptor and its putative, membrane-anchored interaction partners Jagged1, Delta-like (DLL 1 and DLL4 in stromal cells whereas other Notch receptors and ligands were absent from these cells. During in vitro differentiation with estrogen/progesterone (E2P4 and/or cyclic adenosine monophosphate (cAMP HDSC constitutively expressed Notch2 and weakly downregulated Jagged1 mRNA and protein, measured by quantitative PCR (qPCR and Western blotting, respectively. However, increased levels of DLL1 and DLL4 were observed in the decidualizing cultures. Transfection of a Notch luciferase reporter and qPCR of the Notch target gene hairy and enhancer of split 1 (HES1 revealed an induction of canonical Notch activity during in vitro differentiation. In contrast, treatment of HDSC with a chemical Notch/γ-secretase inhibitor decreased cAMP/E2P4-stimulated Notch luciferase activity, HES1 transcript levels and mRNA expression of the decidual marker genes prolactin (PRL and insulin-like growth factor binding protein 1 (IGFBP1. Similarly, siRNA-mediated gene silencing or antibody-mediated blocking of Notch2 diminished HES1, PRL and IGFBP1 mRNA levels as well as secreted PRL protein. In summary, the data suggest that canonical, Notch2-dependent signaling plays a role in human decidualization.

  3. Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate.

    Science.gov (United States)

    Heathman, Thomas R J; Stolzing, Alexandra; Fabian, Claire; Rafiq, Qasim A; Coopman, Karen; Nienow, Alvin W; Kara, Bo; Hewitt, Christopher J

    2016-04-01

    The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 10(5) and 1.02 ± 0.005 × 10(5) cells/mL compared with 0.79 ± 0.05 × 10(5) and 0.36 ± 0.04 × 10(5) cells/mL in FBS-containing medium. This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  4. Immunophenotyping reveals the diversity of human dental pulp mesenchymal stromal cells in vivo and their evolution upon in vitro amplification

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    Maxime DUCRET

    2016-11-01

    Full Text Available Mesenchymal stromal/stem cells (MSCs from human dental pulp (DP can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31- cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31- DP cell population contained 1.4% of CD56+, 1.5% of CD146+, 2.4% of CD271+ and 6.3% of MSCA-1+ cells but very few Stro-1+ cells (≤1%. CD56+, CD146+, CD271+ and MSCA-1+ cell subpopulations expressed various levels of these markers. CD146+MSCA-1+, CD271+MSCA-1+ and CD146+CD271+ cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56+, CD271+, MSCA-1+ and Stro-1+ cells, and MSCA-1 by 15-30% of CD56+, CD146+, CD271+ and Stro-1+ cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by less than 1% of the expanded cell populations. Interestingly, the percentage of CD56+ cells strongly increased from P1 (25% to P4 (80% both in all sub-populations studied. CD146+CD56+, MSCA-1+CD56+ and CD146+MSCA-1+ cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products.

  5. Endometrial stromal cells of women with recurrent miscarriage fail to discriminate between high- and low-quality human embryos.

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    Charlotte H E Weimar

    Full Text Available BACKGROUND: The aetiology of recurrent miscarriage (RM remains largely unexplained. Women with RM have a shorter time to pregnancy interval than normally fertile women, which may be due to more frequent implantation of non-viable embryos. We hypothesized that human endometrial stromal cells (H-EnSCs of women with RM discriminate less effectively between high-and low-quality human embryos and migrate more readily towards trophoblast spheroids than H-EnSCs of normally fertile women. METHODOLOGY/PRINCIPAL FINDINGS: Monolayers of decidualized H-EnSCs were generated from endometrial biopsies of 6 women with RM and 6 fertile controls. Cell-free migration zones were created and the effect of the presence of a high-quality (day 5 blastocyst, n = 13, a low-quality (day 5 blastocyst with three pronuclei or underdeveloped embryo, n = 12 or AC-1M88 trophoblast cell line spheroid on H-ESC migratory activity was analyzed after 18 hours. In the absence of a spheroid or embryo, migration of H-EnSCs from fertile or RM women was similar. In the presence of a low-quality embryo in the zone, the migration of H-EnSCs of control women was inhibited compared to the basal migration in the absence of an embryo (P<0.05 and compared to the migration in the presence of high-quality embryo (p<0.01. Interestingly, the migratory response H-EnSCs of women with RM did not differ between high- and low-quality embryos. Furthermore, in the presence of a spheroid their migration was enhanced compared to the H-EnSCs of controls (p<0.001. CONCLUSIONS: H-EnSCs of fertile women discriminate between high- and low-quality embryos whereas H-EnSCs of women with RM fail to do so. H-EnSCs of RM women have a higher migratory response to trophoblast spheroids. Future studies will focus on the mechanisms by which low-quality embryos inhibit the migration of H-EnSCs and how this is deregulated in women with RM.

  6. Direct analysis of thymic function in children with Down's syndrome

    Directory of Open Access Journals (Sweden)

    Meschiari Liviana

    2005-02-01

    Full Text Available Abstract Background Down's syndrome (DS is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes (the so called "recent thymic emigrants", RTE in children with DS. Methods We studied 8 children affected by DS, aged 2–7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE, i.e. peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC. Results In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found. Conclusions The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects.

  7. [Human umbilic mesenchymal stromal cells repairs diabetic foot in rats associated with VEGF expressional change].

    Science.gov (United States)

    Wang, Ying; Dan, Qi-Qin; Wang, Qing-Ping; Zhou, Ning; Jin, Xing-Fang; Hou, Zong-Liua; Peng, Bao-Kun; Wang, Ting-Hua

    2014-01-01

    OBJECTITVE: To explore the mechanism of human umbilic mesenchymal cells (HUMSCs) implantation for the treatment of diabetic foot in rats associate with vascular endothelia growth factor (VEGF) expression changes. After diabetic foot model in rats were established by administration of streptozotozin (STZ) in intraperitoneal injection (2 weeks), ulceration in foot was induced by incision injury combined with swearing staphylococcus aureas. Then, HUMSCs were smeared on the ulceration of foot in diabetic rats. Ten days later, the densities of blood vessel and the level of VEGF expression were determined by using immunohistochemistry, RT-PCR and Western blot. HUMSC grafts reduced significantly the volume of ulceration in diabetic foot rats (P < 0.05). RT-PCR and Western blot showed that VEGF and its mRNA were significantly upregulated (P < 0.05). VEGF immunstaining was found in blood vessels and the densities of blood vessels in HUMSC group were increased significantly (P < 0.05). HUMSC implantation showed a positive role in promoting the recovery of the ulceration in foot with diabetic rats.

  8. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    Science.gov (United States)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  9. Update on Aire and thymic negative selection.

    Science.gov (United States)

    Passos, Geraldo A; Speck-Hernandez, Cesar A; Assis, Amanda F; Mendes-da-Cruz, Daniella A

    2018-01-01

    Twenty years ago, the autoimmune regulator (Aire) gene was associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, and was cloned and sequenced. Its importance goes beyond its abstract link with human autoimmune disease. Aire identification opened new perspectives to better understand the molecular basis of central tolerance and self-non-self distinction, the main properties of the immune system. Since 1997, a growing number of immunologists and molecular geneticists have made important discoveries about the function of Aire, which is essentially a pleiotropic gene. Aire is one of the functional markers in medullary thymic epithelial cells (mTECs), controlling their differentiation and expression of peripheral tissue antigens (PTAs), mTEC-thymocyte adhesion and the expression of microRNAs, among other functions. With Aire, the immunological tolerance became even more apparent from the molecular genetics point of view. Currently, mTECs represent the most unusual cells because they express almost the entire functional genome but still maintain their identity. Due to the enormous diversity of PTAs, this uncommon gene expression pattern was termed promiscuous gene expression, the interpretation of which is essentially immunological - i.e. it is related to self-representation in the thymus. Therefore, this knowledge is strongly linked to the negative selection of autoreactive thymocytes. In this update, we focus on the most relevant results of Aire as a transcriptional and post-transcriptional controller of PTAs in mTECs, its mechanism of action, and its influence on the negative selection of autoreactive thymocytes as the bases of the induction of central tolerance and prevention of autoimmune diseases. © 2017 John Wiley & Sons Ltd.

  10. General Information about Thymoma and Thymic Carcinoma

    Science.gov (United States)

    ... the body against infections . Enlarge Anatomy of the thymus gland. The thymus gland is a small organ that lies in the upper chest under the breastbone. It makes white blood cells, called lymphocytes, ... of the thymus. Thymomas and thymic carcinomas are rare tumors of ...

  11. Stages of Thymoma and Thymic Carcinoma

    Science.gov (United States)

    ... the body against infections . Enlarge Anatomy of the thymus gland. The thymus gland is a small organ that lies in the upper chest under the breastbone. It makes white blood cells, called lymphocytes, ... of the thymus. Thymomas and thymic carcinomas are rare tumors of ...

  12. Treatment Options for Thymoma and Thymic Carcinoma

    Science.gov (United States)

    ... the body against infections . Enlarge Anatomy of the thymus gland. The thymus gland is a small organ that lies in the upper chest under the breastbone. It makes white blood cells, called lymphocytes, ... of the thymus. Thymomas and thymic carcinomas are rare tumors of ...

  13. PICTORIAL ESSAY Thymic masses: A radiological review

    African Journals Online (AJOL)

    pericardium, aortic arch, left innominate vein, and trachea. ... Thymic masses: A radiological review. M K Mittal, MD, FICR; B Sureka, MD, DNB; M Sinha, MD; A Mittal, MD, DNB; B B Thukral, MD, FICR. Department of Radiodiagnosis and Imaging, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India.

  14. In vivo mechanisms of acquired thymic tolerance

    DEFF Research Database (Denmark)

    Chen, W; Issazadeh-Navikas, Shohreh; Sayegh, M H

    1997-01-01

    Injection of antigen into the thymus of adult animals induces specific systemic tolerance, but the mechanisms of acquired thymic tolerance are not well understood. To investigate these mechanisms we used a model of intrathymic injection of ovalbumin (OVA) in BALB/c mice. We show an antigen-specif...

  15. Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b

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    Brosens Jan J

    2009-07-01

    Full Text Available Abstract Background Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line. Methods Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro. Results St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82. Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages. Conclusion St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural

  16. Delivery of dimethyloxallyl glycine in mesoporous bioactive glass scaffolds to improve angiogenesis and osteogenesis of human bone marrow stromal cells.

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    Wu, Chengtie; Zhou, Yinghong; Chang, Jiang; Xiao, Yin

    2013-11-01

    Development of hypoxia-mimicking bone tissue engineering scaffolds is of great importance in stimulating angiogenesis for bone regeneration. Dimethyloxallyl glycine (DMOG) is a cell-permeable, competitive inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH), which can stabilize hypoxia-inducible factor 1α (HIF-1α) expression. The aim of this study was to develop hypoxia-mimicking scaffolds by delivering DMOG in mesoporous bioactive glass (MBG) scaffolds and to investigate whether the delivery of DMOG could induce a hypoxic microenvironment for human bone marrow stromal cells (hBMSC). MBG scaffolds with varied mesoporous structures (e.g. surface area and mesopore volume) were prepared by controlling the contents of mesopore-template agent. The composition, large-pore microstructure and mesoporous properties of MBG scaffolds were characterized. The effect of mesoporous properties on the loading and release of DMOG in MBG scaffolds was investigated. The effects of DMOG delivery on the cell morphology, cell viability, HIF-1α stabilization, vascular endothelial growth factor (VEGF) secretion and bone-related gene expression (alkaline phosphatase, ALP; osteocalcin, OCN; and osteopontin, OPN) of hBMSC in MBG scaffolds were systematically investigated. The results showed that the loading and release of DMOG in MBG scaffolds can be efficiently controlled by regulating their mesoporous properties via the addition of different contents of mesopore-template agent. DMOG delivery in MBG scaffolds had no cytotoxic effect on the viability of hBMSC. DMOG delivery significantly induced HIF-1α stabilization, VEGF secretion and bone-related gene expression of hBMSC in MBG scaffolds in which DMOG counteracted the effect of HIF-PH and stabilized HIF-1α expression under normoxic condition. Furthermore, it was found that MBG scaffolds with slow DMOG release significantly enhanced the expression of bone-related genes more than those with instant DMOG release. The results

  17. Human decidual stromal cells secrete soluble pro-apoptotic factors during decidualization in a cAMP-dependent manner.

    Science.gov (United States)

    Leno-Durán, E; Ruiz-Magaña, M J; Muñoz-Fernández, R; Requena, F; Olivares, E G; Ruiz-Ruiz, C

    2014-10-10

    Is there a relationship between decidualization and apoptosis of decidual stromal cells (DSC)? Decidualization triggers the secretion of soluble factors that induce apoptosis in DSC. The differentiation and apoptosis of DSC during decidualization of the receptive decidua are crucial processes for the controlled invasion of trophoblasts in normal pregnancy. Most DSC regress in a time-dependent manner, and their removal is important to provide space for the embryo to grow. However, the mechanism that controls DSC death is poorly understood. The apoptotic response of DSC was analyzed after exposure to different exogenous agents and during decidualization. The apoptotic potential of decidualized DSC supernatants and prolactin (PRL) was also evaluated. DSC lines were established from samples of decidua from first trimester pregnancies. Apoptosis was assayed by flow cytometry. PRL production, as a marker of decidualization, was determined by enzyme-linked immunosorbent assay. DSCs were resistant to a variety of apoptosis-inducing substances. Nevertheless, DSC underwent apoptosis during decidualization in culture, with cAMP being essential for both apoptosis and differentiation. In addition, culture supernatants from decidualized DSC induced apoptosis in undifferentiated DSC, although paradoxically these supernatants decreased the spontaneous apoptosis of decidual lymphocytes. Exogenously added PRL did not induce apoptosis in DSC and an antibody that neutralized the PRL receptor did not decrease the apoptosis induced by supernatants. Further studies are needed to examine the involvement of other soluble factors secreted by decidualized DSC in the induction of apoptosis. The present results indicate that apoptosis of DSC occurs in parallel to differentiation, in response to decidualization signals, with soluble factors secreted by decidualized DSC being responsible for triggering cell death. These studies are relevant in the understanding of how the regression of decidua

  18. Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Shani Samuel

    2016-09-01

    Full Text Available Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs. However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval of 15% non-activated platelet-rich concentrate (PRC in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM. Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of

  19. Safety and biodistribution profile of placental-derived mesenchymal stromal cells (PLX-PAD) following intramuscular delivery.

    Science.gov (United States)

    Ramot, Yuval; Meiron, Moran; Toren, Amir; Steiner, Michal; Nyska, Abraham

    2009-08-01

    The administration of mesenchymal stromal cells (MSCs) provides an exciting emerging therapeutic modality for the treatment of peripheral arterial disease, a condition that is associated with critical limb ischemia as its end stage. Placental-derived MSCs, termed PLX-PAD cells, are stable adhesive stromal cells isolated from full-term human placentae, cultured on carriers, and expanded in a bioreactor called the PluriX. These cells can be expanded in vitro without phenotypic or karyotypic changes. We studied the safety and biodistribution properties of PLX-PAD cells following intramuscular administration in NOD/SCID mice. No significant clinical signs, hematological and biochemical parameters, or major pathological changes were found in PLX-PAD-treated animals in comparison to vehicle controls. Several animals in the control and PLX-PAD-treated groups developed thymic malignant lymphoma, first seen after one month, as expected in this mouse strain. In addition, both groups developed spontaneous mesenteric vessel inflammation. Real-time quantitative polymerase chain reaction (RT-qPCR) demonstrated that distribution of PLX-PAD cells was confined to the injection site. Placental-derived MSCs remained in this site with gradual decrease in concentration during a three-month period. In view of these data, we conclude that the administration of PLX-PAD cells is not associated with any adverse effects in NOD/SCID mice.

  20. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures

    DEFF Research Database (Denmark)

    Justesen, J; Dokkedahl, Karin Stenderup; Eriksen, E F

    2002-01-01

    Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the ......Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP...... phosphatase (AP+), and adipocytic colonies containing adipocytes (Ad+) were quantitated. In addition, steady state mRNA levels of gene markers of adipocytic and osteoblastic phenotypes were determined using reverse-transcriptase polymerase chain reaction (RT-PCR). The adipogenic and osteogenic media induced...

  1. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.

    Science.gov (United States)

    Naaijkens, B A; Niessen, H W M; Prins, H-J; Krijnen, P A J; Kokhuis, T J A; de Jong, N; van Hinsbergh, V W M; Kamp, O; Helder, M N; Musters, R J P; van Dijk, A; Juffermans, L J M

    2012-04-01

    Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

  2. Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo.

    Science.gov (United States)

    Chan, Michael C W; Kuok, Denise I T; Leung, Connie Y H; Hui, Kenrie P Y; Valkenburg, Sophie A; Lau, Eric H Y; Nicholls, John M; Fang, Xiaohui; Guan, Yi; Lee, Jae W; Chan, Renee W Y; Webster, Robert G; Matthay, Michael A; Peiris, J S Malik

    2016-03-29

    Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

  3. Evaluation of early stage human bone marrow stromal proliferation, cell migration and osteogenic differentiation on μ-MIM structured stainless steel surfaces.

    Science.gov (United States)

    Bitar, Malak; Benini, Fausta; Brose, Claudia; Friederici, Vera; Imgrund, Philipp; Bruinink, Arie

    2013-05-01

    It is well established that surface topography greatly affect cell-surface interactions. In a recent study we showed that microstructured stainless steel surfaces characterized by the presence of defined hexagonally arranged hemisphere-like structures significantly affected cell architecture (shape and focal adhesion size) of primary human bone mesenchymal stromal cells. This study aimed at further investigating the influence these microstructures (microcline protruding hemispheres) on critical aspects of cell behaviour namely; proliferation, migration and osteogenic differentiation. As with previously reported data, we used primary human bone mesenchymal stromal cells to investigate such effects at an early stage in vitro. Cells of different patients were utilised for cell migration studies. Our data showed that an increase in cell proliferation was exhibited as a function of surface topography (hemispheres). Cell migration velocity also varied as a function of surface topography on patient specific basis and seems to relate to the differentiated state of the seeded cell population (as demonstrated by bALP positivity). Osteogenic differentiation, however, did not exhibit significant variations (both up and down-regulation) as a function of both surface topography and time in culture.

  4. Pigment epithelium derived factor inhibits the growth of human endometrial implants in nude mice and of ovarian endometriotic stromal cells in vitro.

    Directory of Open Access Journals (Sweden)

    Yanmei Sun

    Full Text Available Angiogenesis is a prerequisite for the formation and development of endometriosis. Pigment epithelium derived factor (PEDF is a natural inhibitor of angiogenesis. We previously demonstrated a reduction of PEDF in the peritoneal fluid, serum and endometriotic lesions from women with endometriosis compared with women without endometriosis. Here, we aim to investigate the inhibitory effect of PEDF on human endometriotic cells in vivo and in vitro. We found that PEDF markedly inhibited the growth of human endometrial implants in nude mice and of ovarian endometriotic stromal cells in vitro by up-regulating PEDF expression and down-regulating vascular endothelial growth factor (VEGF expression. Moreover, apoptotic index was significantly increased in endometriotic lesions in vivo and endometriotic stromal cells in vitro when treated with PEDF. In mice treated with PEDF, decreased microvessel density labeled by Von Willebrand factor but not by α-Smooth Muscle Actin was observed in endometriotic lesions. And it showed no increase in PEDF expression of the ovary and uterus tissues. These findings suggest that PEDF gene therapy may be a new treatment for endometriosis.

  5. Pigment epithelium derived factor inhibits the growth of human endometrial implants in nude mice and of ovarian endometriotic stromal cells in vitro.

    Science.gov (United States)

    Sun, Yanmei; Che, Xuan; Zhu, Libo; Zhao, Mengdan; Fu, Guofang; Huang, Xiufeng; Xu, Hong; Hu, Fuqiang; Zhang, Xinmei

    2012-01-01

    Angiogenesis is a prerequisite for the formation and development of endometriosis. Pigment epithelium derived factor (PEDF) is a natural inhibitor of angiogenesis. We previously demonstrated a reduction of PEDF in the peritoneal fluid, serum and endometriotic lesions from women with endometriosis compared with women without endometriosis. Here, we aim to investigate the inhibitory effect of PEDF on human endometriotic cells in vivo and in vitro. We found that PEDF markedly inhibited the growth of human endometrial implants in nude mice and of ovarian endometriotic stromal cells in vitro by up-regulating PEDF expression and down-regulating vascular endothelial growth factor (VEGF) expression. Moreover, apoptotic index was significantly increased in endometriotic lesions in vivo and endometriotic stromal cells in vitro when treated with PEDF. In mice treated with PEDF, decreased microvessel density labeled by Von Willebrand factor but not by α-Smooth Muscle Actin was observed in endometriotic lesions. And it showed no increase in PEDF expression of the ovary and uterus tissues. These findings suggest that PEDF gene therapy may be a new treatment for endometriosis.

  6. Stepwise development of thymic microenvironments in vivo is regulated by thymocyte subsets

    NARCIS (Netherlands)

    W. van Ewijk (Willem); G. Hollander; C. Terhorst; B. Wang (Baoping)

    2000-01-01

    textabstractT-cell development is under the tight control of thymic microenvironments. Conversely, the integrity of thymic microenvironments depends on the physical presence of developing thymocytes, a phenomenon designated as 'thymic crosstalk'. We now show, using

  7. Thymic factors and T cell maturation in vitro : A comparison of the effects of thymic epithelial cultures with thymic extracts and thymus dependent serum factors

    NARCIS (Netherlands)

    Kruisbeek, A.M.

    1979-01-01

    In the present review the results obtained so far with thymic epithelial culture supernatants (TES) or cocultivation of potential precursor T cells with TE cells will be discussed and compared with the effects reported for other thymic factors in studies on T cell differentiation in vitro. These

  8. The International Thymic Malignancy Interest Group thymic initiative: a state-of-the-art study of thymic malignancies.

    Science.gov (United States)

    Detterbeck, Frank; Korst, Robert

    2014-01-01

    Thymic malignancies are relatively rare tumors. A general lack of knowledge, misconceptions about benignancy, confusion about the definition of terms, and variability in reporting of outcomes have further hampered progress in these diseases. The International Thymic Malignancy Interest Group has emerged to counter these challenges and has brought together a worldwide multidisciplinary community determined to improve outcomes for these patients. Although the organization is young (initiated in 2010), major early accomplishments have created a foundation and infrastructure for scientific research. These include consensus definitions of terms, an unprecedented global database, development of practical clinical resources and, together with the International Association for the Study of Lung Cancer, development of proposals for the first formal stage classification of these malignant tumors. Many articles have been published or are under way, and a second phase of projects building on the early success is proceeding. The greatest accomplishment of the International Thymic Malignancy Interest Group lies in the establishment of an open culture of collaboration and the engagement of a broad group of individuals united by a common mission. It is a testament to what can be achieved, despite ongoing and inherent challenges, by determination and a collective effort. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Serum-free human MSC medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture.

    Science.gov (United States)

    Schubert, Susanna; Brehm, Walter; Hillmann, Aline; Burk, Janina

    2017-09-19

    For clinical applications of multipotent mesenchymal stromal cells (MSCs), serum-free culture is preferable to standardize cell products and prevent contamination with pathogens. In contrast to human MSCs, knowledge on serum-free culture of large animal MSCs is limited, despite its relevance for preclinical studies and development of veterinary cellular therapeutics. This study aimed to evaluate the suitability of a commercially available serum-free human MSC medium for culturing equine adipose-derived MSCs in comparison with human adipose MSCs. Enzyme-free isolation by explant technique and expansion of equine and human cells in the serum-free medium were feasible. However, serum-free culture altered the morphology and complicated handling of equine MSCs, with cell aggregation and spontaneous detachment of multilayers, compared to culture in standard medium supplemented with fetal bovine serum. Furthermore, proliferation and the surface immunophenotype of equine cells were more variable compared to the controls and appeared to depend on the lot of the serum-free medium. Particularly the expression of CD90 was different between experimental groups (P cultured in serum-free medium (5.21-83.40%) compared to standard medium (86.20-99.50%). Additionally, small subpopulations expressing MSC exclusion markers such as CD14 (0.28-11.60%), CD34 (0.00-9.87%), CD45 (0.35-10.50%), or MHCII (0.00-3.67%) were found in equine samples after serum-free culture. In contrast, human samples displayed a more consistent morphology and a consistent CD29+ (98.60-99.90%), CD73+ (94.60-98.40%), CD90+ (99.60-99.90%), and CD105+ (97.40-99.80%) immunophenotype after culture in serum-free medium. The obtained data demonstrate that the serum-free medium was suitable for human MSC culture but did not lead to entirely satisfactory results in equine MSCs. This underlines that requirements regarding serum-free culture conditions are species-specific, indicating a need for serum-free media to be

  10. Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Bernardi, Martina; Albiero, Elena; Alghisi, Alberta; Chieregato, Katia; Lievore, Chiara; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2013-08-01

    A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Effects of TGFβ1, PDGF-BB, and bFGF, on human corneal fibroblasts proliferation and differentiation during stromal repair.

    Science.gov (United States)

    Gallego-Muñoz, Patricia; Ibares-Frías, Lucía; Valsero-Blanco, María Cruz; Cantalapiedra-Rodriguez, Roberto; Merayo-Lloves, Jesús; Martínez-García, M Carmen

    2017-08-01

    In an effort to improve the regenerative nature of corneal repair, this study reports the use of an in vitro human corneal fibroblasts (HCFs) wound model after treatment with three of the main growth factors (GFs) involved in corneal healing: transforming growth factor beta 1 (TGFβ1), platelet-derived growth factor BB-isoform (PDGF-BB), and basic fibroblast growth factor (bFGF) in order to delve in cell proliferation and differentiation processes. HCFs were mechanically wounded. The individual effect of TGFβ1, PDGF-BB, and bFGF on cell proliferation and differentiation during the repair process was studied at different time points until wound closure. Wound dimensions and morphological changes were evaluated by microscopy. Cell proliferation and myofibroblast differentiation were analyzed by immunofluorescence cytochemistry. Changes in cell morphology were apparent at Day 4. PDGF-BB- and bFGF-treated cells had fibroblast-like morphology. TGFβ1 stimulated proliferation in the wound edge and surrounding area, induced myofibroblast differentiation and inhibited cellular migration. PDGF-BB induced rapid wound closure due to proliferation, high motility, and late myofibroblast differentiation. The time course of closure induced by bFGF was similar to that for PDGF-BB, but was mostly due to proliferation in the wound area, and inhibited myofibroblast differentiation. Each of the GFs induced increases in responses promoting stromal repair differently. This study provides insight regarding how to optimize the outcome of stromal repair following corneal injury. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

    Directory of Open Access Journals (Sweden)

    Djida Ghoubay-Benallaoua

    Full Text Available Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC and epithelial limbal (LSC stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8, E8 supplemented with EGF (E8+ or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

  13. Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

    Science.gov (United States)

    Ghoubay-Benallaoua, Djida; de Sousa, Céline; Martos, Raphaël; Latour, Gaël; Schanne-Klein, Marie-Claire; Dupin, Elisabeth; Borderie, Vincent

    2017-01-01

    Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

  14. Human adipose stromal cells (ASC for the regeneration of injured cartilage display genetic stability after in vitro culture expansion.

    Directory of Open Access Journals (Sweden)

    Simona Neri

    Full Text Available Mesenchymal stromal cells are emerging as an extremely promising therapeutic agent for tissue regeneration due to their multi-potency, immune-modulation and secretome activities, but safety remains one of the main concerns, particularly when in vitro manipulation, such as cell expansion, is performed before clinical application. Indeed, it is well documented that in vitro expansion reduces replicative potential and some multi-potency and promotes cell senescence. Furthermore, during in vitro aging there is a decrease in DNA synthesis and repair efficiency thus leading to DNA damage accumulation and possibly inducing genomic instability. The European Research Project ADIPOA aims at validating an innovative cell-based therapy where autologous adipose stromal cells (ASCs are injected in the diseased articulation to activate regeneration of the cartilage. The primary objective of this paper was to assess the safety of cultured ASCs. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype and microsatellite instability analysis. In addition, RT-PCR array-based evaluation of the expression of genes related to DNA damage signaling pathways was performed. Finally, the senescence and replicative potential of cultured cells was evaluated by telomere length and telomerase activity assessment, whereas anchorage-independent clone development was tested in vitro by soft agar growth. We found that cultured ASCs do not show genetic alterations and replicative senescence during the period of observation, nor anchorage-independent growth, supporting an argument for the safety of ASCs for clinical use.

  15. The role of simvastatin in the osteogenesis of injectable tissue-engineered bone based on human adipose-derived stromal cells and platelet-rich plasma.

    Science.gov (United States)

    Zhou, Yongsheng; Ni, Yongwei; Liu, Yunsong; Zeng, Baijin; Xu, Yongwei; Ge, Wenshu

    2010-07-01

    An injectable tissue-engineered bone (ITB) composed of human adipose-derived stromal cells (hADSCs) and platelet-rich plasma (hPRP) was preliminarily constructed, but its osteogenic capability needs improving. This study aimed to evaluate if simvastatin can be applied as a bone anabolic agent for this ITB. We found 0.01 microm, 0.1 microm, and 1 microm simvastatin could induce hADSCs' osteoblastic differentiation in vitro that accompanied with non-inhibition on cell proliferation, high alkaline phosphatase activity, more mineralization deposition and more expression of osteoblast-related genes such as osteocalcin, core binding factor alpha1, bone morphogenetic protein-2, vascular endothelial growth factor, and basic fibroblast growth factor. Simvastatin at 1 mum seemed the most optimal concentration due to its high osteocalcin secretion in media (P investigation. Copyright 2010 Elsevier Ltd. All rights reserved.

  16. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

    NARCIS (Netherlands)

    B. Naaijkens (Benno); H.W.M. Niessen (Hans ); H.-J. Prins (H.); P.A.J. Krijnen (Paul); T.J.A. Kokhuis (Tom); N. de Jong (Nico); V.W.M. van Hinsbergh (Victor); O. Kamp (Otto); K. Helder MScN (Onno); R.J.P. Musters (René); A. van Dijk (Annemieke); L.J.M. Juffermans (Lynda)

    2012-01-01

    textabstractAdipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

  17. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

    NARCIS (Netherlands)

    Naaijkens, B.A.; Niessen, H.W.M.; Prins, H.J.; Krijnen, P.A.J.; Kokhuis, T.J.A.; de Jong, N.; van Hinsbergh, V.W.M.; Kamp, O.; Helder, M.N.; Musters, R.J.P.; van Dijk, A.; Juffermans, L.J.M.

    2012-01-01

    Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

  18. Human ciliary neurotrophic factor-overexpressing stable bone marrow stromal cells in the treatment of a rat model of traumatic spinal cord injury.

    Science.gov (United States)

    Abbaszadeh, Hojjat-Allah; Tiraihi, Taki; Noori-Zadeh, Ali; Delshad, Ali Reza; Sadeghizade, Majid; Taheri, Taher

    2015-07-01

    Traumatic injury to the central nervous system (CNS) often causes motor dysfunctions. However, because of the CNS complexity and variability in the clinical presentations, efforts to repair damaged CNS tissue and restoring its functions are particularly demanding. On the other hand, recent progress in the regenerative therapy field have led to novel approaches for the treatment of traumatic CNS injury and renewed hopes to overcome the obstacles. It appears that the balance between neurite re-growth-inhibiting and neurite re-growth-inducing molecules determines the axonal re-growth fate. Neurotrophic factors can tilt this balance and indeed promote cell survival and axonal re-growth over neurodegeneration. One of the promising neurotrophic factors in this field is ciliary neurotrophic factor (CNTF). We transfected rat bone marrow stromal cells with a mammalian expression vector-inserted human CNTF gene through the use of a non-viral method to prepare human CNTF-overexpressing stem cells under ex vivo conditions. We transplanted these modified cells to the rat model of spinal cord traumatic injury to explore functional recovery after contusion induction. Our data from immunocytochemistry and behavioral tests showed that such cells can act as a powerful potential approach to treat traumatic CNS injuries because these modified cells improved the behavioral test scores in the rat model of spinal cord injury. CNTF-overexpressing bone marrow stromal cells can ameliorate spinal cord traumatic injury and can be used in the treatment of traumatic CNS injuries in the near future. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  19. Immortalized human fetal bone marrow-derived mesenchymal stromal cell expressing suicide gene for anti-tumor therapy in vitro and in vivo.

    Science.gov (United States)

    Lee, Wayne Y W; Zhang, Ting; Lau, Carol P Y; Wang, C C; Chan, Kai-Ming; Li, Gang

    2013-12-01

    Cancer is one of the greatest health challenges facing the world today with >10 million new cases of cancer every year. The self-renewal, tumor-homing ability and low immunogenicity of mesenchymal stromal cells (MSCs) make them potential delivery candidates for suicide genes for anti-tumor therapy. However, unstable supply and short life span of adult MSCs in vitro have limited this therapeutic potential. In this study, we aimed to evaluate if immortalization of human fetal bone marrow-derived mesenchymal stromal cells by simian virus 40 (SV40-hfBMSCs) could be a stable source of MSCs for clinical application of suicide gene therapy. Transduction of SV40 and herpes simplex virus thymidine kinase-IRES-green fluorescent protein (TK-GFP) did not cause significant change in the stem cell properties of hfBMSCs. The anti-tumor effect of SV40-TK-hfBMSCs in the presence of the prodrug ganciclovir was demonstrated in vitro and in nude mice bearing human prostate cancer cells, DU145 and PC3, which had been transduced with luciferase and GFP for imaging evaluation by an in vivo live imaging system (IVIS 200 imaging system; Caliper Life Sciences). Repeated injection of low doses (1 × 10(6) cells/kg) of SV40-TK-hfBMSCs was as effective as previously reported and did not cause observable harmful side effects in multiple organs. Mixed lymphocyte reaction showed that SV40-TK-hfBMSCs did not induce significant proliferation of lymphocytes isolated from healthy adults. Taken together, immortalized hfBMSCs represent a reliable and safe source of MSCs for further clinical translational study. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  20. A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue.

    Science.gov (United States)

    Carmelo, Joana G; Fernandes-Platzgummer, Ana; Diogo, Maria Margarida; da Silva, Cláudia Lobato; Cabral, Joaquim M S

    2015-08-01

    Human mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and the development of microcarrier-based cultures in scalable bioreactors with well-defined xenogeneic-free components represent important milestones towards the clinical-scale production of these cells. In this work, we optimized our previously developed xeno-free microcarrier-based system for the scalable expansion of human MSC isolated from bone marrow (BM MSC) and adipose-derived stem/stromal cells (ASC). By adapting the agitation/feeding protocol at the initial cell seeding/cultivation stage in spinner flasks, we were able to maximize cell expansion rate and final cell yield. Maximal cell densities of 3.6 × 10(5) and 1.9 × 10(5) cells/mL were obtained for BM MSC (0.60 ± 0.04 day(-1) ) and ASC (0.9 ± 0.1 day(-1) ) cultures, upon seven and eight days of cultivation, respectively. Ready-to-use microcarriers Synthemax® II and Enhanced Attachment® supported identical expansion performance of BM MSC, turning those effective alternatives to the pre-coated plastic microcarriers used in our xeno-free scalable culture system. Importantly, expanded MSC maintained their immunophenotype and multilineage differentiation potential. Moreover, secretome analysis suggested a priming effect of stirred culture conditions on cytokine production by MSC. This culture system yielded considerable final cell densities that can be scaled-up to controlled large-scale bioreactors allowing a more efficient, safe and cost-effective MSC production for clinical settings. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

    Energy Technology Data Exchange (ETDEWEB)

    Sugino, Noriko [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Miura, Yasuo, E-mail: ym58f5@kuhp.kyoto-u.ac.jp [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Yao, Hisayuki [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Iwasa, Masaki; Fujishiro, Aya [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Division of Gastroenterology and Hematology, Shiga University of Medical Science, Shiga 520-2192 (Japan); Fujii, Sumie [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Hirai, Hideyo [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Takaori-Kondo, Akifumi [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Ichinohe, Tatsuo [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Maekawa, Taira [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan)

    2016-01-22

    Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34{sup +} hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation. - Highlights: • Human BM-MSCs in an early osteoinductive stage (e-MSCs) support hematopoiesis. • Adhesion- and chemotaxis-associated gene signatures are altered in e-MSCs. • Expression of CXCL12 and VCAM1 is remarkably decreased in e-MSCs. • e-MSCs are at differentiation stage between MSCs and osteoprogenitor cells. • Osteoinductive treatment

  2. CD54-Mediated Interaction with Pro-inflammatory Macrophages Increases the Immunosuppressive Function of Human Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Nicolas Espagnolle

    2017-04-01

    Full Text Available Mesenchymal stromal cells (MSCs sense and modulate inflammation and represent potential clinical treatment for immune disorders. However, many details of the bidirectional interaction of MSCs and the innate immune compartment are still unsolved. Here we describe an unconventional but functional interaction between pro-inflammatory classically activated macrophages (M1MΦ and MSCs, with CD54 playing a central role. CD54 was upregulated and enriched specifically at the contact area between M1MФ and MSCs. Moreover, the specific interaction induced calcium signaling and increased the immunosuppressive capacities of MSCs dependent on CD54 mediation. Our data demonstrate that MSCs can detect an inflammatory microenvironment via a direct and physical interaction with innate immune cells. This finding opens different perspectives for MSC-based cell therapy.

  3. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François, E-mail: fberthia@rci.rutgers.edu

    2015-02-27

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

  4. The JAZF1-SUZ12 fusion protein disrupts PRC2 complexes and impairs chromatin repression during human endometrial stromal tumorogenesis.

    Science.gov (United States)

    Ma, Xianyong; Wang, Jinglan; Wang, Jianhui; Ma, Charles X; Gao, Xiaobin; Patriub, Vytas; Sklar, Jeffrey L

    2017-01-17

    The Polycomb repressive complex 2 (PRC2), which contains three core proteins EZH2, EED and SUZ12, controls chromatin compaction and transcription repression through trimethylation of lysine 27 on histone 3. The (7;17)(p15;q21) chromosomal translocation present in most cases of endometrial stromal sarcomas (ESSs) results in the in-frame fusion of the JAZF1 and SUZ12 genes. We have investigated whether and how the fusion protein JAZF1-SUZ12 functionally alters PRC2. We found that the fusion protein exists at high levels in ESS containing the t(7;17). Co-transient transfection assay indicated JAZF1-SUZ12 destabilized PRC2 components EZH2 and EED, resulting in decreased histone methyl transferase (HMT) activity, which was confirmed by in vitro studies using reconstituted PRC2 and nucleosome array substrates. We also demonstrated the PRC2 containing the fusion protein decreased the binding affinity to target chromatin loci. In addition, we found that trimethylation of H3K27 was decreased in ESS samples with the t(7;17), but there was no detectable change in H3K9 in these tissues. Moreover, re-expression of SUZ12 in Suz12 (-/-) ES cells rescued the neuronal differentiation while the fusion protein failed to restore this function and enhanced cell proliferation. In summary, our studies reveal that JAZF1-SUZ12 fusion protein disrupts the PRC2 complex, abolishes HMT activity and subsequently activates chromatin/genes normally repressed by PRC2. Such dyesfunction of PRC2 inhibits normal neural differentiation of ES cell and increases cell proliferation. Related changes induced by the JAZF-SUZ12 protein in endometrial stromal cells may explain the oncogenic effect of the t(7;17) in ESS.

  5. Thymic B cells and central T cell tolerance

    Directory of Open Access Journals (Sweden)

    Ludger eKlein

    2015-07-01

    Full Text Available Central T cell tolerance is believed to be mainly induced by thymic dendritic cells and medullary thymic epithelial cells. The thymus also harbors substantial numbers of B cells. These may arise though intrathymic B lymphopoiesis or immigration from the bloodstream. Importantly, and in contrast to resting ‘mainstream’ B cells in the periphery, thymic B cells display elevated levels of MHC class II and constitutively express CD80. Arguably their most unexpected feature is the expression of Aire. These unique features of thymic B cells result from a licensing process that involves cross-talk with CD4 single-positive T cells and CD40 signaling. Together, these recent findings suggest that B cells play a more prominent role as thymic APCs than previously appreciated.

  6. Thymic Epithelial Tumor with Heart Metastasis in a Horse

    Directory of Open Access Journals (Sweden)

    Farshid Shahriar

    2010-01-01

    Full Text Available Thymic malignancy is rare in horses. Thymic epithelial tumor was diagnosed in an 18-year-old mare with invasion and metastasis to the pericardium and heart. At necropsy, the cranial thoracic cavity was obliterated by a large mass located in the thymic region and the right atrium was also expanded and effaced by a similar mass. Histologically, the neoplasm was composed of sheets of spindle cells with intraparenchymal Hassall's corpuscles and formation of pseudorosettes around blood vessels compatible with type A thymic epithelial tumor according to World Health Organization classification. The neoplastic cells were diffusely immunoreactive for cytokeratin and negative for vimentin, S100, neuron specific enolase, glial fibrillar acidic protein, chromogranin A, synaptophysin, CD3 and CD79a markers. To the authors' knowledge, cardiac invasion and distinct histological pattern of pseudorosette formation have not been described in equine thymic epithelial tumors previously.

  7. Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding.

    Directory of Open Access Journals (Sweden)

    Ozlem Guzeloglu Kayisli

    Full Text Available Use of long-acting progestin only contraceptives (LAPCs offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understanding to mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s in human endometrial stromal cells (HESCs. Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2 or E2+ medroxyprogesterone acetate (MPA or E2+ etonogestrel (ETO or E2+ progesterone (P4 were analyzed by quantitative Real-time (q-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depot MPA (DMPA treated women and ovariectomized guinea pigs (GPs treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67 gene group regulated by all three progestins, whereas a 235 gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR activation as one of upstream regulators of the 235 MPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both MPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-DMPA versus pre-DMPA administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting PR and GR

  8. Cilostazol Improves Proangiogenesis Functions in Human Early Endothelial Progenitor Cells through the Stromal Cell-Derived Factor System and Hybrid Therapy Provides a Synergistic Effect In Vivo.

    Science.gov (United States)

    Tseng, Shih-Ya; Chao, Ting-Hsing; Li, Yi-Heng; Cho, Chung-Lung

    2016-01-01

    This study investigated the effect of cilostazol on proangiogenesis functions in human early endothelial progenitor cells (EPCs) in vitro and the therapeutic implication of hybrid therapy with cilostazol and human early EPCs in vivo. Cilostazol significantly increased colony-forming units and enhanced differentiation of EPCs toward endothelial lineage. Treatments resulted in antiapoptotic effects and stimulated proliferation and migration and in vitro vascular tube formation through activation of stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4)/phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. Blood flow recovery and capillary density in murine ischemic hindlimbs were significantly improved in cilostazol-treated, human early EPCs-treated, and cotreatment groups. The effects were attenuated with SDF-1α inhibition. Plasma SDF-1α levels were significantly higher in 3 active treatment groups after surgery, with greatest effects observed in hybrid therapy. The angiogenic effects of transplanted EPCs pretreated with cilostazol ex vivo were superior to untreated EPCs using in vivo Matrigel assay. Implanted EPCs were incorporated into the capillary, with pretreatment or cotreatment with cilostazol resulting in enhanced effects. Taken together, cilostazol promotes a large number of proangiogenic functions in human early EPCs through activation of SDF-1/CXCR4/PI3K/Akt signaling, and hybrid therapy provides a synergistic effect in vivo. Cotreatment may be beneficial in ischemic disease.

  9. Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

    Science.gov (United States)

    Park, Tea Soon; Huo, Jeffrey S.; Peters, Ann; Talbot, C. Conover; Verma, Karan; Zimmerlin, Ludovic; Kaplan, Ian M.; Zambidis, Elias T.

    2012-01-01

    Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%–0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33+CD45+CD34− myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG+TRA-1-81+ hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self

  10. Human Bone Marrow Mesenchymal stromal cell derived Osteoblasts Promote the Expansion of Hematopoietic Progenitors through Beta-Catenin and Notch Signaling Pathways.

    Science.gov (United States)

    Michalicka, Matthew; Boisjoli, Gavin; Jahan, Suria; Hovey, Owen; Doxtator, Emily; Abu-Khader, Ahmad; Pasha, Roya; Pineault, Nicolas

    2017-10-19

    Coculture of hematopoietic stem cells (HSC) with primary stromal cells from HSC niches supports the maintenance and expansion of HSC and progenitors ex vivo. However, a major drawback is the availability of primary human samples for research and clinical applications. We investigated the use of in vitro derived osteoblasts as a new source of feeder cells and characterized the molecular pathways that mediate their growth promoting activities. First, we compared the growth and differentiation modulating activities of mesenchymal stromal cells (MSC)-derived osteoblasts (M-OST) to that of their undifferentiated precursor on umbilical cord blood (UCB) progenitors. Feeder-free cultures were also included as baseline control. Cell growth and expansion of hematopoietic progenitors were significantly enhanced by both feeder cell types. However, progenitor cell growth was considerably greater with M-OST. Coculture also promoted the maintenance of immature CD34+ progenitor subsets and modulated in a positive fashion the expression of several homing-related cell surface receptors, in a feeder-specific fashion. Serial transplantation experiments revealed that M-OST coculture supported the maintenance of long-term lympho-myeloid reconstituting HSC that provided engraftment levels generally superior to that from MSC cocultures. Mechanistically, we found that coculture with M-OST was associated with enhanced -catenin (-Cat) activity in UCB cells and that abrogation of -Cat/TCF activity blunted the growth promoting activity of the M-OST coculture. Conversely, Notch inhibition reduced UCB cell expansion but to a much lesser extent. In conclusion, this study demonstrates that M-OST are excellent feeder cells for HSC and progenitors, and identify key molecular pathways responsible for the growth enhancing activities of osteoblasts on UCB progenitors.

  11. Osteogenic potential of human adipose-derived stromal cells on 3-dimensional mesoporous TiO{sub 2} coating with magnesium impregnation

    Energy Technology Data Exchange (ETDEWEB)

    Cecchinato, Francesca, E-mail: francesca.cecchinato@mah.se [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Karlsson, Johan [Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg (Sweden); Ferroni, Letizia; Gardin, Chiara [Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova (Italy); Galli, Silvia; Wennerberg, Ann [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Zavan, Barbara [Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova (Italy); Andersson, Martin [Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg (Sweden); Jimbo, Ryo [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki (Japan)

    2015-07-01

    The aim of this study was to evaluate the osteogenic response of human adipose-derived stromal cells (ADScs) to mesoporous titania (TiO{sub 2}) coatings produced with evaporation-induced self-assembly method (EISA) and loaded with magnesium. Our emphasis with the magnesium release functionality was to modulate progenitor cell osteogenic differentiation under standard culture conditions. Osteogenic properties of the coatings were assessed for stromal cells by means of scanning electron microscopy (SEM) imaging, colorimetric mitochondrial viability assay (MTT), colorimetric alkaline phosphates activity (ALP) assay and real time RT-polymerase chain reaction (PCR). Using atomic force microscopy (AFM) it was shown that the surface expansion area (S{sub dr}) was strongly enhanced by the presence of magnesium. From MTT results it was shown that ADSc viability was significantly increased on mesoporous surfaces compared to the non-porous one at a longer cell culture time. However, no differences were observed between the magnesium impregnated and non-impregnated surfaces. The alkaline phosphatase activity confirmed that ADSc started to differentiate into the osteogenic phenotype after 2 weeks of culturing. The gene expression profile at 2 weeks of cell growth showed that such coatings were capable to incorporate specific osteogenic markers inside their interconnected nano-pores and, at 3 weeks, ADSc differentiated into osteoblasts. Interestingly, magnesium significantly promoted the osteopontin gene expression, which is an essential gene for the early biomaterial–cell osteogenic interaction. - Highlights: • The magnesium loading presents a transitory effect on mesoporous TiO{sub 2} surface topography • The mesoporous structure promotes cellular attachment and spreading • The mesoporous structure activates osteogenesis of mesenchymal stem cells in absence of osteogenic promoters • The physical adsorbed magnesium is suggested to be involved in the expression of

  12. Systemic Administration of Human Bone Marrow-Derived Mesenchymal Stromal Cell Extracellular Vesicles Ameliorates Aspergillus Hyphal Extract-Induced Allergic Airway Inflammation in Immunocompetent Mice.

    Science.gov (United States)

    Cruz, Fernanda F; Borg, Zachary D; Goodwin, Meagan; Sokocevic, Dino; Wagner, Darcy E; Coffey, Amy; Antunes, Mariana; Robinson, Kristen L; Mitsialis, S Alex; Kourembanas, Stella; Thane, Kristen; Hoffman, Andrew M; McKenna, David H; Rocco, Patricia R M; Weiss, Daniel J

    2015-11-01

    An increasing number of studies demonstrate that administration of either conditioned media (CM) or extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) derived from bone marrow and other sources are as effective as the MSCs themselves in mitigating inflammation and injury. The goal of the current study was to determine whether xenogeneic administration of CM or EVs from human bone marrow-derived MSCs would be effective in a model of mixed Th2/Th17, neutrophilic-mediated allergic airway inflammation, reflective of severe refractory asthma, induced by repeated mucosal exposure to Aspergillus hyphal extract (AHE) in immunocompetent C57Bl/6 mice. Systemic administration of both CM and EVs isolated from human and murine MSCs, but not human lung fibroblasts, at the onset of antigen challenge in previously sensitized mice significantly ameliorated the AHE-provoked increases in airway hyperreactivity (AHR), lung inflammation, and the antigen-specific CD4 T-cell Th2 and Th17 phenotype. Notably, both CM and EVs from human MSCs (hMSCs) were generally more potent than those from mouse MSCs (mMSCs) in most of the outcome measures. The weak cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride was found to inhibit release of both soluble mediators and EVs, fully negating effects of systemically administered hMSCs but only partly inhibited the ameliorating effects of mMSCs. These results demonstrate potent xenogeneic effects of CM and EVs from hMSCs in an immunocompetent mouse model of allergic airway inflammation and they also show differences in mechanisms of action of hMSCs versus mMSCs to mitigate AHR and lung inflammation in this model. There is a growing experience demonstrating benefit of mesenchymal stromal cell (MSC)-based cell therapies in preclinical models of asthma. In the current study, conditioned media (CM) and, in particular, the extracellular vesicle fraction obtained from the CM were as potent as the MSCs

  13. Adipose Tissue-Derived Stromal Cells Inhibit TGF-beta 1-Induced Differentiation of Human Dermal Fibroblasts and Keloid Scar-Derived Fibroblasts in a Paracrine Fashion

    NARCIS (Netherlands)

    Spiekman, Maroesjka; Przybyt, Ewa; Plantinga, Josee A.; Gibbs, Susan; van der Lei, Berend; Harmsen, Martin C.

    2014-01-01

    Background: Adipose tissue-derived stromal cells augment wound healing and skin regeneration. It is unknown whether and how they can also influence dermal scarring. The authors hypothesized that adipose tissue-derived stromal cells inhibit adverse differentiation of dermal fibroblasts induced by the

  14. Native thymic extracellular matrix improves in vivo thymic organoid T cell output, and drives in vitro thymic epithelial cell differentiation.

    Science.gov (United States)

    Hun, Michael; Barsanti, Marco; Wong, Kahlia; Ramshaw, John; Werkmeister, Jerome; Chidgey, Ann P

    2017-02-01

    Although the thymus is a primary lymphoid organ, its function is compromised by an age-induced loss of resident epithelial cells, which results in reduced naïve T cell output. This has important implications for immune recovery in aged and elderly patients following damage from cytoablative therapies. As thymic architecture plays a crucial role in naïve T cell development, a tissue specific scaffold that provides essential supporting matrix may assist in stem cell-based thymus regeneration to recreate complex organoids. Here we investigate thymus decellularization approaches that preserve major extracellular matrix components and support thymic epithelial cells for the generation of a functional thymic microenvironment with improved T cell output. We also established an in vitro, serum-free culture system that both maintains a progenitor thymic epithelial cell pool and drives their differentiation in the presence of decellularized thymic matrix. This approach enables further dissection of key cellular and niche components involved in thymic epithelial stem cell maintenance and T cell production. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

    Energy Technology Data Exchange (ETDEWEB)

    Tatrai, Peter, E-mail: peter.tatrai@biomembrane.hu [Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest (Hungary); Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Szepesi, Aron, E-mail: aron.szepesi@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Matula, Zsolt, E-mail: matula.zsolt@gmail.com [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Szigeti, Anna, E-mail: anna.szigeti@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Buchan, Gyoengyi, E-mail: buchan@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Madi, Andras, E-mail: madi@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Stem Cell, Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Uher, Ferenc, E-mail: uher@biomembrane.hu [Stem Cell Laboratory, Hungarian National Blood Transfusion Service, Dioszegi ut 64, H-1113 Budapest (Hungary); and others

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer We immortalized human adipose stromal cells (ASCs) with hTERT, Bmi-1, and SV40T. Black-Right-Pointing-Pointer hTERT-only ASCs are prone to transformation, while Bmi-only ASCs become senescent. Black-Right-Pointing-Pointer SV40T introduced along with hTERT abrogates proliferation control and multipotency. Black-Right-Pointing-Pointer hTERT combined with Bmi-1 yields stable phenotype up to 140 population doublings. -- Abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC{sup hTERT}, ASC{sup Bmi-1}, ASC{sup Bmi-1+hTERT} and ASC{sup SV40T+hTERT} were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC{sup Bmi-1} had limited replicative potential, while the rapidly proliferating ASC{sup SV40T+hTERT} acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC{sup hTERT} and ASC{sup hTERT+Bmi-1}, on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC{sup hTERT} also acquired aberrant karyotype and showed signs of transformation after long-term culture

  16. Syndecan-1 Acts as an Important Regulator of CXCL1 Expression and Cellular Interaction of Human Endometrial Stromal and Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Dunja Maria Baston-Buest

    2017-01-01

    Full Text Available Successful implantation of the embryo into the human receptive endometrium is substantial for the establishment of a healthy pregnancy. This study focusses on the role of Syndecan-1 at the embryo-maternal interface, the multitasking coreceptor influencing ligand concentration, release and receptor presentation, and cellular morphology. CXC motif ligand 1, being involved in chemotaxis and angiogenesis during implantation, is of special interest as a ligand of Syndecan-1. Human endometrial stromal cells with and without Syndecan-1 knock-down were decidualized and treated with specific inhibitors to evaluate signaling pathways regulating CXC ligand 1 expression. Western blot analyses of MAPK and Wnt members were performed, followed by analysis of spheroid interactions between human endometrial cells and extravillous trophoblast cells. By mimicking embryo contact using IL-1β, we showed less ERK and c-Jun activation by depletion of Syndecan-1 and less Frizzled 4 production as part of the canonical Wnt pathway. Additionally, more beta-catenin was phosphorylated and therefore degraded after depletion of Syndecan-1. Secretion of CXC motif ligand 1 depends on MEK-1 with respect to Syndecan-1. Regarding the interaction of endometrial and trophoblast cells, the spheroid center-to-center distances were smaller after depletion of Syndecan-1. Therefore, Syndecan-1 seems to affect signaling processes relevant to signaling and intercellular interaction at the trophoblast-decidual interface.

  17. Fibulin-5 localisation in human endometrial cancer shifts from epithelial to stromal with increasing tumour grade, and silencing promotes endometrial epithelial cancer cell proliferation.

    Science.gov (United States)

    Winship, Amy Louise; Rainczuk, Kate; Ton, Amanda; Dimitriadis, Eva

    2016-07-01

    Endometrial cancer is the most common invasive gynaecological malignancy. While endocrine, genetic and inflammatory factors are thought to contribute to its pathogenesis, its precise etiology and molecular regulators remain poorly understood. Fibulin-5 is an extracellular matrix (ECM) protein that inhibits cell growth and invasion in several cancer cell types and is downregulated in a number of types of human cancer. However, it is unknown whether fibulin-5 plays a role in endometrial tumourigenesis. In the current report, the expression and localisation of fibulin-5 in type I endometrioid human endometrial cancers of grades (G) 1-3 was investigated using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Fibulin-5 mRNA was found to be significantly reduced in whole tumour tissues from women across G1-3 compared with benign endometrium (Pendometrial epithelial cancer cells expressing fibulin-5 stimulated cell adhesion and proliferation in vitro . Fibulin-5 mRNA expression in Ishikawa cells was induced by transforming growth factor-β and fibulin-5 in turn activated extracellular signal-regulated kinases (ERK1/2), suggesting that it may act via the mitogen-activated protein kinase pathway. In summary, the present study identified fibulin-5 as a downregulated ECM gene in human endometrial cancer and observed a shift from epithelial to stromal protein localisation with increasing tumour grade in women. These data suggest that loss of fibulin-5 function may promote endometrial cancer progression by enhancing epithelial cell adhesion and proliferation.

  18. The Influence of IL-10 and TNFα on Chondrogenesis of Human Mesenchymal Stromal Cells in Three-Dimensional Cultures

    Directory of Open Access Journals (Sweden)

    Michal Jagielski

    2014-09-01

    Full Text Available Chondrogenic differentiated mesenchymal stromal cells (MSCs are a promising cell source for articular cartilage repair. This study was undertaken to determine the effectiveness of two three-dimensional (3D culture systems for chondrogenic MSC differentiation in comparison to primary chondrocytes and to assess the effect of Interleukin (IL-10 and Tumor Necrosis Factor (TNFα on chondrogenesis by MSCs in 3D high-density (H-D culture. MSCs were isolated from femur spongiosa, characterized using a set of typical markers and introduced in scaffold-free H-D cultures or non-woven polyglycolic acid (PGA scaffolds for chondrogenic differentiation. H-D cultures were stimulated with recombinant IL-10, TNFα, TNFα + IL-10 or remained untreated. Gene and protein expression of type II collagen, aggrecan, sox9 and TNFα were examined. MSCs expressed typical cell surface markers and revealed multipotency. Chondrogenic differentiated cells expressed cartilage-specific markers in both culture systems but to a lower extent when compared with articular chondrocytes. Chondrogenesis was more pronounced in PGA compared with H-D culture. IL-10 and/or TNFα did not impair the chondrogenic differentiation of MSCs. Moreover, in most of the investigated samples, despite not reaching significance level, IL-10 had a stimulatory effect on the type II collagen, aggrecan and TNFα expression when compared with the respective controls.

  19. Human mesenchymal stromal cells enhance the immunomodulatory function of CD8+CD28− regulatory T cells

    Science.gov (United States)

    Liu, Qiuli; Zheng, Haiqing; Chen, Xiaoyong; Peng, Yanwen; Huang, Weijun; Li, Xiaobo; Li, Gang; Xia, Wenjie; Sun, Qiquan; Xiang, Andy Peng

    2015-01-01

    One important aspect of mesenchymal stromal cells (MSCs)-mediated immunomodulation is the recruitment and induction of regulatory T (Treg) cells. However, we do not yet know whether MSCs have similar effects on the other subsets of Treg cells. Herein, we studied the effects of MSCs on CD8+CD28− Treg cells and found that the MSCs could not only increase the proportion of CD8+CD28− T cells, but also enhance CD8+CD28−T cells' ability of hampering naive CD4+ T-cell proliferation and activation, decreasing the production of IFN-γ by activated CD4+ T cells and inducing the apoptosis of activated CD4+ T cells. Mechanistically, the MSCs affected the functions of the CD8+CD28− T cells partially through moderate upregulating the expression of IL-10 and FasL. The MSCs had no distinct effect on the shift from CD8+CD28+ T cells to CD8+CD28− T cells, but did increase the proportion of CD8+CD28− T cells by reducing their rate of apoptosis. In summary, this study shows that MSCs can enhance the regulatory function of CD8+CD28− Treg cells, shedding new light on MSCs-mediated immune regulation. PMID:25482073

  20. Thymic Hyperplasia after Lung Transplantation Imitating Posttransplant Lymphoproliferative Disorder

    Directory of Open Access Journals (Sweden)

    Christina Maria Steger

    2011-01-01

    Full Text Available Thymic hyperplasia is usually associated with the treatment of malignant tumours and is sometimes linked with endocrine diseases. For the first time, we report a case of thymic hyperplasia in a patient 2 years after bilateral lung transplantation. Contrast-enhanced chest CT scan was highly suspicious for a posttransplant lymphoma or thymoma. Therefore, the patient received total thymectomy. Excised specimens were sent to the Department of Pathology. Unexpectedly, the histological examination revealed hyperplastic thymic tissue without evidence for a posttransplant lymphoproliferative disorder or malignancy.

  1. Murine neonatal recent thymic emigrants are phenotypically and functionally distinct from adult recent thymic emigrants

    OpenAIRE

    Opiela, Shannon J.; Koru-Sengul, Tulay; Adkins, Becky

    2009-01-01

    In contrast to adults, the murine neonatal CD4+ compartment contains a high frequency of recent thymic emigrants (RTEs). However, the functional capabilities of these cells in neonates are relatively unknown. Moreover, it has not been determined whether RTEs from neonates and adults are comparable. Here we have directly compared neonatal and adult CD4+ RTEs for the first time, using a transgenic mouse strain that allows for the identification and purification of RTEs. Our data demonstrate tha...

  2. Human platelet lysate is an alternative to fetal bovine serum for large-scale expansion of bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Gottipamula, Sanjay; Sharma, Archana; Krishnamurthy, Sagar; Majumdar, Anish Sen; Seetharam, Raviraja N

    2012-07-01

    Human platelet lysate (HPL) was evaluated as an alternative to fetal bovine serum (FBS) in large-scale culturing of bone marrow-derived mesenchymal stromal cells (BM-MSCs) for therapeutic applications. Dulbecco's modified Eagle medium (DMEM)of low glucose (LG) and Knock Out (KO) were used with human platelet lysate (HPL) as LG-HPL and KO-HPL, and with FBS as LG-FBS and KO-FBS to culture the BM-MSCs. HPL at 10 % (v/v) supported BM-MSCs growth and subsequent isolation efficiency generated >90 × 10(6) MSCs in LG-HPL. Population doublings (PDs) and population doubling times of LG-HPL and KO-HPL (PDT) were not significantly different but LG-HPL showed a significant clonogenic potential and HPL cultures had an average PDT of 36.5 ± 6.5 h and an average PDs of 5 ± 0.7/passage. BM-MSCs cultured with LG-HPL had significantly higher immunosuppression compared to LG-FBS, but KO-HPL and KO-FBS-grown cultures were not significantly different. HPL is therefore alternative to FBS for large-scale production of BM-MSCs for therapeutic applications.

  3. Blood Vessel Formation and Bone Regeneration Potential of the Stromal Vascular Fraction Seeded on a Calcium Phosphate Scaffold in the Human Maxillary Sinus Floor Elevation Model

    Directory of Open Access Journals (Sweden)

    Elisabet Farré-Guasch

    2018-01-01

    Full Text Available Bone substitutes are used as alternatives for autologous bone grafts in patients undergoing maxillary sinus floor elevation (MSFE for dental implant placement. However, bone substitutes lack osteoinductive and angiogenic potential. Addition of adipose stem cells (ASCs may stimulate osteogenesis and osteoinduction, as well as angiogenesis. We aimed to evaluate the vascularization in relation to bone formation potential of the ASC-containing stromal vascular fraction (SVF of adipose tissue, seeded on two types of calcium phosphate carriers, within the human MSFE model, in a phase I study. Autologous SVF was obtained from ten patients and seeded on β-tricalcium phosphate (n = 5 or biphasic calcium phosphate carriers (n = 5, and used for MSFE in a one-step surgical procedure. After six months, biopsies were obtained during dental implant placement, and the quantification of the number of blood vessels was performed using histomorphometric analysis and immunohistochemical stainings for blood vessel markers, i.e., CD34 and alpha-smooth muscle actin. Bone percentages seemed to correlate with blood vessel formation and were higher in study versus control biopsies in the cranial area, in particular in β-tricalcium phosphate-treated patients. This study shows the safety, feasibility, and efficiency of the use of ASCs in the human MSFE, and indicates a pro-angiogenic effect of SVF.

  4. Regulating Chondrogenesis of Human Mesenchymal Stromal Cells with a Retinoic Acid Receptor-Beta Inhibitor: Differential Sensitivity of Chondral Versus Osteochondral Development

    Directory of Open Access Journals (Sweden)

    Solvig Diederichs

    2014-05-01

    Full Text Available Aim: Main objective was to investigate whether the synthetic retinoic acid receptor (RAR-β antagonist LE135 is able to drive in vitro chondrogenesis of human mesenchymal stromal cells (MSCs or improve differentiation by suppressing hypertrophic chondrocyte development. Methods: Chondrogenesis of human bone marrow and adipose tissue-derived MSCs was induced in micromass pellet culture for six weeks. Effects of LE135 alone and in combinatorial treatment with TGF-β on deposition of cartilaginous matrix including collagen type II and glycosaminoglycans, on deposition of non-hyaline cartilage collagens type I and X, and on hypertrophy markers including alkaline phosphatase (ALP, indian hedghehog (IHH and matrix metalloproteinase (MMP-13 were assessed. Results: LE135 was no inducer of chondrogenesis and failed to stimulate deposition of collagen type II and glycosaminoglycans. Moreover, addition of LE135 to TGF-β-treated pellets inhibited cartilaginous matrix deposition and gene expression of COL2A1. In contrast, non-hyaline cartilage collagens were less sensitive to LE135 and hypertrophy markers remained unaffected. Conclusion: This demonstrates a differential sensitivity of chondral versus endochondral differentiation pathways to RARβ signaling; however, opposite to the desired direction. The relevance of trans-activating versus trans-repressing RAR signaling, including effects on activator protein (AP-1 is discussed and implications for overcoming current limits of hMSC chondrogenesis are considered.

  5. A 3D culture system enhances the ability of human bone marrow stromal cells to support the growth of limbal stem/progenitor cells.

    Science.gov (United States)

    González, Sheyla; Mei, Hua; Nakatsu, Martin N; Baclagon, Elfren R; Deng, Sophie X

    2016-03-01

    The standard method of cultivating limbal epithelial progenitor/stem cells (LSCs) on a monolayer of mouse 3T3 feeder cells possesses the risk of cross-contamination in clinical applications. Human feeder cells have been used to eliminate this risk; however, efficiency from xenobiotic-free cultures on a monolayer appears to be lower than in the standard method using 3T3 cells. We investigated whether bone marrow stromal cells (BMSCs), also known as bone marrow-derived mesenchymal stem cells, could serve as feeder cells for the expansion of LSCs in the 3-dimensional (3D) system. Primary single human LSCs on a monolayer of 3T3s served as the control. Very poor growth was observed when single LSCs were cultured on BMSCs. When LSC clusters were cultured on a BMSC monolayer (CC-BM), 3D culture system (3D CC-BM) and fibrin 3D system (fibrin 3D CC-BM), the 3D CC-BM method supported a greater LSC expansion. The 3D CC-BM system produced a 2.5-fold higher cell growth rate than the control (p0.05), whereas the proportion of K12(+) cells was lower (p3D culture. Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

  6. In situ normoxia enhances survival and proliferation rate of human adipose tissue-derived stromal cells without increasing the risk of tumourigenesis.

    Directory of Open Access Journals (Sweden)

    Jane Ru Choi

    Full Text Available Adipose tissue-derived stromal cells (ASCs natively reside in a relatively low-oxygen tension (i.e., hypoxic microenvironment in human body. Low oxygen tension (i.e., in situ normoxia, has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O2 concentration (21% O2 or in situ normoxia (2% O2. We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O2 concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O2 as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.

  7. Genetic profiling and surface proteome analysis of human atrial stromal cells and rat ventricular epicardium-derived cells reveals novel insights into their cardiogenic potential

    Directory of Open Access Journals (Sweden)

    Sebastian Temme

    2017-12-01

    Full Text Available Epicardium-derived cells (EPDC and atrial stromal cells (ASC display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73, CD90, CD44, CD105, cardiac markers (Gata4, Tbx5, troponin T and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM, cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases.

  8. Choice of xenogenic-free expansion media significantly influences the myogenic differentiation potential of human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Brun, Juliane; Abruzzese, Tanja; Rolauffs, Bernd; Aicher, Wilhelm K; Hart, Melanie L

    2016-03-01

    Mesenchymal stromal cells (MSCs) have great potential for use in cell-based therapies for restoration of structure and function of many tissue types including smooth muscle. We compared proliferation, immunophenotype, differentiation capability and gene expression of bone marrow-derived MSCs expanded in different media containing human serum, plasma and platelet lysate in combination with commonly used protocols for myogenic, osteogenic, chondrogenic and adipogenic differentiation. Moreover, we developed a xenogenic-free protocol for myogenic differentiation of MSCs. Expansion of MSCs in media complemented with serum, serum + platelet lysate or plasma + platelet lysate were multipotent because they differentiated toward four mesenchymal (myogenic, osteogenic, chondrogenic, adipogenic) lineages. Addition of platelet lysate to expansion media increased the proliferation of MSCs and their expression of CD146. Incubation of MSCs in medium containing human serum or plasma plus 5% human platelet lysate in combination with smooth muscle cell (SMC)-inducing growth factors TGFβ1, PDGF and ascorbic acid induced high expression of ACTA2, TAGLN, CNN1 and/or MYH11 contractile SMC markers. Osteogenic, adipogenic and chondrogenic differentiations served as controls. Our study provides novel data on the myogenic differentiation potential of human MSCs toward the SMC lineage using different xenogenic-free cell culture expansion media in combination with distinct differentiation medium compositions. We show that the choice of expansion medium significantly influences the differentiation potential of human MSCs toward the smooth muscle cell, as well as osteogenic, adipogenic and chondrogenic lineages. These results can aid in designing studies using MSCs for tissue-specific therapeutic applications. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Proteomic characterisation reveals active Wnt-signalling by human multipotent stromal cells as a key regulator of beta cell survival and proliferation.

    Science.gov (United States)

    Kuljanin, Miljan; Bell, Gillian I; Sherman, Stephen E; Lajoie, Gilles A; Hess, David A

    2017-10-01

    Novel strategies to stimulate the expansion of beta cell mass in situ are warranted for diabetes therapy. The aim of this study was to elucidate the secretome of human bone marrow (BM)-derived multipotent stromal cells (MSCs) with documented islet regenerative paracrine function. We hypothesised that regenerative MSCs will secrete a unique combination of protein factors that augment islet regeneration. Human BM-derived MSCs were examined for glucose-lowering capacity after transplantation into streptozotocin-treated NOD/severe combined immunodeficiency (SCID) mice and segregated into samples with regenerative (MSC(R)) vs nonregenerative (MSC(NR)) capacity. Secreted proteins associated with islet regenerative function were identified using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics. To functionally validate the importance of active Wnt signalling, we stimulated the Wnt-signalling pathway in MSC(NR) samples during ex vivo expansion using glycogen synthase kinase 3 (GSK3) inhibition (CHIR99201), and the conditioned culture media (CM) generated was tested for the capacity to support cultured human islet cell survival and proliferation in vitro. MSC(R) showed increased secretion of proteins associated with cell growth, matrix remodelling, immunosuppressive and proangiogenic properties. In contrast, MSC(NR) uniquely secreted proteins known to promote inflammation and negatively regulate angiogenesis. Most notably, MSC(R) maintained Wnt signalling via Wnt5A/B (~2.5-fold increase) autocrine activity during ex vivo culture, while MSC(NR) repressed Wnt signalling via Dickkopf-related protein (DKK)1 (~2.5-fold increase) and DKK3 secretion. Inhibition of GSK3 activity in MSC(NR) samples increased the accumulation of nuclear β-catenin and generated CM that augmented beta cell survival (13% increases) and proliferation when exposed to cultured human islets. Maintenance of active Wnt signalling within human MSCs promotes the

  10. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Kevin Dzobo

    2016-08-01

    Full Text Available Mesenchymal stromal/stem cells (MSCs represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4, SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

  11. Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization.

    Science.gov (United States)

    Camden, Alison J; Szwarc, Maria M; Chadchan, Sangappa B; DeMayo, Francesco J; O'Malley, Bert W; Lydon, John P; Kommagani, Ramakrishna

    2017-09-01

    Is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) required for progesterone-driven endometrial stromal cell decidualization? GREB1 is a novel progesterone-responsive gene required for progesterone-driven human endometrial stromal cell (HESC) decidualization. Successful establishment of pregnancy requires HESCs to transform from fibroblastic to epithelioid cells in a process called decidualization. This process depends on the hormone progesterone, but the molecular mechanisms by which it occurs have not been determined. Primary and transformed HESCs in which GREB1 expression was knocked down were decidualized in culture for up to 6 days. Wild-type and progesterone receptor (PR) knockout mice were treated with progesterone, and their uteri were assessed for levels of GREB1 expression. Analysis of previous data included data mining of expression profile data sets and in silico transcription factor-binding analysis. Endometrial biopsies obtained from healthy women of reproductive age during the proliferative phase (Days 8-12) of their menstrual cycle were used for isolating HESCs. Experiments were carried out with early passage (no more than four passages) HESCs isolated from at least three subjects. Transcript levels of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were detected by quantitative RT-PCR as readouts for HESC decidualization. Cells were also imaged by phase-contrast microscopy. To assess the requirement for GREB1, PR and SRC-2, cells were transfected with specifically targeted small interfering RNAs. Results are shown as mean and SE from three replicates of one representative patient-derived primary endometrial cell line. Experiments were also conducted with transformed HESCs. Progesterone treatment of mice and transformed HESCs led to an ~5-fold (5.6 ± 0.81, P endometrial function and dysfunction should be assessed by using knock-out mouse models. Identification and functional analysis of

  12. Expression and function of the autoimmune regulator (Aire) gene in non-thymic tissue

    Science.gov (United States)

    Eldershaw, S A; Sansom, D M; Narendran, P

    2011-01-01

    Educational immune tolerance to self-antigens is induced primarily in the thymus where tissue-restricted antigens (TRAs) are presented to T lymphocytes by cells of the thymic stroma – a process known as central tolerance. The expression of these TRAs is controlled in part by a transcription factor encoded by the autoimmune regulatory (Aire) gene. Patients with a mutation of this gene develop a condition known as autoimmune–polyendocrinopathy–candidiasis–ectodermal–dystrophy (APECED), characterized by autoimmune destruction of endocrine organs, fungal infection and dental abnormalities. There is now evidence for TRA expression and for mechanisms of functional tolerance outside the thymus. This has led to a number of studies examining Aire expression and function at these extra-thymic sites. These investigations have been conducted across different animal models using different techniques and have often shown discrepant results. Here we review the studies of extra thymic Aire and discuss the evidence for its expression and function in both human and murine systems. PMID:21303359

  13. Expression and function of the autoimmune regulator (Aire) gene in non-thymic tissue.

    Science.gov (United States)

    Eldershaw, S A; Sansom, D M; Narendran, P

    2011-03-01

    Educational immune tolerance to self-antigens is induced primarily in the thymus where tissue-restricted antigens (TRAs) are presented to T lymphocytes by cells of the thymic stroma - a process known as central tolerance. The expression of these TRAs is controlled in part by a transcription factor encoded by the autoimmune regulatory (Aire) gene. Patients with a mutation of this gene develop a condition known as autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED), characterized by autoimmune destruction of endocrine organs, fungal infection and dental abnormalities. There is now evidence for TRA expression and for mechanisms of functional tolerance outside the thymus. This has led to a number of studies examining Aire expression and function at these extra-thymic sites. These investigations have been conducted across different animal models using different techniques and have often shown discrepant results. Here we review the studies of extra thymic Aire and discuss the evidence for its expression and function in both human and murine systems. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

  14. Thy28 protects against anti-CD3-mediated thymic cell death in vivo.

    Science.gov (United States)

    Toyota, H; Sudo, K; Kojima, K; Yanase, N; Nagao, T; Takahashi, R H; Iobe, H; Kuwabara, T; Kakiuchi, T; Mizuguchi, J

    2015-04-01

    Apoptotic cell death plays a pivotal role in the development and/or maintenance of several tissues including thymus. Deregulated thymic cell death is associated with autoimmune diseases including experimental autoimmune encephalomyelitis (EAE), a prototype murine model for analysis of human multiple sclerosis. Because Thy28 expression is modulated during thymocyte development, we tested whether Thy28 affects induction of EAE as effectively as antigen-induced thymocyte deletion using Thy28 transgenic (TG) mice. Thy28 TG mice showed partial resistance to anti-CD3 monoclonal antibody (mAb)-induced thymic cell death in vivo, as assessed by annexin V-expression and loss of mitochondrial membrane potential. The resistance to anti-CD3 mAb-induced cell death in Thy28 TG mice appeared to correlate with a decreased c-Jun N-terminal kinase phosphorylation and reduced down-regulation of Bcl-xL. Moreover, thymic hyperplasia was detected in Thy28 TG mice, although thymocyte development was unaltered. Development of peripheral lymphoid tissues including spleen and lymph nodes was also unaltered. Thy28 TG spleen T cells showed an increased production of IFN-γ, but not IL-17, in response to both anti-CD3 and anti-CD28 mAbs. Finally, Thy28 TG mice displayed accelerated induction of EAE as assessed by disease incidence, clinical score, and pathology following immunization with myelin oligodendrocyte glycoprotein compared with control WT mice. These findings suggest that modulation of Thy28 expression plays a crucial role in the determination of thymic cell fate, which may contribute to the development of EAE through proinflammatory cytokine production.

  15. Thymic masses: A radiological review | Mittal | SA Journal of Radiology

    African Journals Online (AJOL)

    Abstract. Various thymic masses and their normal variations have different pathological and management consequences. Radiologists and clinicians should be aware of these entities so that appropriate and timely treatment can be given.

  16. Antiretroviral therapy increases thymic output in children with HIV

    DEFF Research Database (Denmark)

    Schou Sandgaard, Katrine; Lewis, Joanna; Adams, Stuart

    2014-01-01

    OBJECTIVE: Disease progression and response to antiretroviral therapy (ART) in HIV-infected children is different to that of adults. Immune reconstitution in adults is mainly from memory T cells, whereas in children it occurs predominantly from the naive T-cell pool. It is unclear however what...... and cannot in themselves be used as quantitative estimates of thymic output. DESIGN: To compare thymic output in HIV-infected children on ART, HIV-infected children not on ART and uninfected children of different ages. METHOD: Combined T-cell receptor excision circle (TREC) and proliferation data are used...... with a recently described mathematical model to give explicit measures of thymic output. RESULTS: We found that age-adjusted thymic output is reduced in untreated children with HIV, which increases significantly with length of time on ART. CONCLUSION: Our results suggest that a highly active thymus in early...

  17. Differential effect of platelet-rich plasma and fetal calf serum on bone marrow-derived human mesenchymal stromal cells expanded in vitro.

    Science.gov (United States)

    Goedecke, Anja; Wobus, Manja; Krech, Mathias; Münch, Nadine; Richter, Katja; Hölig, Kristina; Bornhauser, Martin

    2011-08-01

    Mesenchymal stromal cells (MSCs) derived from various sources have great potential for use in cell-based therapies. Since the proportion of primary MSCs contained in bone marrow or adipose tissue is low, plastic adherence and in vitro expansion are necessary to expand MSCs prior to clinical application. Human platelet-rich plasma has been introduced as an alternative serum source but functional differences have so far not been described. Here we cultured MSCs derived from human bone marrow in medium supplemented with either 10% fetal calf serum (FCS) or 5% and 10% platelet-rich plasma (PRP) until the first or second passage. Parameters under investigation were cell yield, clonogenicity, phenotype as well as migratory and differentiation potential. In addition, the secretion of SDF-1α and the induced migration of CD34(+) haematopoietic stem cells (HSCs) were investigated with regard to the different serum source. The use of PRP resulted in a significantly higher expansion rate and yield at passages 0 and 1. In addition, the level of secreted SDF-1α was significantly increased in the supernatant of MSCs cultured with FCS instead of human PRP. Consistent with this, the migration capacity of MSCs cultured with 10% FCS as well as their capability to induce the migration of CD34(+) haematopoietic progenitors in a transwell assay was higher. Our results demonstrate that human PRP can be seen as an alternative serum source to FCS for MSC cultivation. However, the requirements of the specific clinical application must be carefully considered before the respective serum source is selected. Copyright © 2010 John Wiley & Sons, Ltd.

  18. Manufacturing and use of human placenta-derived mesenchymal stromal cells for phase I clinical trials: Establishment and evaluation of a protocol

    Directory of Open Access Journals (Sweden)

    Ilić Nina

    2014-01-01

    Full Text Available Background/Aim. Mesenchymal stromal cells (MSCs have been utilised in many clinical trials as an experimental treatment in numerous clinical settings. Bone marrow remains the traditional source tissue for MSCs but is relatively hard to access in large volumes. Alternatively, MSCs may be derived from other tissues including the placenta and adipose tissue. In an initial study no obvious differences in parameters such as cell surface phenotype, chemokine receptor display, mesodermal differentiation capacity or immunosuppressive ability, were detected when we compared human marrow derived- MSCs to human placenta-derived MSCs. The aim of this study was to establish and evaluate a protocol and related processes for preparation placenta-derived MSCs for early phase clinical trials. Methods. A full-term placenta was taken after delivery of the baby as a source of MSCs. Isolation, seeding, incubation, cryopreservation of human placentaderived MSCs and used production release criteria were in accordance with the complex regulatory requirements applicable to Code of Good Manufacturing Practice manufacturing of ex vivo expanded cells. Results. We established and evaluated instructions for MSCs preparation protocol and gave an overview of the three clinical areas application. In the first trial, MSCs were co-transplanted iv to patient receiving an allogeneic cord blood transplant as therapy for treatmentrefractory acute myeloid leukemia. In the second trial, MSCs were administered iv in the treatment of idiopathic pulmonary fibrosis and without serious adverse effects. In the third trial, MSCs were injected directly into the site of tendon damage using ultrasound guidance in the treatment of chronic refractory tendinopathy. Conclusion. Clinical trials using both allogeneic and autologous cells demonstrated MSCs to be safe. A described protocol for human placenta-derived MSCs is appropriate for use in a clinical setting, relatively inexpensive and can be

  19. Platelet lysate suppresses the expression of lipocalin-type prostaglandin D2 synthase that positively controls adipogenic differentiation of human mesenchymal stromal cells.

    Science.gov (United States)

    Lange, Claudia; Brunswig-Spickenheier, Bärbel; Eissing, Leah; Scheja, Ludger

    2012-11-01

    Mesenchymal stromal cells (MSCs) have been shown to display a considerable therapeutic potential in cellular therapies. However, harmful adipogenic maldifferentiation of transplanted MSCs may seriously threaten the success of this therapeutic approach. We have previously demonstrated that using platelet lysate (PL) instead of widely used fetal calf serum (FCS) diminished lipid accumulation in adipogenically stimulated human MSCs and identified, among others, lipocalin-type prostaglandin D2 synthase (L-PGDS) as a gene suppressed in PL-supplemented MSCs. Here, we investigated the role of PL and putatively pro-adipogenic L-PGDS in human MSC adipogenesis. Next to strongly reduced levels of L-PGDS we show that PL-supplemented MSCs display markedly decreased expression of adipogenic master regulators and differentiation markers, both before and after induction of adipocyte differentiation. The low adipogenic differentiation capability of PL-supplemented MSCs could be partially restored by exogenous addition of L-PGDS protein. Conversely, siRNA-mediated downregulation of L-PGDS in FCS-supplemented MSCs profoundly reduced adipocyte differentiation. In contrast, inhibiting endogenous prostaglandin synthesis by aspirin did not reduce differentiation, suggesting that a mechanism such as lipid shuttling but not the prostaglandin D2 synthase activity of L-PGDS is critical for adipogenesis. Our data demonstrate that L-PGDS is a novel pro-adipogenic factor in human MSCs which might be of relevance in adipocyte metabolism and disease. L-PGDS gene expression is a potential quality marker for human MSCs, as it might predict unwanted adipogenic differentiation after MSC transplantation. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma.

    Science.gov (United States)

    de Castro, Ligia Lins; Xisto, Debora Gonçalves; Kitoko, Jamil Zola; Cruz, Fernanda Ferreira; Olsen, Priscilla Christina; Redondo, Patricia Albuquerque Garcia; Ferreira, Tatiana Paula Teixeira; Weiss, Daniel Jay; Martins, Marco Aurélio; Morales, Marcelo Marcos; Rocco, Patricia Rieken Macedo

    2017-06-24

    Asthma is a chronic inflammatory disease that can be difficult to treat due to its complex pathophysiology. Most current drugs focus on controlling the inflammatory process, but are unable to revert the changes of tissue remodeling. Human mesenchymal stromal cells (MSCs) are effective at reducing inflammation and tissue remodeling; nevertheless, no study has evaluated the therapeutic effects of extracellular vesicles (EVs) obtained from human adipose tissue-derived MSCs (AD-MSC) on established airway remodeling in experimental allergic asthma. C57BL/6 female mice were sensitized and challenged with ovalbumin (OVA). Control (CTRL) animals received saline solution using the same protocol. One day after the last challenge, each group received saline, 10 5 human AD-MSCs, or EVs (released by 10 5  AD-MSCs). Seven days after treatment, animals were anesthetized for lung function assessment and subsequently euthanized. Bronchoalveolar lavage fluid (BALF), lungs, thymus, and mediastinal lymph nodes were harvested for analysis of inflammation. Collagen fiber content of airways and lung parenchyma were also evaluated. In OVA animals, AD-MSCs and EVs acted differently on static lung elastance and on BALF regulatory T cells, CD3 + CD4 + T cells, and pro-inflammatory mediators (interleukin [IL]-4, IL-5, IL-13, and eotaxin), but similarly reduced eosinophils in lung tissue, collagen fiber content in airways and lung parenchyma, levels of transforming growth factor-β in lung tissue, and CD3 + CD4 + T cell counts in the thymus. No significant changes were observed in total cell count or percentage of CD3 + CD4 + T cells in the mediastinal lymph nodes. In this immunocompetent mouse model of allergic asthma, human AD-MSCs and EVs effectively reduced eosinophil counts in lung tissue and BALF and modulated airway remodeling, but their effects on T cells differed in lung and thymus. EVs may hold promise for asthma; however, further studies are required to elucidate the different

  1. Chondrogenesis of human bone marrow mesenchymal stromal cells in highly porous alginate-foams supplemented with chondroitin sulfate

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Zhao [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Nooeaid, Patcharakamon [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Kohl, Benjamin [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Roether, Judith A.; Schubert, Dirk W. [Institute of Polymer Materials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Meier, Carola [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Boccaccini, Aldo R. [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Godkin, Owen; Ertel, Wolfgang; Arens, Stephan [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Schulze-Tanzil, Gundula, E-mail: gundula.schulze@pmu.ac.at [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Institute of Anatomy, Paracelsus Medical University, Nuremberg (Germany)

    2015-05-01

    To overcome the limited intrinsic cartilage repair, autologous chondrocyte or bone-marrow-derived mesenchymal stromal cell (BM-MSC) was implanted into cartilage defects. For this purpose suitable biocompatible scaffolds are needed to provide cell retention, chondrogenesis and initial mechanical stability. The present study should indicate whether a recently developed highly porous alginate (Alg) foam scaffold supplemented with chondroitin sulfate (CS) allows the attachment, survival and chondrogenesis of BM-MSCs and articular chondrocytes. The foams were prepared using a freeze-drying method; some of them were supplemented with CS and subsequently characterized for porosity, biodegradation and mechanical profile. BM-MSCs were cultured for 1–2 weeks on the scaffold either under chondrogenic or maintenance conditions. Cell vitality assays, histology, glycosaminoglycan (sGAG) assay, and type II and I collagen immunolabelings were performed to monitor cell growth and extracellular matrix (ECM) synthesis in the scaffolds. Scaffolds had a high porosity ~ 93–95% with a mean pore sizes of 237 ± 48 μm (Alg) and 197 ± 61 μm (Alg/CS). Incorporation of CS increased mechanical strength of the foams providing gradually CS release over 7 days. Most of the cells survived in the scaffolds. BM-MSCs and articular chondrocytes formed rounded clusters within the scaffold pores. The BM-MSCs, irrespective of whether cultured under non/chondrogenic conditions and chondrocytes produced an ECM containing sGAGs, and types II and I collagen. Total collagen and sGAG contents were higher in differentiated BM-MSC cultures supplemented with CS than in CS-free foams after 14 days. The cell cluster formation induced by the scaffolds might stimulate chondrogenesis via initial intense cell–cell contacts. - Highlights: • Alginate foam scaffolds revealed a high porosity and mean pore size of 197–237 μm. • Chondroitin sulfate was released over 14 days by the scaffolds. • Chondrocytes

  2. mRNA-binding protein TIA-1 reduces cytokine expression in human endometrial stromal cells and is down-regulated in ectopic endometrium.

    Science.gov (United States)

    Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D; Kristiansson, Helena; Duke, Cindy M P; Choe, Gina; Flannery, Clare; Kallen, Caleb B; Seli, Emre

    2014-12-01

    Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3'-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Endometrial

  3. Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

    Directory of Open Access Journals (Sweden)

    Chengbo Tan

    2016-01-01

    Full Text Available Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs. Platelet concentrates (PC were harvested from healthy volunteers and made into single donor-derived PL (sPL. The PL mixtures (mPL were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA. The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS. No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

  4. Neuroprotective effects of human umbilical cord-derived mesenchymal stromal cells combined with nimodipine against radiation-induced brain injury through inhibition of apoptosis.

    Science.gov (United States)

    Wang, Gui-Hua; Liu, Yang; Wu, Xiao-Bing; Lu, Ying; Liu, Jin; Qin, Ya-Ru; Li, Tong; Duan, Hai-Feng

    2016-01-01

    Mesenchymal stromal cells (MSCs) possess the ability to repair brain injuries. Additionally, nimodipine is a neuroprotective agent that increases cerebral blood flow and may help with the homing of MSCs to the injury site. Here we investigate the effectiveness of a combined human umbilical cord-derived MSCs and nimodipine therapy in radiation-induced brain injury (RIBI). Female mice received whole brain irradiation (WBI) and were treated with saline, nimodipine, hUC-MSCs, or hUC-MSCs combined with nimodipine. Body weight was measured weekly. An open field test for locomotor activity and a step-down avoidance test for learning and memory function were conducted at week 4 and week 12 post-WBI. The histological damage was evaluated by hematoxylin and eosin staining and glial fibrillary acidic protein immunohistochemistry. Quantitative polymerase chain reaction and Western blotting were used to detect apoptosis-related mediators (p53, Bax and Bcl-2). In mice receiving the hUC-MSCs or the combined treatment, their body weight recovered, their locomotor and cognitive ability improved, and the percentage of necrotic neurons and astrocytes was reduced. The combined therapy was significantly (P nimodipine therapy is due to apoptosis inhibition and enhancing homing of MSCs to the injured brain. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  5. Flow cytometric cell sorting and in vitro pre-osteoinduction are not requirements for in vivo bone formation by human adipose-derived stromal cells.

    Science.gov (United States)

    Liu, Yunsong; Zhao, Yan; Zhang, Xiao; Chen, Tong; Zhao, Xianghui; Ma, Gui-e; Zhou, Yongsheng

    2013-01-01

    Human adipose-derived stromal cells (hASCs) are a promising cell source for bone tissue engineering. However, before the clinical application of hASCs for the treatment of bone defects, key questions require answers, including whether pre-osteoinduction (OI) and flow cytometric cell purification are indispensible steps for in vivo bone formation by hASCs. In this study, hASCs were purified by flow cytometric cell sorting (FCCS). The osteogenic capabilities of hASCs and purified hASCs with or without pre-osteoinduction were examined through in vitro and in vivo experiments. We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs. However, 8 weeks after in vivo implantation, there were no significant differences between hASCs and hASCs that had undergone OI (hASCs+OI) or between purified hASCs and purified hASCs+OI (P>0.05). Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo. These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

  6. Flow cytometric cell sorting and in vitro pre-osteoinduction are not requirements for in vivo bone formation by human adipose-derived stromal cells.

    Directory of Open Access Journals (Sweden)

    Yunsong Liu

    Full Text Available Human adipose-derived stromal cells (hASCs are a promising cell source for bone tissue engineering. However, before the clinical application of hASCs for the treatment of bone defects, key questions require answers, including whether pre-osteoinduction (OI and flow cytometric cell purification are indispensible steps for in vivo bone formation by hASCs. In this study, hASCs were purified by flow cytometric cell sorting (FCCS. The osteogenic capabilities of hASCs and purified hASCs with or without pre-osteoinduction were examined through in vitro and in vivo experiments. We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs. However, 8 weeks after in vivo implantation, there were no significant differences between hASCs and hASCs that had undergone OI (hASCs+OI or between purified hASCs and purified hASCs+OI (P>0.05. Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo. These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

  7. Effects of Intermittent Administration of Parathyroid Hormone (1-34 on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

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    Xiaoxiao Wang

    2016-01-01

    Full Text Available Periodontitis is the most common cause of tooth loss and bone destruction in adults worldwide. Human periodontal ligament stem cells (hPDLSCs may represent promising new therapeutic biomaterials for tissue engineering applications. Stromal precursor antigen-1 (STRO-1 has been shown to have roles in adherence, proliferation, and multipotency. Parathyroid hormone (PTH has been shown to enhance proliferation in osteoblasts. Therefore, in this study, we aimed to compare the functions of STRO-1(+ and STRO-1(− hPDLSCs and to investigate the effects of PTH on the osteogenic capacity of STRO-1(+ hPDLSCs in order to evaluate their potential applications in the treatment of periodontitis. Our data showed that STRO-1(+ hPDLSCs expressed higher levels of the PTH-1 receptor (PTH1R than STRO-1(− hPDLSCs. In addition, intermittent PTH treatment enhanced the expression of PTH1R and osteogenesis-related genes in STRO-1(+ hPDLSCs. PTH-treated cells also exhibited increased alkaline phosphatase activity and mineralization ability. Therefore, STRO-1(+ hPDLSCs represented a more promising cell resource for biomaterials and tissue engineering applications. Intermittent PTH treatment improved the capacity for STRO-1(+ hPDLSCs to repair damaged tissue and ameliorate the symptoms of periodontitis.

  8. Osteogenic Differentiation of Human Dental Pulp Stromal Cells on 45S5 Bioglass® Based Scaffolds In Vitro and In Vivo

    Science.gov (United States)

    El-Gendy, Reem; Newby, Phillipa J.; Boccaccini, Aldo R.; Kirkham, Jennifer

    2013-01-01

    The increasing clinical demand for bone substitutes has driven significant progress in cell-based therapies for bone tissue engineering. The underpinning goals for success are to identify the most appropriate cell source and to provide three-dimensional (3D) scaffolds that support cell growth and enhance osteogenic potential. In this study, human dental pulp stromal cells (HDPSCs) were cultured under basal or osteogenic conditions either in monolayers or on 3D Bioglass® scaffolds in vitro for 2 or 4 weeks. Cell–scaffold constructs were also implanted intraperitoneally in nude mice for 8 weeks. Osteogenic potential was assessed using quantitative real-time polymerase chain reaction and histological/immunohistochemical assays. In monolayer culture, osteoinductive conditions enhanced HDPSC expression of osteogenic gene markers (COL1A1, RUNX2, OC, and/or OCN) compared with basal conditions while culture of HDPSCs on 3D scaffolds promoted osteogenic gene expression compared with monolayer culture under both basal and osteogenic conditions. These results were confirmed using histological and immunohistochemical analyses. In vivo implantation of the HDPSC 3D Bioglass constructs showed evidence of sporadic woven bone-like spicules and calcified tissue. In conclusion, this study has demonstrated the potential of using a combination of HDPSCs with 3D 45S5 Bioglass scaffolds to promote bone-like tissue formation in vitro and in vivo, offering a promising approach for clinical bone repair and regeneration. PMID:23046092

  9. Macrophage interactions with polylactic acid and chitosan scaffolds lead to improved recruitment of human mesenchymal stem/stromal cells: a comprehensive study with different immune cells

    Science.gov (United States)

    Quelhas, Pedro

    2016-01-01

    Despite the importance of immune cell–biomaterial interactions for the regenerative outcome, few studies have investigated how distinct three-dimensional biomaterials modulate the immune cell-mediated mesenchymal stem/stromal cells (MSC) recruitment and function. Thus, this work compares the response of varied primary human immune cell populations triggered by different model scaffolds and describes its functional consequence on recruitment and motility of bone marrow MSC. It was found that polylactic acid (PLA) and chitosan scaffolds lead to an increase in the metabolic activity of macrophages but not of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells or monocytes. PBMC and NK cells increase their cell number in PLA scaffolds and express a secretion profile that does not promote MSC recruitment. Importantly, chitosan increases IL-8, MIP-1, MCP-1 and RANTES secretion by macrophages while PLA stimulates IL-6, IL-8 and MCP-1 production, all chemokines that can lead to MSC recruitment. This secretion profile of macrophages in contact with biomaterials correlates with the highest MSC invasion. Furthermore, macrophages enhance stem cell motility within chitosan scaffolds by 44% but not in PLA scaffolds. Thus, macrophages are the cells that in contact with engineered biomaterials become activated to secrete bioactive molecules that stimulate MSC recruitment. PMID:27628173

  10. Intra-articular injection of human meniscus stem/progenitor cells promotes meniscus regeneration and ameliorates osteoarthritis through stromal cell-derived factor-1/CXCR4-mediated homing.

    Science.gov (United States)

    Shen, Weiliang; Chen, Jialin; Zhu, Ting; Chen, Longkun; Zhang, Wei; Fang, Zhi; Heng, Boon Chin; Yin, Zi; Chen, Xiao; Ji, Junfeng; Chen, Weishan; Ouyang, Hong-Wei

    2014-03-01

    Meniscus injury is frequently encountered in clinical practice. Current surgical therapy involving partial or complete meniscectomy relieves pain in the short-term but often leads to osteoarthritis (OA) in the long-term. In this study, we report a new strategy of articular cartilage protection by intra-articular injection of novel human meniscus stem/progenitor cells (hMeSPCs). We found that hMeSPCs displayed both mesenchymal stem cell characteristics and high expression levels of collagen II. In the rat meniscus injury model, hMeSPC transplantation not only led to more neo-tissue formation and better-defined shape but also resulted in more rounded cells and matured extracellular matrix. Stromal cell-derived factor-1 (SDF-1) enhanced the migration of hMeSPCs, whereas AMD3100 abolished the chemotactic effects of SDF-1 on hMeSPCs, both in vitro and in vivo. In an experimental OA model, transplantation of hMeSPCs effectively protected articular cartilage, as evidenced by reduced expression of OA markers such as collagen I, collagen X, and hypoxia-inducible factor 2α but increased expression of collagen II. Our study demonstrated for the first time that intra-articular injection of hMeSPCs enhanced meniscus regeneration through the SDF-1/CXCR4 axis. Our study highlights a new strategy of intra-articular injection of hMeSPCs for meniscus regeneration.

  11. The Human Mesenchymal Stromal Cell-Derived Osteocyte Capacity to Modulate Dendritic Cell Functions Is Strictly Dependent on the Culture System.

    Science.gov (United States)

    Trabanelli, Sara; La Manna, Federico; Romano, Marco; Salvestrini, Valentina; Cavo, Michele; Ciciarello, Marilena; Lemoli, Roberto M; Curti, Antonio

    2015-01-01

    In vitro differentiation of mesenchymal stromal cells (MSC) into osteocytes (human differentiated osteogenic cells, hDOC) before implantation has been proposed to optimize bone regeneration. However, a deep characterization of the immunological properties of DOC, including their effect on dendritic cell (DC) function, is not available. DOC can be used either as cellular suspension (detached, Det-DOC) or as adherent cells implanted on scaffolds (adherent, Adh-DOC). By mimicking in vitro these two different routes of administration, we show that both Det-DOC and Adh-DOC can modulate DC functions. Specifically, the weak downregulation of CD80 and CD86 caused by Det-DOC on DC surface results in a weak modulation of DC functions, which indeed retain a high capacity to induce T-cell proliferation and to generate CD4(+)CD25(+)Foxp3(+) T cells. Moreover, Det-DOC enhance the DC capacity to differentiate CD4(+)CD161(+)CD196(+) Th17-cells by upregulating IL-6 secretion. Conversely, Adh-DOC strongly suppress DC functions by a profound downregulation of CD80 and CD86 on DC as well as by the inhibition of TGF-β production. In conclusion, we demonstrate that different types of DOC cell preparation may have a different impact on the modulation of the host immune system. This finding may have relevant implications for the design of cell-based tissue-engineering strategies.

  12. Human umbilical cord perivascular cells exhibited enhanced migration capacity towards hepatocellular carcinoma in comparison with bone marrow mesenchymal stromal cells: a role for autocrine motility factor receptor.

    Science.gov (United States)

    Bayo, Juan; Fiore, Esteban; Aquino, Jorge B; Malvicini, Mariana; Rizzo, Manglio; Peixoto, Estanislao; Alaniz, Laura; Piccioni, Flavia; Bolontrade, Marcela; Podhajcer, Osvaldo; Garcia, Mariana G; Mazzolini, Guillermo

    2014-01-01

    Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Unfortunately, the incidence and mortality associated with HCC are increasing. Therefore, new therapeutic strategies are urgently needed and the use of mesenchymal stromal cells (MSCs) as carrier of therapeutic genes is emerging as a promising option. Different sources of MSCs are being studied for cell therapy and bone marrow-derived cells are the most extensively explored; however, birth associated-tissues represent a very promising source. The aim of this work was to compare the in vitro and in vivo migration capacity between bone marrow MSCs (BM-MSCs) and human umbilical cord perivascular cells (HUCPVCs) towards HCC. We observed that HUCPVCs presented higher in vitro and in vivo migration towards factors released by HCC. The expression of autocrine motility factor (AMF) receptor, genes related with the availability of the receptor on the cell surface (caveolin-1 and -2) and metalloproteinase 3, induced by the receptor activation and important for cell migration, was increased in HUCPVCs. The chemotactic response towards recombinant AMF was increased in HUCPVCs compared to BM-MSCs, and its inhibition in the conditioned medium from HCC induced higher decrease in HUCPVC migration than in BM-MSC. Our results indicate that HUCPVCs could be a useful cellular source to deliver therapeutic genes to HCC.

  13. Potential Biomedical Application of Enzymatically Treated Alginate/Chitosan Hydrosols in Sponges—Biocompatible Scaffolds Inducing Chondrogenic Differentiation of Human Adipose Derived Multipotent Stromal Cells

    Directory of Open Access Journals (Sweden)

    Anna Zimoch-Korzycka

    2016-08-01

    Full Text Available Current regenerative strategies used for cartilage repair rely on biomaterial functionality as a scaffold for cells that may have potential in chondrogenic differentiation. The purpose of the research was to investigate the biocompatibility of enzymatically treated alginate/chitosan hydrosol sponges and their suitability to support chondrogenic differentiation of human adipose derived multipotent stromal cells (hASCs. The alginate/chitosan and enzyme/alginate/chitosan sponges were formed from hydrosols with various proportions and were used as a biomaterial in this study. Sponges were tested for porosity and wettability. The porosity of each sponge was higher than 80%. An equal dose of alginate and chitosan in the composition of sponges improved their swelling ability. It was found that equal concentrations of alginate and chitosan in hydrosols sponges assure high biocompatibility properties that may be further improved by enzymatic treatment. Importantly, the high biocompatibility of these biomaterials turned out to be crucial in the context of hydrosols’ pro-chondrogenic function. After exposure to the chondrogenic conditions, the hASCs in N/A/C and L/A/C sponges formed well developed nodules and revealed increased expression of collagen type II, aggrecan and decreased expression of collagen type I. Moreover, in these cultures, the reactive oxygen species level was lowered while superoxide dismutase activity increased. Based on the obtained results, we conclude that N/A/C and L/A/C sponges may have prospective application as hASCs carriers for cartilage repair.

  14. Distribution and viability of fetal and adult human bone marrow stromal cells in a biaxial rotating vessel bioreactor after seeding on polymeric 3D additive manufactured scaffolds

    Directory of Open Access Journals (Sweden)

    Anne eLeferink

    2015-10-01

    Full Text Available One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow derived mesenchymal stromal cells (MSCs are promising candidates for tissue engineering based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix (ECM distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation.

  15. Comparative miRNA-Based Fingerprinting Reveals Biological Differences in Human Olfactory Mucosa- and Bone-Marrow-Derived Mesenchymal Stromal Cells

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    Susan Louise Lindsay

    2016-05-01

    Full Text Available Previously we reported that nestin-positive human mesenchymal stromal cells (MSCs derived from the olfactory mucosa (OM enhanced CNS myelination in vitro to a greater extent than bone-marrow-derived MSCs (BM-MSCs. miRNA-based fingerprinting revealed the two MSCs were 64% homologous, with 26 miRNAs differentially expressed. We focused on miR-146a-5p and miR-140-5p due to their reported role in the regulation of chemokine production and myelination. The lower expression of miR-140-5p in OM-MSCs correlated with higher secretion of CXCL12 compared with BM-MSCs. Addition of CXCL12 and its pharmacological inhibitors to neural co-cultures supported these data. Studies on related miR-146a-5p targets demonstrated that OM-MSCs had lower levels of Toll-like receptors and secreted less pro-inflammatory cytokines, IL-6, IL-8, and CCL2. OM-MSCs polarized microglia to an anti-inflammatory phenotype, illustrating potential differences in their inflammatory response. Nestin-positive OM-MSCs could therefore offer a cell transplantation alternative for CNS repair, should these biological behaviors be translated in vivo.

  16. Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition

    Science.gov (United States)

    Rothdiener, Miriam; Hegemann, Miriam; Uynuk-Ool, Tatiana; Walters, Brandan; Papugy, Piruntha; Nguyen, Phong; Claus, Valentin; Seeger, Tanja; Stoeckle, Ulrich; Boehme, Karen A.; Aicher, Wilhelm K.; Stegemann, Jan P.; Hart, Melanie L.; Kurz, Bodo; Klein, Gerd; Rolauffs, Bernd

    2016-10-01

    Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype.

  17. Analysis of surface protein expression in human bone marrow stromal cells: new aspects of culture-induced changes, inter-donor differences and intracellular expression.

    Science.gov (United States)

    Schäck, Luisa Marilena; Noack, Sandra; Weist, Ramona; Jagodzinski, Michael; Krettek, Christian; Buettner, Manuela; Hoffmann, Andrea

    2013-12-15

    The most widely used technique for isolation of human bone marrow stromal cells (hBMSCs) from bone marrow includes density gradient centrifugation, recovery of the mononuclear cell population, and subsequent isolation of hBMSCs by virtue of their plastic adherence. During subsequent in vitro cultivation, they may lose their original characteristics since in vitro the stem cell niche cannot yet be properly mimicked. To further characterize these culture-induced changes in regard to mRNA and extra- and intracellular protein expression, as well as potential differences between hBMSCs from different donors, we investigated a panel of CD antigens for their presence on in vitro cultured hBMSCs. Interestingly, after culture-induced downregulation of their extracellular expression, both CD146 and CD271 persist intracellularly, which hints at the possibility that culture-induced changes may be reversed by appropriate stimuli. Further, CD34-a protein whose expression on hBMSCs is still controversial-is expressed at the intracellular level in hBMSCs of all donors independently of passage number. CD34 mRNA levels are significantly higher in female than in male donors. In summary, we further elucidate phenotypical changes induced by in vitro culture of hBMSCs, highlight interindividual differences in the phenotype of these cells and for the first time show the intracellular expression of CD34.

  18. Genome-wide analysis of gene expression during adipogenesis in human adipose-derived stromal cells reveals novel patterns of gene expression during adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Melvin Anyasi Ambele

    2016-05-01

    Full Text Available We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived stromal cells (ASCs induced to differentiate into adipocytes in vitro. Gene expression was assessed on days 1, 7, 14 and 21 post-induction and genes differentially expressed numbered 128, 218, 253 and 240 respectively. Up-regulated genes were associated with blood vessel development, leukocyte migration, as well as tumor growth, invasion and metastasis. They also shared common pathways with certain obesity-related pathophysiological conditions. Down-regulated genes were enriched for immune response processes. KLF15, LMO3, FOXO1 and ZBTB16 transcription factors were up-regulated throughout the differentiation process. CEBPA, PPARG, ZNF117, MLXIPL, MMP3 and RORB were up-regulated only on days 14 and 21, which coincide with the maturation of adipocytes and could possibly serve as candidates for controlling fat accumulation and the size of mature adipocytes. In summary, we have identified genes that were up-regulated only on days 1 and 7 or days 14 and 21 that could serve as potential early and late-stage differentiation markers.

  19. Human Bone Marrow Stromal Cells can Differentiate to a Retinal Pigment Epithelial Phenotype when Co-Cultured with Pig Retinal Pigment Epithelium using a Transwell System

    Directory of Open Access Journals (Sweden)

    Ping Duan

    2013-05-01

    Full Text Available Background: There is an increasing interest in generating retinal pigment epithelial (RPE cells from stem cells for therapy against degenerative eye diseases. Human bone marrow stromal cells (hBMSCs can be induced to express retinal neuron-specific markers when co-cultured with retinal neurons, however, whether hBMSCs can differentiate into RPE-like cells in a co-culture system has not been clarified. Methods: The induction of hBMSCs into RPE-like cells was performed by combining hBMSCs and pig RPE cells in a transwell system. The biomarkers of hBMSCs-derived RPE cells were determined by quantitative RT-PCR and immunofluorescence. The function of induced cells was assayed by ELISA for secretion of neurotrophic factors. Results: Intracellular pigment granules and many RPE markers existed in hBMSCs-derived RPE cells after co-culturing with pig RPE cells for 14 days. Typical RPE functions, such as phagocytosis of photoreceptor outer segments and secretion of the trophic factors, brain-derived neurotrophic factor (BDNF and glia-derived neurotrophic factor (GDNF, were observed in these induced cells. Conclusion: hBMSCs can be induced toward functional RPE cells simply by transwell-based co-culture with RPE cells.

  20. Human Umbilical Cord Perivascular Cells Exhibited Enhanced Migration Capacity towards Hepatocellular Carcinoma in Comparison with Bone Marrow Mesenchymal Stromal Cells: A Role for Autocrine Motility Factor Receptor

    Directory of Open Access Journals (Sweden)

    Juan Bayo

    2014-01-01

    Full Text Available Hepatocellular carcinoma (HCC is the third cause of cancer-related death worldwide. Unfortunately, the incidence and mortality associated with HCC are increasing. Therefore, new therapeutic strategies are urgently needed and the use of mesenchymal stromal cells (MSCs as carrier of therapeutic genes is emerging as a promising option. Different sources of MSCs are being studied for cell therapy and bone marrow-derived cells are the most extensively explored; however, birth associated-tissues represent a very promising source. The aim of this work was to compare the in vitro and in vivo migration capacity between bone marrow MSCs (BM-MSCs and human umbilical cord perivascular cells (HUCPVCs towards HCC. We observed that HUCPVCs presented higher in vitro and in vivo migration towards factors released by HCC. The expression of autocrine motility factor (AMF receptor, genes related with the availability of the receptor on the cell surface (caveolin-1 and -2 and metalloproteinase 3, induced by the receptor activation and important for cell migration, was increased in HUCPVCs. The chemotactic response towards recombinant AMF was increased in HUCPVCs compared to BM-MSCs, and its inhibition in the conditioned medium from HCC induced higher decrease in HUCPVC migration than in BM-MSC. Our results indicate that HUCPVCs could be a useful cellular source to deliver therapeutic genes to HCC.

  1. Lim Mineralization Protein 3 Induces the Osteogenic Differentiation of Human Amniotic Fluid Stromal Cells through Kruppel-Like Factor-4 Downregulation and Further Bone-Specific Gene Expression

    Directory of Open Access Journals (Sweden)

    Marta Barba

    2012-01-01

    Full Text Available Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs, can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.

  2. Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

    Science.gov (United States)

    Wuchter, Patrick; Vetter, Marcel; Saffrich, Rainer; Diehlmann, Anke; Bieback, Karen; Ho, Anthony D; Horn, Patrick

    2016-06-01

    Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  3. Long-term efficiency of mesenchymal stromal cell-mediated CD-MSC/5FC therapy in human melanoma xenograft model.

    Science.gov (United States)

    Kucerova, L; Skolekova, S; Demkova, L; Bohovic, R; Matuskova, M

    2014-10-01

    Mesenchymal stromal cells (MSC) can be exploited as cellular delivery vehicles for the enzymes converting non-toxic prodrugs to toxic substances. Because of their inherent chemoresistance, they exert potent bystander and antitumor effect. Here we show that the human adipose tissue-derived MSC expressing fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) in combination with 5-fluorocytosine (5FC) mediated a long-term tumor-free survival in the 83.3% of tumor-bearing animals. CD-MSC/5FC treatment induced cytotoxicity against model human melanoma cells EGFP-A375. Only 4% of the therapeutic CD-MSC cells eliminated >98.5% of the tumor cells in vitro. Long-term tumor-free survival was confirmed in 15 out of the 18 animals. However, repeatedly used CD-MSC/5FC therapeutic regimen generated more aggressive and metastatic variant of the melanoma cells EGFP-A375/Rel3. These cells derived from the refractory xenotransplants exhibited increased resistance to the CD-MSC/5FC treatment, altered cell adhesion, migration, tumorigenic and metastatic properties. However, long-term curative effect was achieved by the augmentation of the CD-MSC/5FC regimen along with the inhibition of c-Met/hepatocyte growth factor signaling axis in this aggressive melanoma derivative. In summary, the CD-MSC/5FC regimen can be regarded as a very effective antitumor approach to achieve long-term tumor-free survival as demonstrated on a mouse model of aggressive human melanoma xenografts.

  4. Enhanced osteoblastic differentiation and bone formation in co-culture of human bone marrow mesenchymal stromal cells and peripheral blood mononuclear cells with exogenous VEGF.

    Science.gov (United States)

    Joensuu, K; Uusitalo, L; Alm, J J; Aro, H T; Hentunen, T A; Heino, T J

    2015-05-01

    Despite recent advances in bone tissue engineering, efficient bone formation and vascularization remains a challenge for clinical applications. The aim of this study was to investigate if the osteoblastic differentiation of human mesenchymal stromal cells (MSCs) can be enhanced by co-culturing them with peripheral blood (PB) mononuclear cells (MNCs), with and without vascular endothelial growth factor (VEGF), a coupling factor of bone formation and angiogenesis. Human bone marrow (BM) derived MSCs were co-cultured with PB-MNCs in osteogenic medium with or without VEGF. Osteoblastic differentiation and mineral deposition were studied by staining for alkaline phosphatase (ALP), and von Kossa, respectively, and measurements for ALP activity and calcium concentration (Ca). Cell proliferation was assayed with Alamar blue. The mechanism(s) were further studied by Transwell(®) cell culture experiments. Both ALP and mineralization (von Kossa and Ca) were significantly higher in the MSC-MNC co-cultures compared to plain MSC cultures. VEGF alone had no effect on osteoblastic differentiation of MSCs, but further enhanced differentiation in co-culture settings. The mechanism was shown to require cell-cell contact between MSCs and MNCs and the factors contributing to further differentiation appear to be soluble. No differences were observed in cell proliferation. Our study demonstrates that the in vitro ALP activity and mineralization of human BM-MSCs is more efficient in the presence of PB-MNCs, and exogenously added VEGF further enhances the stimulatory effect. This indicates that PB-MNCs could be a potential cell source in development of co-culture systems for novel tissue engineering applications for enhanced bone healing. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  5. High rate of unnecessary thymectomy and its cause. Can computed tomography distinguish thymoma, lymphoma, thymic hyperplasia, and thymic cysts?

    Energy Technology Data Exchange (ETDEWEB)

    Ackman, Jeanne B., E-mail: jackman@mgh.harvard.edu [MGH Department of Radiology, Division of Thoracic Imaging and Intervention, Founders House 202, 55 Fruit Street, Boston, MA 02114 (United States); Verzosa, Stacey, E-mail: sverzosa@mgh.harvard.edu [Massachusetts General Hospital, Harvard Medical School (United States); Kovach, Alexandra E., E-mail: akovach@mgh.harvard.edu [Massachusetts General Hospital, Harvard Medical School (United States); Louissaint, Abner, E-mail: alouissaint@mgh.harvard.edu [Massachusetts General Hospital, Harvard Medical School (United States); Lanuti, Michael, E-mail: mlanuti@mgh.harvard.edu [Massachusetts General Hospital, Harvard Medical School (United States); Wright, Cameron D., E-mail: cdwright@mgh.harvard.edu [Massachusetts General Hospital, Harvard Medical School (United States); Shepard, Jo-Anne O., E-mail: jshepard@mgh.harvard.edu [Massachusetts General Hospital, Harvard Medical School (United States); Halpern, Elkan F., E-mail: elk@mgh-ita.org [Massachusetts General Hospital, Harvard Medical School (United States)

    2015-03-15

    Highlights: •The unnecessary thymectomy rate of 44% was due to concern for thymoma, based on CT findings. •It was comprised of lymphoma, thymic cysts, thymic hyperplasia, and reactive or atrophic tissue. •There are significant differentiating features of these lesions on CT. •Knowledge of these CT features may help avert unnecessary thymectomy. •Shortcomings of CT in the evaluation of these lesions remain; in such cases, MRI or biopsy can help. -- Abstract: Purpose: To determine the non-therapeutic thymectomy rate in a recent six-year consecutive thymectomy cohort, the etiology of these unnecessary thymectomies, and the differentiating CT features of thymoma, lymphoma, thymic hyperplasia, and thymic cysts. Materials and methods: Electronic data base query of all thymectomies performed at the Massachusetts General Hospital from 2006 to 2012 yielded 160 thymectomy cases, 124 of which had available imaging. The non-therapeutic thymectomy rate (includes thymectomy for lymphoma and benign disease) was calculated. Preoperative clinical and CT imaging features were assessed by review of the in-house electronic medical record by 2 thoracic surgeons and 2 pathology-blinded radiologists, respectively. Results: The non-therapeutic thymectomy rate of 43.8% (70/160) was largely secondary to concern for thymoma and was comprised of lymphoma (54.3%, 38/70), thymic bed cysts (24.3%, 17/70), thymic hyperplasia (17.1%, 12/70), and reactive or atrophic tissue (4.3%, 3/70). Among these four lesions, there were significant differences in location with respect to midline, morphology, circumscription, homogeneity of attenuation, fatty intercalation, coexistent lymphadenopathy, overt pericardial invasion, and mass effect (p < 0.001). True thymic cysts ranged in attenuation from −20 to 58 Hounsfield units (HU), with a mean attenuation of 23 HU. Conclusion: The high rate of unnecessary thymectomy was due to misinterpretation of thymic cysts, thymic hyperplasia, and lymphoma as

  6. Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens.

    Science.gov (United States)

    Cepeda, Sergio; Cantu, Carolina; Orozco, Stephanie; Xiao, Yangming; Brown, Zoe; Semwal, Manpreet K; Venables, Thomas; Anderson, Mark S; Griffith, Ann V

    2018-01-30

    Although autoimmune disorders are a significant source of morbidity and mortality in older individuals, the mechanisms governing age-associated increases in susceptibility remain incompletely understood. Central T cell tolerance is mediated through presentation of self-antigens by cells constituting the thymic microenvironment, including epithelial cells, dendritic cells, and B cells. Medullary thymic epithelial cells (mTECs) and B cells express distinct cohorts of self-antigens, including tissue-restricted self-antigens (TRAs), such that developing T cells are tolerized to antigens from peripheral tissues. We find that expression of the TRA transcriptional regulator Aire, as well as Aire-dependent genes, declines with age in thymic B cells in mice and humans and that cell-intrinsic and cell-extrinsic mechanisms contribute to the diminished capacity of peripheral B cells to express Aire within the thymus. Our findings indicate that aging may diminish the ability of thymic B cells to tolerize T cells, revealing a potential mechanistic link between aging and autoimmunity. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens

    Directory of Open Access Journals (Sweden)

    Sergio Cepeda

    2018-01-01

    Full Text Available Although autoimmune disorders are a significant source of morbidity and mortality in older individuals, the mechanisms governing age-associated increases in susceptibility remain incompletely understood. Central T cell tolerance is mediated through presentation of self-antigens by cells constituting the thymic microenvironment, including epithelial cells, dendritic cells, and B cells. Medullary thymic epithelial cells (mTECs and B cells express distinct cohorts of self-antigens, including tissue-restricted self-antigens (TRAs, such that developing T cells are tolerized to antigens from peripheral tissues. We find that expression of the TRA transcriptional regulator Aire, as well as Aire-dependent genes, declines with age in thymic B cells in mice and humans and that cell-intrinsic and cell-extrinsic mechanisms contribute to the diminished capacity of peripheral B cells to express Aire within the thymus. Our findings indicate that aging may diminish the ability of thymic B cells to tolerize T cells, revealing a potential mechanistic link between aging and autoimmunity.

  8. Thymic irradiation inhibits the rapid recovery of TH1 but not TH2-like functions of CD4+ T cells after total lymphoid irradiation

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    Bass, H.; Adkins, B.; Strober, S. (Department of Medicine, Stanford University School of Medicine, CA (United States))

    1991-10-15

    Four to six weeks after total lymphoid irradiation (TLI), there is a selective deficit in the CD4+ T cells which secrete IL-2, proliferate in the MLR, and induce GVHD (Th1-like functions). A similar deficit in CD4+ T cells which secrete IL-4 and help antibody responses (Th2-like functions) is not observed. In the present study, shielding of the thymus with lead during TLI increased the Th1-like functions of CD4+ cells. Mice without thymus shields showed a marked selective reduction in the medullary stromal cells identified with the monoclonal antibody, MD1, and the severe reduction was prevented with thymus shields. Thus, shielding the thymus prevents the depletion of thymic medullary stromal cells and allows for a rapid recovery of Th1-like functions in the mouse spleen after TLI. Th2-like functions recover rapidly after TLI whether or not the thymus is irradiated.

  9. DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells.

    Science.gov (United States)

    Wu, Guoying; Hirabayashi, Keiji; Sato, Shinya; Akiyama, Nobuko; Akiyama, Taishin; Shiota, Kunio; Yagi, Shintaro

    2012-11-02

    Medullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model. We identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire(-/-) mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire(-/-) mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire(+/+) mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire(+/+) and Aire(-/-) mTECs but distinct from that of athymic cells and tissues. These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the

  10. DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells

    Directory of Open Access Journals (Sweden)

    Wu Guoying

    2012-11-01

    Full Text Available Abstract Background Medullary thymic epithelial cells (mTECs are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire, which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs, and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model. Results We identified the T-DMRs of mTECs (mTEC-T-DMRs via genome-wide DNA methylation analysis of Aire−/− mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire−/− mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire+/+ mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire+/+ and Aire−/− mTECs but distinct from that of athymic cells and tissues. Conclusions These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic

  11. The role of exchange protein directly activated by cyclic AMP 2-mediated calreticulin expression in the decidualization of human endometrial stromal cells.

    Science.gov (United States)

    Kusama, Kazuya; Yoshie, Mikihiro; Tamura, Kazuhiro; Nakayama, Takahiro; Nishi, Hirotaka; Isaka, Keiichi; Tachikawa, Eiichi

    2014-01-01

    Decidualization of human endometrial stromal cells (ESCs) accompanied by the production of prolactin (PRL) and IGF-binding protein (IGFBP) 1 and rounded-cell morphology is indispensable for the establishment and maintenance of pregnancy. Protein kinase A (PKA)-mediated cAMP signaling is known to be crucial for decidualization. We previously reported that activation of a cAMP mediator, called Exchange protein directly activated by cAMP (EPAC) promotes cAMP analog- or ovarian steroid-induced decidualization in cultured human ESCs. In addition, small interfering RNA-mediated knock-down of the EPAC subtypes, EPAC1 or EPAC2, or knock-down of Rap1, a downstream factor of EPAC signaling, blocked functional and morphological decidualization of ESCs. However, factors downstream of EPAC2 other than Rap1 have not been determined. The present study was undertaken to identify additional downstream targets of EPAC2 associated with decidualization. Using proteomic analysis, we identified calreticulin (CRT) as a potential target of EPAC2. Knock-down of CRT expression in cultured ESCs significantly inhibited PKA-selective cAMP analog- or PKA-selective cAMP analog plus EPAC-selective cAMP analog-induced PRL and IGFBP1 expression. Furthermore, CRT knock-down suppressed the ovarian steroid-stimulated PRL and IGFBP1 expression and morphological differentiation, and silencing of EPAC2 or CRT significantly increased senescence-associated β-galactosidase activity with enhanced p21 expression and decreased p53 expression. These results suggest that EPAC2 and CRT are associated with cellular senescence in ESCs. In conclusion, we demonstrate here that EPAC2-mediated CRT expression is essential for the functional and morphological differentiation of ESCs into decidual cells. Furthermore, both EPAC2 and CRT might prevent ESCs from undergoing abnormal cellular senescence during decidualization.

  12. Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions.

    Science.gov (United States)

    Castiglia, Sara; Mareschi, Katia; Labanca, Luciana; Lucania, Graziella; Leone, Marco; Sanavio, Fiorella; Castello, Laura; Rustichelli, Deborah; Signorino, Elena; Gunetti, Monica; Bergallo, Massimiliano; Bordiga, Anna Maria; Ferrero, Ivana; Fagioli, Franca

    2014-06-01

    Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). Bone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed. The MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed. We demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice-compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  13. CXC chemokine ligand 12/stromal cell-derived factor-1 regulates cell adhesion in human colon cancer cells by induction of intercellular adhesion molecule-1.

    Science.gov (United States)

    Tung, Shui-Yi; Chang, Shun-Fu; Chou, Ming-Hui; Huang, Wen-Shih; Hsieh, Yung-Yu; Shen, Chien-Heng; Kuo, Hsing-Chun; Chen, Cheng-Nan

    2012-10-25

    The CXC chemokine ligand 12 (CXCL12)/stromal cell-derived factor-1 (SDF-1) and CXC receptor 4 (CXCR4) axis is involved in human colorectal cancer (CRC) carcinogenesis and can promote the progression of CRC. Interaction between CRC cells and endothelium is a key event in tumor progression. The aim of this study was to investigate the effect of SDF-1 on the adhesion of CRC cells. Human CRC DLD-1 cells were used to study the effect of SDF-1 on intercellular adhesion molecule-1 (ICAM-1) expression and cell adhesion to endothelium. SDF-1 treatment induced adhesion of DLD-1 cells to the endothelium and increased the expression level of the ICAM-1. Inhibition of ICAM-1 by small interfering RNA (siRNA) and neutralizing antibody inhibited SDF-1-induced cell adhesion. By using specific inhibitors and short hairpin RNA (shRNA), we demonstrated that the activation of ERK, JNK and p38 pathways is critical for SDF-1-induced ICAM-1 expression and cell adhesion. Promoter activity and transcription factor ELISA assays showed that SDF-1 increased Sp1-, C/EBP-β- and NF-κB-DNA binding activities in DLD-1 cells. Inhibition of Sp1, C/EBP-β and NF-κB activations by specific siRNA blocked the SDF-1-induced ICAM-1 promoter activity and expression. The effect of SDF-1 on cell adhesion was mediated by the CXCR4. Our findings support the hypothesis that ICAM-1 up-regulation stimulated by SDF-1 may play an active role in CRC cell adhesion.

  14. Hexane extract of aged black garlic reduces cell proliferation and attenuates the expression of ICAM-1 and VCAM‑1 in TNF-α-activated human endometrial stromal cells.

    Science.gov (United States)

    Kim, Ki-Hyung; Park, Jin Kyeong; Choi, Young-Whan; Kim, Youn-Han; Lee, Eun Na; Lee, Ja-Rang; Kim, Heui-Soo; Baek, Sun-Yong; Kim, Bong-Seon; Lee, Kyu-Sup; Yoon, Sik

    2013-07-01

    Increasing evidence indicates the potentially crucial roles of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the pathological process underlying endometriosis. The present study aimed to investigate the effects of a hexane extract of aged black garlic (HEABG) on the proliferation and expression of ICAM-1 and VCAM-1 in tumor necrosis factor-α (TNF-α)-activated human endometrial stromal cells (HESCs) isolated from patients with endometriosis. HESCs were isolated from endometriotic tissues obtained from women with advanced endometriosis who underwent laparoscopic surgery for ovarian endometrioma (n=18). Cell proliferation and cell cycle analysis were assessed by WST-1 assay and flow cytometry, respectively. The expression of ICAM-1 and VCAM-1 was measured by flow cytometry, immunofluorescence staining, immunoblotting and quantitative reverse transcriptase-PCR. The secretion of interleukin-6 (IL-6) was determined by enzyme-linked immunosorbent assay (ELISA). The activation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) was detected by electrophoretic mobility shift assay (EMSA) and the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 MAPK was analyzed by immunoblotting. Cell proliferation and cell cycle progression were significantly suppressed by HEABG in the TNF-α-induced HESCs through the inhibition of the ERK and JNK signaling pathways. Remarkably, the treatment of the HESCs with HEABG potently suppressed the TNF-α-induced ICAM-1 and VCAM-1 transcript and protein expression by inhibiting the activation of NF-κB and AP-1 transcription factors. Our results suggest that HEABG may be effective in the prevention and treatment of endometriosis in humans.

  15. Human Wharton's jelly-derived mesenchymal stromal cells engineered to secrete Epstein-Barr virus interleukin-10 show enhanced immunosuppressive properties.

    Science.gov (United States)

    Quaranta, Paola; Focosi, Daniele; Di Iesu, Marilena; Cursi, Chiara; Zucca, Alessandra; Curcio, Michele; Lapi, Simone; Boldrini, Linda; Stampacchia, Giulia; Paolicchi, Aldo; Scatena, Fabrizio; Freer, Giulia; Pistello, Mauro

    2016-02-01

    Mesenchymal stromal cells (MSCs) modulate the immune response and represent a potential treatment for inflammatory and autoimmune diseases. We hypothesized that this feature could be potentiated by co-administering anti-inflammatory cytokines. In this article, we asked whether engineering of Wharton Jelly-derived human MSCs (WJ-hMSCs) to express an anti-inflammatory cytokine increases cell immunomodulatory properties without altering their native features. We used Epstein-Barr virus-derived interleukin-10 (vIL-10), which shares some immunosuppressive properties with human IL-10 but lacks immunostimulatory activity. Engineering was accomplished by transducing WJ-hMSCs with a self-inactivating feline immunodeficiency virus-derived vector co-expressing vIL-10 and herpes simplex virus type-1 thymidine kinase (TK). TK was added to allow future tracking of WJ-hMSC in vivo by positron electron tomography (PET). The results show that (i) expression of TK and/or vIL-10 does not change WJ-hMSC phenotypic and functional properties; (ii) vIL-10 is secreted, biologically active and enhances the immunosuppressing functions of WJ-hMSCs; (iii) v-IL10 and TK can be produced simultaneously by the same cells and do not interfere with each other. WJ-hMSCs engineered to secrete vIL-10 could be a powerful tool for adoptive cell therapy of immune-mediated diseases, and therefore, additional studies are warranted to confirm their efficacy in suitable animal disease models. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  16. TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

    Science.gov (United States)

    Colombier, Pauline; Clouet, Johann; Boyer, Cécile; Ruel, Maëva; Bonin, Gaëlle; Lesoeur, Julie; Moreau, Anne; Fellah, Borhane-Hakim; Weiss, Pierre; Lescaudron, Laurent; Camus, Anne; Guicheux, Jérôme

    2016-03-01

    Degenerative disc disease (DDD) primarily affects the central part of the intervertebral disc namely the nucleus pulposus (NP). DDD explains about 40% of low back pain and is characterized by massive cellular alterations that ultimately result in the disappearance of resident NP cells. Thus, repopulating the NP with regenerative cells is a promising therapeutic approach and remains a great challenge. The objectives of this study were to evaluate the potential of growth factor-driven protocols to commit human adipose stromal cells (hASCs) toward NP-like cell phenotype and the involvement of Smad proteins in this differentiation process. Here, we demonstrate that the transforming growth factor-β1 and the growth differentiation factor 5 synergistically drive the nucleopulpogenic differentiation process. The commitment of the hASCs was robust and highly specific as attested by the expression of NP-related genes characteristic of young healthy human NP cells. In addition, the engineered NP-like cells secreted an abundant aggrecan and type II collagen rich extracellular matrix comparable with that of native NP. Furthermore, we demonstrate that these in vitro engineered cells survived, maintained their specialized phenotype and secretory activity after in vivo transplantation in nude mice subcutis. Finally, we provide evidence suggesting that the Smad 2/3 pathway mainly governed the acquisition of the NP cell molecular identity while the Smad1/5/8 pathway controlled the NP cell morphology. This study offers valuable insights for the development of biologically-inspired treatments for DDD by generating adapted and exhaustively characterized autologous regenerative cells. © 2015 AlphaMed Press.

  17. Cytoskeletal proteins and stem cell markers gene expression in human bone marrow mesenchymal stromal cells after different periods of simulated microgravity

    Science.gov (United States)

    Gershovich, P. M.; Gershovich, J. G.; Zhambalova, A. P.; Romanov, Yu. A.; Buravkova, L. B.

    2012-01-01

    Mesenchymal stem (stromal) cells (MSCs) are present in a variety of tissues during prenatal and postnatal human development. In adult organism, they are prevalent in bone marrow and supposed to be involved in space-flight induced osteopenia. We studied expression of various genes in human bone marrow MSCs after different terms of simulated microgravity (SMG) provided by Random Positioning Machine. Simulated microgravity induced transient changes in expression level of genes associated with actin cytoskeleton, especially after 48 h of SMG. However, after 120 h exposure in SMG partial restoration of gene expression levels (relative to the control) was found. Similar results were obtained with bmMSCs subjected to 24 h readaptation in static state after 24 h in SMG. Analysis of 84 genes related to identification, growth and differentiation of stem cells revealed that expression of nine genes was changed slightly after 48 h in SMG. More pronounced changes in gene expression of "stem cells markers" were observed after 120 h of simulated microgravity. Among 84 investigated genes, 30 were up-regulated and 24 were down-regulated. Finally, MSCs osteogenesis induced by long-term (10-20 days) simulation of microgravity was accompanied by down-regulation of gene expression of the main osteogenic differentiation markers ( ALPL, OMD) and master transcription osteogenic factor of MSCs ( Runx2). Thus, our study demonstrated that changes in expression level of some genes associated with actin cytoskeleton and stem cell markers are supposed to be one of the mechanisms, which contribute to precursor's cellular adaptation to the microgravity conditions. These results can clarify genomic mechanisms through which SMG reduces osteogenic differentiation of bmMSCs.

  18. Endothelial cells are essential for ovarian stromal tissue restructuring after xenotransplantation of isolated ovarian stromal cells.

    Science.gov (United States)

    Dath, C; Dethy, A; Van Langendonckt, A; Van Eyck, A S; Amorim, C A; Luyckx, V; Donnez, J; Dolmans, M M

    2011-06-01

    Grafting of isolated follicles represents an approach to prevent the risk of reimplanting malignant cells with cryopreserved ovarian fragments. Optimal conditions and cell types required to sustain human follicular growth need to be identified. To help improve the grafting technique, we investigated whether short-term xenografting of a suspension containing ovarian stromal and endothelial cells without follicles could enhance graft survival and revascularization. In human ovary, CD34 selectively labels endothelial cells of blood vessels. A CD34-replete ovarian stromal cell group, including stromal and endothelial cells, was obtained after enzymatic digestion of fresh human ovarian cortex. Magnetic-activated cell sorting was used to establish a CD34-depleted ovarian stromal cell group. Proportions of CD34-positive cells were evaluated by flow cytometry and immunocytochemistry. Cell suspensions were embedded in human plasma clots and grafted (n = 10 for each group, 7 days) to the ovarian bursa of nude mice. Angiogenesis was quantified after human/mouse CD34 immunostaining. CD34-replete grafts had a well-organized and vascularized stromal structure, containing tubular components staining for human CD34 and corresponding to functional vessels, as evidenced by intraluminal red blood cells. CD34-depleted grafts tended to be smaller than CD34-replete grafts and poorly vascularized with central necrosis. Global microvessel density was higher in the CD34-replete than depleted group (337.9 versus 187.3 vessels/mm(2), P ovarian endothelial and stromal cells to ensure the formation of a well-vascularized and structured ovarian-like stroma after short-term xenografting, for future application in the transplantation of isolated follicles.

  19. Adult thymic epithelial cell (TEC) progenitors and TEC stem cells: Models and mechanisms for TEC development and maintenance.

    Science.gov (United States)

    Hamazaki, Yoko

    2015-11-01

    The thymus is the primary lymphoid organ for generating self-restricted and self-tolerant functional T cells. Its two distinct anatomical regions, the cortex and the medulla, are involved in positive and negative selection, respectively. Thymic epithelial cells (TECs) constitute the framework of this tissue and function as major stromal components. Extensive studies for more than a decade have revealed how TECs are generated during organogenesis; progenitors specific for medullary TECs (mTECs) and cortical TECs (cTECs) as well as bipotent progenitors for both lineages have been identified, and the signaling pathways required for the development and maturation of mTECs have been elucidated. However, little is known about the initial commitment of mTECs and cTECs during ontogeny, and how regeneration of both lineages is sustained in the postnatal/adult thymus. Recently, stem cell activities in TECs have been demonstrated, and TEC progenitors have been identified in the postnatal thymus. In this review, recent advances in studying the development and maintenance of TECs are summarized, and the possible mechanisms of thymic regeneration and involution are discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Comparing the Gene Expression Profile of Stromal Cells from Human Cord Blood and Bone Marrow: Lack of the Typical “Bone” Signature in Cord Blood Cells

    Directory of Open Access Journals (Sweden)

    Julia Bosch

    2013-01-01

    Full Text Available With regard to the bone-regenerative capacity, bone marrow stromal cells (BMSC can still be termed the “gold standard.” Nevertheless, neonatal stromal cells from cord blood (CB feature advantages concerning availability, immaturity, and proliferation potential. The detailed gene expression analysis and overexpression of genes expressed differentially provide insight into the inherent capacity of stromal cells. Microarray and qRT-PCR analyses revealed closely related gene expression patterns of two stromal cell populations derived from CB. In contrast to the CB-derived cell types, BMSC displayed high expression levels of BSP, OSX, BMP4, OC, and PITX2. Lentiviral overexpression of BSP but not of OSX in CB-cells increased the capacity to form a mineralized matrix. BMP4 induced the secretion of proteoglycans during chondrogenic pellet culture and extended the osteogenic but reduced the adipogenic differentiation potential. BMSC revealed the typical osteogenic gene expression signature. In contrast, the CB-derived cell types exhibited a more immature gene expression profile and no predisposition towards skeletal development. The absence of BSP and BMP4—which were defined as potential key players affecting the differentiation potential—in neonatal stromal cells should be taken into consideration when choosing a cell source for tissue regeneration approaches.

  1. Induction of Thymic Stromal Lymphopoietin Production by Nonanoic Acid and Exacerbation of Allergic Inflammation in Mice

    Directory of Open Access Journals (Sweden)

    Saori Yamashita

    2013-01-01

    Conclusions: Nonanoic acid and certain fatty acids induced TSLP production, resulting in the exacerbation of allergic inflammation. We propose that TSLP-inducing chemical compounds such as nonanoic acid be recognized as chemical allergo-accelerators.

  2. A Distinct Sensitization Pattern Associated with Asthma and the Thymic Stromal Lymphopoietin (TSLP Genotype

    Directory of Open Access Journals (Sweden)

    Hiroaki Iijima

    2013-01-01

    Conclusions: Cluster analysis identified the presence of distinct sensitization patterns to common inhaled allergens. TSLP may cause asthma by promoting innate allergic responses to indoor allergens and this contribution is significantly modified by smoking.

  3. Intrauterine human chorionic gonadotropin infusion in oocyte donors promotes endometrial synchrony and induction of early decidual markers for stromal survival: a randomized clinical trial.

    Science.gov (United States)

    Strug, Michael R; Su, Renwei; Young, James E; Dodds, William G; Shavell, Valerie I; Díaz-Gimeno, Patricia; Ruíz-Alonso, Maria; Simón, Carlos; Lessey, Bruce A; Leach, Richard E; Fazleabas, Asgerally T

    2016-07-01

    Does a single intrauterine infusion of human chorionic gonadotropin (hCG) at the time corresponding to a Day 3 embryo transfer in oocyte donors induce favorable molecular changes in the endometrium for embryo implantation? Intrauterine hCG was associated with endometrial synchronization between endometrial glands and stroma following ovarian stimulation and the induction of early decidual markers associated with stromal cell survival. The clinical potential for increasing IVF success rates using an intrauterine hCG infusion prior to embryo transfer remains unclear based on previously reported positive and non-significant findings. However, infusion of CG in the non-human primate increases the expression of pro-survival early decidual markers important for endometrial receptivity, including α-smooth muscle actin (α-SMA) and NOTCH1. Oocyte donors (n=15) were randomly assigned to receive an intrauterine infusion of 500 IU hCG (n=7) or embryo culture media vehicle (n=8) 3 days following oocyte retrieval during their donor stimulation cycle. Endometrial biopsies were performed 2 days later, followed by either RNA isolation or tissue fixation in formalin and paraffin embedding. Reverse transcription of total RNA from endometrial biopsies generated cDNA, which was used for analysis in the endometrial receptivity array (ERA; n = 5/group) or quantitative RT-PCR to determine relative expression of ESR1, PGR, C3 and NOTCH1. Tissue sections were stained with hematoxylin and eosin followed by blinded staging analysis for dating of endometrial glands and stroma. Immunostaining for ESR1, PGR, α-SMA, C3 and NOTCH1 was performed to determine their tissue localization. Intrauterine hCG infusion was associated with endometrial synchrony and reprograming of stromal development following ovarian stimulation. ESR1 and PGR were significantly elevated in the endometrium of hCG-treated patients, consistent with earlier staging. The ERA did not predict an overall positive impact of

  4. Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

    2001-01-01

    While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insulin......-like growth factors (IGFs) and IGF-binding proteins (IGFBPs). Thus, we studied the effects of TGF-beta1 on IGFs and IGFBPs in human marrow stromal (hMS) osteoblast precursor cells. TGF-beta1 increased the steady-state mRNA level of IGF-I up to 8.5+/-0.6-fold (P...

  5. Self-Antigen-Driven Thymic B Cell Class Switching Promotes T Cell Central Tolerance

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    Jason Perera

    2016-10-01

    Full Text Available B cells are unique antigen-presenting cells because their antigen presentation machinery is closely tied to the B cell receptor. Autoreactive thymic B cells can efficiently present cognate self-antigens to mediate CD4+ T cell-negative selection. However, the nature of thymocyte-thymic B cell interaction and how this interaction affects the selection of thymic B cell repertoire and, in turn, the T cell repertoire are not well understood. Here we demonstrate that a large percentage of thymic B cells have undergone class switching intrathymically. Thymic B cell class switching requires cognate interaction with specific T cells. Class-switched thymic B cells have a distinct repertoire compared with unswitched thymic B cells or splenic B cells. Particularly, autoreactive B cell specificities preferentially expand in the thymus by undergoing class switching, and these enriched, class-switched autoreactive thymic B cells play an important role in CD4 T cell tolerance.

  6. Upper thymic prolongation simulating mediastinal lymphadenomegaly

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    Cristiane Wosny

    2009-09-01

    Full Text Available The thymus is located in the anterior portion of the upper mediastinum, immediately behind the sternal manubrium, and extends to the anterior mediastinum, anteriorly to the pericardium. Two patients were evaluated due to nodulations at the transition from the cervical region to the anterior mediastinum, which simulated lymphadenomegaly. The first patient, a seven-year-old male, presented with a rhabdomyosarcoma of the masticatory space; during progressive follow-up, a nodule was noted with FDG uptake on the positron emission tomography coupled with the computed tomography (PET-CT. The second patient, a 51-year-old female, presented with a nodulation characterized on the magnetic resonance image for follow-up of a papilliferous carcinoma of the thyroid. In both cases, the nodulation displayed an upper prolongation of the thymus. These nodulations showed the same density on the computed tomography and the same signal intensity on the magnetic resonance image as the adjacent thymic tissue, and there was no adipose tissue layer between the nodulations and the thymus. Knowledge of the upper prolongation of the thymus as an anatomical variation is vital for differentiating it from mediastinal lymphadenomegaly, thus avoiding unnecessary biopsies or procedures.

  7. Expansion on stromal cells preserves the undifferentiated state of human hematopoietic stem cells despite compromised reconstitution ability.

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    Mattias Magnusson

    Full Text Available Lack of HLA-matched hematopoietic stem cells (HSC limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC stroma that protects human hematopoietic stem/progenitor cells (HSPC from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38-CD90+ characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38-CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.

  8. Identification of EPSTI1, a novel gene induced by epithelial-stromal interaction in human breast cancer

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    Nielsen, Helga Lind; Rønnov-Jessen, Lone; Villadsen, René

    2002-01-01

    reading frame (ORF) encoding a putative 307-amino-acid protein, and mapped to chromosome 13q13.3. EPSTI1 was highly upregulated in invasive breast carcinomas compared with normal breast. In a tissue mRNA panel the most prominent expression of EPSTI1 was found in placenta. Thus, EPSTI1 is a novel human...

  9. Presence of donor-derived thymic epithelial cells in [B6-->MRL/lpr] mice after allogeneic intra-bone marrow-bone marrow transplantation (IBM-BMT).

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    Takaki, Takashi; Hosaka, Naoki; Miyake, Takashi; Cui, Wenhao; Nishida, Teruhisa; Inaba, Muneo; Ikehara, Susumu

    2008-12-01

    We have previously shown that allogeneic intra-bone marrow-bone marrow transplantation (IBM-BMT) can be used to treat autoimmune diseases in MRL/lpr (H-2(K)) mice with replacing not only hematolymphoid cells but also stromal cells by normal C57BL/6 (B6: H-2(b)) mouse cells. In the present study, we examined for existence of donor-derived thymic epithelial cells (TECs) in the host thymus using green fluorescent protein (GFP)-B6 (H-2(b)) mice. In [GFP-B6-->MRL/lpr] chimeric mice, splenocytes and thymocytes were completely replaced by donor-type cells, and levels of serum autoantibodies and proteinuria were significantly - reduced to those levels of normal donors. Interestingly, GFP-expressing TECs - not only medullary TECs, which express mouse thymus stromal (MTS)-10, but also cortical TECs, which express cytokeratin 18 - were found. Also, the number of autoimmune regulator (AIRE) expressing TECs, which regulates tissue-specific antigens to delete autoreactive cells, was reduced in the chimeric mice to that of the donor, whereas the number of forkhead box N1 (FOXN1) expressing TECs, which are crucial in the terminal differentiation of TECs, remained unchanged. These findings suggest that BMCs contain the precursors of functional TECs, and that they can differentiate into TECs, thereby correcting thymic function.

  10. Human Wharton’s jelly-derived mesenchymal stromal cells reduce renal fibrosis through induction of native and foreign hepatocyte growth factor synthesis in injured tubular epithelial cells

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    2013-01-01

    Introduction Based on some well-documented reports, we attempted to clarify the antifibrotic mechanisms of human Wharton’s-jelly-derived mesenchymal stromal cells (WJ-MSCs) from the perspective of induction of hepatocyte growth factor (HGF) expression in tubular epithelial cells (TECs). Methods A rat model of acute kidney injury (AKI) was established through unilateral renal ischemia for 1 hour. Two days later, a single intravenous cell or vehicle injection, or contralateral nephrectomy, was performed. Rats were sacrificed at 1 day, 1 week, 4 weeks, or 6 weeks after the intervention. Renal fibrosis was evaluated by Masson trichrome staining and Sircol collagen assay. The upregulation of α-smooth muscle actin (α-SMA) versus E-cadherin expression was adopted as an indicator of tubular epithelial-mesenchymal transition (EMT). Gene and protein expression of HGF or transforming growth factor-beta1 (TGF-β1) was determined by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. HGF expression in TECs was detected with immunostaining. In vitro, rat TECs subjected to hypoxia injury were incubated with or without conditioned medium (CM) from WJ-MSCs for 1, 3, 24, or 48 hours. Rat or human HGF synthesis in TECs was assessed with immunostaining, RT-PCR, or ELISA. Results Cell delivery or nephrectomy led to abrogation of renal scarring. At the incipient period of AKI, through induction of HGF expression, either of them remarkably promoted the upregulation of HGF versus TGF-β1 expression in damaged kidney. Rat TECs were not only the principal cells expressing HGF but also exhibited human HGF expression after cell infusion. During fibrogenesis, the downregulation of HGF versus TGF-β1 expression was greatly prevented by WJ-MSCs or kidney removal, thereby resulting in tubular EMT delay. In vitro, after 24 or 48 hours of incubation, CM not only robustly induced the upregulation of rat HGF gene expression in TECs but substantially amplified the release

  11. Platelet lysate and granulocyte-colony stimulating factor serve safe and accelerated expansion of human bone marrow stromal cells for stroke therapy.

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    Yamauchi, Tomohiro; Saito, Hisayasu; Ito, Masaki; Shichinohe, Hideo; Houkin, Kiyohiro; Kuroda, Satoshi

    2014-12-01

    Autologous human bone marrow stromal cells (hBMSCs) should be expanded in the animal serum-free condition within clinically relevant periods in order to secure safe and effective cell therapy for ischemic stroke. This study was aimed to assess whether the hBMSCs enhance their proliferation capacity and provide beneficial effect in the infarct brain when cultured with platelet lysate (PL) and granulocyte-colony stimulating factor (G-CSF). The hBMSCs were cultured in the fetal calf serum (FCS)-, PL-, or PL/G-CSF-containing medium. Cell growth kinetics was analyzed. The hBMSCs-PL, hBMSC-PL/G-CSF, or vehicle was stereotactically transplanted into the ipsilateral striatum of the rats subjected to permanent middle cerebral artery occlusion 7 days after the insult. Motor function was assessed for 8 weeks, and the fate of transplanted hBMSCs was examined using immunohistochemistry. As the results, the hBMSCs-PL/G-CSF showed more enhanced proliferation than the hBMSCs-FCS and hBMSCs-PL. Transplantation of hBMSCs expanded with the PL- or PL/G-CSF-containing medium equally promoted functional recovery compared with the vehicle group. Histological analysis revealed that there were no significant differences in their migration, survival, and neural differentiation in the infarct brain between the hBMSCs-PL and hBMSCs-PL/G-CSF. These findings strongly suggest that the combination of PL and G-CSF may accelerate hBMSC expansion and serve safe cell therapy for patients with ischemic stroke at clinically relevant timing.

  12. Human Tubal-Derived Mesenchymal Stromal Cells Associated with Low Level Laser Therapy Significantly Reduces Cigarette Smoke-Induced COPD in C57BL/6 mice.

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    Jean Pierre Schatzmann Peron

    Full Text Available Cigarette smoke-induced chronic obstructive pulmonary disease is a very debilitating disease, with a very high prevalence worldwide, which results in a expressive economic and social burden. Therefore, new therapeutic approaches to treat these patients are of unquestionable relevance. The use of mesenchymal stromal cells (MSCs is an innovative and yet accessible approach for pulmonary acute and chronic diseases, mainly due to its important immunoregulatory, anti-fibrogenic, anti-apoptotic and pro-angiogenic. Besides, the use of adjuvant therapies, whose aim is to boost or synergize with their function should be tested. Low level laser (LLL therapy is a relatively new and promising approach, with very low cost, no invasiveness and no side effects. Here, we aimed to study the effectiveness of human tube derived MSCs (htMSCs cell therapy associated with a 30mW/3J-660 nm LLL irradiation in experimental cigarette smoke-induced chronic obstructive pulmonary disease. Thus, C57BL/6 mice were exposed to cigarette smoke for 75 days (twice a day and all experiments were performed on day 76. Experimental groups receive htMSCS either intraperitoneally or intranasally and/or LLL irradiation either alone or in association. We show that co-therapy greatly reduces lung inflammation, lowering the cellular infiltrate and pro-inflammatory cytokine secretion (IL-1β, IL-6, TNF-α and KC, which were followed by decreased mucus production, collagen accumulation and tissue damage. These findings seemed to be secondary to the reduction of both NF-κB and NF-AT activation in lung tissues with a concomitant increase in IL-10. In summary, our data suggests that the concomitant use of MSCs + LLLT may be a promising therapeutic approach for lung inflammatory diseases as COPD.

  13. Metformin inhibits StAR expression in human endometriotic stromal cells via AMPK-mediated disruption of CREB-CRTC2 complex formation.

    Science.gov (United States)

    Xu, Jia-Ning; Zeng, Cheng; Zhou, Yan; Peng, Chao; Zhou, Ying-Fang; Xue, Qing

    2014-08-01

    Endometriosis is an estrogen-dependent disease affecting reproductive women. Metformin could have a therapeutic effect on endometriosis through regulation of local estrogen production. The aim of this study was to investigate the molecular and cellular mechanism by which metformin regulates StAR expression in human endometriotic stromal cells (ESCs). ESCs derived from ovarian endometriomas were cultured with metformin and prostaglandin E2 (PGE2). StAR mRNA was measured by quantitative PCR; pregnenolone, progesterone, and estrogen production were measured by ELISA kits; steroidogenic acute regulatory protein (StAR), AMP-activated protein kinase, cAMP response element binding protein (CREB), and CREB-regulated transcription coactivator 2 (CRTC2) protein expression were measured by Western blot assay; and CRTC2 translocation and its association with CREB were assessed by coimmunoprecipitation assay and CRTC2-CREB complex binding by a chromatin immunoprecipitation assay. 1) StAR mRNA levels in ESCs are 264 times higher than those in endometrial cells. 2) Metformin downregulates the StAR mRNA expression (maximum 31.7%) stimulated by PGE2 (2.4-fold) in ESCs. 3) PGE2 induces CRTC2 translocation and enhances its association with CREB to form a transcription complex that binds to the StAR promoter region. 4) Metformin prevents the nuclear translocation of CRTC2 by increasing AMP-activated protein kinase phosphorylation. This inhibits transcription of StAR by disrupting formation of the CREB-CRTC2 complex, involved in activation of the StAR promoter cAMP response element. We have demonstrated a detailed mechanistic analysis of StAR expression regulated by metformin in ESCs. Our data highlight a role for CRTC2 in the mechanism by which metformin inhibits StAR expression.

  14. Fate of tenogenic differentiation potential of human bone marrow stromal cells by uniaxial stretching affected by stretch-activated calcium channel agonist gadolinium.

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    Hui Yin Nam

    Full Text Available The role for mechanical stimulation in the control of cell fate has been previously proposed, suggesting that there may be a role of mechanical conditioning in directing mesenchymal stromal cells (MSCs towards specific lineage for tissue engineering applications. Although previous studies have reported that calcium signalling is involved in regulating many cellular processes in many cell types, its role in managing cellular responses to tensile loading (mechanotransduction of MSCs has not been fully elucidated. In order to establish this, we disrupted calcium signalling by blocking stretch-activated calcium channel (SACC in human MSCs (hMSCs in vitro. Passaged-2 hMSCs were exposed to cyclic tensile loading (1 Hz + 8% for 6, 24, 48, and 72 hours in the presence of the SACC blocker, gadolinium. Analyses include image observations of immunochemistry and immunofluorescence staining from extracellular matrix (ECM production, and measuring related tenogenic and apoptosis gene marker expression. Uniaxial tensile loading increased the expression of tenogenic markers and ECM production. However, exposure to strain in the presence of 20 μM gadolinium reduced the induction of almost all tenogenic markers and ECM staining, suggesting that SACC acts as a mechanosensor in strain-induced hMSC tenogenic differentiation process. Although cell death was observed in prolonged stretching, it did not appear to be apoptosis mediated. In conclusion, the knowledge gained in this study by elucidating the role of calcium in MSC mechanotransduction processes, and that in prolonged stretching results in non-apoptosis mediated cell death may be potential useful for regenerative medicine applications.

  15. Potential role of 20S proteasome in maintaining stem cell integrity of human bone marrow stromal cells in prolonged culture expansion

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Zhang, Wei-Guo; Liu, Xue-Qin; Zhu, Qian; Cheng, Xiao-Long; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Anatomy, University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Xiao, Zhi-Cheng, E-mail: zhicheng.xiao@monash.edu [Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical College, Kunming 650031 (China); Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Melbourne 3800 (Australia)

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Prolonged culture expansion retards proliferation and induces senescence of hBMSCs. Black-Right-Pointing-Pointer Reduced 20S proteasomal activity and expression potentially contribute to cell aging. Black-Right-Pointing-Pointer MG132-mediated 20S proteasomal inhibition induces senescence-like phenotype. Black-Right-Pointing-Pointer 18{alpha}-GA stimulates proteasomal activity and restores replicative senescence. Black-Right-Pointing-Pointer 18{alpha}-GA retains differentiation without affecting stem cell characterizations. -- Abstract: Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18{alpha}-glycyrrhetinic acid (18{alpha}-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.

  16. Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation

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    Nagarajan Selvamurugan

    2017-01-01

    Full Text Available Pulsed electromagnetic fields (PEMFs have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs’ cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-β signaling pathway and microRNA 21 (miR21 were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-β signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-β signaling pathway, was found to be miR21-5p’s putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-β signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

  17. Green synthesis of silver nanoparticles using Pimpinella anisum seeds: antimicrobial activity and cyt