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Sample records for adhesion proliferation differentiation

  1. Effect of Water-Glass Coating on HA and HA-TCP Samples for MSCs Adhesion, Proliferation, and Differentiation

    PubMed Central

    Bajpai, Indu; Kim, Duk Yeon; Kyong-Jin, Jung; Song, In-Hwan

    2016-01-01

    Ca-P and silicon based materials have become very popular as bone tissue engineering materials. In this study, water-glass (also known as sodium silicate glass) was coated on sintered hydroxyapatite (HA) and HA-TCP (TCP stands for tricalcium phosphate) samples and subsequently heat-treated at 600°C for 2 hrs. X-rays diffraction showed the presence of β- and α-TCP phases along with HA in the HA-TCP samples. Samples without coating, with water-glass coating, and heat-treated after water-glass coating were used to observe the adhesion and proliferation response of bone marrow derived-mesenchymal stem cells (MSCs). Cell culture was carried out for 4 hrs, 1 day, and 7 days. Interestingly, all samples showed similar response for cell adhesion and proliferation up to 7-day culture but fibronectin, E-cadherin, and osteogenic differentiation related genes (osteocalcin and osteopontin) were significantly induced in heat-treated water-glass coated HA-TCP samples. A water-glass coating on Ca-P samples was not found to influence the cell proliferation response significantly but activated some extracellular matrix genes and induced osteogenic differentiation in the MSCs. PMID:27429988

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

    PubMed

    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.

  3. The influence of high intensity terahertz radiation on mammalian cell adhesion, proliferation and differentiation

    NASA Astrophysics Data System (ADS)

    Williams, Rachel; Schofield, Amy; Holder, Gareth; Downes, Joan; Edgar, David; Harrison, Paul; Siggel-King, Michele; Surman, Mark; Dunning, David; Hill, Stephen; Holder, David; Jackson, Frank; Jones, James; McKenzie, Julian; Saveliev, Yuri; Thomsen, Neil; Williams, Peter; Weightman, Peter

    2013-01-01

    Understanding the influence of exposure of biological systems to THz radiation is becoming increasingly important. There is some evidence to suggest that THz radiation can influence important activities within mammalian cells. This study evaluated the influence of the high peak power, low average power THz radiation produced by the ALICE (Daresbury Laboratory, UK) synchrotron source on human epithelial and embryonic stem cells. The cells were maintained under standard tissue culture conditions, during which the THz radiation was delivered directly into the incubator for various exposure times. The influence of the THz radiation on cell morphology, attachment, proliferation and differentiation was evaluated. The study demonstrated that there was no difference in any of these parameters between irradiated and control cell cultures. It is suggested that under these conditions the cells are capable of compensating for any effects caused by exposure to THz radiation with the peak powers levels employed in these studies.

  4. Modulation of keratin in adhesion, proliferation, adipogenic, and osteogenic differentiation of porcine adipose-derived stem cells.

    PubMed

    Wu, Yen-Lin; Lin, Che-Wei; Cheng, Nai-Chen; Yang, Kai-Chiang; Yu, Jiashing

    2017-01-01

    Recently, keratin attracts tremendous interest because of its intrinsic ability to interact with different cells. It has the potential to serve as a controllable extracellular matrix protein that can be used to demonstrate cell mechanism and cell-matrix interaction. However, there have been relatively few studies on the effects of keratin on stem cells. In the present work, we study the effects of human keratin on porcine adipose-derived stem cells (pASCs) and a series of selective cell lines: 3T3 fibroblasts, Madin-Darby canine kidney (MDCK) cells, and MG63 osteoblasts. Relative to un-treated culture plate, our results showed that keratin coating substrates promote cell adhesion and proliferation to above cell lines. Keratin also improved pASCs adhesion, proliferation, and enhanced cell viability. Evaluation of genetic markers showed that adipogenic and osteogenic differentiations of pASCs can be successfully induced, thus demonstrating that keratin did not influence the stemness of pASCs. Furthermore, keratin improved adipogenic differentiations of pASCs in terms of up-regulations in lipoprotein lipase, peroxisome proliferator-activated receptor gamma, and CCAAT-enhancer-binding protein alpha. The osteogenic markers type I collagen, runt-related transcription factor 2, and vitamin D receptor were also upregulated when pASCs cultured on keratin substrates. Therefore, keratin can serve as a biological derived material for surface modification and scaffold fabrication for biomedical purpose. The combination of keratin with stem cells may be a potential candidate for tissue repair in the field of regenerative medicine. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 180-192, 2017.

  5. Mimicking bone extracellular matrix: integrin-binding peptidomimetics enhance osteoblast-like cells adhesion, proliferation, and differentiation on titanium.

    PubMed

    Fraioli, Roberta; Rechenmacher, Florian; Neubauer, Stefanie; Manero, José M; Gil, Javier; Kessler, Horst; Mas-Moruno, Carlos

    2015-04-01

    Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins αvβ3 and α5β1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials.

  6. Influence of different modifications of a calcium phosphate bone cement on adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells.

    PubMed

    Vater, Corina; Lode, Anja; Bernhardt, Anne; Reinstorf, Antje; Heinemann, Christiane; Gelinsky, Michael

    2010-03-15

    Collagen and noncollagenous proteins of the extracellular bone matrix are able to stimulate bone cell activities and bone healing. The modification of calcium phosphate bone cements used as temporary bone replacement materials with these proteins seems to be a promising approach to accelerate new bone formation. In this study, we investigated adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells (hBMSC) on Biocement D/collagen composites which have been modified with osteocalcin and O-phospho-L-serine. Modification with osteocalcin was carried out by its addition to the cement precursor before setting as well as by functionalization of the cement samples after setting and sterilization. hBMSC were cultured on these samples for 28 days with and without osteogenic supplements. We found a positive impact especially of the phosphoserine-modifications but also of both osteocalcin-modifications on differentiation of hBMSC indicated by higher expression of the osteoblastic markers matrix metalloproteinase-13 and bone sialo protein II. For hBMSC cultured on phosphoserine-containing composites, an increased proliferation has been observed. However, in case of the osteocalcin-modified samples, only osteocalcin adsorbed after setting and sterilization of the cement samples was able to promote initial adhesion and proliferation of hBMSC. The addition of osteocalcin before setting results in a finer microstructure but the biological activity of osteocalcin might be impaired due to the sterilization process. Thus, our data indicate that the initial adhesion and proliferation of hBMSC is enhanced rather by the biological activity of osteocalcin than by the finer microstructure.

  7. Indirect coating of RGD peptides using a poly-L-lysine spacer enhances jaw periosteal cell adhesion, proliferation, and differentiation into osteogenic tissue.

    PubMed

    Ardjomandi, N; Klein, C; Kohler, K; Maurer, A; Kalbacher, H; Niederländer, J; Reinert, S; Alexander, D

    2012-08-01

    The aim of our study was to generate a biofunctionalized, three-dimensional (3D) biomaterial to enhance jaw periosteal cell (JPC) adhesion and differentiation into osteogenic tissue. Therefore, open-cell polylactic acid (OPLA) scaffolds were coated covalently with different RGD peptides (a conserved recognition sequence of the most ECM proteins--arginine-glycine-asparagine) and different coating variants. The linear and cyclic RGD peptides were either applied directly or indirectly via a poly-L-lysine (PLL) spacer. JPCs were analyzed on coated constructs in 2D and 3D cultures and showed enhanced rates for indirectly coated scaffolds using the PLL spacer. By gene expression, we detected significantly increased levels of osteogenic marker genes, such as alkaline phosphatase, RUNX2, and AMELY in JPCs seeded onto PLL/linear RGD constructs compared to the otherwise-coated constructs. An analysis of the JPC mineralization capacity revealed the highest amounts of calcium-phosphate precipitates in cells growing within the PLL/linear scaffolds. Additionally, the JPC adhesion behavior on OPLA scaffolds seems to be mediated by ITGB3, ITGB1, and ITGAV, as shown by blocking assays. We concluded that coating of OPLA constructs with linear RGD peptides via PLL represents a suitable approach for functionalizing the polymer surface and enhancing adhesion, proliferation, and mineralization of JPCs.

  8. Effects of decellularized matrices derived from periodontal ligament stem cells and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro.

    PubMed

    Heng, Boon Chin; Zhu, Shaoyue; Xu, Jianguang; Yuan, Changyong; Gong, Ting; Zhang, Chengfei

    2016-04-01

    A major bottleneck to the therapeutic applications of dental pulp stem cells (DPSC) are their limited proliferative capacity ex vivo and tendency to undergo senescence. This may be partly due to the sub-optimal in vitro culture milieu, which could be improved by an appropriate extracellular matrix substratum. This study therefore examined decellularized matrix (DECM) from stem cells derived from human exfoliated deciduous teeth (SHED) and periodontal ligament stem cells (PDLSC), as potential substrata for DPSC culture. Both SHED-DECM and PDLSC-DECM promoted rapid adhesion and spreading of newly-seeded DPSC compared to bare polystyrene (TCPS), with vinculin immunocytochemistry showing expression of more focal adhesions by newly-adherent DPSC cultured on DECM versus TCPS. Culture of DPSC on SHED-DECM and PDLSC-DECM yielded higher proliferation of cell numbers compared to TCPS. The qRT-PCR data showed significantly higher expression of nestin by DPSC cultured on DECM versus the TCPS control. Osteogenic differentiation of DPSC was enhanced by culturing on PDLSC-DECM and SHED-DECM versus TCPS, as demonstrated by alizarin red S staining for mineralized calcium deposition, alkaline phosphatase assay and qRT-PCR analysis of key osteogenic marker expression. Hence, both SHED-DECM and PDLSC-DECM could enhance the ex vivo culture of DPSC under both non-inducing and osteogenic-inducing conditions.

  9. MiR-9-5p, miR-675-5p and miR-138-5p Damages the Strontium and LRP5-Mediated Skeletal Cell Proliferation, Differentiation, and Adhesion.

    PubMed

    Sun, Tianhao; Leung, Frankie; Lu, William W

    2016-02-15

    This study was designed to evaluate the effects of strontium on the expression levels of microRNAs (miRNAs) and to explore their effects on skeletal cell proliferation, differentiation, adhesion, and apoptosis. The targets of these miRNAs were also studied. Molecular cloning, cell proliferation assay, cell apoptosis assay, quantitative real-time PCR, and luciferase reporter assay were used. Strontium altered the expression levels of miRNAs in vitro and in vivo. miR-9-5p, miR-675-5p, and miR-138-5p impaired skeletal cell proliferation, cell differentiation and cell adhesion. miR-9-5p and miR-675-5p induced MC3T3-E1 cell apoptosis more specifically than miR-138-5p. miR-9-5p, miR-675-5p, and miR-138-5p targeted glycogen synthase kinase 3 β (GSK3β), ATPase Aminophospholipid Transporter Class I Type 8A Member 2 (ATP8A2), and Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1), respectively. Low-density lipoprotein receptor-related protein 5 (LRP5) played a positive role in skeletal development. miR-9-5p, miR-675-5p, and miR-138-5p damage strontium and LRP5-mediated skeletal cell proliferation, differentiation, and adhesion, and induce cell apoptosis by targeting GSK3β, ATP8A2, and EIF4EBP1, respectively.

  10. Effect of various concentrations of Ti in hydrocarbon plasma polymer films on the adhesion, proliferation and differentiation of human osteoblast-like MG-63 cells

    NASA Astrophysics Data System (ADS)

    Vandrovcova, Marta; Grinevich, Andrey; Drabik, Martin; Kylian, Ondrej; Hanus, Jan; Stankova, Lubica; Lisa, Vera; Choukourov, Andrei; Slavinska, Danka; Biederman, Hynek; Bacakova, Lucie

    2015-12-01

    Hydrocarbon polymer films (ppCH) enriched with various concentrations of titanium were deposited on microscopic glass slides by magnetron sputtering from a Ti target. The maximum concentration of Ti (about 20 at.%) was achieved in a pure argon atmosphere. The concentration of Ti decreased rapidly after n-hexane vapors were introduced into the plasma discharge, and reached zero values at n-hexane flow of 0.66 sccm. The decrease in Ti concentration was associated with decreasing oxygen and titanium carbide concentration in the films, decreasing wettability (the water drop contact angle increased from 20° to 91°) and decreasing root-mean-square roughness (from 3.3 nm to 1.0 nm). The human osteoblast-like MG-63 cells cultured on pure ppCH films and on films with 20 at.% of Ti showed relatively high concentrations of ICAM-1, a marker of cell immune activation. Lower concentrations of Ti (mainly 5 at.%) improved cell adhesion and osteogenic differentiation, as revealed by higher concentrations of talin, vinculin and osteocalcin. Higher Ti concentrations (15 at.%) supported cell growth, as indicated by the highest final cell population densities on day 7 after seeding. Thus, enrichment of ppCH films with appropriate concentrations of Ti makes these films more suitable for potential coatings of bone implants.

  11. Adhesion and proliferation of human mesenchymal stem cells from dental pulp on porous silicon scaffolds.

    PubMed

    Collart-Dutilleul, Pierre-Yves; Secret, Emilie; Panayotov, Ivan; Deville de Périère, Dominique; Martín-Palma, Raúl J; Torres-Costa, Vicente; Martin, Marta; Gergely, Csilla; Durand, Jean-Olivier; Cunin, Frédérique; Cuisinier, Frédéric J

    2014-02-12

    In regenerative medicine, stem-cell-based therapy often requires a scaffold to deliver cells and/or growth factors to the injured site. Porous silicon (pSi) is a promising biomaterial for tissue engineering as it is both nontoxic and bioresorbable. Moreover, surface modification can offer control over the degradation rate of pSi and can also promote cell adhesion. Dental pulp stem cells (DPSC) are pluripotent mesenchymal stem cells found within the teeth and constitute a readily source of stem cells. Thus, coupling the good proliferation and differentiation capacities of DPSC with the textural and chemical properties of the pSi substrates provides an interesting approach for therapeutic use. In this study, the behavior of human DPSC is analyzed on pSi substrates presenting pores of various sizes, 10 ± 2 nm, 36 ± 4 nm, and 1.0 ± 0.1 μm, and undergoing different chemical treatments, thermal oxidation, silanization with aminopropyltriethoxysilane (APTES), and hydrosilylation with undecenoic acid or semicarbazide. DPSC adhesion and proliferation were followed for up to 72 h by fluorescence microscopy, scanning electron microscopy (SEM), enzymatic activity assay, and BrdU assay for mitotic activity. Porous silicon with 36 nm pore size was found to offer the best adhesion and the fastest growth rate for DPSC compared to pSi comporting smaller pore size (10 nm) or larger pore size (1 μm), especially after silanization with APTES. Hydrosilylation with semicarbazide favored cell adhesion and proliferation, especially mitosis after cell adhesion, but such chemical modification has been found to led to a scaffold that is stable for only 24-48 h in culture medium. Thus, semicarbazide-treated pSi appeared to be an appropriate scaffold for stem cell adhesion and immediate in vivo transplantation, whereas APTES-treated pSi was found to be more suitable for long-term in vitro culture, for stem cell proliferation and differentiation.

  12. Nanomechanics controls neuronal precursors adhesion and differentiation.

    PubMed

    Migliorini, Elisa; Ban, Jelena; Grenci, Gianluca; Andolfi, Laura; Pozzato, Alessandro; Tormen, Massimo; Torre, Vincent; Lazzarino, Marco

    2013-08-01

    The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell-substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine.

  13. Functionalization of CoCr surfaces with cell adhesive peptides to promote HUVECs adhesion and proliferation

    NASA Astrophysics Data System (ADS)

    Castellanos, Maria Isabel; Mas-Moruno, Carlos; Grau, Anna; Serra-Picamal, Xavier; Trepat, Xavier; Albericio, Fernando; Joner, Michael; Gil, Francisco Javier; Ginebra, Maria Pau; Manero, Jose María; Pegueroles, Marta

    2017-01-01

    Biomimetic surface modification with peptides that have specific cell-binding moieties is a promising approach to improve endothelialization of metal-based stents. In this study, we functionalized CoCr surfaces with RGDS, REDV, YIGSR peptides and their combinations to promote endothelial cells (ECs) adhesion and proliferation. An extensive characterization of the functionalized surfaces was performed by XPS analysis, surface charge and quartz crystal microbalance with dissipation monitoring (QCM-D), which demonstrated the successful immobilization of the peptides to the surface. Cell studies demonstrated that the covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represents the most powerful strategy to enhance the early stages of ECs adhesion and proliferation, indicating a positive synergistic effect between the two peptide motifs. Although these peptide sequences slightly increased smooth muscle cells (SMCs) adhesion, these values were ten times lower than those observed for ECs. The combination of RGDS with the REDV sequence did not show synergistic effects in promoting the adhesion or proliferation of ECs. The strategy presented in this study holds great potential to overcome clinical limitations of current metal stents by enhancing their capacity to support surface endothelialization.

  14. Critical role of heparin binding domains of ameloblastin for dental epithelium cell adhesion and ameloblastoma proliferation.

    PubMed

    Sonoda, Akira; Iwamoto, Tsutomu; Nakamura, Takashi; Fukumoto, Emiko; Yoshizaki, Keigo; Yamada, Aya; Arakaki, Makiko; Harada, Hidemitsu; Nonaka, Kazuaki; Nakamura, Seiji; Yamada, Yoshihiko; Fukumoto, Satoshi

    2009-10-02

    AMBN (ameloblastin) is an enamel matrix protein that regulates cell adhesion, proliferation, and differentiation of ameloblasts. In AMBN-deficient mice, ameloblasts are detached from the enamel matrix, continue to proliferate, and form a multiple cell layer; often, odontogenic tumors develop in the maxilla with age. However, the mechanism of AMBN functions in these biological processes remains unclear. By using recombinant AMBN proteins, we found that AMBN had heparin binding domains at the C-terminal half and that these domains were critical for AMBN binding to dental epithelial cells. Overexpression of full-length AMBN protein inhibited proliferation of human ameloblastoma AM-1 cells, but overexpression of heparin binding domain-deficient AMBN protein had no inhibitory effect. In full-length AMBN-overexpressing AM-1 cells, the expression of Msx2, which is involved in the dental epithelial progenitor phenotype, was decreased, whereas the expression of cell proliferation inhibitors p21 and p27 was increased. We also found that the expression of enamelin, a marker of differentiated ameloblasts, was induced, suggesting that AMBN promotes odontogenic tumor differentiation. Thus, our results suggest that AMBN promotes cell binding through the heparin binding sites and plays an important role in preventing odontogenic tumor development by suppressing cell proliferation and maintaining differentiation phenotype through Msx2, p21, and p27.

  15. Cell proliferation and differentiation in chemical leukemogenesis

    NASA Technical Reports Server (NTRS)

    Irons, R. D.; Stillman, W. S.; Clarkson, T. W. (Principal Investigator)

    1993-01-01

    In tissues such as bone marrow with normally high rates of cell division, proliferation is tightly coordinated with cell differentiation. Survival, proliferation and differentiation of early hematopoietic progenitor cells depend on the growth factors, interleukin 3 (IL-3) and/or granulocyte-macrophage colony stimulating factor (GM-CSF) and their synergism with other cytokines. We provide evidence that a characteristic shared by a diverse group of compounds with demonstrated leukemogenic potential is the ability to act synergistically with GM-CSF. This results in an increase in recruitment of a resting population of hematopoietic progenitor cells normally unresponsive to the cytokine and a twofold increase in the size of the proliferating cell population normally regarded to be at risk of transformation in leukemogenesis. These findings support the possibility that transient alterations in hematopoietic progenitor cell differentiation may be an important factor in the early stages of development of leukemia secondary to chemical or drug exposure.

  16. Cell Adhesion and Proliferation on Sulfonated and Non-Modified Chitosan Films.

    PubMed

    Martínez-Campos, Enrique; Civantos, Ana; Redondo, Juan Alfonso; Guzmán, Rodrigo; Pérez-Perrino, Mónica; Gallardo, Alberto; Ramos, Viviana; Aranaz, Inmaculada

    2016-09-15

    Three types of chitosan-based films have been prepared and evaluated: a non-modified chitosan film bearing cationizable aliphatic amines and two films made of N-sulfopropyl chitosan derivatives bearing both aliphatic amines and negative sulfonate groups at different ratios. Cell adhesion and proliferation on chitosan films of C2C12 pre-myoblastic cells and B16 cells as tumoral model have been tested. A differential cell behavior has been observed on chitosan films due to their different surface modification. B16 cells have shown lower vinculin expression when cultured on sulfonated chitosan films. This study shows how the interaction among cells and material surface can be modulated by physicochemical characteristics of the biomaterial surface, altering tumoral cell adhesion and proliferation processes.

  17. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation.

    PubMed

    Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi; Patel, Vyomesh; Fukumoto, Satoshi; Yamada, Yoshihiko

    2014-12-15

    The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development.

  18. Cell adhesion and proliferation on polyethylene grafted with Au nanoparticles

    NASA Astrophysics Data System (ADS)

    Kasálková, N. Slepičková; Slepička, P.; Kolská, Z.; Sajdl, P.; Bačáková, L.; Rimpelová, S.; Švorčík, V.

    2012-02-01

    Plasma treatment and subsequent Au nano-particles grafting of polyethylene (PE) lead to changes in surface morphology, roughness and wettability, significantly increasing the attractiveness of the material for cells. The PE samples were exposed to argon plasma. Plasma modified PE was chemically grafted by immersion to biphenyldithiol and consequently into solution of Au nano-particles. Changes in chemical structure of the modified PE were studied using X-ray Photoelectron Spectroscopy (XPS) and electrokinetic analysis ( ζ-potential). The surface wettability of the modified PE samples was examined by measurement of the contact angle by standard goniometry. The surface morphology of the plasma modified PE and that grafted with Au nano-particles was studied by Atomic Force Microscopy (AFM). The modified PE samples were seeded with rat vascular smooth muscle cells (VSMCs) and their adhesion and proliferation were studied. Chemically bounded biphenyldithiol increases the number of the incorporated gold nano-particles and changes sample surface properties. The presence of the biphenyldithiol and the gold nano-particles on the PE surface influences dramatically adhesion and proliferation of VSMCs.

  19. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro.

    PubMed

    Zhao, Ningbo; Wang, Xin; Qin, Lei; Guo, Zhengze; Li, Dehua

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation.

  20. Disruption of the novel gene fad104 causes rapid postnatal death and attenuation of cell proliferation, adhesion, spreading and migration

    SciTech Connect

    Nishizuka, Makoto; Kishimoto, Keishi; Kato, Ayumi; Ikawa, Masahito; Okabe, Masaru; Sato, Ryuichiro; Niida, Hiroyuki; Nakanishi, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2009-03-10

    The molecular mechanisms at the beginning of adipogenesis remain unknown. Previously, we identified a novel gene, fad104 (factor for adipocyte differentiation 104), transiently expressed at the early stage of adipocyte differentiation. Since the knockdown of the expression of fad104 dramatically repressed adipogenesis, it is clear that fad104 plays important roles in adipocyte differentiation. However, the physiological roles of fad104 are still unknown. In this study, we generated fad104-deficient mice by gene targeting. Although the mice were born in the expected Mendelian ratios, all died within 1 day of birth, suggesting fad104 to be crucial for survival after birth. Furthermore, analyses of mouse embryonic fibroblasts (MEFs) prepared from fad104-deficient mice provided new insights into the functions of fad104. Disruption of fad104 inhibited adipocyte differentiation and cell proliferation. In addition, cell adhesion and wound healing assays using fad104-deficient MEFs revealed that loss of fad104 expression caused a reduction in stress fiber formation, and notably delayed cell adhesion, spreading and migration. These results indicate that fad104 is essential for the survival of newborns just after birth and important for cell proliferation, adhesion, spreading and migration.

  1. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

    SciTech Connect

    Zhao, Ningbo Wang, Xin Qin, Lei Guo, Zhengze Li, Dehua

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.

  2. On the role of differential adhesion in gangliogenesis in the enteric nervous system.

    PubMed

    Hackett-Jones, Emily J; Landman, Kerry A; Newgreen, Donald F; Zhang, Dongcheng

    2011-10-21

    A defining characteristic of the normal development of the enteric nervous system (ENS) is the existence of mesoscale patterned entities called ganglia. Ganglia are clusters of neurons with associated enteric neural crest (ENC) cells, which form in the simultaneously growing gut wall. At first the precursor ENC cells proliferate and gradually differentiate to produce the enteric neurons; these neurons form clusters with ENC scattered around and later lying on the periphery of neuronal clusters. By immunolabelling neural cell-cell adhesion molecules, we infer that the adhesive capacity of neurons is greater than that of ENC cells. Using a discrete mathematical model, we test the hypothesis that local rules governing differential adhesion of neuronal agents and ENC agents will produce clusters which emulate ganglia. The clusters are relatively stable, relatively uniform and small in size, of fairly uniform spacing, with a balance between the number of neuronal and ENC agents. These features are attained in both fixed and growing domains, reproducing respectively organotypic in vitro and in vivo observations. Various threshold criteria governing ENC agent proliferation and differentiation and neuronal agent inhibition of differentiation are important for sustaining these characteristics. This investigation suggests possible explanations for observations in normal and abnormal ENS development.

  3. Keratinocytes display normal proliferation, survival and differentiation in conditional beta4-integrin knockout mice.

    PubMed

    Raymond, Karine; Kreft, Maaike; Janssen, Hans; Calafat, Jero; Sonnenberg, Arnoud

    2005-03-01

    The alpha6beta4 integrin is located at the basal surface of keratinocytes, in hemidesmosomal structures that mediate stable adhesion of epidermal cells to the underlying basement membrane component laminin-5. The absence of alpha6beta4 integrin causes junctional epidermolysis bullosa, a severe blistering disease of the skin leading to perinatal death, confirming its essential role in mediating strong keratinocyte adhesion. Several studies have suggested that alpha6beta4 integrin can also regulate signaling cascades that control cell proliferation, survival and migration through a mechanism independent of its adhesive function. We have generated a conditional knockout mouse strain, in which the gene encoding the beta4 integrin subunit (Itgb4) was inactivated only in small stretches of the skin. These mice were viable and permitted an accurate analysis of the consequences of the loss of beta4 on various biological processes by comparing beta4-positive and -negative parts of the skin in the same animal. Despite the complete loss of hemidesmosomes in regions lacking alpha6beta4 integrin, the distribution of a range of adhesion receptors and basement membrane proteins was unaltered. Moreover, loss of alpha6beta4 did not affect squamous differentiation, proliferation or survival, except for areas in which keratinocytes had detached from the basement membrane. These in vivo observations were confirmed in vitro by using immortalized keratinocytes - derived from beta4-subunit conditional knockout mice - from which the gene encoding beta4 had been deleted by Cre-mediated recombination. Consistent with the established role of alpha6beta4 in adhesion strengthening, its loss from cells was found to increase their motility. Our findings clearly demonstrate that, after birth, epidermal differentiation, proliferation and survival all proceed normally in the absence of alpha6beta4, provided that cell adhesion is not compromised.

  4. Effects of SOX2 on Proliferation, Migration and Adhesion of Human Dental Pulp Stem Cells.

    PubMed

    Liu, Pengfei; Cai, Jinglei; Dong, Delu; Chen, Yaoyu; Liu, Xiaobo; Wang, Yi; Zhou, Yulai

    2015-01-01

    As a key factor for cell pluripotent and self-renewing phenotypes, SOX2 has attracted scientists' attention gradually in recent years. However, its exact effects in dental pulp stem cells (DPSCs) are still unclear. In this study, we mainly investigated whether SOX2 could affect some biological functions of DPSCs. DPSCs were isolated from the dental pulp of human impacted third molar. SOX2 overexpressing DPSCs (DPSCs-SOX2) were established through retroviral infection. The effect of SOX2 on cell proliferation, migration and adhesion ability was evaluated with CCK-8, trans-well system and fibronectin-induced cell attachment experiment respectively. Whole genome expression of DPSCs-SOX2 was analyzed with RNA microarray. Furthermore, a rescue experiment was performed with SOX2-siRNA in DPSC-SOX2 to confirm the effect of SOX2 overexpression in DPSCs. We found that SOX2 overexpression could result in the enhancement of cell proliferation, migration, and adhesion in DPSCs obviously. RNA microarray analysis indicated that some key genes in the signal pathways associated with cell cycle, migration and adhesion were upregulated in different degree, and the results were further confirmed with qPCR and western-blot. Finally, DPSC-SOX2 transfected with SOX2-siRNA showed a decrease of cell proliferation, migration and adhesion ability, which further confirmed the biological effect of SOX2 in human DPSCs. This study indicated that SOX2 could improve the cell proliferation, migration and adhesion ability of DPSCs through regulating gene expression about cell cycle, migration and adhesion, and provided a novel strategy to develop seed cells with strong proliferation, migration and adhesion ability for tissue engineering.

  5. Control of mesenchymal stem cell phenotype and differentiation depending on cell adhesion mechanism.

    PubMed

    Kang, J; Park, H M; Kim, Y W; Kim, Y H; Varghese, S; Seok, H K; Kim, Y G; Kim, S H

    2014-11-25

    Control of cell-matrix adhesion has become an important issue in the regulation of stem cell function. In this study, a maltose-binding protein (MBP)-linked basic fibroblast growth factor (FGF2)-immobilised polystyrene surface (PS-MBP-FGF2) was applied as an artificial matrix to regulate integrin-mediated signalling. We sought to characterise human mesenchymal-stem cell (hMSC) behaviour in response to two different mechanisms of cell adhesion; (i) FGF2-heparan sulphate proteoglycan (HSPG)-mediated adhesion vs. (ii) fibronectin (FN)-integrin-mediated adhesion. Heparin inhibited hMSC adhesion to PS-MBP-FGF2 but not to FN-coated surface. The phosphorylation of focal adhesion kinase, cytoskeletal re-organisation, and cell proliferation were restricted in hMSCs adhering to PS-MBP-FGF2 compared to FN-coated surface. Expression of MSC markers, such as CD105, CD90 and CD166, decreased in hMSCs expanded on PS-MBP-FGF2 compared to expression in cells expanded on FN-coated surface. hMSCs that were expanded on FN-coated surface differentiated into osteogenic and adipogenic cells more readily than those that were expanded on PS-MBP-FGF2. Furthermore, we characterised the N-linked glycan structures of hMSCs depending on the cell adhesion mechanism using mass spectrometry (MS)-based quantitative techniques. MS analysis revealed that 2,3-sialylated glycans, a potential marker of stem cell function, were more abundant on hMSCs expanded on FN-coated surface than on those expanded on PS-MBP-FGF2. Thus, the differentiation potential of hMSCs is controlled by the type of adhesion substrate that might provide an idea for the design of biomaterials to control stem cell fate. Elucidation of the glycan structure on the cell membrane may help characterise hMSC function.

  6. Endothelial cell responses in terms of adhesion, proliferation, and morphology to stiffness of polydimethylsiloxane elastomer substrates.

    PubMed

    Ataollahi, Forough; Pramanik, Sumit; Moradi, Ali; Dalilottojari, Adel; Pingguan-Murphy, Belinda; Wan Abas, Wan Abu Bakar; Abu Osman, Noor Azuan

    2015-07-01

    Extracellular environments can regulate cell behavior because cells can actively sense their mechanical environments. This study evaluated the adhesion, proliferation and morphology of endothelial cells on polydimethylsiloxane (PDMS)/alumina (Al2 O3 ) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 2.5, 5, 7.5, and 10 wt % Al2 O3 at a curing temperature of 50°C for 4 h. The substrates were then characterized by mechanical, structural, and morphological analyses. The cell adhesion, proliferation, and morphology of cultured bovine aortic endothelial (BAEC) cells on substrate materials were evaluated by using resazurin assay and 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The composites (PDMS/2.5, 5, 7.5, and 10 wt % Al2 O3 ) exhibited higher stiffness than the pure PDMS substrate. The results also revealed that stiffer substrates promoted endothelial cell adhesion and proliferation and also induced spread morphology in the endothelial cells compared with lesser stiff substrates. Statistical analysis showed that the effect of time on cell proliferation depended on stiffness. Therefore, this study concludes that the addition of different Al2 O3 percentages to PDMS elevated substrate stiffness which in turn increased endothelial cell adhesion and proliferation significantly and induced spindle shape morphology in endothelial cells.

  7. Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane

    SciTech Connect

    Rodríguez-Fuentes, Nayeli; Rodríguez-Hernández, Ana G.; Enríquez-Jiménez, Juana; Alcántara-Quintana, Luz E.; Fuentes-Mera, Lizeth; Piña-Barba, María C.; Zepeda-Rodríguez, Armando; and others

    2013-05-10

    Highlights: •Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells. •Nukbone induced osteoblastic differentiation of human mesenchymal stem cells. •Results showed that Nukbone offer an excellent option for bone tissue regeneration due to properties. -- Abstract: Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

  8. Surface modifications of photocrosslinked biodegradable elastomers and their influence on smooth muscle cell adhesion and proliferation.

    PubMed

    Ilagan, Bernadette G; Amsden, Brian G

    2009-09-01

    Photocrosslinked, biodegradable elastomers based on aliphatic polyesters have many desirable features as scaffolds for smooth muscle tissue engineering. However, they lack cell adhesion motifs. To address this shortcoming, two different modification procedures were studied utilizing a high and a low crosslink density elastomer: base etching and the incorporation of acryloyl-poly(ethylene glycol) (PEG)-Gly-Arg-Gly-Asp-Ser (GRGDS) into the elastomer network during photocrosslinking. Base etching improved surface hydrophilicity without altering surface topography, but did not improve bovine aortic smooth muscle cell adhesion. Incorporation of PEG-GRGDS into the elastomer network significantly improved cell adhesion for both high and low crosslink density elastomers, with a greater effect with the higher crosslink density elastomer. Incorporation of GRGDS into the high crosslink density elastomer also enhanced smooth muscle cell proliferation, while proliferation on the low crosslink density unmodified, base etched, and PEG-GRGDS incorporated elastomers was significantly greater than on the high crosslink density unmodified and base etched elastomer.

  9. Bioinspired superhydrophobic poly(L-lactic acid) surfaces control bone marrow derived cells adhesion and proliferation.

    PubMed

    Alves, Natália M; Shi, Jun; Oramas, Elena; Santos, José L; Tomás, Helena; Mano, João F

    2009-11-01

    The aptitude of a cell to adhere, migrate, and differentiate on a compact substrate or scaffold is important in the field of tissue engineering and biomaterials. It is well known that cell behavior can be controlled and guided through the change in micro- and nano-scale topographic features. In this work, we intend to demonstrate that special topographic features that control wettability may also have an important role in the biological performance of biodegradable substrates. Poly(L-lactic acid) surfaces with superhydrophobic characteristics were produced, based on the so-called Lotus effect, exhibiting dual micro- and nano-scale roughness. The water contact angle could be higher than 150 degrees and a value of that order could be kept even upon immersion in a simulated body fluid solution for more than 20 days. Such water repellent surfaces were found to prevent adhesion and proliferation of bone marrow derived cells previously isolated from the femurs of 6-week-old male Wistar rats, when compared with smoother surfaces prepared by simple solvent casting. Such results demonstrate that these superhydrophobic surfaces may be used to control cell behavior onto biodegradable substrates.

  10. miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

    PubMed Central

    Amelio, Ivano; Lena, Anna Maria; Viticchiè, Giuditta; Shalom-Feuerstein, Ruby; Terrinoni, Alessandro; Dinsdale, David; Russo, Giandomenico; Fortunato, Claudia; Bonanno, Elena; Spagnoli, Luigi Giusto; Aberdam, Daniel; Knight, Richard Austen

    2012-01-01

    During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis. PMID:23071155

  11. Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

    PubMed

    Patrulea, V; Hirt-Burri, N; Jeannerat, A; Applegate, L A; Ostafe, V; Jordan, O; Borchard, G

    2016-05-20

    RGD peptide sequences are known to regulate cellular activities by interacting with α5β1, αvβ5 and αvβ3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3 μg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation.

  12. Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation.

    PubMed

    Mobasseri, Rezvan; Tian, Lingling; Soleimani, Masoud; Ramakrishna, Seeram; Naderi-Manesh, Hossein

    2017-01-29

    Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on different substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation.

  13. Simulation of proliferation and differentiation of cells in a stem-cell niche

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2008-10-01

    Stem-cell niches represent microscopic compartments formed of environmental cells that nurture stem cells and enable them to maintain tissue homeostasis. The spatio-temporal kinetics of proliferation and differentiation of cells in such niches depend on the specifics of the niche structure and on adhesion and communication between cells and may also be influenced by spatial constraints on cell division. We propose a generic lattice model, taking all these factors into account, and systematically illustrate their role. The model is motivated by the experimental data available for the niches located in the subventricular zone of adult mammalian brain. The general conclusions drawn from our Monte Carlo simulations are applicable to other niches as well. One of our main findings is that the kinetics under consideration are highly stochastic due to a relatively small number of cells proliferating and differentiating in a niche and the autocatalytic character of the symmetric cell division. In particular, the kinetics exhibit huge stochastic bursts especially if the adhesion between cells is taken into account. In addition, the results obtained show that despite the small number of cells present in stem-cell niches, their arrangement can be predetermined to appreciable extent provided that the adhesion of different cells is different so that they tend to segregate.

  14. The effects of micro arc oxidation of gamma titanium aluminide surfaces on osteoblast adhesion and differentiation.

    PubMed

    Santiago-Medina, Pricilla; Sundaram, Paul A; Diffoot-Carlo, Nanette

    2014-06-01

    The adhesion and proliferation of human fetal osteoblasts, hFOB 1.19, on micro arc oxidized (MAO) gamma titanium aluminide (γTiAl) surfaces were examined in vitro. Cells were seeded on MAO treated γTiAl disks and incubated for 3 days at 33.5 °C and subsequently for 7 days at 39.5 °C. Samples were then analyzed by scanning electron microscopy (SEM) and alkaline phosphatase assay (ALP) to evaluate cell adhesion and differentiation, respectively. Similar Ti-6Al-4V alloy samples were used for comparison. Untreated γTiAl and Ti-6Al-4V disks to study the effect of micro arc oxidation and glass coverslips as cell growth controls were also incubated concurrently. The ALP Assay results, at 10 days post seeding, showed significant differences in cell differentiation, with P values <0.05 between MAO γTiAl and MAO Ti-6Al-4V with respect to the corresponding untreated alloys. While SEM images showed that hFOB 1.19 cells adhered and proliferated on all MAO and untreated surfaces, as well as on glass coverslips at 10 days post seeding, cell differentiation, determined by the ALP assay, was significantly higher for the MAO alloys.

  15. The Effects Of Micro Arc Oxidation Of Gamma Titanium Aluminide Surfaces On Osteoblast Adhesion And Differentiation

    PubMed Central

    Santiago-Medina, Pricilla; Sundaram, Paul A.; Diffoot-Carlo, Nanette

    2014-01-01

    The adhesion and proliferation of human fetal osteoblasts, hFOB 1.19, on micro arc oxidized (MAO) gamma titanium aluminide (γTiAl) surfaces were examined in vitro. Cells were seeded on MAO treated γTiAl disks and incubated for 3 days at 33.5°C and subsequently for 7 days at 39.5°C. Samples were then analyzed by Scanning Electron Microscopy (SEM) and the Alkaline Phosphatase Assay (ALP) to evaluate cell adhesion and differentiation, respectively. Similar Ti-6Al-4V alloy samples were used for comparison. Untreated γTiAl and Ti-6Al-4V disks, to study the effect of micro arc oxidation and glass coverslips as cell growth controls were also incubated concurrently. The ALP Assay results, at 10 days post seeding, showed significant differences in cell differentiation, with p values < 0.05 between MAO γTiAl and MAO Ti-6Al-4V with respect to the corresponding untreated alloys. While SEM images showed that hFOB 1.19 cells adhered and proliferated on all MAO and untreated surfaces, as well as on glass coverslips at 10 days post seeding, cell differentiation, determined by the ALP assay, was significantly higher for the MAO alloys. PMID:24577944

  16. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation.

    PubMed

    Kobayashi, Tatsuya; Lu, Jun; Cobb, Bradley S; Rodda, Stephen J; McMahon, Andrew P; Schipani, Ernestina; Merkenschlager, Matthias; Kronenberg, Henry M

    2008-02-12

    Small noncoding RNAs, microRNAs (miRNAs), bind to messenger RNAs through base pairing to suppress gene expression. Despite accumulating evidence that miRNAs play critical roles in various biological processes across diverse organisms, their roles in mammalian skeletal development have not been demonstrated. Here, we show that Dicer, an essential component for biogenesis of miRNAs, is essential for normal skeletal development. Dicer-null growth plates show a progressive reduction in the proliferating pool of chondrocytes, leading to severe skeletal growth defects and premature death of mice. The reduction of proliferating chondrocytes in Dicer-null growth plates is caused by two distinct mechanisms: decreased chondrocyte proliferation and accelerated differentiation into postmitotic hypertrophic chondrocytes. These defects appear to be caused by mechanisms downstream or independent of the Ihh-PTHrP signaling pathway, a pivotal signaling system that regulates chondrocyte proliferation and differentiation. Microarray analysis of Dicer-null chondrocytes showed limited expression changes in miRNA-target genes, suggesting that, in the majority of cases, chondrocytic miRNAs do not directly regulate target RNA abundance. Our results demonstrate the critical role of the Dicer-dependent pathway in the regulation of chondrocyte proliferation and differentiation during skeletal development.

  17. Peptides Regulate Cortical Thymocytes Differentiation, Proliferation, and Apoptosis

    PubMed Central

    Khavinson, V. Kh.; Polyakova, V. O.; Linkova, N. S.; Dudkov, A. V.; Kvetnoy, I. M.

    2011-01-01

    The processes of differentiation, proliferation, and apoptosis were studied in a cell culture of human cortical thymocytes under the influence of short peptides T-32 (Glu-Asp-Ala) and T-38 (Lys-Glu-Asp). Peptides T-32 and T-38 amplified cortical thymocytes differentiation towards regulatory T cells, increased their proliferative activity, and decreased the level of apoptosis. Moreover, peptides under study stimulated proliferative and antiapoptotic activity of the mature regulatory T cells. PMID:22312461

  18. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

    SciTech Connect

    Takabe, Piia; Bart, Geneviève; Ropponen, Antti; Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

    2015-09-10

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.

  19. Cholic acid functionalized star poly(DL-lactide) for promoting cell adhesion and proliferation.

    PubMed

    Fu, Hui-Li; Zou, Tao; Cheng, Si-Xue; Zhang, Xian-Zheng; Zhuo, Ren-Xi

    2007-01-01

    Cholic acid functionalized star poly(DL-lactide) was synthesized through the ring-opening polymerization of DL-lactide initiated by cholic acid. The properties and cell behaviour of the cholic acid functionalized star poly(DL-lactide) were investigated as compared with linear poly(DL-lactide)s with different molecular weights and a star poly(DL-lactide) initiated by glycerol. In comparison to linear poly(DL-lactide)s, the cholic acid functionalized star poly(DL-lactide) had better wettability and slightly higher surface energy. The cell adhesion and proliferation on different materials were evaluated using two types of cells, 3T3 mouse fibroblasts and ECV304 human endothelial cells. Compared with the linear poly(DL-lactide)s, the cholic acid functionalized star poly(DL-lactide) showed obviously improved property for cell adhesion. The cell proliferation on the cholic acid functionalized star poly(DL-lactide) was also enhanced. The improvement in cell proliferation was not so significant as compared with the improvement in cell adhesion. This modification strategy provides an effective and simple way to promote cell attachment and growth in tissue engineering.

  20. Interleukin-1 regulates proliferation and differentiation of oligodendrocyte progenitor cells.

    PubMed

    Vela, José M; Molina-Holgado, Eduardo; Arévalo-Martín, Angel; Almazán, Guillermina; Guaza, Carmen

    2002-07-01

    Interleukin-1 (IL-1) is a pleiotropic cytokine expressed during normal CNS development and in inflammatory demyelinating diseases, but remarkably little is known about its effect on oligodendroglial cells. In this study we explored the role of IL-1beta in oligodendrocyte progenitors and differentiated oligodendrocytes. The effects of IL-1beta were compared to those of IL-1 receptor antagonist, the specific inhibitor of IL-1 activity, since progenitors and differentiated oligodendrocytes produce IL-1beta and express IL-1 receptors. Unlike other proinflammatory cytokines (TNFalpha and IFNgamma), IL-1beta was not toxic for oligodendrocyte lineage cells. However, this cytokine inhibited proliferation of oligodendrocyte progenitors in the presence of growth factors (PDGF plus bFGF). This was evidenced by a significant decrease in both cells incorporating bromodeoxyuridine (45%) and total cell numbers (57%) after 6 days of treatment. Interestingly, IL-1beta blocked proliferation at the late progenitor/prooligodendrocyte (O4+) stage but did not affect proliferation of early progenitors (A2B5+). Inhibition of proliferation paralleled with promotion of differentiation, as revealed by the increased percentage of R-mab+ cells (6.7-fold). Moreover, when oligodendrocyte progenitors were allowed to differentiate in the absence of growth factors, treatment with IL-1beta promoted maturation to the MBP+ stage (4.2-fold) and survival of differentiating oligodendrocytes (2.1-fold). Regarding intracellular signaling, IL-1beta activated the p38 mitogen-activated protein kinase (MAPK) but not the p42/p44 MAPK and, when combined with growth factors, intensified p38 activation but inhibited the growth-factor-induced p42/p44 activation. IL-1beta also induced a time-dependent inhibition of PFGF-Ralpha gene expression. These results support a role for IL-1beta in promoting mitotic arrest and differentiation of oligodendrocyte progenitors as well as maturation and survival of differentiating

  1. Hydroxyapatite promotes superior keratocyte adhesion and proliferation in comparison with current keratoprosthesis skirt materials

    PubMed Central

    Mehta, J S; Futter, C E; Sandeman, S R; Faragher, R G A F; Hing, K A; Tanner, K E; Allan, B D S

    2005-01-01

    Aim: Published clinical series suggest the osteoodontokeratoprosthesis (OOKP) may have a lower extrusion rate than current synthetic keratoprostheses. The OOKP is anchored in the eye wall by autologous tooth. The authors’ aim was to compare adhesion, proliferation, and morphology for telomerase transformed keratocytes seeded on calcium hydroxyapatite (the principal mineral constituent of tooth) and materials used in the anchoring elements of commercially available synthetic keratoprostheses. Methods: Test materials were hydroxyapatite, polytetrafluoroethylene (PTFE), polyhydroxyethyl methacrylate (HEMA), and glass (control). Cell adhesion and viability were quantified at 4 hours, 24 hours, and 1 week using a calcein-AM/EthD-1 viability/cytotoxicity assay. Focal contact expression and cytoskeletal organisation were studied at 24 hours by confocal microscopy with immunoflourescent labelling. Further studies of cell morphology were performed using light and scanning electron microscopy. Results: Live cell counts were significantly greater on hydroxyapatite surfaces at each time point (p<0.04). Dead cell counts were significantly higher for PTFE at 7 days (p<0.002). ß1 integrin expression was highest on hydroxyapatite. Adhesion structures were well expressed in flat, spread out keratocytes on both HA and glass. Keratocytes tended to be thinner and spindle shaped on PTFE. The relatively few keratocytes visible on HEMA test surfaces were rounded and poorly adherent. Conclusions: Keratocyte adhesion, spreading, and viability on hydroxyapatite test surfaces is superior to that seen on PTFE and HEMA. Improving the initial cell adhesion environment in the skirt element of keratoprostheses may enhance tissue integration and reduce device failure rates. PMID:16170132

  2. Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation.

    PubMed

    Malaisse, Jérémy; Pendaries, Valérie; Hontoir, Fanny; De Glas, Valérie; Van Vlaender, Daniel; Simon, Michel; Lambert de Rouvroit, Catherine; Poumay, Yves; Flamion, Bruno

    2016-03-18

    Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal.

  3. Effect of surface potential on epithelial cell adhesion, proliferation and morphology.

    PubMed

    Chang, Hsun-Yun; Kao, Wei-Lun; You, Yun-Wen; Chu, Yi-Hsuan; Chu, Kuo-Jui; Chen, Peng-Jen; Wu, Chen-Yi; Lee, Yu-Hsuan; Shyue, Jing-Jong

    2016-05-01

    Cell adhesion is the basis of individual cell survival, division and motility. Hence, understanding the effects that the surface properties have on cell adhesion, proliferation and morphology are crucial. In particular, surface charge/potential has been identified as an important factor that affects cell behavior. However, how cells respond to incremental changes in surface potential remains unclear. By using binary self-assembled monolayer (SAM) modified Au surfaces that are similar in mechanical/chemical properties and provide a series of surface potentials, the effect of surface potential on the behavior of cells can be studied. In this work, the effect of surface potential on epithelial cells, including human embryonic kidney (HEK293T) and human hepatocellular carcinoma (HepG2), were examined. The results showed that the adhesion density of epithelial cells increased with increasing surface potential, which is similar to but varied more significantly compared with fibroblasts. The proliferation rate is found to be independent of surface potential in both cell types. Furthermore, epithelial cells show no morphological change with respect to surface potential, whereas the morphology of the fibroblasts clearly changed with the surface potential. These differences between the cell types were rationalized by considering the difference in extracellular matrix composition. Laminin-dominant epithelial cells showed higher adhesion density and less morphological change than did fibronectin-dominant fibroblasts because the more significant adsorption of positively charged laminin on the surface enhanced the adhesion of epithelial cells. In contrast, due to the dominance of negatively charged fibronectin that adsorbed weakly on the surface, fibroblasts had to change their morphology to fit the inhomogeneous fibronectin-adsorbed area.

  4. Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

    PubMed Central

    2014-01-01

    Introduction We previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined. Methods Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed. Results Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation. Conclusions These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA. PMID:24467809

  5. Research progress on the proliferation and differentiation of

    NASA Astrophysics Data System (ADS)

    An, A.; Tan, B.

    Space environments such as microgravity magnetic field radiation and heavy metal ions affects the development and functions of human and mammalian cells To study these influences and the corresponding metabolisms is in favour of knowing about the development and differentiation process of organism cells In recent years researches on the differentiation of stem cells induced in vitro provide a new pathway for the repair of tissue lesion and therapy of human diseases Stem cells are potential in capable of differentiating into different functional cells But there has no reliable methods to induce the stem cells differentiating forward specific cells and to gain enough cells for transplantation which limited their application on clinical therapy It has been indicated that microgravity influenced embryonic development hematopoietic and mesenchymal stem cells and so on Hematopoietic stem cell migration and its differentiation were affected by microgravity The specific differentiation of hematopoietic stem cells was inhibited under microgravity The expression of proteins regulating cell cycle period also changed Mesenchymal stem cells provide a source of cells for the repair of musculoskeletal tissue in ground experiment While under microgravity the proliferation and differentiation of mesenchymal stem cells were influenced along with the differentiated cells function changed Furthermore in the differentiation process of stem cells under microgravity the mechanism of signal transport was also affected and the specific differentiation

  6. In vitro microdistraction of preosteoblasts: distraction promotes proliferation and oscillation promotes differentiation.

    PubMed

    Gabbay, Joubin S; Zuk, Patricia A; Tahernia, Amir; Askari, Morad; O'Hara, Catherine M; Karthikeyan, Tharun; Azari, Kodi; Hollinger, Jeffery O; Bradley, James P

    2006-11-01

    Osteoblast biology is influenced in vivo by a 3-dimensional (3D) extracellular matrix that mediates their adhesion and interaction and by a constant state of compressive and tensile forces. To study the role of mechanical stress on osteoblasts in vitro, these parameters must be addressed. Therefore, this study describes the use of a novel, in vitro system that subjects cells to distractive and compressive forces in a 3D environment. This system, termed a microdistractor system, was used to apply linear forces to 3D collagen type I gels containing preosteoblasts. Gels were induced for up to 16 days in osteogenic medium and subjected to either constant linear distraction (distraction gels) or to repeating cycles of distraction and compression (oscillation gels). The effect of these stresses was evaluated over time by measuring proliferation rates, protein synthesis (i.e., cellular activity), and osteogenic differentiation levels. While linear forces in general appeared to increase protein synthesis, force-specific effects on proliferation and differentiation were observed. Specifically, distraction forces appeared to enhance MC3T3 proliferation while distraction/compressive forces appeared to accelerate their osteogenic differentiation program. Therefore, these results suggest that the microdistraction system may be an appropriate in vitro system for the study of mechanobiology in osteoblast phenotype.

  7. Effect of resveratrol on proliferation and differentiation of embryonic cardiomyoblasts

    SciTech Connect

    Leong, C.-W.; Wong, C.H.; Lao, S.-C.; Leong, Emilia Conceicao; Lao, Iok Fong; Law, Patrick Tik Wan; Fung, Kwok Pui; Tsang, Kam Sze; Waye, Mary Miu-Yee; Tsui, Stephen Kwok-Wing; Wang Yitao . E-mail: YTWang@umac.mo; Lee, Simon Ming-Yuen . E-mail: simonlee@umac.mo

    2007-08-17

    Resveratrol (trans-3,5,4'-trihydroxystilbene), a polyphenolic compound found largely in the skins of red grapes, has been used as a nutritional supplement or an investigational new drug for prevention of cardiovascular diseases. Previous reports showed that resveratrol had a protective effect against oxidative agent-induced cell injury. Our studies indicate that resveratrol plays a role in the differentiation of cardiomyoblasts. The cardiomyoblast cell line, H9c2, was exposed to 30-120 {mu}M resveratrol for up to 5 days. Resveratrol inhibits cardiomyoblast proliferation without causing cells injury. Moreover, resveratrol treatment modulated the differentiation of morphological characteristics including elongation and cell fusion in cardiomyoblasts. Proliferation and differentiation of H9c2 cells were further revealed by measurement of the mRNA expression of a cell cycle marker (CDK2), a differentiation marker (myogenin), and a contractile apparatus protein (MLC-2). Gene expression analysis revealed that resveratrol promoted entry into cell cycle arrest but extended the myogenic differentiation progress. These results have implications for the role of resveratrol in modulating cell cycle control and differentiation in cardiomyoblasts.

  8. TiN x O y coatings facilitate the initial adhesion of osteoblasts to create a suitable environment for their proliferation and the recruitment of endothelial cells.

    PubMed

    Moussa, M; Banakh, O; Wehrle-Haller, B; Fontana, P; Scherrer, S; Cattani, M; Wiskott, A; Durual, S

    2017-02-28

    Titanium-nitride-oxide coatings (TiN x O y ) improve osseointegration of endosseous implants. The exact mechanisms by which these effects are mediated are poorly understood except for an increase of osteoblast proliferation while a high degree of differentiation is maintained. One hypothesis holds that TiN x O y facilitates the initial spreading and adhesion of the osteoblasts. The aim of this work was to investigate the molecular mechanisms of osteoblast adhesion on TiN x O y as compared to microrough titanium SLA. A global view of the osseointegrative process, that is, taking into account other cell groups, especially endothelial cells, is also presented. To this aim, gene expression and focal adhesion analysis, cocultures and wound assays were performed early after seeding, from 6 h to 3 days. We demonstrated that TiN x O y coatings enhance osteoblast adhesion and spreading when compared to the standard microrough titanium. The integrin β1, either in association with α1 or with α2 plays a central role in these mechanisms. TiN x O y coatings optimize the process of osseointegration by acting at several levels, especially by upregulating osteoblast adhesion and proliferation, but also by supporting neovascularization and the development of a suitable inflammatory environment.

  9. Obestatin changes proliferation, differentiation and apoptosis of porcine preadipocytes.

    PubMed

    Tang, Shengqiu; Dong, Xiaoying; Zhang, Wei

    2014-02-01

    Obestatin, originally identified and purified from rat stomach extracts, was reported to bind to orphan G protein-coupled receptor, GPR39, and inhibit appetite and gastric motility. This study was conducted to investigate the effects of porcine obestatin on proliferation, differentiation and apoptosis of porcine preadipocytes isolated from subcutaneous fat of piglets. At indicated times of culture, morphology of preadipocytes and accumulated lipid droplets within the cells were identified by invert microscope. After treating with obestatin (0, 0.1, 1, 10 and 100nM), cell proliferation was measured by MTT method and protein expression of CCAAT/enhancer binding protein-α (C/EBPα), peroxisome proliferator-activated receptor-γ (PPARγ), Caspase-7 and Caspase-9 was determined by Western Blot, mRNA expression of GPR39 and Caspase-3 was analyzed by RT-PCR, and the activity of Caspase-3 was measured by spectrophotometric method. The results showed that obestatin had no effect on GPR39 expression, while promotes the optical density (OD) value of cells, enhanced protein expression of PPARγ and C/EBPa, decreased mRNA expression and activity of Caspase-3, and inhibited protein expression of Caspase-7 and Caspase-9 in a dose-dependent manner. These results suggested that obestatin enhances proliferation and differentiation of preadipocytes promoting PPARγ and C/EBPa expression, and inhibiting preadipocyte apoptosis by decreasing expression of Caspase-3, Caspase-7 and Caspase-9.

  10. Differential migration and proliferation of geometrical ensembles of cell clusters

    SciTech Connect

    Kumar, Girish; Chen, Bo; Co, Carlos C.; Ho, Chia-Chi

    2011-06-10

    Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell-cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.

  11. Control of keratinocyte proliferation and differentiation by p63.

    PubMed

    Truong, Amy B; Khavari, Paul A

    2007-02-01

    The p53 family member p63 has been implicated in both the development and maintenance of stratified epithelial tissues, including the epidermis. Increasing data support p63 function in the regenerative capacity of basal keratinocytes by maintaining cell proliferation. Recent studies further suggest this regulation relies on inhibition of p53 activity. In addition, p63 appears to exert separate control over epidermal differentiation, which may involve control of such key signaling molecules as IKKalpha and Notch. While studies over the past decade have greatly expanded our knowledge of p63 function, much remains to be understood regarding how p63 regulates epidermal homeostasis. Future efforts to identify and validate direct p63 target genes as well as to understand the expression and function of individual p63 isoforms will be important to further define how p63 functions in the control of keratinocyte proliferation and differentiation.

  12. ETO2 coordinates cellular proliferation and differentiation during erythropoiesis

    PubMed Central

    Goardon, Nicolas; Lambert, Julie A; Rodriguez, Patrick; Nissaire, Philippe; Herblot, Sabine; Thibault, Pierre; Dumenil, Dominique; Strouboulis, John; Romeo, Paul-Henri; Hoang, Trang

    2006-01-01

    The passage from proliferation to terminal differentiation is critical for normal development and is often perturbed in malignancies. To define the molecular mechanisms that govern this process during erythropoiesis, we have used tagging/proteomics approaches and characterized protein complexes nucleated by TAL-1/SCL, a basic helix–loop–helix transcription factor that specifies the erythrocytic lineage. In addition to known TAL-1 partners, GATA-1, E2A, HEB, LMO2 and Ldb1, we identify the ETO2 repressor as a novel component recruited to TAL-1 complexes through interaction with E2A/HEB. Ectopic expression and siRNA knockdown experiments in hematopoietic progenitor cells show that ETO2 actively represses erythroid TAL-1 target genes and governs the expansion of erythroid progenitors. At the onset of erythroid differentiation, a change in the stoichiometry of ETO2 within the TAL-1 complex activates the expression of known erythroid-specific TAL-1 target genes and of Gfi-1b and p21Cip, encoding two essential regulators of erythroid cell proliferation. These results suggest that the dynamics of ETO2 recruitment within nuclear complexes couple cell proliferation to cell differentiation and determine the onset of terminal erythroid maturation. PMID:16407974

  13. Amphiphilic Macromolecule Nanoassemblies Suppress Smooth Muscle Cell Proliferation and Platelet Adhesion

    PubMed Central

    Chan, Jennifer W.; Lewis, Daniel R.; Petersen, Latrisha K.; Moghe, Prabhas V.; Uhrich, Kathryn E.

    2016-01-01

    While the development of second- and third-generation drug-eluting stents (DES) have significantly improved patient outcomes by reducing smooth muscle cell (SMC) proliferation, DES have also been associated with an increased risk of late-stent thrombosis due to delayed re-endothelialization and hypersensitivity reactions from the drug-polymer coating. Furthermore, DES anti-proliferative agents do not counteract the upstream oxidative stress that triggers the SMC proliferation cascade. In this study, we investigate biocompatible amphiphilic macromolecules (AMs) that address high oxidative lipoprotein microenvironments by competitively binding oxidized lipid receptors and suppressing SMC proliferation with minimal cytotoxicity. To determine the influence of nanoscale assembly on proliferation, micelles and nanoparticles were fabricated from AM unimers containing a phosphonate or carboxylate end-group, sugar-based hydrophobic domain, and a hydrophilic poly(ethylene glycol) domain. The results indicate that when SMCs are exposed to high levels of oxidized lipid stimuli, nanotherapeutics inhibit lipid uptake, downregulate scavenger receptor expression, and attenuate scavenger receptor gene transcription in SMCs, and thus significantly suppress proliferation. Although both functional end-groups were similarly efficacious, nanoparticles suppressed oxidized lipid uptake and scavenger receptor expression more effectively compared to micelles, indicating the relative importance of formulation characteristics (e.g., higher localized AM concentrations and nanotherapeutic stability) in scavenger receptor binding as compared to AM end-group functionality. Furthermore, AM coatings significantly prevented platelet adhesion to metal, demonstrating its potential as an anti-platelet therapy to treat thrombosis. Thus, AM micelles and NPs can effectively repress early stage SMC proliferation and thrombosis through non-cytotoxic mechanisms, highlighting the promise of nanomedicine for

  14. Amphiphilic macromolecule nanoassemblies suppress smooth muscle cell proliferation and platelet adhesion.

    PubMed

    Chan, Jennifer W; Lewis, Daniel R; Petersen, Latrisha K; Moghe, Prabhas V; Uhrich, Kathryn E

    2016-04-01

    While the development of second- and third-generation drug-eluting stents (DES) have significantly improved patient outcomes by reducing smooth muscle cell (SMC) proliferation, DES have also been associated with an increased risk of late-stent thrombosis due to delayed re-endothelialization and hypersensitivity reactions from the drug-polymer coating. Furthermore, DES anti-proliferative agents do not counteract the upstream oxidative stress that triggers the SMC proliferation cascade. In this study, we investigate biocompatible amphiphilic macromolecules (AMs) that address high oxidative lipoprotein microenvironments by competitively binding oxidized lipid receptors and suppressing SMC proliferation with minimal cytotoxicity. To determine the influence of nanoscale assembly on proliferation, micelles and nanoparticles were fabricated from AM unimers containing a phosphonate or carboxylate end-group, a sugar-based hydrophobic domain, and a hydrophilic poly(ethylene glycol) domain. The results indicate that when SMCs are exposed to high levels of oxidized lipid stimuli, nanotherapeutics inhibit lipid uptake, downregulate scavenger receptor expression, and attenuate scavenger receptor gene transcription in SMCs, and thus significantly suppress proliferation. Although both functional end-groups were similarly efficacious, nanoparticles suppressed oxidized lipid uptake and scavenger receptor expression more effectively compared to micelles, indicating the relative importance of formulation characteristics (e.g., higher localized AM concentrations and nanotherapeutic stability) in scavenger receptor binding as compared to AM end-group functionality. Furthermore, AM coatings significantly prevented platelet adhesion to metal, demonstrating its potential as an anti-platelet therapy to treat thrombosis. Thus, AM micelles and NPs can effectively repress early stage SMC proliferation and thrombosis through non-cytotoxic mechanisms, highlighting the promise of nanomedicine for

  15. 3D Surface Topology Guides Stem Cell Adhesion and Differentiation

    PubMed Central

    Viswanathan, Priyalakshmi; Ondeck, Matthew G.; Chirasatitsin, Somyot; Nghamkham, Kamolchanok; Reilly, Gwendolen C.; Engler, Adam J.; Battaglia, Giuseppe

    2015-01-01

    Polymerized high internal phase emulsion (polyHIPE) foams are extremely versatile materials for investigating cell-substrate interactions in vitro. Foam morphologies can be controlled by polymerization conditions to result in either open or closed pore structures with different levels of connectivity, consequently enabling the comparison between 2D and 3D matrices using the same substrate with identical surface chemistry conditions. Additionally, here we achieve the control of pore surface topology (i.e. how different ligands are clustered together) using amphiphilic block copolymers as emulsion stabilisers. We demonstrate that adhesion of human mesenchymal progenitor (hES-MP) cells cultured on polyHIPE foams is dependent on foam surface topology and chemistry but is independent of porosity and interconnectivity. We also demonstrate that the interconnectivity, architecture and surface topology of the foams has an effect on the osteogenic differentiation potential of hES-MP cells. Together these data demonstrate that the adhesive heterogeneity of a 3D scaffold could regulate not only mesenchymal stem cell attachment but also cell behavior in the absence of soluble growth factors. PMID:25818420

  16. Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

    SciTech Connect

    Capkovic, Katie L.; Stevenson, Severin; Johnson, Marc C.; Thelen, Jay J.; Cornelison, D.D.W.

    2008-04-15

    Although recent advances in broad-scale gene expression analysis have dramatically increased our knowledge of the repertoire of mRNAs present in multiple cell types, it has become increasingly clear that examination of the expression, localization, and associations of the encoded proteins will be critical for determining their functional significance. In particular, many signaling receptors, transducers, and effectors have been proposed to act in higher-order complexes associated with physically distinct areas of the plasma membrane. Adult muscle stem cells (satellite cells) must, upon injury, respond appropriately to a wide range of extracellular stimuli: the role of such signaling scaffolds is therefore a potentially important area of inquiry. To address this question, we first isolated detergent-resistant membrane fractions from primary satellite cells, then analyzed their component proteins using liquid chromatography-tandem mass spectrometry. Transmembrane and juxtamembrane components of adhesion-mediated signaling pathways made up the largest group of identified proteins; in particular, neural cell adhesion molecule (NCAM), a multifunctional cell-surface protein that has previously been associated with muscle regeneration, was significant. Immunohistochemical analysis revealed that not only is NCAM localized to discrete areas of the plasma membrane, it is also a very early marker of commitment to terminal differentiation. Using flow cytometry, we have sorted physically homogeneous myogenic cultures into proliferating and differentiating fractions based solely upon NCAM expression.

  17. Receptor FGFRL1 does not promote cell proliferation but induces cell adhesion

    PubMed Central

    YANG, XIAOCHEN; STEINBERG, FLORIAN; ZHUANG, LEI; BESSEY, RALPH; TRUEB, BEAT

    2016-01-01

    Fibroblast growth factor receptor (FGFR)-like protein 1 (FGFRL1) is the most recently discovered member of the FGFR family. Owing to the fact that it interacts with FGF ligands, but lacks the intracellular tyrosine kinase domain, several researchers have speculated that it may function as a decoy receptor and exert a negative effect on cell proliferation. In this study, we performed overexpression experiments with TetOn-inducible cell clones and downregulation experiments with siRNA oligonucleotides, and found that FGFRL1 had absolutely no effect on cell growth and proliferation. Likewise, we did not observe any influence of FGFRL1 on ERK1/2 activation and on the phosphorylation of 250 other signaling proteins analyzed by the Kinexus antibody microarray. On the other hand, with bacterial petri dishes, we observed a clear effect of FGFRL1 on cell adhesion during the initial hours after cell seeding. Our results suggest that FGFRL1 is a cell adhesion protein similar to the nectins rather than a signaling receptor similar to FGFR1-FGFR4. PMID:27220341

  18. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion.

    PubMed

    Takabe, Piia; Bart, Geneviève; Ropponen, Antti; Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

    2015-09-10

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells.

  19. Role of HLA-G1 in trophoblast cell proliferation, adhesion and invasion

    SciTech Connect

    Jiang, Feng; Zhao, Hongxi; Wang, Li; Guo, Xinyu; Wang, Xiaohong; Yin, Guowu; Hu, Yunsheng; Li, Yi; Yao, Yuanqing

    2015-02-27

    Trophoblast cells are important in embryo implantation and fetomaternal tolerance. HLA-G is specifically expressed at the maternal–fetal interface and is a regulator in pregnancy. The aim of the present study was to detect the effect of HLA-G1 on trophoblast cell proliferation, adhesion, and invasion. Human trophoblast cell lines (JAR and HTR-8/SVneo cells) were infected with HLA-G1-expressing lentivirus. After infection, HLA-G1 expression of the cells was detected by western blotting. Cell proliferation was detected by the BrdU assay. The cell cycle and apoptosis of JAR and HTR-8/SVneo cells was measured by flow cytometry (FCM). The invasion of the cells under different conditions was detected by the transwell invasion chamber assay. HLA-G1 didn't show any significant influence on the proliferation, apoptosis, adhesion, and invasion of trophocytes in normal culture conditions. However, HLA-G1 inhibited JAR and HTR-8/SVneo cells invasion induced by hepatocyte growth factor (HGF) under normal oxygen conditions. In conditions of hypoxia, HLA-G1 couldn't inhibit the induction of cell invasion by HGF. HLA-G1 is not an independent factor for regulating the trophocytes. It may play an indirect role in embryo implantation and formation of the placenta. - Highlights: • HLA-G1 could not influence trophocytes under normal conditions. • HLA-G1 inhibited cell invasion induced by HGF under normal oxygen condition. • HLA-G1 could not influence cell invasion under hypoxia conditions.

  20. Ligand targeting of EphA2 enhances keratinocyte adhesion and differentiation via desmoglein 1.

    PubMed

    Lin, Samantha; Gordon, Kristin; Kaplan, Nihal; Getsios, Spiro

    2010-11-15

    EphA2 is a receptor tyrosine kinase that is engaged and activated by membrane-linked ephrin-A ligands residing on adjacent cell surfaces. Ligand targeting of EphA2 has been implicated in epithelial growth regulation by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2)-mitogen activated protein kinase (MAPK) pathway. Although contact-dependent EphA2 activation was required for dampening Erk1/2-MAPK signaling after a calcium switch in primary human epidermal keratinocytes, the loss of this receptor did not prevent exit from the cell cycle. Incubating keratinocytes with a soluble ephrin-A1-Fc peptide mimetic to target EphA2 further increased receptor activation leading to its down-regulation. Moreover, soluble ligand targeting of EphA2 restricted the lateral expansion of epidermal cell colonies without limiting proliferation in these primary cultures. Rather, ephrin-A1-Fc peptide treatment promoted epidermal cell colony compaction and stratification in a manner that was associated with increased keratinocyte differentiation. The ligand-dependent increase in keratinocyte adhesion and differentiation relied largely upon the up-regulation of desmoglein 1, a desmosomal cadherin that maintains the integrity and differentiated state of suprabasal keratinocytes in the epidermis. These data suggest that keratinocytes expressing EphA2 in the basal layer may respond to ephrin-A1-based cues from their neighbors to facilitate entry into a terminal differentiation pathway.

  1. Cell-substrate adhesion during Trypanosoma cruzi differentiation

    PubMed Central

    1988-01-01

    The transformation of Trypanosoma cruzi epimastigotes to the mammal infective metacyclic trypomastigotes (metacyclogenesis) can be performed in vitro under chemically defined conditions. Under these conditions, differentiating epimastigotes adhere to a surface before their transformation into metacyclic trypomastigotes. Scanning and transmission electron microscopy of adhered and non-adhered parasites during the metacyclogenesis process show that only epimastigotes and few transition forms are found in the first population, whereas metacyclic trypomastigotes are exclusively found in the cell culture supernatant. PAGE analysis of the [35S]methionine metabolic labeling products of adhered and non-adhered parasites shows that although most of the polypeptides are conserved, adhered parasites express specifically four polypeptides in the range of 45-50 kD with an isoelectric point of 4.8. These proteins might be involved in the adhesion process and are recognized by an antiserum against total adhered parasite proteins. This antiserum also recognized a group of 45- 50 kD in the iodine-radiolabeled surface proteins of differentiating cells, providing direct evidence that these components are indeed surface antigens. The results suggest that epimastigotes must adhere to a substrate before their transformation to metacyclic trypomastigotes, being released to the medium as the metacyclogenesis process is accomplished. This could correspond to the process naturally occurring within the triatomine invertebrate host. PMID:3283152

  2. Virtual-tissue computer simulations define the roles of cell adhesion and proliferation in the onset of kidney cystic disease

    PubMed Central

    Belmonte, Julio M.; Clendenon, Sherry G.; Oliveira, Guilherme M.; Swat, Maciej H.; Greene, Evan V.; Jeyaraman, Srividhya; Glazier, James A.; Bacallao, Robert L.

    2016-01-01

    In autosomal dominant polycystic kidney disease (ADPKD), cysts accumulate and progressively impair renal function. Mutations in PKD1 and PKD2 genes are causally linked to ADPKD, but how these mutations drive cell behaviors that underlie ADPKD pathogenesis is unknown. Human ADPKD cysts frequently express cadherin-8 (cad8), and expression of cad8 ectopically in vitro suffices to initiate cystogenesis. To explore cell behavioral mechanisms of cad8-driven cyst initiation, we developed a virtual-tissue computer model. Our simulations predicted that either reduced cell–cell adhesion or reduced contact inhibition of proliferation triggers cyst induction. To reproduce the full range of cyst morphologies observed in vivo, changes in both cell adhesion and proliferation are required. However, only loss-of-adhesion simulations produced morphologies matching in vitro cad8-induced cysts. Conversely, the saccular cysts described by others arise predominantly by decreased contact inhibition, that is, increased proliferation. In vitro experiments confirmed that cell–cell adhesion was reduced and proliferation was increased by ectopic cad8 expression. We conclude that adhesion loss due to cadherin type switching in ADPKD suffices to drive cystogenesis. Thus, control of cadherin type switching provides a new target for therapeutic intervention. PMID:27193300

  3. Effect of testosterone incorporation on cell proliferation and differentiation for polymer-bioceramic composites.

    PubMed

    da Costa, Kelen Jorge Rodrigues; Passos, Joel J; Gomes, Alinne D M; Sinisterra, Rubén D; Lanza, Célia R M; Cortés, Maria Esperanza

    2012-11-01

    In the current study, we characterized the polycaprolactone (PCL), poly(lactic acid-co-glycolic acid) (PLGA), and biphasic calcium phosphate (BCP) composites coated with testosterone propionate (T) using Fourier transform infrared spectroscopy (FTIR) and powder X-ray diffraction (XRD). Osteoblastic cells were seeded with PCL/BCP, PCL/BCP/T, PLGA/PCL/BCP and PLGA/PCL/BCP/T scaffolds, and cell viability, proliferation, differentiation and adhesion were analyzed. The results of physic-chemical experiments showed no displacements or suppression of bands in the FTIR spectra of scaffolds. The XRD patterns of the scaffolds showed an amorphous profile. The osteoblastic cells viability and proliferation increased in the presence of composites with testosterone over 72 h, and were significantly greater when PLGA/PCL/BCP/T scaffold was tested against PCL/BCP/T. Furthermore alkaline phosphatase production was significantly greater in the same group. In conclusion, the PLGA/PCL/BCP scaffold with testosterone could be a promising option for bone tissue applications due to its biocompatibility and its stimulatory effect on cell proliferation.

  4. Keeping the Balance Between Proliferation and Differentiation: The Primary Cilium

    PubMed Central

    Irigoín, Florencia; Badano, Jose L

    2011-01-01

    Primary cilia are post-mitotic cellular organelles that are present in the vast majority of cell types in the human body. An extensive body of data gathered in recent years is demonstrating a crucial role for this organelle in a number of cellular processes that include mechano and chemo-sensation as well as the transduction of signaling cascades critical for the development and maintenance of different tissues and organs. Consequently, cilia are currently viewed as cellular antennae playing a critical role at the interphase between cells and their environment, integrating a range of stimuli to modulate cell fate decisions including cell proliferation, migration and differentiation. Importantly, this regulatory role is not just a consequence of their participation in signal transduction but is also the outcome of both the tight synchronization/regulation of ciliogenesis with the cell cycle and the role of individual ciliary proteins in cilia-dependent and independent processes. Here we review the role of primary cilia in the regulation of cell proliferation and differentiation and illustrate how this knowledge has provided insight to understand the phenotypic consequences of ciliary dysfunction. PMID:22131874

  5. Megakaryocytes are mechanically responsive and influence osteoblast proliferation and differentiation

    PubMed Central

    Soves, Constance P.; Miller, Joshua D.; Begun, Dana L.; Taichman, Russell S.; Hankenson, Kurt D.; Goldstein, Steven A.

    2014-01-01

    Maintenance of bone mass and geometry is influenced by mechanical stimuli. Paradigms suggest that osteocytes embedded within the mineralized matrix and osteoblasts on the bone surfaces are the primary responders to physical forces. However, other cells within the bone marrow cavity, such as megakaryocytes (MKs), are also subject to mechanical forces. Recent studies have highlighted the potent effects of MKs on osteoblast proliferation as well as bone formation in vivo. We hypothesize that MKs are capable of responding to physical forces and that the interactions between these cells and osteoblasts can be influenced by mechanical stimulation. In this study, we demonstrate that two MK cell lines respond to fluid shear stress in culture. Furthermore, using laser capture microdissection, we isolated MKs from histologic sections of murine tibiae that were exposed to compressive loads in vivo. C-fos, a transcription factor shown to be upregulated in response to load in various tissue types, was increased in MKs from loaded relative to non-loaded limbs at a level comparable to that of osteocytes from the same limbs. We also developed a co-culture system to address whether mechanical stimulation of MKs in culture would impact osteoblast proliferation and differentiation. The presence of MKs in co-culture, but not conditioned media, had dramatic effects on proliferation of preosteoblast MC3T3-E1 cells in culture. Our data suggests a minimal decrease in proliferation as well as an increase in mineralization capacity of osteoblasts co-cultured with MKs exposed to shear compared to co-cultures with unstimulated MKs. PMID:24882736

  6. Adhesion and proliferation of human endothelial cells on photochemically modified polytetrafluoroethylene.

    PubMed

    Gumpenberger, T; Heitz, J; Bäuerle, D; Kahr, H; Graz, I; Romanin, C; Svorcik, V; Leisch, F

    2003-12-01

    We studied the adhesion and proliferation of human endothelial cells on photochemically modified polytetrafluoroethylene samples. The polymer surfaces were modified by exposure to the ultraviolet light of a Xe(2)(*)-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere. Treatment times were between 10 and 20 min. The endothelial cell density was determined 1, 3 and 8 days after seeding by image analysis. Surface modification of the samples resulted in a significant increase in the number of adhering cells and in the formation of a confluent cell layer after 3-8 days. The results were comparable than those obtained on polystyrene Petri dishes, which are used as standard substrates in cell cultivation. Thus modified PTFE appears to be a promising material for the fabrication of artificial vascular prostheses coated with endothelial cells.

  7. Surface chemistry and polymer film thickness effects on endothelial cell adhesion and proliferation

    PubMed Central

    Bhattacharyya, Dhiman; Xu, Hao; Deshmukh, Rajendra R.; Timmons, Richard B.; Nguyen, Kytai T.

    2010-01-01

    Adherence and growth rates of human aortic endothelial cells (HAEC) on plasma polymerized poly(vinylacetic acid) films were measured as functions of the surface density of —COOH groups and plasma deposited film thickness. Pulsed plasma polymerization was employed to produce films containing 3.6 to 9% —COOH groups, expressed as a percent of total carbon content. Endothelial cells exhibited increased cell adherence and proliferation with increasing —COOH surface densities. Additionally, and unexpectedly, cell growth was also dependent on the film thicknesses, which ranged from 25 to 200 nm. The results indicate that optimization of the functional group surface density and film thickness could produce significant enhancements in initial adhesion and subsequent growth of the HAEC cells. PMID:20213813

  8. The effect of elastin on chondrocyte adhesion and proliferation on poly (ɛ-caprolactone)/elastin composites.

    PubMed

    Annabi, Nasim; Fathi, Ali; Mithieux, Suzanne M; Martens, Penny; Weiss, Anthony S; Dehghani, Fariba

    2011-02-01

    The aim of this study was to demonstrate the effect of elastin on chondrocyte adhesion and proliferation within the structure of poly (ɛ-caprolactone) (PCL)/elastin composites. The homogenous 3D structure composites were constructed by using high pressure CO(2) in two stages. Porous PCL structures with average pore sizes of 540 ± 21 μm and a high degree of interconnectivity were produced using gas foaming/salt leaching. The PCL scaffolds were then impregnated with elastin and cross-linked with glutaraldehyde (GA) under high pressure CO(2). The effects of elastin and cross-linker concentrations on the characteristics of composites were investigated. Increasing the elastin concentration from 25mg/ml to 100mg/ml elevated the amount of cross-linked elastin inside the macropores of PCL. Fourier transform infrared (FTIR) analysis showed that elastin was homogeneously distributed throughout the 3D structure of all composites. The weight gain of composites increased 2-fold from 15.8 ± 0.3 to 38.3 ± 0.7 (w/w) % by increasing the elastin concentration from 25mg/ml to 50mg/ml and approached a plateau above this concentration. The presence of elastin within the pores of PCL improved the water uptake properties of PCL scaffolds; the water uptake ratio of PCL was enhanced 100-fold from 0.030 ± 0.005g liquid/g polymer to 11.80 ± 0.01g liquid/g polymer, when the elastin solution concentration was 50mg/ml. These composites exhibited lower compressive modulus and energy loss compared to pure PCL scaffolds due to their higher water content and elasticity. In vitro studies show that these composites can support primary articular cartilage chondrocyte adhesion and proliferation within the 3D structures. These results demonstrate the potential of using PCL/elastin composites for cartilage repair.

  9. Exploring cellular adhesion and differentiation in a micro-/nano-hybrid polymer scaffold.

    PubMed

    Cheng, Ke; Kisaalita, William S

    2010-01-01

    Polymer scaffolds play an important role in three dimensional (3-D) cell culture and tissue engineering. To best mimic the archiecture of natural extracellular matrix (ECM), a nano-fibrous and micro-porous combined (NFMP) scaffold was fabricated by combining phase separation and particulate leaching techniques. The NFMP scaffold possesses architectural features at two levels, including the micro-scale pores and nano-scale fibers. To evaluate the advantages of micro/nano combination, control scaffolds with only micro-pores or nano-fibers were fabricated. Cell grown in NFMP and control scaffolds were characterized with respect to morphology, proliferation rate, diffentiation and adhesion. The NFMP scaffold combined the advantages of micro- and nano-scale structures. The NFMP scaffold nano-fibers promoted neural differentiation and induced "3-D matrix adhesion", while the NFMP scaffold micro-pores facilitated cell infiltration. This study represents a systematic comparison of cellular activities on micro-only, nano-only and micro/nano combined scaffolds, and demonstrates the unique advantages of the later.

  10. The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro.

    PubMed

    Schlie-Wolter, Sabrina; Ngezahayo, Anaclet; Chichkov, Boris N

    2013-06-10

    Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions-which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance.

  11. The effect of graphene substrate on osteoblast cell adhesion and proliferation.

    PubMed

    Aryaei, Ashkan; Jayatissa, Ahalapitiya H; Jayasuriya, Ambalangodage C

    2014-09-01

    Understanding the effect of graphene substrate on graphene-cell interaction is important for considering graphene as a potential candidate for biomedical applications. In this article, biocompatibility of few layers of graphene film transferred to different substrates was evaluated using osteoblasts. The substrates were oxidized silicon wafer (SiO2/Si stack), soda lime glass, and stainless steel. Chemical vapor deposition method was employed to synthesize graphene on copper substrate using methane and hydrogen as precursors. The quality and the thickness of graphene films on different substrates were estimated by Raman spectra, whereas the thickness of graphene film was confirmed by reflectance and transmittance spectroscopy. The study was also focused on cell attachment and morphology at two time points. The results show that graphene does not have any toxic effect on osteoblasts. The cell adhesion improves with graphene coated substrate than the substrate alone. It seems that graphene substrate properties play a dominant role in cell adhesion. The result of this study suggests that a layer of graphene on bone implants will be beneficial for osteoblast attachment and proliferation.

  12. Hydrophilic PCU scaffolds prepared by grafting PEGMA and immobilizing gelatin to enhance cell adhesion and proliferation.

    PubMed

    Shi, Changcan; Yuan, Wenjie; Khan, Musammir; Li, Qian; Feng, Yakai; Yao, Fanglian; Zhang, Wencheng

    2015-05-01

    Gelatin contains many functional motifs which can modulate cell specific adhesion, so we modified polycarbonate urethane (PCU) scaffold surface by immobilization of gelatin. PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatins onto the surface of aminated PCU scaffolds. To increase the immobilization amount of gelatin, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto PCU scaffolds by surface initiated atom transfer radical polymerization. Then, following amination and immobilization, PCU-g-PEGMA-g-gelatin scaffolds were obtained. Both modified scaffolds were characterized by chemical and biological methods. After immobilization of gelatin, the microfiber surface became rough, but the original morphology of scaffolds was maintained successfully. PCU-g-PEGMA-g-gelatin scaffolds were more hydrophilic than PCU-g-gelatin scaffolds. Because hydrophilic PEGMA and gelatin were grafted and immobilized onto the surface, the PCU-g-PEGMA-g-gelatin scaffolds showed low platelet adhesion, perfect anti-hemolytic activity and excellent cell growth and proliferation capacity. It could be envisioned that PCU-g-PEGMA-g-gelatin scaffolds might have potential applications in tissue engineering artificial scaffolds.

  13. Toward Cell Selective Surfaces: Cell Adhesion and Proliferation on Breath Figures with Antifouling Surface Chemistry.

    PubMed

    Martínez-Campos, Enrique; Elzein, Tamara; Bejjani, Alice; García-Granda, Maria Jesús; Santos-Coquillat, Ana; Ramos, Viviana; Muñoz-Bonilla, Alexandra; Rodríguez-Hernández, Juan

    2016-03-01

    We report the preparation of microporous functional polymer surfaces that have been proven to be selective surfaces toward eukaryotic cells while maintaining antifouling properties against bacteria. The fabrication of functional porous films has been carried out by the breath figures approach that allowed us to create porous interfaces with either poly(ethylene glycol) methyl ether methacrylate (PEGMA) or 2,3,4,5,6-pentafluorostyrene (5FS). For this purpose, blends of block copolymers in a polystyrene homopolymer matrix have been employed. In contrast to the case of single functional polymer, using blends enables us to vary the chemical distribution of the functional groups inside and outside the formed pores. In particular, fluorinated groups were positioned at the edges while the hydrophilic PEGMA groups were selectively located inside the pores, as demonstrated by TOF-SIMS. More interestingly, studies of cell adhesion, growth, and proliferation on these surfaces confirmed that PEGMA functionalized interfaces are excellent candidates to selectively allow cell growth and proliferation while maintaining antifouling properties.

  14. Investigation of In Vitro Bone Cell Adhesion and Proliferation on Ti Using Direct Current Stimulation.

    PubMed

    Bodhak, Subhadip; Bose, Susmita; Kinsel, William C; Bandyopadhyay, Amit

    2012-12-01

    Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 µA, were used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell-materials interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 µA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 µA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell-materials interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model.

  15. Adhesion, Proliferation and Migration of NIH/3T3 Cells on Modified Polyaniline Surfaces

    PubMed Central

    Rejmontová, Petra; Capáková, Zdenka; Mikušová, Nikola; Maráková, Nela; Kašpárková, Věra; Lehocký, Marián; Humpolíček, Petr

    2016-01-01

    Polyaniline shows great potential and promises wide application in the biomedical field thanks to its intrinsic conductivity and material properties, which closely resemble natural tissues. Surface properties are crucial, as these predetermine any interaction with biological fluids, proteins and cells. An advantage of polyaniline is the simple modification of its surface, e.g., by using various dopant acids. An investigation was made into the adhesion, proliferation and migration of mouse embryonic fibroblasts on pristine polyaniline films and films doped with sulfamic and phosphotungstic acids. In addition, polyaniline films supplemented with poly (2-acrylamido-2-methyl-1-propanesulfonic) acid at various ratios were tested. Results showed that the NIH/3T3 cell line was able to adhere, proliferate and migrate on the pristine polyaniline films as well as those films doped with sulfamic and phosphotungstic acids; thus, utilization of said forms in biomedicine appears promising. Nevertheless, incorporating poly (2-acrylamido-2-methyl-1-propanesulfonic) acid altered the surface properties of the polyaniline films and significantly affected cell behavior. In order to reveal the crucial factor influencing the surface/cell interaction, cell behavior is discussed in the context of the surface energy of individual samples. It was clearly demonstrated that the lesser the difference between the surface energy of the sample and cell, the more cyto-compatible the surface is. PMID:27649159

  16. Thymus vulgaris (thyme) inhibits proliferation, adhesion, migration, and invasion of human colorectal cancer cells.

    PubMed

    Al-Menhali, Afnan; Al-Rumaihi, Aisha; Al-Mohammed, Hana; Al-Mazrooey, Hana; Al-Shamlan, Maryam; AlJassim, Meaad; Al-Korbi, Noof; Eid, Ali Hussein

    2015-01-01

    Colorectal cancer (CRC) remains one of the most common malignancies and a leading cause of cancer-related deaths. Its prognosis remains poor for patients with several grades of this disease. This underscores the need for alternative modalities, such as herbal medicines, to treat this disease. A commonly used plant that appears to be of high medicinal value is Thymus vulgaris L. However, the effects of this plant on the malignant behavior of human CRC cells remains poorly investigated. This study was undertaken to determine the anticancer efficacy of T. vulgaris extract (TVE) in CRC cells. Our results show that TVE inhibits proliferation in a concentration- and time-dependent fashion. This decreased proliferation was concomitant with increased apoptotic cell death as evidenced by increased caspase3/7 activity. Moreover, TVE also decreased adhesion to fibronectin in a concentration-dependent manner. The migratory and invasive capacities of HCT116 cells were significantly inhibited by TVE. Taken together, these data suggest that the TVE inhibits malignant phenotype of colon cancer cells. Therefore, T. vulgaris could have an anticancer effect and that some of its bioactive compounds may prove to be effective treatment modalities for human CRC.

  17. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons.

    PubMed

    Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A

    2014-10-17

    During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia-NCAMs) modulate cell-cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia-NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb's to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell-cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

  18. Tumor suppressor KAI1 affects integrin {alpha}v{beta}3-mediated ovarian cancer cell adhesion, motility, and proliferation

    SciTech Connect

    Ruseva, Zlatna; Geiger, Pamina Xenia Charlotte; Hutzler, Peter; Kotzsch, Matthias; Luber, Birgit; Schmitt, Manfred; Gross, Eva; Reuning, Ute

    2009-06-10

    The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin {alpha}v{beta}3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin {alpha}v{beta}3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with {beta}1-integrins, also colocalizes with integrin {alpha}v{beta}3. Functionally, elevated KAI1 levels drastically increased integrin {alpha}v{beta}3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin {alpha}v{beta}3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin {alpha}v{beta}3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.

  19. Insulin signaling via Akt2 switches plakophilin 1 function from stabilizing cell adhesion to promoting cell proliferation.

    PubMed

    Wolf, Annika; Rietscher, Katrin; Glaß, Markus; Hüttelmaier, Stefan; Schutkowski, Mike; Ihling, Christian; Sinz, Andrea; Wingenfeld, Aileen; Mun, Andrej; Hatzfeld, Mechthild

    2013-04-15

    Downregulation of adherens junction proteins is a frequent event in carcinogenesis. How desmosomal proteins contribute to tumor formation by regulating the balance between adhesion and proliferation is not well understood. The desmosomal protein plakophilin 1 can increase intercellular adhesion by recruiting desmosomal proteins to the plasma membrane or stimulate proliferation by enhancing translation rates. Here, we show that these dual functions of plakophilin 1 are regulated by growth factor signaling. Insulin stimulation induced the phosphorylation of plakophilin 1, which correlated with reduced intercellular adhesion and an increased activity of plakophilin 1 in the stimulation of translation. Phosphorylation was mediated by Akt2 at four motifs within the plakophilin 1 N-terminal domain. A plakophilin 1 phospho-mimetic mutant revealed reduced intercellular adhesion and accumulated in the cytoplasm, where it increased translation and proliferation rates and conferred the capacity of anchorage-independent growth. The cytoplasmic accumulation was mediated by the stabilization of phosphorylated plakophilin 1, which displayed a considerably increased half-life, whereas non-phosphorylated plakophilin 1 was more rapidly degraded. Our data indicate that upon activation of growth factor signaling, plakophilin 1 switches from a desmosome-associated growth-inhibiting to a cytoplasmic proliferation-promoting function. This supports the view that the deregulation of plakophilin 1, as observed in several tumors, directly contributes to hyperproliferation and carcinogenesis in a context-dependent manner.

  20. Interplay of Anionic Charge, Poly(ethylene glycol), and Iodinated Tyrosine Incorporation within Tyrosine-derived Polycarbonates: Effects on Vascular Smooth Muscle Cell Adhesion, Proliferation and Motility

    PubMed Central

    Johnson, Patrick A.; Luk, Arnold; Demtchouk, Aleksey; Patel, Hiral; Sung, Hak-Joon; Treiser, Matthew D.; Gordonov, Simon; Sheihet, Larisa; Bolikal, Das; Kohn, Joachim; Moghe, Prabhas V.

    2009-01-01

    Regulation of smooth muscle cell adhesion, proliferation, and motility on biomaterials is critical to the performance of blood-contacting implants and vascular tissue engineering scaffolds. The goal of this study was to examine the underlying substrate-smooth muscle cell response relations, using a selection of polymers representative of an expansive library of multifunctional, tyrosine-derived polycarbonates. Three chemical components within the polymer structure were selectively varied through copolymerization: 1) the content of iodinated tyrosine to achieve X-ray visibility; 2) the content of poly(ethylene glycol) (PEG) to decrease protein adsorption and cell adhesivity; and 3) the content of desaminotyrosyl-tyrosine (DT) which regulates the rate of polymer degradation. Using quartz crystal microbalance with dissipation, we quantified differential serum protein adsorption behavior due to the chemical components DT, iodinated tyrosine, and PEG: increased PEG content within the polymer structure progressively decreased protein adsorption but the simultaneous presence of both DT and iodinated tyrosine reversed the effects of PEG. The complex interplay of these components was next tested on the adhesion, proliferation, and motility behavior cultured human aortic smooth muscle cells. The incorporation of PEG into the polymer reduced cell attachment, which was reversed in the presence of iodinated tyrosine. Further, we found that as little as 10% DT content was sufficient to negate the PEG effect in polymers containing iodinated tyrosine while in non-iodinated polymers the PEG effect on cell attachment was reversed. Cross-functional analysis of motility and proliferation revealed divergent substrate chemistry related cell response regimes. For instance, within the series of polymers containing both iodinated tyrosine and 10% of DT, increasing PEG levels lowered smooth muscle cell motility without a change in the rate of cell proliferation. In contrast, for non

  1. TIEG1-null tenocytes display age-dependent differences in their gene expression, adhesion, spreading and proliferation properties

    SciTech Connect

    Haddad, Oualid; Gumez, Laurie; Hawse, John R.; Subramaniam, Malayannan; Spelsberg, Thomas C.; Bensamoun, Sabine F.

    2011-07-15

    The remodeling of extracellular matrix is a crucial mechanism in tendon development and the proliferation of fibroblasts is a key factor in this process. The purpose of this study was to further elucidate the role of TIEG1 in mediating important tenocyte properties throughout the aging process. Wildtype and TIEG1 knockout tenocytes adhesion, spreading and proliferation were characterized on different substrates (fibronectin, collagen type I, gelatin and laminin) and the expression levels of various genes known to be involved with tendon development were analyzed by RT-PCR. The experiments revealed age-dependent and substrate-dependent properties for both wildtype and TIEG1 knockout tenocytes. Taken together, our results indicate an important role for TIEG1 in regulating tenocytes adhesion, spreading, and proliferation throughout the aging process. Understanding the basic mechanisms of TIEG1 in tenocytes may provide valuable information for treating multiple tendon disorders.

  2. Adhesion and proliferation of skeletal muscle cells on single layer poly(lactic acid) ultra-thin films.

    PubMed

    Ricotti, Leonardo; Taccola, Silvia; Pensabene, Virginia; Mattoli, Virgilio; Fujie, Toshinori; Takeoka, Shinji; Menciassi, Arianna; Dario, Paolo

    2010-10-01

    An increasing interest in bio-hybrid systems and cell-material interactions is evident in the last years. This leads towards the development of new nano-structured devices and the assessment of their biocompatibility. In the present study, the development of free-standing single layer poly(lactic acid) (PLA) ultra-thin films is described, together with the analysis of topography and roughness properties. The biocompatibility of the PLA films has been tested in vitro, by seeding C2C12 skeletal muscle cells, and thus assessing cells shape, density and viability after 24, 48 and 72 h. The results show that free-standing flexible PLA nanofilms represent a good matrix for C2C12 cells adhesion, spreading and proliferation. Early differentiation into myotubes is also allowed. The biocompatibility of the novel ultra-thin films as substrates for cell growth promotes their application in the fields of regenerative medicine, muscle tissue engineering, drug delivery, and-in general-in the field of bio-hybrid devices.

  3. Increasing α7β1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression

    PubMed Central

    Liu, Jianming; Burkin, Dean J.; Kaufman, Stephen J.

    2008-01-01

    The dystrophin-glycoprotein complex maintains the integrity of skeletal muscle by associating laminin in the extracellular matrix with the actin cytoskeleton. Several human muscular dystrophies arise from defects in the components of this complex. The α7β1-integrin also binds laminin and links the extracellular matrix with the cytoskeleton. Enhancement of α7-integrin levels alleviates pathology in mdx/utrn−/− mice, a model of Duchenne muscular dystrophy, and thus the integrin may functionally compensate for the absence of dystrophin. To test whether increasing α7-integrin levels affects transcription and cellular functions, we generated α7-integrin-inducible C2C12 cells and transgenic mice that overexpress the integrin in skeletal muscle. C2C12 myoblasts with elevated levels of integrin exhibited increased adhesion to laminin, faster proliferation when serum was limited, resistance to staurosporine-induced apoptosis, and normal differentiation. Transgenic expression of eightfold more integrin in skeletal muscle did not result in notable toxic effects in vivo. Moreover, high levels of α7-integrin in both myoblasts and in skeletal muscle did not disrupt global gene expression profiles. Thus increasing integrin levels can compensate for defects in the extracellular matrix and cytoskeleton linkage caused by compromises in the dystrophin-glycoprotein complex without triggering apparent overt negative side effects. These results support the use of integrin enhancement as a therapy for muscular dystrophy. PMID:18045857

  4. Proliferation, differentiation and apoptosis in connexin43-null osteoblasts

    NASA Technical Reports Server (NTRS)

    Furlan, F.; Lecanda, F.; Screen, J.; Civitelli, R.

    2001-01-01

    Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). We have shown that interference with Cx43 expression or function disrupts transcriptional regulation of osteoblast genes, and that deletion of Cx43 in the mouse causes skeletal malformations, delayed mineralization, and osteoblast dysfunction. Here, we studied the mechanisms by which genetic deficiency of Cx43 alters osteoblast development. While cell proliferation rates were similar in osteoblastic cells derived from calvaria of Cx43-null and wild type mice, camptothecin-induced apoptosis was 3-fold higher in mutant compared to wild type osteoblasts. When grown in mineralizing medium, Cx43-null cells were able to produce mineralized matrix but it took one week longer to reach the same mineralization levels as in normal cells. Likewise, expression of alkaline phosphatase activity per cell--a marker of osteoblast differentiation--was maximal only 2 weeks later in Cx43-null relative to wild-type cells. These observations suggest that Cx43 is important for a normal and timely development of the osteoblastic phenotype. Delayed differentiation and increase programmed cell death may explain the skeletal phenotype of Cx43-null mice.

  5. Adhesions

    MedlinePlus

    Adhesions are bands of scar-like tissue. Normally, internal tissues and organs have slippery surfaces so they can shift easily as the body moves. Adhesions cause tissues and organs to stick together. They ...

  6. Adhesion

    MedlinePlus

    ... the intestines, adhesions can cause partial or complete bowel obstruction . Adhesions inside the uterine cavity, called Asherman syndrome , ... 1. Read More Appendicitis Asherman syndrome Glaucoma Infertility Intestinal obstruction Review Date 4/5/2016 Updated by: Irina ...

  7. Nanomaterial scaffolds for stem cell proliferation and differentiation in tissue engineering.

    PubMed

    Zhao, Chunyan; Tan, Aaron; Pastorin, Giorgia; Ho, Han Kiat

    2013-01-01

    Tissue engineering is a clinically driven field and has emerged as a potential alternative to organ transplantation. The cornerstone of successful tissue engineering rests upon two essential elements: cells and scaffolds. Recently, it was found that stem cells have unique capabilities of self-renewal and multilineage differentiation to serve as a versatile cell source, while nanomaterials have lately emerged as promising candidates in producing scaffolds able to better mimic the nanostructure in natural extracellular matrix and to efficiently replace defective tissues. This article, therefore, reviews the key developments in tissue engineering, where the combination of stem cells and nanomaterial scaffolds has been utilized over the past several years. We consider the high potential, as well as the main issues related to the application of stem cells and nanomaterial scaffolds for a range of tissues including bone, cartilage, nerve, liver, eye etc. Promising in vitro results such as efficient attachment, proliferation and differentiation of stem cells have been compiled in a series of examples involving different nanomaterials. Furthermore, the merits of the marriage of stem cells and nanomaterial scaffolds are also demonstrated in vivo, providing early successes to support subsequent clinical investigations. This progress simultaneously drives mechanistic research into the mechanotransduction process responsible for the observations in order to optimize the process further. Current understanding is chiefly reported to involve the interaction of stem cells and the anchoring nanomaterial scaffolds by activating various signaling pathways. Substrate surface characteristics and scaffold bulk properties are also reported to influence not only short term stem cell adhesion, spreading and proliferation, but also longer term lineage differentiation, functionalization and viability. It is expected that the combination of stem cells and nanomaterials will develop into an

  8. The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro

    SciTech Connect

    Schlie-Wolter, Sabrina; Ngezahayo, Anaclet; Chichkov, Boris N.

    2013-06-10

    Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions—which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance. -- Highlights: • We analyse the impact of ECM components on cell behaviour in vitro. • We compare five different cell types, using the same culture conditions. • The ECM significantly guides all cell responses. • Cell behaviour follows a cell specific ligand-priority ranking. • This gives insight in tissue formation and is essential for biomedical applications.

  9. Decreased astroglial cell adhesion and proliferation on zinc oxide nanoparticle polyurethane composites

    PubMed Central

    Seil, Justin T; Webster, Thomas J

    2008-01-01

    Nanomaterials offer a number of properties that are of interest to the field of neural tissue engineering. Specifically, materials that exhibit nanoscale surface dimensions have been shown to promote neuron function while simultaneously minimizing the activity of cells such as astrocytes that inhibit central nervous system regeneration. Studies demonstrating enhanced neural tissue regeneration in electrical fields through the use of conductive materials have led to interest in piezoelectric materials (or those materials which generate a transient electrical potential when mechanically deformed) such as zinc oxide (ZnO). It has been speculated that ZnO nanoparticles possess increased piezoelectric properties over ZnO micron particles. Due to this promise in neural applications, the objective of the present in vitro study was, for the first time, to assess the activity of astroglial cells on ZnO nanoparticle polymer composites. ZnO nanoparticles embedded in polyurethane were analyzed via scanning electron microscopy to evaluate nanoscale surface features of the composites. The surface chemistry was characterized via X-ray photoelectron spectroscopy. Astroglial cell response was evaluated based on cell adhesion and proliferation. Astrocyte adhesion was significantly reduced on ZnO nanoparticle/polyurethane (PU) composites with a weight ratio of 50:50 (PU:ZnO) wt.%, 75:25 (PU:ZnO) wt.%, and 90:10 (PU:ZnO) wt.% in comparison to pure PU. The successful production of ZnO nanoparticle composite scaffolds suitable for decreasing astroglial cell density demonstrates their potential as a nerve guidance channel material with greater efficiency than what may be available today. PMID:19337420

  10. Proliferation and osteogenic differentiation of rat BMSCs on a novel Ti/SiC metal matrix nanocomposite modified by friction stir processing

    NASA Astrophysics Data System (ADS)

    Zhu, Chenyuan; Lv, Yuting; Qian, Chao; Qian, Haixin; Jiao, Ting; Wang, Liqiang; Zhang, Fuqiang

    2016-12-01

    The aims of this study were to fabricate a novel titanium/silicon carbide (Ti/SiC) metal matrix nanocomposite (MMNC) by friction stir processing (FSP) and to investigate its microstructure and mechanical properties. In addition, the adhesion, proliferation and osteogenic differentiation of rat bone marrow stromal cells (BMSCs) on the nanocomposite surface were investigated. The MMNC microstructure was observed by both scanning and transmission electron microscopy. Mechanical properties were characterized by nanoindentation and Vickers hardness testing. Integrin β1 immunofluorescence, cell adhesion, and MTT assays were used to evaluate the effects of the nanocomposite on cell adhesion and proliferation. Osteogenic and angiogenic differentiation were evaluated by alkaline phosphatase (ALP) staining, ALP activity, PCR and osteocalcin immunofluorescence. The observed microstructures and mechanical properties clearly indicated that FSP is a very effective technique for modifying Ti/SiC MMNC to contain uniformly distributed nanoparticles. In the interiors of recrystallized grains, characteristics including twins, fine recrystallized grains, and dislocations formed concurrently. Adhesion, proliferation, and osteogenic and angiogenic differentiation of rat BMSCs were all enhanced on the novel Ti/SiC MMNC surface. In conclusion, nanocomposites modified using FSP technology not only have superior mechanical properties under stress-bearing conditions but also provide improved surface and physicochemical properties for cell attachment and osseointegration.

  11. Proliferation and osteogenic differentiation of rat BMSCs on a novel Ti/SiC metal matrix nanocomposite modified by friction stir processing

    PubMed Central

    Zhu, Chenyuan; Lv, Yuting; Qian, Chao; Qian, Haixin; Jiao, Ting; Wang, Liqiang; Zhang, Fuqiang

    2016-01-01

    The aims of this study were to fabricate a novel titanium/silicon carbide (Ti/SiC) metal matrix nanocomposite (MMNC) by friction stir processing (FSP) and to investigate its microstructure and mechanical properties. In addition, the adhesion, proliferation and osteogenic differentiation of rat bone marrow stromal cells (BMSCs) on the nanocomposite surface were investigated. The MMNC microstructure was observed by both scanning and transmission electron microscopy. Mechanical properties were characterized by nanoindentation and Vickers hardness testing. Integrin β1 immunofluorescence, cell adhesion, and MTT assays were used to evaluate the effects of the nanocomposite on cell adhesion and proliferation. Osteogenic and angiogenic differentiation were evaluated by alkaline phosphatase (ALP) staining, ALP activity, PCR and osteocalcin immunofluorescence. The observed microstructures and mechanical properties clearly indicated that FSP is a very effective technique for modifying Ti/SiC MMNC to contain uniformly distributed nanoparticles. In the interiors of recrystallized grains, characteristics including twins, fine recrystallized grains, and dislocations formed concurrently. Adhesion, proliferation, and osteogenic and angiogenic differentiation of rat BMSCs were all enhanced on the novel Ti/SiC MMNC surface. In conclusion, nanocomposites modified using FSP technology not only have superior mechanical properties under stress-bearing conditions but also provide improved surface and physicochemical properties for cell attachment and osseointegration. PMID:27958394

  12. Investigation of In Vitro Bone Cell Adhesion and Proliferation on Ti Using Direct Current Stimulation

    PubMed Central

    Bodhak, Subhadip; Bose, Susmita; Kinsel, William C.; Bandyopadhyay, Amit

    2012-01-01

    Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 µA, were used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell–materials interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 µA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 µA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell-materials interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model. PMID:23144532

  13. Videography supported adhesion, and proliferation behavior of MG-63 osteoblastic cells on 2.5D titania nanotube matrices.

    PubMed

    Manurung, Robeth Viktoria; Fu, Pei-Wen; Chu, Yeh-Shiu; Lo, Chun-Min; Chattopadhyay, Surojit

    2016-04-01

    Human osteosarcoma cells MG-63 were cultured on anodically etched titania nanotubes (TiO2 NT), with diameters ranging from 40-100 nm, to study the correlations between cell proliferation and adhesion on the 2.5 dimensional (2.5D) extracellular matrix (ECM). Unlike other reports, mostly based on mouse stem cells, and 2D cell culture, our studies indicate that the 2.5D NT promote higher proliferation and activity, but less 2D adhesion. Proliferation of the MG-63 cells was significantly higher in the NTs, the best being the 70 nm diameter sample, compared to planar titania (control). This is consistent with previous studies. However, cellular adhesion was stronger on TiO2 NT with increasing diameter, and highest on the control as obtained from shear stress measurement, paxilin imaging, and western blot measurements probing focal adhesion kinase, p130 CAS, and extracellular-regulated kinase, in addition to cell morphology imaging by fluorescence microscopy. We provide direct videography of cell migration, and cell speed data indicating faster filopodial activity on the TiO2 NT surfaces having lower adhesion. This evidence was not available previously. The NT matrices promote cells with smaller surface area, because of less 2D stretching. In contrast, on comparatively planar 2D-like surfaces uniaxial stretching of the cell body with strong anchoring of the filopodia, resulted in larger cell surface area, and demonstrated stronger adhesion. The difference in the results, with those previously published, may be generally attributed to, among others, the use of mouse stem cells (human osteosarcoma used here), and unannealed as-grown TiO2 NTs used previously (annealed ECMs used here).

  14. The role of adhesion strength in human mesenchymal stem cell osteoblastic differentiation on biodegradable polymers

    NASA Astrophysics Data System (ADS)

    Krizan, Sylva Jana

    Human mesenchymal stem cells (hMSC) are promising candidates for promoting bone growth on biodegradable polymer scaffolds however little is known about early hMSC-polymer interactions. Adhesion is highly dynamic and during adhesive reinforcement, numerous proteins form adhesion plaques linking the cell's cytoskeleton with the extracellular environment. These proteins are known to affect cellular function but their role in hMSC differentiation is less clear. Adhesion plaques are associated with adhesive force, still a detachment force of hMSC on polycaprolactone (PCL), poly-lactide-co-glycolide (PLGA) or alginate has never been described or shown to affect downstream function. We demonstrate that hMSC attached to PCL, PLGA and alginate exhibit different adhesion strengths (tau50) as determined by both fluid shear and spinning disk systems, with PLGA demonstrating the greatest tau 50. Elastic modulus and hydrophobicity were characterized for these surfaces and correlated positively with tau50 to an optimum. Attachment studies of hMSC showed that adhesion plateau timespans were independent of cell line and surface but both morphology and focal adhesion expression varied by polymer type. Differentiation studies of hMSC on PLGA and PCL showed a strong association between markers of differentiation (alkaline phosphatase activity and mineral content) and tau50 within polymer groups, but a poor relationship was found between tau50 and differentiation across polymer groups, suggesting that other polymer properties may be important for differentiation. Subsequently, we examined the role of focal adhesion kinase (FAK) and Rho-GTPase (RhoA) on hMSC adhesion and differentiation when plated onto PLGA. hMSC were retrovirally transduced with mutant constructs of FAK and RhoA cDNA. Alternatively, hMSC were treated with Rho-kinase inhibitor, Y27632. Both cells transduced with mutant RhoA or FAK constructs, or those treated with Y27632 displayed aberrant cell morphology and changes

  15. Enteric glia modulate epithelial cell proliferation and differentiation through 15-deoxy-Δ12,14-prostaglandin J2

    PubMed Central

    Bach-Ngohou, Kalyane; Mahé, Maxime M; Aubert, Philippe; Abdo, Hind; Boni, Sébastien; Bourreille, Arnaud; Denis, Marc G; Lardeux, Bernard; Neunlist, Michel; Masson, Damien

    2010-01-01

    The enteric nervous system (ENS) and its major component, enteric glial cells (EGCs), have recently been identified as a major regulator of intestinal epithelial barrier functions. Indeed, EGCs inhibit intestinal epithelial cell (IEC) proliferation and increase barrier resistance and IEC adhesion via the release of EGC-derived soluble factors. Interestingly, EGC regulation of intestinal epithelial barrier functions is reminiscent of previously reported peroxisome proliferator-activated receptor γ (PPARγ)-dependent functional effects. In this context, the present study aimed at identifying whether EGC could synthesize and release the main PPARγ ligand, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), and regulate IEC functions such as proliferation and differentiation via a PPARγ dependent pathway. First, we demonstrated that the lipocalin but not the haematopoetic form for prostaglandin D synthase (PGDS), the enzyme responsible of 15dPGJ2 synthesis, was expressed in EGCs of the human submucosal plexus and of the subepithelium, as well as in rat primary culture of ENS and EGC lines. Next, 15dPGJ2 was identified in EGC supernatants of various EGC lines. 15dPGJ2 reproduced EGC inhibitory effects upon IEC proliferation, and inhibition of lipocalin PGDS expression by shRNA abrogated these effects. Furthermore, EGCs induced nuclear translocation of PPARγ in IEC, and both EGC and 15dPGJ2 effects upon IEC proliferation were prevented by the PPARγ antagonist GW9662. Finally, EGC induced differentiation-related gene expression in IEC through a PPARγ-dependent pathway. Our results identified 15dPGJ2 as a novel glial-derived mediator involved in the control of IEC proliferation/differentiation through activation of PPARγ. They also suggest that alterations of glial PGDS expression may modify intestinal epithelial barrier functions and be involved in the development of pathologies such as cancer or inflammatory bowel diseases. PMID:20478974

  16. Proliferation and differentiation of neural stem cells irradiated with X-rays in logarithmic growth phase.

    PubMed

    Isono, Mayu; Otsu, Masahiro; Konishi, Teruaki; Matsubara, Kana; Tanabe, Toshiaki; Nakayama, Takashi; Inoue, Nobuo

    2012-07-01

    Exposure of the fetal brain to ionizing radiation causes congenital brain abnormalities. Normal brain formation requires regionally and temporally appropriate proliferation and differentiation of neural stem cells (NSCs) into neurons and glia. Here, we investigated the effects of X-irradiation on proliferating homogenous NSCs prepared from mouse ES cells. Cells irradiated with X-rays at a dose of 1Gy maintained the capabilities for proliferation and differentiation but stopped proliferation temporarily. In contrast, the cells ceased proliferation following irradiation at a dose of >5Gy. These results suggest that irradiation of the fetal brain at relatively low doses may cause congenital brain abnormalities as with relatively high doses.

  17. Nanoceramics on osteoblast proliferation and differentiation in bone tissue engineering.

    PubMed

    S, Sai Nievethitha; N, Subhapradha; D, Saravanan; N, Selvamurugan; Tsai, Wei-Bor; N, Srinivasan; R, Murugesan; A, Moorthi

    2017-05-01

    Bone, a highly dynamic connective tissue, consist of a bioorganic phase comprising osteogenic cells and proteins which lies over an inorganic phase predominantly made of CaPO4 (biological apatite). Injury to bone can be due to mechanical, metabolic or inflammatory agents also owing pathological conditions like fractures, osteomyelitis, osteolysis or cysts may arise in enameloid, chondroid, cementum, or chondroid bone which forms the intermediate tissues of the body. Bone tissue engineering (BTE) applies bioactive scaffolds, host cells and osteogenic signals for restoring damaged or diseased tissues. Various bioceramics used in BTE can be bioactive (like glass ceramics and hydroxyapatite bioactive glass), bioresorbable (like tricalcium phosphates) or bioinert (like zirconia and alumina). Limiting the size of these materials to nano-scale has resulted in a higher surface area to volume ratio thereby improving multi-functionality, solubility, surface catalytic activity, high heat and electrical conductivity. Nanoceramics have been found to induce osteoconduction, osteointegration, osteogenesis and osteoinduction. The present review aims at summarizing the interactions of nanoceramics and osteoblast/stem cells for promoting the proliferation and differentiation of the osteoblast cells by nanoceramics as superior bone substitutes in bone tissue engineering applications.

  18. Tracking autophagy during proliferation and differentiation of Trypanosoma brucei

    PubMed Central

    Proto, William R.; Jones, Nathaniel G.; Coombs, Graham H.; Mottram, Jeremy C.

    2014-01-01

    Autophagy is a lysosome-dependent degradation mechanism that sequesters target cargo into autophagosomal vesicles. The Trypanosoma brucei genome contains apparent orthologues of several autophagy-related proteins including an ATG8 family. These ubiquitin-like proteins are required for autophagosome membrane formation, but our studies show that ATG8.3 is atypical. To investigate the function of other ATG proteins, RNAi compatible T. brucei were modified to function as autophagy reporter lines by expressing only either YFP-ATG8.1 or YFP-ATG8.2. In the insect procyclic lifecycle stage, independent RNAi down-regulation of ATG3 or ATG7 generated autophagy-defective mutants and confirmed a pro-survival role for autophagy in the procyclic form nutrient starvation response. Similarly, RNAi depletion of ATG5 or ATG7 in the bloodstream form disrupted autophagy, but did not impede proliferation. Further characterisation showed bloodstream form autophagy mutants retain the capacity to undergo the complex cellular remodelling that occurs during differentiation to the procyclic form and are equally susceptible to dihydroxyacetone-induced cell death as wild type parasites, not supporting a role for autophagy in this cell death mechanism. The RNAi reporter system developed, which also identified TOR1 as a negative regulator controlling YFP-ATG8.2 but not YFP-ATG8.1 autophagosome formation, will enable further targeted analysis of the mechanisms and function of autophagy in the medically relevant bloodstream form of T. brucei. PMID:28357206

  19. The Hedgehog pathway: role in cell differentiation, polarity and proliferation.

    PubMed

    Jia, Yanfei; Wang, Yunshan; Xie, Jingwu

    2015-02-01

    Hedgehog (Hh) is first described as a genetic mutation that has "spiked" phenotype in the cuticles of Drosophila in later 1970s. Since then, Hh signaling has been implicated in regulation of differentiation, proliferation, tissue polarity, stem cell population and carcinogenesis. The first link of Hh signaling to cancer was established through discovery of genetic mutations of Hh receptor gene PTCH1 being responsible for Gorlin syndrome in 1996. It was later shown that Hh signaling is associated with many types of cancer, including skin, leukemia, lung, brain and gastrointestinal cancers. Another important milestone for the Hh research field is the FDA approval for the clinical use of Hh inhibitor Erivedge/Vismodegib for treatment of locally advanced and metastatic basal cell carcinomas. However, recent clinical trials of Hh signaling inhibitors in pancreatic, colon and ovarian cancer all failed, indicating a real need for further understanding of Hh signaling in cancer. In this review, we will summarize recent progress in the Hh signaling mechanism and its role in human cancer.

  20. Differential adhesion of microspheres mediated by DNA hybridization I: experiment.

    PubMed

    Zhang, Ying; Milam, Valeria T; Graves, David J; Hammer, Daniel A

    2006-06-01

    We have developed a novel method to study collective behavior of multiple hybridized DNA chains by measuring the adhesion of DNA-coated micron-scale beads under hydrodynamic flow. Beads coated with single-stranded DNA probes are linked to surfaces coated with single target strands through DNA hybridization, and hydrodynamic shear forces are used to discriminate between strongly and weakly bound beads. The adhesiveness of microspheres depends on the strength of interaction between DNA chains on the bead and substrate surfaces, which is a function of the degree of DNA chain overlap, the fidelity of the match between hybridizing pairs, and other factors that affect the hybridization energy, such as the salt concentration in the hybridization buffer. The force for bead detachment is linearly proportional to the degree of chain overlap. There is a detectable drop in adhesion strength when there is a single base mismatch in one of the hybridizing chains. The effect of single nucleotide mismatch was tested with two different strand chemistries, with mutations placed at several different locations. All mutations were detectable, but there was no comprehensive rule relating the drop in adhesive strength to the location of the defect. Since adhesiveness can be coupled to the strength of overlap, the method holds promise to be a novel methodology for oligonucleotide detection.

  1. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee.

    PubMed

    Advani, Alexander S; Chen, Annie Y; Babbitt, Courtney C

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors.

  2. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee

    PubMed Central

    Advani, Alexander S.; Chen, Annie Y.; Babbitt, Courtney C.

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors. PMID:26971204

  3. Evaluation of magnesium alloys with alternative surface finishing for the proliferation and chondro-differentiation of human mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Trinidad, J.; Arruebarrena, G.; Sáenz De Argandoña, E.; Ruiz De Eguino, G.; Infante, A.; Rodríguez, C. I.

    2010-11-01

    Articular cartilage has little capacity for self-repair. As a result, continuous mechanical stress can lead to the degradation of articular cartilage, culminating in progressive damage and joint degeneration. Tissue engineering has arisen as a promising therapeutic approach to cartilage repair. Magnesium alloys are one of the most important metallic biomaterials emerging in this area due to their biocompatibility, bio-absorbability and especially to their mechanical properties. These properties make magnesium alloys a promising biomaterial in the regeneration of cartilage tissue. Objective. This study was undertaken to analyze the influence of surface characteristics of magnesium alloys in the adhesion, proliferation and differentiation of human mesenchymal stem cells (MSCs). Methods. Two commercial magnesium alloys (AZ31B and ZM21) were subjected to different treatments in order to obtain four different surfaces in each alloy. Human MSCs were seeded into the magnesium alloys and analyzed for their proliferation and chondrogenesis differentiation ability. Results. Human MSCs showed a greater proliferation and chondro-differentiation when cultured in the ZM21 magnesium alloy with a surface finishing of fine sanding, polishing, and etching.

  4. β-eudesmol, a sesquiterpene from Teucrium ramosissimum, inhibits superoxide production, proliferation, adhesion and migration of human tumor cell.

    PubMed

    Ben Sghaier, Mohamed; Mousslim, Mohamed; Pagano, Alessandra; Ammari, Youssef; Luis, José; Kovacic, Hervé

    2016-09-01

    Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non phagocytic cells. These new homologues (Nox1-Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. In this study we examined the effect of β-eudesmol, a sesquiterpenoid alcohol isolated from Teucrium ramosissimum leaves, on proliferation, superoxide anion production, adhesion and migration of human lung (A549) and colon (HT29 and Caco-2) cancer cell lines. Proliferation of tumor cells was inhibited by β-eudesmol. It also significantly inhibited superoxide production in A549 cells. Furthermore, β-eudesmol inhibited adhesion and migration of A549 and HT29 cell. These results demonstrate that β-eudesmol may be a novel anticancer agent for the treatment of lung and colon cancer by different ways: by inhibition of superoxide production or by blocking proliferation, adhesion and migration.

  5. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

    SciTech Connect

    Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A.

    2014-10-17

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

  6. Pak2 regulates hematopoietic progenitor cell proliferation, survival and differentiation

    PubMed Central

    Zeng, Yi; Broxmeyer, Hal E.; Staser, Karl; Chitteti, Brahmananda Reddy; Park, Su-Jung; Hahn, Seongmin; Cooper, Scott; Sun, Zejin; Jiang, Li; Yang, XianLin; Yuan, Jin; Kosoff, Rachelle; Sandusky, George; Srour, Edward F.; Chernoff, Jonathan; Clapp, Wade

    2015-01-01

    p21-activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long-term hematopoiesis and lineage commitment remain unreported. Utilizing a conditional Pak2 knock out (KO) mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short- and long-term competitive hematopoiesis than wild type (WT) cells, it does not affect HSC self-renewal per se. Pak2 disruption decreased the survival and proliferation of multi-cytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by 1) a three to six-fold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte-monocyte progenitors (GMPs) in mice transplanted with Pak2-disrupted BM; 2) Pak2-disrupted BM and c-kit+ cells yielded higher numbers of more mature subsets of granulocyte-monocyte colonies and polymophonuclear neutrophils (PMNs), respectively, when cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF). Pak2 disruption resulted respectively in decreased and increased gene expression of transcription factors JunB and c-Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte-monocyte lineage commitment. Furthermore, Pak2 disruption led to 1) higher percentage of CD4+CD8+ double positive T cells and lower percentages of CD4+CD8− or CD4−CD8+ single positive T cells in thymus and 2) decreased numbers of mature B cells and increased numbers of Pre-Pro B cells in BM, suggesting defects in lymphopoiesis. PMID:25586960

  7. Six family genes control the proliferation and differentiation of muscle satellite cells

    SciTech Connect

    Yajima, Hiroshi; Motohashi, Norio; Ono, Yusuke; Sato, Shigeru; Ikeda, Keiko; Masuda, Satoru; Yada, Erica; Kanesaki, Hironori; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Kawakami, Kiyoshi

    2010-10-15

    Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4{sup +/-}Six5{sup -/-} mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.

  8. The Antiaging Gene Klotho Regulates Proliferation and Differentiation of Adipose-Derived Stem Cells

    PubMed Central

    Fan, Jun; Sun, Zhongjie

    2017-01-01

    Klotho was originally discovered as an aging-suppressor gene. The purpose of this study was to investigate whether secreted Klotho (SKL) affects the proliferation and differentiation of adipose-derived stem cells (ADSCs). RT-PCR and Western blot analysis showed that short-form Klotho was expressed in mouse ADSCs. The Klotho gene mutation KL(−/−) significantly decreased proliferation of ADSCs and expression of pluripotent transcription factors (Nanog, Sox-2, and Oct-4) in mice. The adipogenic differentiation of ADSCs was also decreased in KL(−/−) mice. Incubation with Klotho-deficient medium decreased ADSC proliferation, pluripotent transcription factor levels, and adipogenic differentiation, which is similar to what was found in KL(−/−) mice. These results indicate that Klotho deficiency suppresses ADSC proliferation and differentiation. Interestingly, treatment with recombinant SKL protein rescued the Klotho deficiency-induced impairment in ADSC proliferation and adipogenic differentiation. SKL also regulated ADSCs’ differentiation to other cell lineages (osteoblasts, myofibroblasts), indicating that SKL maintains stemness of ADSCs. It is intriguing that overexpression of SKL significantly increased PPAR-γ expression and lipid formation in ADSCs following adipogenic induction, indicating enhanced adipogenic differentiation. Overexpression of SKL inhibited expression of TGFβ1 and its downstream signaling mediator Smad2/3. This study demonstrates, for the first time, that SKL is essential to the maintenance of normal proliferation and differentiation in ADSCs. Klotho regulates adipogenic differentiation in ADSCs, likely via inhibition of TGFβ1 and activation of PPAR-γ. PMID:26865060

  9. Effects of hierarchical micro/nano-topographies on the morphology, proliferation and differentiation of osteoblast-like cells.

    PubMed

    Huang, Qianli; Elkhooly, Tarek A; Liu, Xujie; Zhang, Ranran; Yang, Xing; Shen, Zhijian; Feng, Qingling

    2016-09-01

    Coating the surfaces of titanium-based implants with appropriate hierarchical micro/nano-topographies resembling the structure of natural bone significantly enhances their biological performance. However, the relationship between nanostructures surfaces and their effects on modulating cellular response is not clearly understood. Moreover, it is not clear whether the surface chemistry or topography is the main factor on modulating cellular behavior, because the commonly used surface modification techniques for titanium-based implants simultaneously modify surface topography and chemistry. The aim of this study is to investigate osteoblast-like cell adhesion, proliferation and differentiation on hierarchical micro/nano-topographies with similar surface chemistry but different nano-scale features. Micro-arc oxidation and post hydrothermal treatment were employed to fabricate micro/nano-topographies on titanium. According to the morphological features, they were classified as microcrater (micro-topography), nanoplate (hierarchical topography with nanoplates) and nanoleaf (hierarchical topography with nanoleaves). The response of osteoblast like cells (SaOS-2) was studied on each surface after sputtering with a thin layer of gold (Au) to minimize the influence of surface chemistry. The morphological evaluation after histochemical staining revealed that the adherent cells were polygonal-shaped on microcrater surface, roundish on nanoplate surface and elongated on nanoleaf surface. Additionally, compared to microcrater surface, nanoplate surface slowed down cell proliferation and exhibited no enhancement on cell differentiation. However, nanoleaf surface supported cell proliferation and promoted cell differentiation. The results indicate that tuning morphological features of nanostructures on micro-topography can serve as a promising strategy to specifically modulate cellular response, such as cell morphology, proliferation, differentiation and mineralization.

  10. Computer simulations of cell sorting due to differential adhesion.

    PubMed

    Zhang, Ying; Thomas, Gilberto L; Swat, Maciej; Shirinifard, Abbas; Glazier, James A

    2011-01-01

    The actions of cell adhesion molecules, in particular, cadherins during embryonic development and morphogenesis more generally, regulate many aspects of cellular interactions, regulation and signaling. Often, a gradient of cadherin expression levels drives collective and relative cell motions generating macroscopic cell sorting. Computer simulations of cell sorting have focused on the interactions of cells with only a few discrete adhesion levels between cells, ignoring biologically observed continuous variations in expression levels and possible nonlinearities in molecular binding. In this paper, we present three models relating the surface density of cadherins to the net intercellular adhesion and interfacial tension for both discrete and continuous levels of cadherin expression. We then use then the Glazier-Graner-Hogeweg (GGH) model to investigate how variations in the distribution of the number of cadherins per cell and in the choice of binding model affect cell sorting. We find that an aggregate with a continuous variation in the level of a single type of cadherin molecule sorts more slowly than one with two levels. The rate of sorting increases strongly with the interfacial tension, which depends both on the maximum difference in number of cadherins per cell and on the binding model. Our approach helps connect signaling at the molecular level to tissue-level morphogenesis.

  11. Effect of dentin treatment on proliferation and differentiation of human dental pulp stem cells

    PubMed Central

    Park, Minjeong; Pang, Nan-Sim

    2015-01-01

    Objectives Sodium hypochlorite (NaOCl) is an excellent bactericidal agent, but it is detrimental to stem cell survival, whereas intracanal medicaments such as calcium hydroxide (Ca[OH]2) promote the survival and proliferation of stem cells. This study evaluated the effect of sequential NaOCl and Ca[OH]2 application on the attachment and differentiation of dental pulp stem cells (DPSCs). Materials and Methods DPSCs were obtained from human third molars. All dentin specimens were treated with 5.25% NaOCl for 30 min. DPSCs were seeded on the dentin specimens and processed with additional 1 mg/mL Ca[OH]2, 17% ethylenediaminetetraacetic acid (EDTA) treatment, file instrumentation, or a combination of these methods. After 7 day of culture, we examined DPSC morphology using scanning electron microscopy and determined the cell survival rate with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We measured cell adhesion gene expression levels after 4 day of culture and odontogenic differentiation gene expression levels after 4 wk using quantitative real-time polymerase chain reaction. Results DPSCs did not attach to the dentin in the NaOCl-treated group. The gene expression levels of fibronectin-1 and secreted phosphoprotein-1 gene in both the Ca[OH]2- and the EDTA-treated groups were significantly higher than those in the other groups. All Ca[OH]2-treated groups showed higher expression levels of dentin matrix protein-1 than that of the control. The dentin sialophosphoprotein level was significantly higher in the groups treated with both Ca[OH]2 and EDTA. Conclusions The application of Ca[OH]2 and additional treatment such as EDTA or instrumentation promoted the attachment and differentiation of DPSCs after NaOCl treatment. PMID:26587415

  12. Metabolic profiling of hematopoietic stem and progenitor cells during proliferation and differentiation into red blood cells.

    PubMed

    Daud, Hasbullah; Browne, Susan; Al-Majmaie, Rasoul; Murphy, William; Al-Rubeai, Mohamed

    2016-01-25

    An understanding of the metabolic profile of cell proliferation and differentiation should support the optimization of culture conditions for hematopoietic stem and progenitor cell (HSPC) proliferation, differentiation, and maturation into red blood cells. We have evaluated the key metabolic parameters during each phase of HSPC culture for red blood cell production in serum-supplemented (SS) and serum-free (SF) conditions. A simultaneous decrease in growth rate, total protein content, cell size, and the percentage of cells in the S/G2 phase of cell cycle, as well as an increase in the percentage of cells with a CD71(-)/GpA(+) surface marker profile, indicates HSPC differentiation into red blood cells. Compared with proliferating HSPCs, differentiating HSPCs showed significantly lower glucose and glutamine consumption rates, lactate and ammonia production rates, and amino acid consumption and production rates in both SS and SF conditions. Furthermore, extracellular acidification was associated with late proliferation phase, suggesting a reduced cellular metabolic rate during the transition from proliferation to differentiation. Under both SS and SF conditions, cells demonstrated a high metabolic rate with a mixed metabolism of both glycolysis and oxidative phosphorylation (OXPHOS) in early and late proliferation, an increased dependence on OXPHOS activity during differentiation, and a shift to glycolytic metabolism only during maturation phase. These changes indicate that cell metabolism may have an important impact on the ability of HSPCs to proliferate and differentiate into red blood cells.

  13. Effects of wollastonite on proliferation and differentiation of human bone marrow-derived stromal cells in PHBV/wollastonite composite scaffolds.

    PubMed

    Li, Haiyan; Zhai, Wanying; Chang, Jiang

    2009-09-01

    In this study, the effects of wollastonite on proliferation and differentiation of human bone marrow-derived stromal cells (hBMSCs) have been investigated based on a polyhydroxybutyrate-co-hydroxyvalerate (PHBV)/ wollastonite (W) composite scaffolds system. Cell morphology, proliferation, and differentiation were measured. The results showed that the incorporation of wollastonite benefited hBMSCs adhesion, proliferation, and differentiation rate. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composite scaffolds increased from 10 to 20 wt%. Based on our previous studies on PHBV/W composite discs, the differentiation measurements in this paper further proved that the wollastonite itself can stimulate the hBMSCs to differentiate toward osteoblasts without any osteogenic medium, and the ionic products (Ca and Si) released from wollastonite might contribute to this advantage. It is also suggested that the osteogenic differentiation of the hBMSCs can be affected by adjusting the wollastonite content in the composite scaffolds.

  14. Involvement of small G protein RhoB in the regulation of proliferation, adhesion and migration by dexamethasone in osteoblastic cells

    PubMed Central

    Wang, Yan; Li, Yidong; Xu, Weidong; Lu, Jian

    2017-01-01

    Long-term exposure to therapeutic doses of glucocorticoids (GCs) results in bone remodeling, which frequently causes osteoporosis and fracture healing retardation because of the abnormality of osteoblastic proliferation and differentiation. The mechanisms of GCs’ effect on osteoblasts are largely unknown. In this present study, we found that dexamethasone (Dex) could induce the expression of the small G protein, RhoB, in mRNA and protein levels in the osteoblast-derived osteosarcoma cell lines MG-63. The up-regulation of RhoB mRNA by Dex mainly occurs at posttranscriptional level by increasing its mRNA stability through PI-3K/Akt and p38 mitogen-activated protein kinase signaling pathways. Over-expression of RhoB in MG-63 cells magnified while down-regulation of RhoB level by RNA interference impaired Dex-induced growth inhibition but not differentiation. What’s more, over-expression of RhoB mimicked the effect of Dex on cell adhesion and migration. And interfering RhoB expression partially suppressed Dex-induced pro-adhesion and anti-migration in MG-63 cells. In conclusion, these results indicate that RhoB plays an important role in the pathological effect of Dex on osteoblastic growth and migration, which is a part of the mechanisms of GCs’ adverse effect on bone remodeling. PMID:28323887

  15. The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing.

    PubMed

    Rashmi, R N; Eckes, Beate; Glöckner, Gernot; Groth, Marco; Neumann, Sascha; Gloy, Joachim; Sellin, Lorenz; Walz, Gerd; Schneider, Maria; Karakesisoglou, Iakowos; Eichinger, Ludwig; Noegel, Angelika A

    2012-03-01

    Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing.

  16. The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing

    PubMed Central

    Rashmi, R.N.; Eckes, Beate; Glöckner, Gernot; Groth, Marco; Neumann, Sascha; Gloy, Joachim; Sellin, Lorenz; Walz, Gerd; Schneider, Maria; Karakesisoglou, Iakowos; Eichinger, Ludwig; Noegel, Angelika A.

    2012-01-01

    Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing. PMID:22198684

  17. cAMP effects in neuroendocrine tumors: The role of Epac and PKA in cell proliferation and adhesion.

    PubMed

    Vitali, E; Cambiaghi, V; Spada, A; Tresoldi, A; Zerbi, A; Peverelli, E; Carnaghi, C; Mantovani, G; Lania, A G

    2015-12-10

    cAMP effects have been initially attributed to protein kinase A (PKA) activation. Subsequently, two exchange proteins directly activated by cAMP (Epac1/2) have been identified as cAMP targets. Aim of this study was to investigate cAMP effects in pancreatic-NET (P-NET) and bronchial carcinoids and in corresponding cell lines (QGP-1 and H727) on cell proliferation and adhesion and to determine PKA and Epac role in mediating these effects. We found that cAMP increased cyclin D1 expression in P-NET and QGP-1 cells, whereas it had opposite effects on bronchial carcinoids and H727 cells and it promoted cell adhesion in QGP-1 and H727 cells. These effects are mimicked by Epac and PKA specific analogs, activating the small GTPase Rap1. In conclusion, we demonstrated that cAMP exerted divergent effects on proliferation and promoted cell adhesion of different neuroendocrine cell types, these effects being mediated by both Epac and PKA and involving the same effector GTPase Rap1.

  18. Vasostatin-2 inhibits cell proliferation and adhesion in vascular smooth muscle cells, which are associated with the progression of atherosclerosis.

    PubMed

    Hou, Jianghong; Xue, Xiaolin; Li, Junnong

    2016-01-22

    Recently, the serum expression level of vasostatin-2 was found to be reduced and is being studied as an important indicator to assess the presence and severity of coronary artery disease; the functional properties of vasostatin-2 and its relationship with the development of atherosclerosis remains unclear. In this study, we attempted to detect the expression of vasostatin-2 and its impact on human vascular smooth muscle cells (VSMCs). Quantitative real-time PCR (qRT-PCR) and western blot were used to assess the expression level of vasostatin-2 in VSMCs between those from atherosclerosis and disease-free donors; we found that vasostatin-2 was significantly down-regulated in atherosclerosis patient tissues and cell lines. In addition, the over-expression of vasostatin-2 apparently inhibits cell proliferation and migration in VSMCs. Gain-of-function in vitro experiments further show that vasostatin-2 over-expression significantly inhibits inflammatory cytokines release in VSMCs. In addition, cell adhesion experimental analysis showed that soluble adhesion molecules (sICAM-1, sVCAM-1) had decreased expression when vasostatin-2 was over-expressed in VSMCs. Therefore, our results indicate that vasostatin-2 is an atherosclerosis-related factor that can inhibit cell proliferation, inflammatory response and cell adhesion in VSMCs. Taken together, our results indicate that vasostatin-2 could serve as a potential diagnostic biomarker and therapeutic option for human atherosclerosis in the near future.

  19. Synthesis of trace element Si and Sr codoping hydroxyapatite with non-cytotoxicity and enhanced cell proliferation and differentiation.

    PubMed

    Gao, Jianyong; Wang, Ming; Shi, Chao; Wang, Liping; Wang, Dalin; Zhu, Yingchun

    2016-11-01

    The main inorganic minerals in natural bones are non-stoichiometric hydroxyapatite (HA, Ca10[PO4]6[OH]2) doped with various trace elements, which may possess important biochemical effects. To investigate the functions of Sr and Si elements in human hard tissues, non-doped HA, trace Si doped HA, Si and Sr codoped HA with the concentration of natural bones are synthesized by hydrothermal method in this study. The samples are characterized by powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscope (SEM). The biological activities are evaluated via cytotoxicity study, adhesion and proliferation of osteoblast measurement, and alkaline phosphatase (ALP) assay. All the synthesized materials are HA phase, which have hierarchical structures with oriented HA nanorods assembled into the platy particles. These materials are non-cytotoxic against L929 cells line even at 400 μg/ml powder suspension. The results clearly indicate that the proliferation of L929 cells increases with trace elements doping from trace Si-HA to Si + Sr-HA. The adhesion and proliferation of osteoblast measurement illustrates that proliferation of osteoblasts advances about 1.3 times for Si-HA and about 1.8 times for Si + Sr-HA compared with undoped HA. In general, Si-HA with trace Si element shows enhanced cell differentiation, and Si + Sr-HA dual-doped with Si and Sr elements presents increased biological activity compared with Si-HA.

  20. Keratinocytes from APP/APLP2-deficient mice are impaired in proliferation, adhesion and migration in vitro.

    PubMed

    Siemes, Christina; Quast, Thomas; Kummer, Christiane; Wehner, Sven; Kirfel, Gregor; Müller, Ulrike; Herzog, Volker

    2006-07-01

    Growing evidence shows that the soluble N-terminal form (sAPPalpha) of the amyloid precursor protein (APP) represents an epidermal growth factor fostering keratinocyte proliferation, migration and adhesion. APP is a member of a protein family including the two mammalian amyloid precursor-like proteins APLP1 and APLP2. In the mammalian epidermis, only APP and APLP2 are expressed. APP and APLP2-deficient mice die shortly after birth but do not display a specific epidermal phenotype. In this report, we investigated the epidermis of APP and/or APLP2 knockout mice. Basal keratinocytes showed reduced proliferation in vivo by about 40%. Likewise, isolated keratinocytes exhibited reduced proliferation rates in vitro, which could be completely rescued by either exogenously added recombinant sAPPalpha, or by co-culture with dermal fibroblasts derived from APP knockout mice. Moreover, APP-knockout keratinocytes revealed reduced migration velocity resulting from severely compromised cell substrate adhesion. Keratinocytes from double knockout mice died within the first week of culture, indicating essential functions of APP-family members for survival in vitro. Our data indicate that sAPPalpha has to be considered as an essential epidermal growth factor which, however, in vivo can be functionally compensated to a certain extent by other growth factors, e.g., factors released from dermal fibroblasts.

  1. Keratinocytes from APP/APLP2-deficient mice are impaired in proliferation, adhesion and migration in vitro

    SciTech Connect

    Siemes, Christina; Quast, Thomas; Kummer, Christiane; Wehner, Sven; Kirfel, Gregor; Mueller, Ulrike; Herzog, Volker . E-mail: Herzog@uni-bonn.de

    2006-07-01

    Growing evidence shows that the soluble N-terminal form (sAPP{alpha}) of the amyloid precursor protein (APP) represents an epidermal growth factor fostering keratinocyte proliferation, migration and adhesion. APP is a member of a protein family including the two mammalian amyloid precursor-like proteins APLP1 and APLP2. In the mammalian epidermis, only APP and APLP2 are expressed. APP and APLP2-deficient mice die shortly after birth but do not display a specific epidermal phenotype. In this report, we investigated the epidermis of APP and/or APLP2 knockout mice. Basal keratinocytes showed reduced proliferation in vivo by about 40%. Likewise, isolated keratinocytes exhibited reduced proliferation rates in vitro, which could be completely rescued by either exogenously added recombinant sAPP{alpha}, or by co-culture with dermal fibroblasts derived from APP knockout mice. Moreover, APP-knockout keratinocytes revealed reduced migration velocity resulting from severely compromised cell substrate adhesion. Keratinocytes from double knockout mice died within the first week of culture, indicating essential functions of APP-family members for survival in vitro. Our data indicate that sAPP{alpha} has to be considered as an essential epidermal growth factor which, however, in vivo can be functionally compensated to a certain extent by other growth factors, e.g., factors released from dermal fibroblasts.

  2. Inhibition of proliferation and induction of differentiation of glioma cells with Datura stramonium agglutinin.

    PubMed

    Sasaki, T; Yamazaki, K; Yamori, T; Endo, T

    2002-10-07

    We found that a lectin, Datura stramonium agglutinin, induced irreversible differentiation in C6 glioma cells. The differentiated cells had long processes, a low rate of proliferation and a high content of glial fibrillary acidic protein. When the medium was replaced with Datura stramonium agglutinin-free medium after 1 h, cell proliferation continued to be inhibited. Experiments with several other lectins indicated that both recognition of linear N-acetyllactosamine repeats and recognition of multiantennary units of cell-surface glycans were required for the inhibition of C6 proliferation. Proliferation of four human glial tumour cells was also inhibited by Datura stramonium agglutinin. Further, these differentiated human glial tumour cells had long processes and a high content of glial fibrillary acidic protein similar to differentiated C6 glioma cells. Taken together, these observations suggest that Datura stramonium agglutinin may be useful as a new therapy for treating glioma without side effects.

  3. Silencing myotubularin related protein 7 enhances proliferation and early differentiation of C2C12 myoblast.

    PubMed

    Yuan, Zhuning; Chen, Yaosheng; Zhang, Xumeng; Zhou, Xingyu; Li, Mingsen; Chen, Hu; Wu, Ming; Zhang, Ying; Mo, Delin

    2017-03-11

    Myotubularin related protein 7 (MTMR7) is a key member of the highly conserved myotubularin related proteins (MTMRs) family, which has phosphatase activity. MTMR7 was increased during myoblast differentiation and exhibited high expression level at primary fibers formation stages in pigs. This suggests that MTMR7 may be involved in myogenesis. In our study, we investigated the roles of MTMR7 on proliferation and differentiation of C2C12 myoblasts. Knocking down MTMR7 not only enhanced myoblast early differentiation via altering the expression of Myf5, but also promoted myoblast proliferation through increasing cyclinA2 expression. The improved proliferation capacity was related to the increased phosphorylation of AKT. Taken together, our research demonstrates that MTMR7 plays an important role in proliferation and early differentiation of C2C12 myoblast.

  4. MAPs/bFGF-PLGA microsphere composite-coated titanium surfaces promote increased adhesion and proliferation of fibroblasts.

    PubMed

    Wang, Zhongshan; Wu, Guofeng; Bai, Shizhu; Feng, Zhihong; Dong, Yan; Zhou, Jian; Qin, Haiyan; Zhao, Yimin

    2014-06-01

    Infection and epithelial downgrowth are two major problems with maxillofacial transcutaneous implants, and both are mainly due to lack of stable closure of soft tissues at transcutaneous sites. Fibroblasts have been shown to play a key role in the formation of biological seals. In this work, titanium (Ti) model surfaces were coated with mussel adhesive proteins (MAPs) utilizing its unique adhesion ability on diverse inorganic and organic surfaces in wet environments. Prepared basic fibroblast growth factor (bFGF)-poly(lactic-co-glycolic acid) (PLGA) microspheres can be easily synthesized and combined onto MAPs-coated Ti surfaces, due to the negative surface charges of microspheres in aqueous solution, which is in contrast to the positive charges of MAPs. Titanium model surfaces were divided into three groups. Group A: MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces. Group B: MAPs-coated Ti surfaces. Group C: uncoated Ti surfaces. The effects of coated Ti surfaces on adhesion of fibroblasts, cytoskeletal organization, proliferation, and extracellular matrix (ECM)-related gene expressions were examined. The results revealed increased adhesion (P < 0.05), enhanced actin cytoskeletal organization, and up-regulated ECM-related gene expressions in groups A and B compared with group C. Increased proliferation of fibroblasts during five days of incubation was observed in group A compared with groups B and C (P < 0.05). Collectively, the results from this in vitro study demonstrated that MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces had the ability to increase fibroblast functionality. In addition, MAPs/bFGF-PLGA microsphere composite-coated Ti surfaces should be studied further as a method of promoting formation of stable biological seals around transcutaneous sites.

  5. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model

    PubMed Central

    Mousavi, Seyed Jamaleddin; Hamdy Doweidar, Mohamed

    2015-01-01

    Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs) can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal) together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa), intermediate (20-25 kPa) or hard (30-45 kPa) substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis. PMID:25933372

  6. Regulation of protein kinase D during differentiation and proliferation of primary mouse keratinocytes.

    PubMed

    Ernest Dodd, M; Ristich, Vladimir L; Ray, Sagarika; Lober, Robert M; Bollag, Wendy B

    2005-08-01

    Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.

  7. The Fto Gene Regulates the Proliferation and Differentiation of Pre-Adipocytes in Vitro.

    PubMed

    Jiao, Yang; Zhang, Jingying; Lu, Lunjie; Xu, Jiaying; Qin, Liqiang

    2016-02-19

    The highly regulated differentiation and proliferation of pre-adipocytes play a key role in the initiation of obesity. Fat mass and obesity associated (FTO) is a novel gene strongly associated with the risk of obesity. A deficiency of FTO may cause growth retardation in addition to fat mass and adipocyte size reduction in vivo. To investigate the potential role of Fto gene on the proliferation and differentiation of pre-adipocytes, we generated Fto-knockdown and overexpressed 3T3-L1 cells. Using numerous proliferation assays our results suggest that Fto knockdown leads to suppression of proliferation, lower mitochondrial membrane potential, less cellular ATP, and decreased and smaller intracellular lipid droplets compared with controls (p < 0.05). Western blot analysis demonstrated that Fto knockdown can significantly suppress peroxisome proliferator-activated receptor gamma (PPARγ) and glucose transporter type 4 (GLUT4) expression and inhibit Akt phosphorylation. By contrast, overexpression of Fto had the opposing effect on proliferation, mitochondrial membrane potential, ATP generation, in vitro differentiation, Akt phosphorylation, and PPARγ and GLUT4 expression. Moreover, we demonstrated that Wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, could inhibit phospho-Akt in Fto overexpressed 3T3-L1 cells. Taken together, the results suggest that Fto regulates the proliferation and differentiation of 3T3-L1 cells via multiple mechanisms, including PPARγ and PI3K/Akt signaling.

  8. Keratinocyte proliferation, differentiation, and apoptosis-Differential mechanisms of regulation by curcumin, EGCG and apigenin

    SciTech Connect

    Balasubramanian, Sivaprakasam; Eckert, Richard L.

    2007-11-01

    We have proposed that it is important to examine the impact of chemopreventive agents on the function of normal human epidermal keratinocytes since these cells comprise the barrier that protects the body from a range of environmental insults. In this context, it is widely appreciated that cancer may be retarded by consumption or topical application of naturally occurring food-derived chemopreventive agents. Our studies show that (-)-epigallocatechin-3-gallate (EGCG), a green tea-derived polyphenol, acts to enhance the differentiation of normal human keratinocytes as evidenced by its ability to increase involucrin (hINV), transglutaminase type 1 (TG1) and caspase-14 gene expression. EGCG also stimulates keratinocyte morphological differentiation. These actions of EGCG are mediated via activation of a nPKC, Ras, MEKK1, MEK3, p38{delta}-ERK1/2 signaling cascade which leads to increased activator protein 1 (AP1) and CAATT enhancer binding protein (C/EBP) transcription factor expression, increased binding of these factors to DNA, and increased gene transcription. In contrast, apigenin, a dietary flavonoid derived from plants and vegetables, and curcumin, an agent derived from turmeric, inhibit differentiation by suppressing MAPK signal transduction and reducing API transcription factor level. Curcumin also acts to enhance apoptosis, although EGCG and apigenin do not stimulate apoptosis. In addition, all of these agents inhibit keratinocyte proliferation. These findings indicate that each of these diet-derived chemopreventive agents has a profound impact on normal human keratinocyte function and that they operate via distinct and sometimes opposing mechanisms. However, all are expected to act as chemopreventive agents.

  9. Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

    SciTech Connect

    Resende, R.R.; Alves, A.S.; Britto, L.R.G; Ulrich, H.

    2008-04-15

    Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G{alpha}{sub q/11}-coupled M{sub 1}, M{sub 3} and M{sub 5} receptors and intracellular calcium stores, whereas G{alpha}{sub i/o}-protein coupled M{sub 2} receptor activity mediated neuronal differentiation.

  10. Osteoblasts Proliferation and Differentiation Stimulating Activities of the Main Components of Epimedii folium

    PubMed Central

    Liu, Mingming; Xu, Haiyan; Ma, Yong; Cheng, Jian; Hua, Zhen; Huang, Guicheng

    2017-01-01

    Background: Osteoporosis is a disease of bones that leads to an increased risk of fracture. Epimedii Folium is commonly used for treating bone fractures and joint diseases for thousands of years in China. Methods: This study was aimed to screen active components, which might have the potency to stimulate osteoblasts proliferation and differentiation in Epimedii Folium. An HPLC method was established to analyze the main components in Epimedii Folium. The MTT and ALP methods were utilized for the assay of osteoblasts proliferation and differentiation activity. Bavachin, a flavonoid compound was treated as the positive control. Results: Totally eight compounds have been identified by comparing their retention time with correspondent standard substances. Icariside I and icariside II significantly stimulated cell proliferation and osteoblasts differentiation. All these compounds were found with a characterized flavonoid structure in each of their molecule backbones. Conclusion: These results lead to a hypothesis that flavonoid monoglycoside structure might be crucial to exhibit the activity. The structure–effect relationship of these compounds with flavonoid monoglycoside structure in mouse primary calvarial osteoblasts needs to be explored in further research. SUMMARY Eight compounds were identified by comparing their retention time with correspondent standard substances.Icariside I and icariside II significantly stimulated cell proliferation and osteoblasts differentiation.Flavonoid monoglycoside structure might be crucial to exhibit the osteoblasts proliferation and differentiation activity. Effects of the main components of Epimedii Folium on osteoblasts proliferation after treating 48 h. Abbreviations used: HPLC: High performance liquid chromatography, MTT: Methylthiazolyldiphenyl - tetrazolium bromide, ALP: Alkaline phosphatase PMID:28216889

  11. Cell-Adhesive Matrices Composed of RGD Peptide-Displaying M13 Bacteriophage/Poly(lactic-co-glycolic acid) Nanofibers Beneficial to Myoblast Differentiation.

    PubMed

    Shin, Yong Cheol; Lee, Jong Ho; Jin, Linhua; Kim, Min Jeong; Kim, Chuntae; Hong, Suck Won; Oh, Jin Woo; Han, Dong-Wook

    2015-10-01

    Recently, there has been considerable effort to develop suitable scaffolds for tissue engineering applications. Cell adhesion is a prerequisite for cells to survive. In nature, the extracellular matrix (ECM) plays this role. Therefore, an ideal scaffold should be structurally similar to the natural ECM and have biocompatibility and biodegradability. In addition, the scaffold should have biofunctionality, which provides the potent ability to enhance the cellular behaviors, such as adhesion, proliferation and differentiation. This study concentrates on fabricating cell-adhesive matrices composed of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) nanofibers. Long rod-shaped M13 bacteriophages are non-toxic and can express many desired proteins on their surface. A genetically engineered M13 phage was constructed to display RGD peptides on its surface. PLGA is a biodegradable polymer with excellent biocompatibility and suitable physicochemical property for adhesive matrices. In this study, RGD-M13 phage/PLGA hybrid nanofiber matrices were fabricated by electrospinning. The physicochemical properties of these matrices were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and contact angle measurement. In addition, the cellular behaviors, such as the initial attachment, proliferation and differentiation, were analyzed by a CCK-8 assay and immunofluorescence staining to evaluate the potential application of these matrices to tissue engineering scaffolds. The RGD-M13 phage/PLGA nanofiber matrices could enhance the cellular behaviors and promote the differentiation of C2C12 myoblasts. These results suggest that the RGD-M13 phage/PLGA nanofiber matrices are beneficial to myoblast differentiation and can serve as effective tissue engineering scaffolds.

  12. mTORC1 regulates PTHrP to coordinate chondrocyte growth, proliferation and differentiation

    PubMed Central

    Yan, Bo; Zhang, Zhongmin; Jin, Dadi; Cai, Chen; Jia, Chunhong; Liu, Wen; Wang, Ting; Li, Shengfa; Zhang, Haiyan; Huang, Bin; Lai, Pinglin; Wang, Hua; Liu, Anling; Zeng, Chun; Cai, Daozhang; Jiang, Yu; Bai, Xiaochun

    2016-01-01

    Precise coordination of cell growth, proliferation and differentiation is essential for the development of multicellular organisms. Here, we report that although the mechanistic target of rapamycin complex 1 (mTORC1) activity is required for chondrocyte growth and proliferation, its inactivation is essential for chondrocyte differentiation. Hyperactivation of mTORC1 via TSC1 gene deletion in chondrocytes causes uncoupling of the normal proliferation and differentiation programme within the growth plate, resulting in uncontrolled cell proliferation, and blockage of differentiation and chondrodysplasia in mice. Rapamycin promotes chondrocyte differentiation and restores these defects in mutant mice. Mechanistically, mTORC1 downstream kinase S6K1 interacts with and phosphorylates Gli2, and releases Gli2 from SuFu binding, resulting in nuclear translocation of Gli2 and transcription of parathyroid hormone-related peptide (PTHrP), a key regulator of bone development. Our findings demonstrate that dynamically controlled mTORC1 activity is crucial to coordinate chondrocyte proliferation and differentiation partially through regulating Gli2/PTHrP during endochondral bone development. PMID:27039827

  13. Periostin Promotes Neural Stem Cell Proliferation and Differentiation following Hypoxic-Ischemic Injury.

    PubMed

    Ma, Si-Min; Chen, Long-Xia; Lin, Yi-Feng; Yan, Hu; Lv, Jing-Wen; Xiong, Man; Li, Jin; Cheng, Guo-Qiang; Yang, Yi; Qiu, Zi-Long; Zhou, Wen-Hao

    2015-01-01

    Neural stem cell (NSC) proliferation and differentiation are required to replace neurons damaged or lost after hypoxic-ischemic events and recover brain function. Periostin (POSTN), a novel matricellular protein, plays pivotal roles in the survival, migration, and regeneration of various cell types, but its function in NSCs of neonatal rodent brain is still unknown. The purpose of this study was to investigate the role of POSTN in NSCs following hypoxia-ischemia (HI). We found that POSTN mRNA levels significantly increased in differentiating NSCs. The proliferation and differentiation of NSCs in the hippocampus is compromised in POSTN knockout mice. Moreover, NSC proliferation and differentiation into neurons and astrocytes significantly increased in cultured NSCs treated with recombinant POSTN. Consistently, injection of POSTN into neonatal hypoxic-ischemic rat brains stimulated NSC proliferation and differentiation in the subventricular and subgranular zones after 7 and 14 days of brain injury. Lastly, POSTN treatment significantly improved the spatial learning deficits of rats subjected to HI. These results suggest that POSTN significantly enhances NSC proliferation and differentiation after HI, and provides new insights into therapeutic strategies for the treatment of hypoxic-ischemic encephalopathy.

  14. Proliferation and Differentiation of Rat Osteoporosis Mesenchymal Stem Cells (MSCs) after Telomerase Reverse Transcriptase (TERT) Transfection

    PubMed Central

    Li, Chao; Wei, Guojun; Gu, Qun; Wang, Qiang; Tao, Shuqin; Xu, Liang

    2015-01-01

    Background The aim of this study was to determine whether MSC are excellent materials for MSCs transplantation in the treatment of osteoporosis. Material/Methods We studied normal, osteoporosis, and TERT-transfected MSC from normal and osteoporosis rats to compare the proliferation and osteogenic differentiation using RT-PCR and Western blot by constructing an ovariectomized rat model of osteoporosis (OVX). The primary MSC from model rats were extracted and cultured to evaluate the proliferation and differentiation characteristics. Results MSCs of osteoporosis rats obviously decreased in proliferation ability and osteogenic differentiation compared to that of normal rats. In contrast, in TERT-transfected MSC, the proliferation and differentiation ability, and especially the ability of osteogenic differentiation, were significantly higher than in osteoporosis MSC. Conclusions TERT-transfected MSCs can help osteoporosis patients in whom MSC proliferation and osteogenic differentiation ability are weak, with an increase in both bone mass and bone density, becoming an effective material for autologous transplantation of MSCs in further treatment of osteoporosis. However, studies are still needed to prove the in vivo effect, biological safety, and molecular mechanism of TERT-osteoporosis treatment. Additionally, because the results are from an animal model, more research is needed in generalizing rat model findings to human osteoporosis patients. PMID:25796354

  15. Adhesive pad differentiation in Drosophila melanogaster depends on the Polycomb group gene Su(z)2.

    PubMed

    Hüsken, Mirko; Hufnagel, Kim; Mende, Katharina; Appel, Esther; Meyer, Heiko; Peisker, Henrik; Tögel, Markus; Wang, Shuoshuo; Wolff, Jonas; Gorb, Stanislav N; Paululat, Achim

    2015-04-15

    The ability of many insects to walk on vertical smooth surfaces such as glass or even on the ceiling has fascinated biologists for a long time, and has led to the discovery of highly specialized adhesive organs located at the distal end of the animals' legs. So far, research has primarily focused on structural and ultrastructural investigations leading to a deeper understanding of adhesive organ functionality and to the development of new bioinspired materials. Genetic approaches, e.g. the analysis of mutants, to achieve a better understanding of adhesive organ differentiation have not been used so far. Here, we describe the first Drosophila melanogaster mutant that develops malformed adhesive organs, resulting in a complete loss of climbing ability on vertical smooth surfaces. Interestingly, these mutants fail to make close contact between the setal tips and the smooth surface, a crucial condition for wet adhesion mediated by capillary forces. Instead, these flies walk solely on their claws. Moreover, we were able to show that the mutation is caused by a P-element insertion into the Su(z)2 gene locus. Remobilization of the P-element restores climbing ability. Furthermore, we provide evidence that the P-element insertion results in an artificial Su(z)2 transcript, which most likely causes a gain-of-function mutation. We presume that this transcript causes deregulation of yet unknown target genes involved in pulvilli differentiation. Our results nicely demonstrate that the genetically treatable model organism Drosophila is highly suitable for future investigations on adhesive organ differentiation.

  16. DNA Replication Fading As Proliferating Cells Advance in Their Commitment to Terminal Differentiation

    PubMed Central

    Estefanía, Monturus Ma.; Ganier, Olivier; Hernández, Pablo; Schvartzman, Jorge B.; Mechali, Marcel; Krimer, Dora B.

    2012-01-01

    Terminal differentiation is the process by which cycling cells stop proliferating to start new specific functions. It involves dramatic changes in chromatin organization as well as gene expression. In the present report we used cell flow cytometry and genome wide DNA combing to investigate DNA replication during murine erythroleukemia-induced terminal cell differentiation. The results obtained indicated that the rate of replication fork movement slows down and the inter-origin distance becomes shorter during the precommitment and commitment periods before cells stop proliferating and accumulate in G1. We propose this is a general feature caused by the progressive heterochromatinization that characterizes terminal cell differentiation. PMID:22359734

  17. Myosin 1e promotes breast cancer malignancy by enhancing tumor cell proliferation and stimulating tumor cell de-differentiation

    PubMed Central

    Ouderkirk-Pecone, Jessica L.; Goreczny, Gregory J.; Chase, Sharon E.; Tatum, Arthur H.; Turner, Christopher E.; Krendel, Mira

    2016-01-01

    Despite advancing therapies, thousands of women die every year of breast cancer. Myosins, actin-dependent molecular motors, are likely to contribute to tumor formation and metastasis via their effects on cell adhesion and migration and may provide promising new targets for cancer therapies. Using the MMTV-PyMT murine model of breast cancer, we identified Myosin 1e (MYO1E) as a novel tumor promoter. Tumor latency in mice lacking MYO1E was significantly increased, and tumors formed in the absence of MYO1E displayed unusual papillary morphology, with well-differentiated layers of epithelial cells covering fibrovascular cores, rather than solid sheets of tumor cells typically observed in this cancer model. These tumors were reminiscent of papillary breast cancer in humans that is typically non-invasive and often cured by tumor excision. MYO1E-null tumors exhibited decreased expression of the markers of cell proliferation, which was recapitulated in primary tumor cells derived from MYO1E-null mice. In agreement with our findings, meta-analysis of patient survival data indicated that MYO1E expression level was associated with reduced recurrence-free survival in basal-like breast cancer. Overall, our data suggests that MYO1E contributes to breast tumor malignancy and regulates the differentiation and proliferation state of breast tumor cells. PMID:27329840

  18. The Wnt signaling pathway in cellular proliferation and differentiation: A tale of two coactivators.

    PubMed

    Teo, Jia-Ling; Kahn, Michael

    2010-09-30

    Wnt signaling pathways play divergent roles during development, normal homeostasis and disease. The responses that result from the activation of the pathway control both proliferation and differentiation. Tight regulation and controlled coordination of the Wnt signaling cascade is required to maintain the balance between proliferation and differentiation. The non-redundant roles of the coactivator proteins CBP and p300, within the context of Wnt signaling are discussed. We highlight their roles as integrators of the various inputs that a cell receives to elicit the correct and coordinated response. We propose that essentially all cellular information - i.e. from other signaling pathways, nutrient levels, etc. - is funneled down into a choice of coactivators usage, either CBP or p300, by their interacting partner beta-catenin (or catenin-like molecules in the absence of beta-catenin) to make the critical decision to either remain quiescent, or once entering cycle to proliferate without differentiation or to initiate the differentiation process.

  19. Increased Cell Proliferation and Gene Expression of Genes Related to Bone Remodeling, Cell Adhesion and Collagen Metabolism in the Periodontal Ligament of Unopposed Molars in Growing Rats

    PubMed Central

    Dorotheou, Domna; Farsadaki, Vassiliki; Bochaton-Piallat, Marie-Luce; Giannopoulou, Catherine; Halazonetis, Thanos D.; Kiliaridis, Stavros

    2017-01-01

    Tooth eruption, the process by which teeth emerge from within the alveolar bone into the oral cavity, is poorly understood. The post-emergent phase of tooth eruption continues throughout life, in particular, if teeth are not opposed by antagonists. The aim of the present study was to better understand the molecular processes underlying post-emergent tooth eruption. Toward this goal, we removed the crowns of the maxillary molars on one side of the mouth of 14 young rats and examined gene expression patterns in the periodontal ligaments (PDLs) of the ipsilateral and contralateral mandibular molars, 3 and 15 days later. Nine untreated rats served as controls. Expression of six genes, Adamts18, Ostn, P4ha3, Panx3, Pth1r, and Tnmd, was upregulated in unopposed molars relative to molars with antagonists. These genes function in osteoblast differentiation and proliferation, cell adhesion and collagen metabolism. Proliferation of PDL cells also increased following loss of the antagonist teeth. Interestingly, mutations in PTH1R have been linked to defects in the post-emergent phase of tooth eruption in humans. We conclude that post-emergent eruption of unopposed teeth is associated with gene expression patterns conducive to alveolar bone formation and PDL remodeling. PMID:28239357

  20. ENO1 promotes tumor proliferation and cell adhesion mediated drug resistance (CAM-DR) in Non-Hodgkin's Lymphomas

    SciTech Connect

    Zhu, Xinghua; Miao, Xiaobing; Wu, Yaxun; Li, Chunsun; Guo, Yan; Liu, Yushan; Chen, Yali; Lu, Xiaoyun; Wang, Yuchan; He, Song

    2015-07-15

    Enolases are glycolytic enzymes responsible for the ATP-generated conversion of 2-phosphoglycerate to phosphoenolpyruvate. In addition to the glycolytic function, Enolase 1 (ENO1) has been reported up-regulation in several tumor tissues. In this study, we investigated the expression and biologic function of ENO1 in Non-Hodgkin's Lymphomas (NHLs). Clinically, by western blot analysis we observed that ENO1 expression was apparently higher in diffuse large B-cell lymphoma than in the reactive lymphoid tissues. Subsequently, immunohistochemical staining of 144 NHLs suggested that the expression of ENO1 was significantly lower in the indolent lymphomas compared with the progressive lymphomas. Further, we identified ENO1 as an independent prognostic factor, and it was significantly correlated with overall survival of NHL patients. In addition, we found that ENO1 could promote cell proliferation, regulate cell cycle associated gene and PI3K/AKT signaling pathway in NHLs. Finally, we verified that ENO1 participated in the process of lymphoma cell adhesion mediated drug resistance (CAM-DR). Adhesion to FN or HS5 cells significantly protected OCI-Ly8 and Daudi cells from cytotoxicity compared with those cultured in suspension, and these effects were attenuated when transfected with ENO1-siRNA. Based on the study, we propose that inhibition of ENO1 expression may be a novel strategy for therapy for NHLs patients, and it may be a target for drug resistance. - Highlights: • ENO1 expression is reversely correlated with clinical outcomes of patients with NHLs. • ENO1 promotes the proliferation of NHL cells. • ENO1 regulates cell adhesion mediated drug resistance.

  1. Effects of heat shock on survival, proliferation and differentiation of mouse neural stem cells.

    PubMed

    Omori, Hiroyuki; Otsu, Masahiro; Suzuki, Asami; Nakayama, Takashi; Akama, Kuniko; Watanabe, Masaru; Inoue, Nobuo

    2014-02-01

    Hyperthermia during pregnancy is a significant cause of reproductive problems ranging from abortion to congenital defects of the central nervous system (CNS), including neural tube defects and microcephaly. Neural stem cells (NSCs) can proliferate and differentiate into neurons and glia, playing a key role in the formation of the CNS. Here, we examined the effects of heat shock on homogeneous proliferating NSCs derived from mouse embryonic stem cells. After heat shock at 42 °C for 20 min, the proliferating NSCs continued to proliferate, although subtle changes were observed in gene expression and cell survival and proliferation. In contrast, heat shock at 43 °C caused a variety of responses: the up-regulation of genes encoding heat shock proteins (HSP), induction of apoptosis, temporal inhibition of cell proliferation and retardation of differentiation. Finally, effects of heat shock at 44 °C were severe, with almost all cells disappearing and the remaining cells losing the capacity to proliferate and differentiate. These temperature-dependent effects of heat shock on NSCs may be valuable in elucidating the mechanisms by which hyperthermia during pregnancy causes various reproductive problems.

  2. Arabidopsis and Tobacco SUPERMAN regulate hormone signalling and mediate cell proliferation and differentiation

    PubMed Central

    Nibau, Candida; Di Stilio, Verónica S.; Wu, Hen-ming; Cheung, Alice Y.

    2011-01-01

    Arabidopsis thaliana SUPERMAN (SUP) plays an important role during flower development by maintaining the boundary between stamens and carpels in the inner two whorls. It was proposed that SUP maintains this boundary by regulating cell proliferation in both whorls, as loss-of-function superman mutants produce more stamens at the expense of carpels. However, the cellular mechanism that underlies SUP function remains unknown. Here Arabidopsis or tobacco (Nicotiana tabacum) SUP was overexpressed in tobacco plants to substantiate SUP's role as a regulator of cell proliferation and boundary definition and provide evidence that its biological role may be mediated via hormonal changes. It was found that moderate levels of SUP stimulated cell growth and proliferation, whereas high levels were inhibitory. SUP stimulated auxin- and cytokinin-regulated processes, and cells overexpressing SUP displayed reduced hormone dependency for proliferation and regeneration into plants. SUP also induced proliferation of female traits in the second and third flower whorls and promoted differentiation of petaloid properties in sepals, further supporting a role for SUP as a boundary regulator. Moreover, cytokinin suppressed stamen development and promoted differentiation of carpeloid tissues, suggesting that SUP may regulate male and female development via its effect on cytokinin signalling. Taken together, these observations suggest a model whereby the effect of SUP on cell growth and proliferation involves the modulation of auxin- and cytokinin-regulated processes. Furthermore, differential SUP expression or different sensitivities of different cell types to SUP may determine whether SUP stimulates or suppresses their proliferation. PMID:20980362

  3. Modelling T cell proliferation: Dynamics heterogeneity depending on cell differentiation, age, and genetic background

    PubMed Central

    2017-01-01

    Cell proliferation is the common characteristic of all biological systems. The immune system insures the maintenance of body integrity on the basis of a continuous production of diversified T lymphocytes in the thymus. This involves processes of proliferation, differentiation, selection, death and migration of lymphocytes to peripheral tissues, where proliferation also occurs upon antigen recognition. Quantification of cell proliferation dynamics requires specific experimental methods and mathematical modelling. Here, we assess the impact of genetics and aging on the immune system by investigating the dynamics of proliferation of T lymphocytes across their differentiation through thymus and spleen in mice. Our investigation is based on single-cell multicolour flow cytometry analysis revealing the active incorporation of a thymidine analogue during S phase after pulse-chase-pulse experiments in vivo, versus cell DNA content. A generic mathematical model of state transition simulates through Ordinary Differential Equations (ODEs) the evolution of single cell behaviour during various durations of labelling. It allows us to fit our data, to deduce proliferation rates and estimate cell cycle durations in sub-populations. Our model is simple and flexible and is validated with other durations of pulse/chase experiments. Our results reveal that T cell proliferation is highly heterogeneous but with a specific “signature” that depends upon genetic origins, is specific to cell differentiation stages in thymus and spleen and is altered with age. In conclusion, our model allows us to infer proliferation rates and cell cycle phase durations from complex experimental 5-ethynyl-2'-deoxyuridine (EdU) data, revealing T cell proliferation heterogeneity and specific signatures. PMID:28288157

  4. Modelling T cell proliferation: Dynamics heterogeneity depending on cell differentiation, age, and genetic background.

    PubMed

    Vibert, Julien; Thomas-Vaslin, Véronique

    2017-03-01

    Cell proliferation is the common characteristic of all biological systems. The immune system insures the maintenance of body integrity on the basis of a continuous production of diversified T lymphocytes in the thymus. This involves processes of proliferation, differentiation, selection, death and migration of lymphocytes to peripheral tissues, where proliferation also occurs upon antigen recognition. Quantification of cell proliferation dynamics requires specific experimental methods and mathematical modelling. Here, we assess the impact of genetics and aging on the immune system by investigating the dynamics of proliferation of T lymphocytes across their differentiation through thymus and spleen in mice. Our investigation is based on single-cell multicolour flow cytometry analysis revealing the active incorporation of a thymidine analogue during S phase after pulse-chase-pulse experiments in vivo, versus cell DNA content. A generic mathematical model of state transition simulates through Ordinary Differential Equations (ODEs) the evolution of single cell behaviour during various durations of labelling. It allows us to fit our data, to deduce proliferation rates and estimate cell cycle durations in sub-populations. Our model is simple and flexible and is validated with other durations of pulse/chase experiments. Our results reveal that T cell proliferation is highly heterogeneous but with a specific "signature" that depends upon genetic origins, is specific to cell differentiation stages in thymus and spleen and is altered with age. In conclusion, our model allows us to infer proliferation rates and cell cycle phase durations from complex experimental 5-ethynyl-2'-deoxyuridine (EdU) data, revealing T cell proliferation heterogeneity and specific signatures.

  5. Enzymatically-tailored pectins differentially influence the morphology, adhesion, cell cycle progression and survival of fibroblasts.

    PubMed

    Nagel, Marie-Danielle; Verhoef, René; Schols, Henk; Morra, Marco; Knox, J Paul; Ceccone, Giacomo; Della Volpe, Claudio; Vigneron, Pascale; Bussy, Cyrill; Gallet, Marlène; Velzenberger, Elodie; Vayssade, Muriel; Cascardo, Giovanna; Cassinelli, Clara; Haeger, Ash; Gilliland, Douglas; Liakos, Ioannis; Rodriguez-Valverde, Miguel; Siboni, Stefano

    2008-01-01

    Improved biocompatibility and performance of biomedical devices can be achieved through the incorporation of bioactive molecules on device surfaces. Five structurally distinct pectic polysaccharides (modified hairy regions (MHRs)) were obtained by enzymatic liquefaction of apple (MHR-B, MHR-A and MHR-alpha), carrot (MHR-C) and potato (MHR-P) cells. Polystyrene (PS) Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of the MHRs. Results clearly demonstrate that MHR-B induces cell adhesion, proliferation and survival, in contrast to the other MHRs. Moreover, MHR-alpha causes cells to aggregate, decrease proliferation and enter into apoptosis. Cells cultured in standard conditions with 1% soluble MHR-B or MHR-alpha show the opposite behaviour to the one observed on MHR-B and -alpha-grafted PS. Fibronectin was similarly adsorbed onto MHR-B and tissue culture polystyrene (TCPS) control, but poorly on MHR-alpha. The Fn cell binding site (RGD sequence) was more accessible on MHR-B than on TCPS control, but poorly on MHR-alpha. The disintegrin echistatin inhibited fibroblast adhesion and spreading on MHR-B-grafted PS, which suggests that MHRs control fibroblast behaviour via serum-adhesive proteins. This study provides a basis for the design of intelligently-tailored biomaterial coatings able to induce specific cell functions.

  6. The interplay of fibronectin functionalization and TGF-β1 presence on fibroblast proliferation, differentiation and migration in 3D matrices.

    PubMed

    Sapudom, Jiranuwat; Rubner, Stefan; Martin, Steve; Thoenes, Stephan; Anderegg, Ulf; Pompe, Tilo

    2015-09-01

    Defined biomimetic three-dimensional (3D) matrices are needed to decipher the complex cellular signalling during wound healing at high resolution in vitro. Soluble factors like TGF-β1 and adhesion promoting structural components of the extracellular matrix (ECM) are known to be key regulators of fibroblast behaviour. The ECM component fibronectin (FN) bears a complex function as adhesion promoter, fibrillar element and soluble factor binder. However, its implementation in biomimetic 3D matrices is frequently ill defined. To study the impact of FN on fibroblast cellular function under differentiating conditions (TGF-β1 stimulation), we functionalized 3D collagen I matrices with FN using two strategies: co-assembly and adsorptive immobilization. In comparison to co-assembly, adsorptive immobilization provided no alteration in collagen microstructure as well as mechanical properties. Moreover, this approach provided a controllable FN amount and a homogenous distribution of FN throughout collagen networks. A strong interplay of FN amount and TGF-β1 stimulation on fibroblast function was found in terms of proliferation, migration and myofibroblast differentiation. High levels of FN alone reduced proliferation and showed no effect on differentiation of fibroblasts, but increased migration. In contrast, fibroblast stimulation with high amounts of FN together with TGF-β1 increased proliferation. Independent of FN, the TGF-β1 stimulation enhanced mRNA expression of matrix components like collagen type I alpha 1 chain (Coll I(a1), FN with extra domain A (EDA-FN) and reduced cell migration. The latter cell behaviour indicated a FN independent differentiation into a myofibroblast phenotype. Overall, our 3D biomimetic matrices allow dissecting the overlapping action of the ECM protein FN and the soluble factor TGF-β1 on fibroblast proliferation, migration and differentiation in 3D microenvironments. Furthermore, this model enables the mimicking of important steps of the

  7. Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells.

    PubMed

    Okeyo, Kennedy Omondi; Kurosawa, Osamu; Yamazaki, Satoshi; Oana, Hidehiro; Kotera, Hidetoshi; Nakauchi, Hiromitsu; Washizu, Masao

    2015-10-01

    Mechanical methods for inducing differentiation and directing lineage specification will be instrumental in the application of pluripotent stem cells. Here, we demonstrate that minimization of cell-substrate adhesion can initiate and direct the differentiation of human pluripotent stem cells (hiPSCs) into cyst-forming trophoblast lineage cells (TLCs) without stimulation with cytokines or small molecules. To precisely control cell-substrate adhesion area, we developed a novel culture method where cells are cultured on microstructured mesh sheets suspended in a culture medium such that cells on mesh are completely out of contact with the culture dish. We used microfabricated mesh sheets that consisted of open meshes (100∼200 μm in pitch) with narrow mesh strands (3-5 μm in width) to provide support for initial cell attachment and growth. We demonstrate that minimization of cell adhesion area achieved by this culture method can trigger a sequence of morphogenetic transformations that begin with individual hiPSCs attached on the mesh strands proliferating to form cell sheets by self-assembly organization and ultimately differentiating after 10-15 days of mesh culture to generate spherical cysts that secreted human chorionic gonadotropin (hCG) hormone and expressed caudal-related homeobox 2 factor (CDX2), a specific marker of trophoblast lineage. Thus, this study demonstrates a simple and direct mechanical approach to induce trophoblast differentiation and generate cysts for application in the study of early human embryogenesis and drug development and screening.

  8. Slowing Down Differentiation of Engrafted Human Myoblasts Into Immunodeficient Mice Correlates With Increased Proliferation and Migration

    PubMed Central

    Riederer, Ingo; Negroni, Elisa; Bencze, Maximilien; Wolff, Annie; Aamiri, Ahmed; Di Santo, James P; Silva-Barbosa, Suse D.; Butler-Browne, Gillian; Savino, Wilson; Mouly, Vincent

    2012-01-01

    We have used a model of xenotransplantation in which human myoblasts were transplanted intramuscularly into immunodeficient Rag2-/-γC-/- mice, in order to investigate the kinetics of proliferation and differentiation of the transplanted cells. After injection, most of the human myoblasts had already differentiated by day 5. This differentiation correlated with reduction in proliferation and limited migration of the donor cells within the regenerating muscle. These results suggest that the precocious differentiation, already detected at 3 days postinjection, is a limiting factor for both the migration from the injection site and the participation of the donor cells to muscle regeneration. When we stimulated in vivo proliferation of human myoblasts, transplanting them in a serum-containing medium, we observed 5 days post-transplantation a delay of myogenic differentiation and an increase in cell numbers, which colonized a much larger area within the recipient's muscle. Importantly, these myoblasts maintained their ability to differentiate, since we found higher numbers of myofibers seen 1 month postengraftment, as compared to controls. Conceptually, these data suggest that in experimental myoblast transplantation, any intervention upon the donor cells and/or the recipient's microenvironment aimed at enhancing proliferation and migration should be done before differentiation of the implanted cells, e.g., day 3 postengraftment. PMID:21934656

  9. Slowing down differentiation of engrafted human myoblasts into immunodeficient mice correlates with increased proliferation and migration.

    PubMed

    Riederer, Ingo; Negroni, Elisa; Bencze, Maximilien; Wolff, Annie; Aamiri, Ahmed; Di Santo, James P; Silva-Barbosa, Suse D; Butler-Browne, Gillian; Savino, Wilson; Mouly, Vincent

    2012-01-01

    We have used a model of xenotransplantation in which human myoblasts were transplanted intramuscularly into immunodeficient Rag2(-/-)γC(-/-) mice, in order to investigate the kinetics of proliferation and differentiation of the transplanted cells. After injection, most of the human myoblasts had already differentiated by day 5. This differentiation correlated with reduction in proliferation and limited migration of the donor cells within the regenerating muscle. These results suggest that the precocious differentiation, already detected at 3 days postinjection, is a limiting factor for both the migration from the injection site and the participation of the donor cells to muscle regeneration. When we stimulated in vivo proliferation of human myoblasts, transplanting them in a serum-containing medium, we observed 5 days post-transplantation a delay of myogenic differentiation and an increase in cell numbers, which colonized a much larger area within the recipient's muscle. Importantly, these myoblasts maintained their ability to differentiate, since we found higher numbers of myofibers seen 1 month postengraftment, as compared to controls. Conceptually, these data suggest that in experimental myoblast transplantation, any intervention upon the donor cells and/or the recipient's microenvironment aimed at enhancing proliferation and migration should be done before differentiation of the implanted cells, e.g., day 3 postengraftment.

  10. Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

    SciTech Connect

    Vezeridis, Peter S.; Chen Qian . E-mail: j.kleinnulend@vumc.nl

    2006-09-29

    Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and L-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation.

  11. The effects and mechanisms of clinorotation on proliferation and differentiation in bone marrow mesenchymal stem cells

    SciTech Connect

    Yan, Ming; Wang, Yongchun; Yang, Min; Liu, Yanwu; Qu, Bo; Ye, Zhengxu; Liang, Wei; Sun, Xiqing; Luo, Zhuojing

    2015-05-01

    Data from human and rodent studies have demonstrated that microgravity induces observed bone loss in real spaceflight or simulated experiments. The decrease of bone formation and block of maturation may play important roles in bone loss induced by microgravity. The aim of this study was to investigate the changes of proliferation and differentiation in bone marrow mesenchymal stem cells (BMSCs) induced by simulated microgravity and the mechanisms underlying it. We report here that clinorotation, a simulated model of microgravity, decreased proliferation and differentiation in BMSCs after exposure to 48 h simulated microgravity. The inhibited proliferation are related with blocking the cell cycle in G2/M and enhancing the apoptosis. While alterations of the osteoblast differentiation due to the decreased SATB2 expression induced by simulated microgravity in BMSCs. - Highlights: • Simulated microgravity inhibited proliferation and differentiation in BMSCs. • The decreased proliferation due to blocked cell cycle and enhanced the apoptosis. • The inhibited differentiation accounts for alteration of SATB2, Hoxa2 and Cbfa1.

  12. Real-Time Discrimination between Proliferation and Neuronal and Astroglial Differentiation of Human Neural Stem Cells

    PubMed Central

    Lee, Rimi; Kim, Il-Sun; Han, Nalae; Yun, Seokhwan; Park, Kook In; Yoo, Kyung-Hwa

    2014-01-01

    Neural stem cells (NSCs) are characterized by a capacity for self-renewal, differentiation into multiple neural lineages, all of which are considered to be promising components for neural regeneration. However, for cell-replacement therapies, it is essential to monitor the process of in vitro NSC differentiation and identify differentiated cell phenotypes. We report a real-time and label-free method that uses a capacitance sensor array to monitor the differentiation of human fetal brain-derived NSCs (hNSCs) and to identify the fates of differentiated cells. When hNSCs were placed under proliferation or differentiation conditions in five media, proliferating and differentiating hNSCs exhibited different frequency and time dependences of capacitance, indicating that the proliferation and differentiation status of hNSCs may be discriminated in real-time using our capacitance sensor. In addition, comparison between real-time capacitance and time-lapse optical images revealed that neuronal and astroglial differentiation of hNSCs may be identified in real-time without cell labeling. PMID:25204726

  13. Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling

    PubMed Central

    Lian, Gewei; Zhang, Jingping; Hecht, Jonathan L.; Sheen, Volney L.

    2014-01-01

    Humans who harbor loss of function mutations in the actin-associated filamin B (FLNB) gene develop spondylocarpotarsal syndrome (SCT), a disorder characterized by dwarfism (delayed bone formation) and premature fusion of the vertebral, carpal and tarsal bones (premature differentiation). To better understand the cellular and molecular mechanisms governing these seemingly divergent processes, we generated and characterized FlnB knockdown ATDC5 cell lines. We found that FlnB knockdown led to reduced proliferation and enhanced differentiation in chondrocytes. Within the shortened growth plate of postnatal FlnB−/− mice long bone, we observed a similarly progressive decline in the number of rapidly proliferating chondrocytes and premature differentiation characterized by an enlarged prehypertrophic zone, a widened Col2a1+/Col10a1+ overlapping region, but relatively reduced hypertrophic zone length. The reduced chondrocyte proliferation and premature differentiation were, in part, attributable to enhanced G2/M phase progression, where fewer FlnB deficient ATDC5 chondrocytes resided in the G2/M phase of the cell cycle. FlnB loss reduced Cdk1 phosphorylation (an inhibitor of G2/M phase progression) and Cdk1 inhibition in chondrocytes mimicked the null FlnB, premature differentiation phenotype, through a β1-integrin receptor- Pi3k/Akt (a key regulator of chondrocyte differentiation) mediated pathway. In this context, the early prehypertrophic differentiation provides an explanation for the premature differentiation seen in this disorder, whereas the progressive decline in proliferating chondrocytes would ultimately lead to reduced chondrocyte production and shortened bone length. These findings begin to define a role for filamin proteins in directing both cell proliferation and differentiation through indirect regulation of cell cycle associated proteins. PMID:24551245

  14. Mycobacterium ulcerans mycolactone interferes with adhesion, migration and proliferation of primary human keratinocytes and HaCaT cell line.

    PubMed

    Graziola, Francesca; Colombo, Elena; Tiberio, Rossana; Leigheb, Giorgio; Bozzo, Chiarella

    2017-04-01

    The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) is closely associated with the secretion of exotoxin mycolactone. The cytotoxicity of mycolactone has been linked to its apoptogenic activity. We explored if low mycolactone concentrations, which are not able to induce apoptosis, can influence other essential activities on two primary human keratinocyte populations, keratinocyte stem cells (KSC) and transit amplifying cells (TAC), and on a human keratinocyte line, HaCaT. We demonstrated that 0.01 and 0.1 ng/ml mycolactone A/B are not able to induce apoptosis in primary human keratinocytes, but interfere with KSC wound repair. Moreover, the same toxin concentrations reduce cell proliferation of KSC and TAC and their ability to adhere to type IV collagen. HaCaT cells are more resistant to the toxin; nevertheless, they show a delayed woud repair when treated with 1 and 10 ng/ml mycolactone A/B. Moreover, these sub-apoptotic concentrations affect their ability to proliferate and adhere to collagen IV. Wound healing is a complex mechanism, which occurs "in vivo" as the outcome of many co-ordinated events. Sub-apoptotic mycolactone concentrations can affect essential mechanisms, which are required to achieve wound repair, such as adhesion, migration and proliferation of human keratinocytes.

  15. Rutin inhibits proliferation, attenuates superoxide production and decreases adhesion and migration of human cancerous cells.

    PubMed

    Ben Sghaier, Mohamed; Pagano, Alessandra; Mousslim, Mohamed; Ammari, Youssef; Kovacic, Hervé; Luis, José

    2016-12-01

    Lung and colorectal cancer are the principal causes of death in the world. Rutin, an active flavonoid compound, is known for possessing a wide range of biological activities. In this study, we examined the effect of rutin on the viability, superoxide anion production, adhesion and migration of human lung (A549) and colon (HT29 and Caco-2) cancer cell lines. In order to control the harmlessness of the tested concentrations of rutin, the viability of cancer cell lines was assessed using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. ROS generation was measured by lucigenin chemiluminescence detecting superoxide ions. To investigate the effect of rutin on the behavior of human lung and colon cancer cell lines, we performed adhesion assays, using various purified extracellular matrix (ECM) proteins. Finally, in vitro cell migration assays were explored using modified Boyden chambers. The viability of cancerous cells was inhibited by rutin. It also significantly attenuated the superoxide production in HT29 cells. In addition, rutin affected adhesion and migration of A549 and HT29 cell. These findings indicate that rutin, a natural molecule, might have potential as anticancer agent against lung and colorectal carcinogenesis.

  16. Skeletal unloading inhibits the in vitro proliferation and differentiation of rat osteoprogenitor cells

    NASA Technical Reports Server (NTRS)

    Kostenuik, P. J.; Halloran, B. P.; Morey-Holton, E. R.; Bikle, D. D.

    1997-01-01

    Loss of weight bearing in the growing rat decreases bone formation, osteoblast numbers, and bone maturation in unloaded bones. These responses suggest an impairment of osteoblast proliferation and differentiation. To test this assumption, we assessed the effects of skeletal unloading using an in vitro model of osteoprogenitor cell differentiation. Rats were hindlimb elevated for 0 (control), 2, or 5 days, after which their tibial bone marrow stromal cells (BMSCs) were harvested and cultured. Five days of hindlimb elevation led to significant decreases in proliferation, alkaline phosphatase (AP) enzyme activity, and mineralization of BMSC cultures. Differentiation of BMSCs was analyzed by quantitative competitive polymerase chain reaction of cDNA after 10, 15, 20, and 28 days of culture. cDNA pools were analyzed for the expression of c-fos (an index of proliferation), AP (an index of early osteoblast differentiation), and osteocalcin (a marker of late differentiation). BMSCs from 5-day unloaded rats expressed 50% less c-fos, 61% more AP, and 35% less osteocalcin mRNA compared with controls. These data demonstrate that cultured osteoprogenitor cells retain a memory of their in vivo loading history and indicate that skeletal unloading inhibits proliferation and differentiation of osteoprogenitor cells in vitro.

  17. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation

    SciTech Connect

    Mizuno, Yosuke; Yagi, Ken; Tokuzawa, Yoshimi; Kanesaki-Yatsuka, Yukiko; Suda, Tatsuo; Katagiri, Takenobu; Fukuda, Toru; Maruyama, Masayoshi; Okuda, Akihiko; Amemiya, Tomoyuki; Kondoh, Yasumitsu; Tashiro, Hideo; Okazaki, Yasushi

    2008-04-04

    Although various microRNAs regulate cell differentiation and proliferation, no miRNA has been reported so far to play an important role in the regulation of osteoblast differentiation. Here we describe the role of miR-125b in osteoblastic differentiation in mouse mesenchymal stem cells, ST2, by regulating cell proliferation. The expression of miR-125b was time-dependently increased in ST2 cells, and the increase in miR-125b expression was attenuated in osteoblastic-differentiated ST2 cells induced by BMP-4. The transfection of exogenous miR-125b inhibited proliferation of ST2 cells and caused inhibition of osteoblastic differentiation. In contrast, when the endogenous miR-125b was blocked by transfection of its antisense RNA molecule, alkaline phosphatase activity after BMP-4 treatment was elevated. These results strongly suggest that miR-125b is involved in osteoblastic differentiation through the regulation of cell proliferation.

  18. The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation.

    PubMed

    Christopherson, Gregory T; Song, Hongjun; Mao, Hai-Quan

    2009-02-01

    Neural stem/progenitor cells (NSCs) are capable of self-renewal and differentiation into all types of neural lineage under different biochemical and topographical cues. In this study, we cultured rat hippocampus-derived adult NSCs (rNSCs) on laminin-coated electrospun Polyethersulfone (PES) fiber meshes with average fiber diameters of 283+/-45 nm, 749+/-153 nm and 1452+/-312 nm; and demonstrated that fiber diameter of PES mesh significantly influences rNSC differentiation and proliferation. Under the differentiation condition (in the presence of 1 microM retinoic acid and 1% fetal bovine serum), rNSCs showed a 40% increase in oligodendrocyte differentiation on 283-nm fibers and 20% increase in neuronal differentiation on 749-nm fibers, in comparison to tissue culture polystyrene surface. SEM imaging revealed that cells stretched multi-directionally to follow underlying 283-nm fibers, but extended along a single fiber axis on larger fibers. When cultured on fiber meshes in serum free medium in the presence of 20 ng/mL of FGF-2, rNSCs showed lower proliferation and more rounded morphology compared to that cultured on laminin-coated 2D surface. As the fiber diameter decreased, higher degree of proliferation and cell spreading and lower degree of cell aggregation were observed. This collective evidence indicates fiber topography can play a vital role in regulating differentiation and proliferation of rNSCs in culture.

  19. Adiponectin stimulates human osteoblasts proliferation and differentiation via the MAPK signaling pathway

    SciTech Connect

    Luo Xianghang; Guo Lijuan; Yuan Lingqing; Xie Hui; Zhou Houde; Wu Xianping; Liao Eryuan . E-mail: eyliao1207@21cn.com

    2005-09-10

    Adipocytes can highly and specifically express adiponectin, and the adiponectin receptor (AdipoR) has been detected in bone-forming cells. The present study was undertaken to investigate the action of adiponectin on osteoblast proliferation and differentiation. AdipoR1 protein was detected in human osteoblasts. Adiponectin promoted osteoblast proliferation and resulted in a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, osteocalcin and type I collagen production, and an increase in mineralized matrix. Suppression of AdipoR1 with small-interfering RNA (siRNA) abolished the adiponectin-induced cell proliferation and ALP expression. Adiponectin induces activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal Kinase (JNK), but not ERK1/2 in osteoblasts, and these effects were blocked by suppression of AdipoR1 with siRNA. Furthermore, pretreatment of osteoblasts with the JNK inhibitor SP600125 abolished the adiponectin-induced cell proliferation. p38 inhibitor SB203580 blocked the adiponectin-induced ALP activity. These data indicate that adiponectin induces human osteoblast proliferation and differentiation, and the proliferation response is mediated by the AdipoR/JNK pathway, while the differentiation response is mediated via the AdipoR/p38 pathway. These findings suggest that osteoblasts are the direct targets of adiponectin.

  20. Effects of Wnt3a on proliferation and differentiation of human epidermal stem cells

    SciTech Connect

    Jia Liwei; Zhou Jiaxi; Peng Sha; Li Juxue; Cao Yujing; Duan Enkui

    2008-04-11

    Epidermal stem cells maintain development and homeostasis of mammalian epidermis throughout life. However, the molecular mechanisms involved in the proliferation and differentiation of epidermal stem cells are far from clear. In this study, we investigated the effects of Wnt3a and Wnt/{beta}-catenin signaling on proliferation and differentiation of human fetal epidermal stem cells. We found both Wnt3a and active {beta}-catenin, two key members of the Wnt/{beta}-catenin signaling, were expressed in human fetal epidermis and epidermal stem cells. In addition, Wnt3a protein can promote proliferation and inhibit differentiation of epidermal stem cells in vitro culture. Our results suggest that Wnt/{beta}-catenin signaling plays important roles in human fetal skin development and homeostasis, which also provide new insights on the molecular mechanisms of oncogenesis in human epidermis.

  1. Histone methyltransferase Setd2 is critical for the proliferation and differentiation of myoblasts.

    PubMed

    Yi, Xin; Tao, Ye; Lin, Xi; Dai, Yuan; Yang, Tingli; Yue, Xiaojing; Jiang, Xuejun; Li, Xiaoyan; Jiang, Ding-Sheng; Andrade, Kelsey C; Chang, Jiang

    2017-04-01

    Skeletal muscle cell proliferation and differentiation are tightly regulated. Epigenetic regulation is a major component of the regulatory mechanism governing these processes. Histone modification is part of the epigenetic code used for transcriptional regulation of chromatin through the establishment of an active or repressive state for genes involved in myogenesis in a temporal manner. Here, we uncovered the function of SET domain containing 2 (Setd2), an essential histone 3 lysine 36 trimethyltransferase, in regulating the proliferation and differentiation of myoblasts. Setd2 was silenced in the skeletal muscle myoblast cell line, C2C12, using the CRISPR/CAS9 system. The mutant cells exhibited defect in myotube formation. The myotube formation marker, myosin heavy chain (MHC), was downregulated earlier in Setd2 silenced cells compared to wild-type myoblasts during differentiation. The deficiency in Setd2 also resulted in repression of Myogenin (MyoG) expression, a key myogenic regulator during differentiation. In addition to the myoblast differentiation defect, decreased proliferation rate with significantly reduced levels of histone 3 phosphorylation, indicative of cell proliferation defect, were observed in the Setd2 silenced cells; suggesting an impaired proliferation phenotype. Furthermore, compromised G1/S- and G2/M-phase transition and decreased expression levels of major regulators of cell cycle G1/S checkpoints, cyclin D1, CDK4, CDK6, and cyclin E2 were detected in Setd2 silenced cells. Consistent with the cell cycle arrested phenotype, cyclin-dependent kinase inhibitor p21 was upregulated in Setd2 silenced cells. Together, this study demonstrates an essential role of Setd2 in myoblast proliferation and differentiation, and uncovers Setd2-mediated molecular mechanism through regulating MyoG and p21.

  2. Human chorionic gonadotropin decreases human breast cancer cell proliferation and promotes differentiation.

    PubMed

    Liao, Xing-Hua; Wang, Yue; Wang, Nan; Yan, Ting-Bao; Xing, Wen-Jing; Zheng, Li; Zhao, Dong-Wei; Li, Yan-Qi; Liu, Long-Yue; Sun, Xue-Guang; Hu, Peng; Zhang, Tong-Cun

    2014-05-01

    Human chorionic gonadotropin (hCG) is a glycoprotein produced by placental trophoblasts. Previous studies indicated that hCG could be responsible for the pregnancy-induced protection against breast cancer in women. It is reported that hCG decreases proliferation and invasion of breast cancer MCF-7 cells. Our research also demonstrates that hCG can reduce the proliferation of MCF-7 cells by downregulating the expression of proliferation markers, proliferating cell nuclear antigen (PCNA), and proliferation-related Ki-67 antigen (Ki-67). Interestingly, we find here that hCG elevates the state of cellular differentiation, as characterized by the upregulation of differentiation markers, β-casein, cytokeratin-18 (CK-18), and E-cadherin. Inhibition of hCG secretion or luteinizing hormone/hCG receptors (LH/hCGRs) synthesis can weaken the effect of hCG on the induction of cell differentiation. Furthermore, hCG can suppress the expression of estrogen receptor alpha. hCG activated receptor-mediated cyclic adenosine monophosphate/protein kinase A signaling pathway. These findings indicated that a protective effect of hCG against breast cancer may be associated with its growth inhibitory and differentiation induction function in breast cancer cells.

  3. Specific β-containing Integrins Exert Differential Control on Proliferation and Two-dimensional Collective Cell Migration in Mammary Epithelial Cells*

    PubMed Central

    Jeanes, Alexa I.; Wang, Pengbo; Moreno-Layseca, Paulina; Paul, Nikki; Cheung, Julia; Tsang, Ricky; Akhtar, Nasreen; Foster, Fiona M.; Brennan, Keith; Streuli, Charles H.

    2012-01-01

    Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the β1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon β1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of β3-integrin in β1-integrin-null cells, indicating that integrins containing different β-subunits exert differential control on mammary epithelial proliferation and migration. β1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in β1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative β1-integrin signals. These results show that β1-integrins promote cell cycle in mammary epithelial cells, whereas β3-integrins are involved in migration. PMID:22511753

  4. B-Myb enhances proliferation and suppresses differentiation of keratinocytes in three-dimensional cell culture.

    PubMed

    Maruyama, Hiroshi; Ishitsuka, Yosuke; Fujisawa, Yasuhiro; Furuta, Junichi; Sekido, Mitsuru; Kawachi, Yasuhiro

    2014-05-01

    B-Myb (Mybl2) is a member of the Myb gene family of transcription factors involved in the control of cell growth, differentiation, and apoptosis. The effects of B-Myb on keratinocyte proliferation and differentiation have not yet been clarified. The present study was performed to examine the role of B-Myb in proliferation and differentiation of the spontaneously immortalized human skin keratinocyte cell line HaCaT and normal human keratinocytes with formation of a stratified epidermoid structure in air-liquid interface three-dimensional culture. B-Myb was expressed specifically in undifferentiated normal keratinocytes and downregulated during differentiation. The constitutive overexpression of B-Myb in HaCaT cells during air exposure-induced differentiation resulted in an undifferentiated phenotype, i.e., thickening of the stratified layers, suppression of differentiation marker expression, and retention of proliferative activity with activation of cell cycle regulatory proteins in the S and G2/M phases. In contrast, suppression of B-Myb caused their downregulation and constrained proliferation with retention of differentiation capacity. These findings suggested that B-Myb plays an important role in maintenance of the undifferentiated phenotype of keratinocytes in the basal epidermal layer.

  5. Overexpression of prothymosin alpha accelerates proliferation and retards differentiation in HL-60 cells.

    PubMed

    Rodríguez, P; Viñuela, J E; Alvarez-Fernández, L; Buceta, M; Vidal, A; Domínguez, F; Gómez-Márquez, J

    1998-05-01

    Prothymosin alpha (ProTalpha) is an acidic nuclear protein the expression of which is related to the proliferation and differentiation processes in mammalian cells. In the present study we have stably transfected HL-60 cells, a biological system that allows the study of both proliferation and differentiation, with recombinant vectors encoding sense and antisense ProTalpha mRNA. In the HL-60 cell clones overexpressing ProTalpha we observed an acceleration in the growth rate, whereas expression of the antisense orientation showed the opposite effect. Moreover, cell-cycle analysis demonstrated that the G1-phase was shortened in the cells expressing the sense construct. Before studying how ProTalpha affects differentiation, we showed that the down-regulation of ProTalpha gene during differentiation occurs in all mammalian cell lines (HL-60, K562, U937, MEL C88, N2A and PC12) analysed. The biological effect evoked by the induction of the ProTalpha sense vector was the retardation of cell differentiation, although expression of the antisense construct showed no effect on differentiation. In conclusion, our findings provide evidence that ProTalpha is directly implicated in cellular proliferation and that the maintenance of high levels of ProTalpha inside HL-60 cells is incompatible with their ability to differentiate.

  6. Overexpression of prothymosin alpha accelerates proliferation and retards differentiation in HL-60 cells.

    PubMed Central

    Rodríguez, P; Viñuela, J E; Alvarez-Fernández, L; Buceta, M; Vidal, A; Domínguez, F; Gómez-Márquez, J

    1998-01-01

    Prothymosin alpha (ProTalpha) is an acidic nuclear protein the expression of which is related to the proliferation and differentiation processes in mammalian cells. In the present study we have stably transfected HL-60 cells, a biological system that allows the study of both proliferation and differentiation, with recombinant vectors encoding sense and antisense ProTalpha mRNA. In the HL-60 cell clones overexpressing ProTalpha we observed an acceleration in the growth rate, whereas expression of the antisense orientation showed the opposite effect. Moreover, cell-cycle analysis demonstrated that the G1-phase was shortened in the cells expressing the sense construct. Before studying how ProTalpha affects differentiation, we showed that the down-regulation of ProTalpha gene during differentiation occurs in all mammalian cell lines (HL-60, K562, U937, MEL C88, N2A and PC12) analysed. The biological effect evoked by the induction of the ProTalpha sense vector was the retardation of cell differentiation, although expression of the antisense construct showed no effect on differentiation. In conclusion, our findings provide evidence that ProTalpha is directly implicated in cellular proliferation and that the maintenance of high levels of ProTalpha inside HL-60 cells is incompatible with their ability to differentiate. PMID:9560301

  7. MicroRNA regulation of myogenic satellite cell proliferation and differentiation.

    PubMed

    Harding, Rachel L; Velleman, Sandra G

    2016-01-01

    Myogenic satellite cells are stem cells responsible for muscle growth and regeneration. MicroRNAs (miRNAs) play significant roles in regulating numerous cellular processes. Two genes essential to satellite cell function are syndecan-4 and glypican-1. To determine if miRNAs influence myogenic satellite cell function, one miRNA predicted to bind syndecan-4 (miR-128) and two predicted to bind glypican-1 (miR-24 and miR-16) were inhibited in vitro by transfection of inhibitors targeting each miRNA. Inhibition of these miRNAs differentially affected the expression of syndecan-4, glypican-1, and myogenic regulatory factors myoD and myogenin. Inhibition of miR-16 reduced proliferation of satellite cells at 72 h. Inhibition of miR-128 and miR-24 did not affect proliferation. Inhibition of miRNAs reduced differentiation of satellite cells into myotubes at 48 and 72 h except for miR-16, which only affected differentiation at 72 h. Inhibition of all three miRNAs decreased myotube width at 24 h of differentiation and increased myotube width at 48 h of differentiation. Inhibiting these miRNAs also increased the number of nuclei per myotube at 72 h of differentiation. These data demonstrate individual miRNAs regulate genes essential for myogenic satellite cell proliferation and differentiation.

  8. Engineered electrospun poly(caprolactone)/polycaprolactone-g-hydroxyapatite nano-fibrous scaffold promotes human fibroblasts adhesion and proliferation.

    PubMed

    Keivani, F; Shokrollahi, P; Zandi, M; Irani, S; F Shokrolahi; Khorasani, S C

    2016-11-01

    Polycaprolactone (PCL)/hydroxyapatite nano-composites are among the best candidates for tissue engineering. However, interactions between nHAp and PCL are difficult to control leading to inhomogeneous dispersion of the bio-ceramic particles. Grafting of polymer chains at high density/chain length while promotes the phase compatibility may result in reduced HAp exposed surface area and therefore, bioactivity is compromised. This issue is addressed here by grafting PCL chains onto HAp nano-particles through ring opening polymerization of ε-caprolactone (PCL-g-HAp). FTIR and TGA analysis showed that PCL (6.9wt%), was successfully grafted on the HAp. PCL/PCL-g-HAp nano-fibrous scaffold showed up to 10 and 33% enhancement in tensile strength and modulus, respectively, compared to those of PCL/HAp. The effects of HAp on the in vitro HAp formation were investigated for both the PCL/HAp and PCL/PCL-g-HAp scaffolds. Precipitation of HAp on the nano-composite scaffolds observed after 15days incubation in simulated body fluid (SBF), as confirmed by scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX). Human fibroblasts were seeded on PCL, PCL/HAp and PCL/PCL-g-HAp scaffolds. According to MTT assay, the highest cell proliferation was recorded for PCL/PCL-g-HAp nano-composite, at all time intervals (1-21days, P<0.001). Fluorescent microscopy (of DAPI stained samples) and electron microscopy images showed that all nano-fibrous scaffolds (PCL, PCL/HAp, and PCL/PCL-g-HAp), were non-toxic against cells, while more cell adhesion, and the most uniform cell distribution observed on the PCL/PCL-g-HAp. Overall, grafting of relatively short chains of PCL on the surface of HAp nano-particles stimulates fibroblasts adhesion and proliferation on the PCL/PCL-g-HAp nano-composite.

  9. The FRK/RAK-SHB signaling cascade: a versatile signal-transduction pathway that regulates cell survival, differentiation and proliferation.

    PubMed

    Annerén, Cecilia; Lindholm, Cecilia K; Kriz, Vitezslav; Welsh, Michael

    2003-06-01

    Recent experiments have unravelled novel signal transduction pathways that involve the SRC homology 2 (SH2) domain adapter protein SHB. SHB is ubiquitously expressed and contains proline rich motifs, a phosphotyrosine binding (PTB) domain, tyrosine phosphorylation sites and an SH2 domain and serves a role in generating signaling complexes in response to tyrosine kinase activation. SHB mediates certain responses in platelet-derived growth factor (PDGF) receptor-, fibroblast growth factor (FGF) receptor-, neural growth factor (NGF) receptor TRKA-, T cell receptor-, interleukin-2 (IL-2) receptor- and focal adhesion kinase- (FAK) signaling. Upstream of SHB in some cells lies the SRC-like FYN-Related Kinase FRK/RAK (also named BSK/IYK or GTK). FRK/RAK and SHB exert similar effects when overexpressed in rat phaeochromocytoma (PC12) and beta-cells, where they both induce PC12 cell differentiation and beta-cell proliferation. Furthermore, beta-cell apoptosis is augmented by these proteins under conditions that cause beta-cell degeneration. The FRK/RAK-SHB responses involve FAK and insulin receptor substrates (IRS) -1 and -2. Besides regulating apoptosis, proliferation and differentiation, SHB is also a component of the T cell receptor (TCR) signaling response. In Jurkat T cells, SHB links several signaling components with the TCR and is thus required for IL-2 production. In endothelial cells, SHB both promotes apoptosis under conditions that are anti-angiogenic, but is also required for proper mitogenicity, spreading and tubular morphogenesis. In embryonic stem cells, dominant-negative SHB (R522K) prevents early cavitation of embryoid bodies and reduces differentiation to cells expressing albumin, amylase, insulin and glucagon, suggesting a role of SHB in development. In summary, SHB is a versatile signal transduction molecule that produces diverse biological responses in different cell types under various conditions. SHB operates downstream of GTK in cells that express

  10. Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells.

    PubMed

    Sun, Bo Kyung; Kim, Ji Hye; Choi, Joon-Seok; Hwang, Sung-Joo; Sung, Jong-Hyuk

    2015-07-22

    Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs) or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA), significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation.

  11. Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

    SciTech Connect

    Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua; Li, Zubing

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Different PTH administration exerts different effects on condylar chondrocyte. Black-Right-Pointing-Pointer Intermittent PTH administration suppresses condylar chondrocyte proliferation. Black-Right-Pointing-Pointer Continuous PTH administration maintains condylar chondrocyte proliferating. Black-Right-Pointing-Pointer Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular

  12. Porous titania surfaces on titanium with hierarchical macro- and mesoporosities for enhancing cell adhesion, proliferation and mineralization.

    PubMed

    Han, Guang; Müller, Werner E G; Wang, Xiaohong; Lilja, Louise; Shen, Zhijian

    2015-02-01

    Titanium received a macroporous titania surface layer by anodization, which contains open pores with average pore diameter around 5 μm. An additional mesoporous titania top layer following the contour of the macropores, of 100-200 nm thickness and with a pore diameter of 10nm, was formed by using the evaporation-induced self-assembly (EISA) method with titanium (IV) tetraethoxide as the precursor. A coherent laminar titania surface layer was thus obtained, creating a hierarchical macro- and mesoporous surface that was characterized by high-resolution electron microscopy. The interfacial bonding between the surface layers and the titanium matrix was characterized by the scratch test that confirmed a stable and strong bonding of titania surface layers on titanium. The wettability to water and the effects on the osteosarcoma cell line (SaOS-2) proliferation and mineralization of the formed titania surface layers were studied systematically by cell culture and scanning electron microscopy. The results proved that the porous titania surface with hierarchical macro- and mesoporosities was hydrophilic that significantly promoted cell attachment and spreading. A synergistic role of the hierarchical macro- and mesoporosities was revealed in terms of enhancing cell adhesion, proliferation and mineralization, compared with the titania surface with solo scale topography.

  13. Testing the differential adhesion hypothesis across the epithelial-mesenchymal transition

    NASA Astrophysics Data System (ADS)

    Pawlizak, Steve; Fritsch, Anatol W.; Grosser, Steffen; Ahrens, Dave; Thalheim, Tobias; Riedel, Stefanie; Kießling, Tobias R.; Oswald, Linda; Zink, Mareike; Manning, M. Lisa; Käs, Josef A.

    2015-08-01

    We analyze the mechanical properties of three epithelial/mesenchymal cell lines (MCF-10A, MDA-MB-231, MDA-MB-436) that exhibit a shift in E-, N- and P-cadherin levels characteristic of an epithelial-mesenchymal transition associated with processes such as metastasis, to quantify the role of cell cohesion in cell sorting and compartmentalization. We develop a unique set of methods to measure cell-cell adhesiveness, cell stiffness and cell shapes, and compare the results to predictions from cell sorting in mixtures of cell populations. We find that the final sorted state is extremely robust among all three cell lines independent of epithelial or mesenchymal state, suggesting that cell sorting may play an important role in organization and boundary formation in tumours. We find that surface densities of adhesive molecules do not correlate with measured cell-cell adhesion, but do correlate with cell shapes, cell stiffness and the rate at which cells sort, in accordance with an extended version of the differential adhesion hypothesis (DAH). Surprisingly, the DAH does not correctly predict the final sorted state. This suggests that these tissues are not behaving as immiscible fluids, and that dynamical effects such as directional motility, friction and jamming may play an important role in tissue compartmentalization across the epithelial-mesenchymal transition.

  14. Testing the differential adhesion hypothesis across the epithelial-mesenchymal transition

    NASA Astrophysics Data System (ADS)

    Pawlizak, Steve; Fritsch, Anatol; Grosser, Steffen; Oswald, Linda; Manning, Lisa; Kas, Josef

    We analyze the properties of three epithelial/mesenchymal cell lines that exhibit a shift in cadherin levels characteristic of an epithelial-mesenchymal transition (EMT) associated with processes such as metastasis, to quantify the role of cell cohesion in cell sorting and compartmentalization. We develop a unique set of methods to measure cell-cell adhesiveness, cell stiffness and cell shapes, and compare the results to predictions from cell sorting in mixtures of cell populations. We find that the final sorted state is extremely robust among all three cell lines independent of epithelial or mesenchymal state, suggesting that cell sorting may play an important role in organization and boundary formation in tumours. We find that surface densities of adhesive molecules do not correlate with measured cell-cell adhesion, but do correlate with cell shapes, cell stiffness and the rate at which cells sort, in accordance with an extended differential adhesion hypothesis (DAH). Surprisingly, the DAH does not correctly predict the final sorted state. This suggests that these tissues are not behaving as immiscible fluids, and that dynamical effects such as directional motility, friction and jamming may play an important role in tissue compartmentalization across the EMT.

  15. MYB36 regulates the transition from proliferation to differentiation in the Arabidopsis root

    PubMed Central

    Liberman, Louisa M.; Sparks, Erin E.; Moreno-Risueno, Miguel A.; Petricka, Jalean J.; Benfey, Philip N.

    2015-01-01

    Stem cells are defined by their ability to self-renew and produce daughter cells that proliferate and mature. These maturing cells transition from a proliferative state to a terminal state through the process of differentiation. In the Arabidopsis thaliana root the transcription factors SCARECROW and SHORTROOT regulate specification of the bipotent stem cell that gives rise to cortical and endodermal progenitors. Subsequent progenitor proliferation and differentiation generate mature endodermis, marked by the Casparian strip, a cell-wall modification that prevents ion diffusion into and out of the vasculature. We identified a transcription factor, MYB DOMAIN PROTEIN 36 (MYB36), that regulates the transition from proliferation to differentiation in the endodermis. We show that SCARECROW directly activates MYB36 expression, and that MYB36 likely acts in a feed-forward loop to regulate essential Casparian strip formation genes. We show that myb36 mutants have delayed and defective barrier formation as well as extra divisions in the meristem. Our results demonstrate that MYB36 is a critical positive regulator of differentiation and negative regulator of cell proliferation. PMID:26371322

  16. Subarachnoid Hemorrhage Promotes Proliferation, Differentiation, and Migration of Neural Stem Cells via BDNF Upregulation

    PubMed Central

    Lee, Wen-Di; Wang, Kuo-Chuan; Tsai, Yi-Fen; Chou, Pin-Chun; Tsai, Li-Kai; Chien, Chung-Liang

    2016-01-01

    Patients who suffer from subarachnoid hemorrhage (SAH) usually have long-term neurological impairments. Endogenous neurogenesis might play a potential role in functional recovery after SAH; however, the underlying neurogenesis mechanism is still unclear. We assessed the extent of neurogenesis in the subventricular zone (SVZ) to better understand the neurogenesis mechanism after SAH. We performed a rat model of SAH to examine the extent of neurogenesis in the SVZ and assessed functional effects of the neurotrophic factors in the cerebrospinal fluid (CSF) on neural stem cells (NSCs) after SAH. In this study, the proliferation, differentiation, and migratory capacities of NSCs in the SVZ were significantly increased on days 5 and 7 post SAH. Furthermore, treatment of cultured rat fetal NSCs with the CSF collected from rats on days 5 and 7 post SAH enhanced their proliferation, differentiation, and migration. Enzyme-linked immunosorbent assay (ELISA) of the CSF detected a marked increase in the concentration of brain-derived neurotrophic factor (BDNF). Treating the cultured NSCs with recombinant BDNF (at the same concentration as that in the CSF) or with CSF from SAH rats, directly, stimulated proliferation, differentiation, and migration to a similar extent. BDNF expression was upregulated in the SVZ of rats on days 5 and 7 post SAH, and BDNF release occurred from NSCs, astrocytes, and microglia in the SVZ. These results indicate that SAH triggers the expression of BDNF, which promotes the proliferation, differentiation, and migration of NSCs in the SVZ after SAH. PMID:27832087

  17. Lipocalin-2 inhibits osteoclast formation by suppressing the proliferation and differentiation of osteoclast lineage cells

    SciTech Connect

    Kim, Hyun-Ju; Yoon, Hye-Jin; Yoon, Kyung-Ae; Gwon, Mi-Ri; Jin Seong, Sook; Suk, Kyoungho; Kim, Shin-Yoon; Yoon, Young-Ran

    2015-06-10

    Lipocalin-2 (LCN2) is a member of the lipocalin superfamily and plays a critical role in the regulation of various physiological processes, such as inflammation and obesity. In this study, we report that LCN2 negatively modulates the proliferation and differentiation of osteoclast precursors, resulting in impaired osteoclast formation. The overexpression of LCN2 in bone marrow-derived macrophages or the addition of recombinant LCN2 protein inhibits the formation of multinuclear osteoclasts. LCN2 suppresses macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursor cells without affecting their apoptotic cell death. Interestingly, LCN2 decreases the expression of the M-CSF receptor, c-Fms, and subsequently blocks its downstream signaling cascades. In addition, LCN2 inhibits RANKL-induced osteoclast differentiation and attenuates the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important modulators in osteoclastogenesis. Mechanistically, LCN2 inhibits NF-κB signaling pathways, as demonstrated by the suppression of IκBα phosphorylation, nuclear translocation of p65, and NF-κB transcriptional activity. Thus, LCN2 is an anti-osteoclastogenic molecule that exerts its effects by retarding the proliferation and differentiation of osteoclast lineage cells. - Highlights: • LCN2 expression is regulated during osteoclast development. • LCN2 suppresses M-CSF-mediated osteoclast precursor proliferation. • LCN2 inhibits RANKL-induced osteoclast differentiation.

  18. Proliferation and Differentiation of Trypanosoma cruzi inside Its Vector Have a New Trigger: Redox Status

    PubMed Central

    Nogueira, Natália P.; Saraiva, Francis M. S.; Sultano, Pedro E.; Cunha, Paula R. B. B.; Laranja, Gustavo A. T.; Justo, Graça A.; Sabino, Kátia C. C.; Coelho, Marsen G. P.; Rossini, Ana; Atella, Georgia C.; Paes, Marcia C.

    2015-01-01

    Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H2O2 and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. β-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle. PMID:25671543

  19. Proliferation and differentiation of Trypanosoma cruzi inside its vector have a new trigger: redox status.

    PubMed

    Nogueira, Natália P; Saraiva, Francis M S; Sultano, Pedro E; Cunha, Paula R B B; Laranja, Gustavo A T; Justo, Graça A; Sabino, Kátia C C; Coelho, Marsen G P; Rossini, Ana; Atella, Georgia C; Paes, Marcia C

    2015-01-01

    Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H2O2 and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. β-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle.

  20. Nano-hydroxyapatite-coated metal-ceramic composite of iron-tricalcium phosphate: Improving the surface wettability, adhesion and proliferation of mesenchymal stem cells in vitro.

    PubMed

    Surmeneva, Maria A; Kleinhans, Claudia; Vacun, Gabriele; Kluger, Petra Juliane; Schönhaar, Veronika; Müller, Michaela; Hein, Sebastian Boris; Wittmar, Alexandra; Ulbricht, Mathias; Prymak, Oleg; Oehr, Christian; Surmenev, Roman A

    2015-11-01

    Thin radio-frequency magnetron sputter deposited nano-hydroxyapatite (HA) films were prepared on the surface of a Fe-tricalcium phosphate (Fe-TCP) bioceramic composite, which was obtained using a conventional powder injection moulding technique. The obtained nano-hydroxyapatite coated Fe-TCP biocomposites (nano-HA-Fe-TCP) were studied with respect to their chemical and phase composition, surface morphology, water contact angle, surface free energy and hysteresis. The deposition process resulted in a homogeneous, single-phase HA coating. The ability of the surface to support adhesion and the proliferation of human mesenchymal stem cells (hMSCs) was studied using biological short-term tests in vitro. The surface of the uncoated Fe-TCP bioceramic composite showed an initial cell attachment after 24h of seeding, but adhesion, proliferation and growth did not persist during 14 days of culture. However, the HA-Fe-TCP surfaces allowed cell adhesion, and proliferation during 14 days. The deposition of the nano-HA films on the Fe-TCP surface resulted in higher surface energy, improved hydrophilicity and biocompatibility compared with the surface of the uncoated Fe-TCP. Furthermore, it is suggested that an increase in the polar component of the surface energy was responsible for the enhanced cell adhesion and proliferation in the case of the nano-HA-Fe-TCP biocomposites.

  1. Dehydroepiandrosterone exerts antiglucocorticoid action on human preadipocyte proliferation, differentiation, and glucose uptake

    PubMed Central

    McNelis, Joanne C.; Manolopoulos, Konstantinos N.; Gathercole, Laura L.; Bujalska, Iwona J.; Stewart, Paul M.; Tomlinson, Jeremy W.

    2013-01-01

    Glucocorticoids increase adipocyte proliferation and differentiation, a process underpinned by the local reactivation of inactive cortisone to active cortisol within adipocytes catalyzed by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). The adrenal sex steroid precursor dehydroepiandrosterone (DHEA) has been shown to inhibit 11β-HSD1 in murine adipocytes; however, rodent adrenals do not produce DHEA physiologically. Here, we aimed to determine the effects and underlying mechanisms of the potential antiglucocorticoid action of DHEA and its sulfate ester DHEAS in human preadipocytes. Utilizing a human subcutaneous preadipocyte cell line, Chub-S7, we examined the metabolism and effects of DHEA in human adipocytes, including adipocyte proliferation, differentiation, 11β-HSD1 expression, and activity and glucose uptake. DHEA, but not DHEAS, significantly inhibited preadipocyte proliferation via cell cycle arrest in the G1 phase independent of sex steroid and glucocorticoid receptor activation. 11β-HSD1 oxoreductase activity in differentiated adipocytes was inhibited by DHEA. DHEA coincubated with cortisone significantly inhibited preadipocyte differentiation, which was assessed by the expression of markers of early (LPL) and terminal (G3PDH) adipocyte differentiation. Coincubation with cortisol, negating the requirement for 11β-HSD1 oxoreductase activity, diminished the inhibitory effect of DHEA. Further consistent with glucocorticoid-opposing effects of DHEA, insulin-independent glucose uptake was significantly enhanced by DHEA treatment. DHEA increases basal glucose uptake and inhibits human preadipocyte proliferation and differentiation, thereby exerting an antiglucocorticoid action. DHEA inhibition of the amplification of glucocorticoid action mediated by 11β-HSD1 contributes to the inhibitory effect of DHEA on human preadipocyte differentiation. PMID:24022868

  2. Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

    PubMed

    Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

    2015-11-01

    Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in

  3. CaMKK2 Suppresses Muscle Regeneration through the Inhibition of Myoblast Proliferation and Differentiation

    PubMed Central

    Ye, Cheng; Zhang, Duo; Zhao, Lei; Li, Yan; Yao, Xiaohan; Wang, Hui; Zhang, Shengjie; Liu, Wei; Cao, Hongchao; Yu, Shuxian; Wang, Yucheng; Jiang, Jingjing; Wang, Hui; Li, Xihua; Ying, Hao

    2016-01-01

    Skeletal muscle has a major role in locomotion and muscle disorders are associated with poor regenerative efficiency. Therefore, a deeper understanding of muscle regeneration is needed to provide a new insight for new therapies. CaMKK2 plays a role in the calcium/calmodulin-dependent kinase cascade; however, its role in skeletal muscle remains unknown. Here, we found that CaMKK2 expression levels were altered under physiological and pathological conditions including postnatal myogensis, freeze or cardiotoxin-induced muscle regeneration, and Duchenne muscular dystrophy. Overexpression of CaMKK2 suppressed C2C12 myoblast proliferation and differentiation, while inhibition of CaMKK2 had opposite effect. We also found that CaMKK2 is able to activate AMPK in C2C12 myocytes. Inhibition of AMPK could attenuate the effect of CaMKK2 overexpression, while AMPK agonist could abrogate the effect of CaMKK2 knockdown on C2C12 cell differentiation and proliferation. These results suggest that CaMKK2 functions as an AMPK kinase in muscle cells and AMPK mediates the effect of CaMKK2 on myoblast proliferation and differentiation. Our data also indicate that CaMKK2 might inhibit myoblast proliferation through AMPK-mediated cell cycle arrest by inducing cdc2-Tyr15 phosphorylation and repress differentiation through affecting PGC1α transcription. Lastly, we show that overexpressing CaMKK2 in the muscle of mice via electroporation impaired the muscle regeneration during freeze-induced injury, indicating that CaMKK2 could serve as a potential target to treat patients with muscle injury or myopathies. Together, our study reveals a new role for CaMKK2 as a negative regulator of myoblast differentiation and proliferation and sheds new light on the molecular regulation of muscle regeneration. PMID:27783047

  4. Differential Adhesive and Bioactive Properties of the Polymeric Surface Coated with Graphene Oxide Thin Film.

    PubMed

    Thampi, Sudhin; Nandkumar, A Maya; Muthuvijayan, Vignesh; Parameswaran, Ramesh

    2017-02-08

    Surface engineering of implantable devices involving polymeric biomaterials has become an essential aspect for medical implants. A surface enhancement technique can provide an array of unique surface properties that improve its biocompatibility and functionality as an implant. Polyurethane-based implants that have found extensively acclaimed usage as an implant in biomedical applications, especially in the area of cardiovascular devices, still lack any mechanism to ward off bacterial or platelet adhesion. To bring out such a defense mechanism we are proposing a surface modification technique. Graphene oxide (GO) in very thin film form was wrapped onto the electrospun fibroporous polycarbonate urethane (PCU) membrane (GOPCU) by a simple method of electrospraying. In the present study, we have developed a simple single-step method for coating a polymeric substrate with a thin GO film and evaluated the novel antiadhesive activity of these films. SEM micrographs after coating showed the presence of very thin GO films over the PCU membrane. On the GOPCU surface, the contact angle was shifted by ∼30°, making the hydrophobic PCU surface slightly hydrophilic, while Raman spectral characterization and mapping showed the presence and distribution of GO over 75% of the membrane. A reduced platelet adhesion on the GOPCU surface was observed; meanwhile, bacterial adhesion also got reduced by 85% for Staphylococcus aureus (Gram positive, cocci) and 64% for Pseudomonas aeruginosa (Gram negative, bacilli). A cell adhesion study conducted using mammalian fibroblast cells projected its proliferation percentage in a MTT assay, with 82% cell survival on PCU and 86% on GOPCU after 24 h culture, while a study for an extended period of 72 h showed 87% of survival on PCU and 88% on GOPCU. This plethora of functionalities by a simple modification technique makes thin GO films a self-sufficient surface engineering material for future biomedical applications.

  5. Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation.

    PubMed

    Zabel, Franziska; Fettelschoss, Antonia; Vogel, Monique; Johansen, Pål; Kündig, Thomas M; Bachmann, Martin F

    2017-03-01

    Several memory B-cell subclasses with distinct functions have been described, of which the most effective is the class-switched (CS) memory B-cell population. We have previously shown, using virus-like particles (VLPs), that the proliferative potential of these CS memory B cells is limited and they fail to re-enter germinal centres (GCs). However, VLP-specific memory B cells quickly differentiated into secondary plasma cells (PCs) with the virtue of elevated antibody production compared with primary PCs. Whereas the induction of VLP(+) memory B cells was strongly dependent on T helper cells, we were wondering whether re-stimulation of VLP(+) memory B cells and their differentiation into secondary PCs would also require T helper cells. Global absence of T helper cells led to strongly impaired memory B cell proliferation and PC differentiation. In contrast, lack of interleukin-21 receptor-dependent follicular T helper cells or CD40 ligand signalling strongly affected proliferation of memory B cells, but differentiation into mature secondary PCs exhibiting increased antibody production was essentially normal. This contrasts with primary B-cell responses, where a strong dependence on CD40 ligand but limited importance of interleukin-21 receptor was seen. Hence, T helper cell dependence differs between primary and secondary B-cell responses as well as between memory B-cell proliferation and PC differentiation.

  6. Magnetically Tuning Tether Mobility of Integrin Ligand Regulates Adhesion, Spreading, and Differentiation of Stem Cells.

    PubMed

    Wong, Dexter S H; Li, Jinming; Yan, Xiaohui; Wang, Ben; Li, Rui; Zhang, Li; Bian, Liming

    2017-03-08

    Cells sense and respond to the surrounding microenvironment through binding of membranous integrin to ligands such as the Arg-Gly-Asp (RGD) peptide. Previous studies show that the RGD tether properties on substrate influence cell adhesion and spreading, but few studies have reported strategies to control the tether mobility of RGD on substrate via a physical and noncontact approach. Herein, we demonstrate a novel strategy to tune the tether mobility of RGD on substrate via magnetic force. We conjugate a monolayer of RGD-bearing magnetic nanoparticles (MNPs) on a glass substrate via the flexible and coiled poly(ethylene glycol) linker of large molecular weight (PEG, average MW: 2000), and this increases the RGD tether mobility, which can be significantly reduced by applying magnetic attraction on MNPs. Our data show that high RGD tether mobility delays the early adhesion and spreading of human mesenchymal stem cells (hMSCs), leading to compromised osteogenic differentiation at later stage. In contrast, hMSCs cultured on substrate with restricted RGD tether mobility, achieved either via a shorter PEG linker (MW: 200) or magnetic force, show significantly better adhesion, spreading, and osteogenic differentiation. The control utilizing RGD-bearing nonmagnetic nanoparticles shows no such enhancing effect of magnetic field on cellular events, further supporting our conjecture of magnetic tuning of RGD tether mobility. We hypothesize that high tether mobility of RGD entails additional time and effort by the cells to fully develop traction force and mechanical feedback, thereby delaying the maturation of FAs and activation of subsequent mechanotransduction signaling. Our staining results of vinculin, a critical component of FAs, and Yes-associated protein (YAP), an important mechanosensitive transcriptional factor, support our hypothesis. We believe that our work not only sheds light on the impact of dynamic presentation of cell adhesive ligands on cellular behaviors

  7. Deoxycholic acid differentially regulates focal adhesion kinase phosphorylation: role of tyrosine phosphatase ShP2.

    PubMed

    Khare, Sharad; Holgren, Cory; Samarel, Allen M

    2006-12-01

    Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.

  8. Prenylation is required for polar cell elongation, cell adhesion, and differentiation in Physcomitrella patens.

    PubMed

    Thole, Julie M; Perroud, Pierre-Francois; Quatrano, Ralph S; Running, Mark P

    2014-05-01

    Protein prenylation is required for a variety of growth and developmental processes in flowering plants. Here we report the consequences of loss of function of all known prenylation subunits in the moss Physcomitrella patens. As in Arabidopsis, protein farnesyltransferase and protein geranylgeranyltransferase type I are not required for viability. However, protein geranylgeranyltransferase type I activity is required for cell adhesion, polar cell elongation, and cell differentiation. Loss of protein geranylgeranyltransferase activity results in colonies of round, single-celled organisms that resemble unicellular algae. The loss of protein farnesylation is not as severe but also results in polar cell elongation and differentiation defects. The complete loss of Rab geranylgeranyltransferase activity appears to be lethal in P. patens. Labeling with antibodies to cell wall components support the lack of polarity establishment and the undifferentiated state of geranylgeranyltransferase type I mutant plants. Our results show that prenylated proteins play key roles in P. patens development and differentiation processes.

  9. rFN/Cad-11-Modified Collagen Type II Biomimetic Interface Promotes the Adhesion and Chondrogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Guo, Hongfeng; Zhang, Yuan; Li, Zhengsheng; Kang, Fei; Yang, Bo; Kang, Xia; Wen, Can; Yan, Yanfei; Jiang, Bo; Fan, Yujiang

    2013-01-01

    Properties of the cell-material interface are determining factors in the successful function of cells for cartilage tissue engineering. Currently, cell adhesion is commonly promoted through the use of polypeptides; however, due to their lack of complementary or modulatory domains, polypeptides must be modified to improve their ability to promote adhesion. In this study, we utilized the principle of matrix-based biomimetic modification and a recombinant protein, which spans fragments 7–10 of fibronectin module III (heterophilic motif ) and extracellular domains 1–2 of cadherin-11 (rFN/Cad-11) (homophilic motif ), to modify the interface of collagen type II (Col II) sponges. We showed that the designed material was able to stimulate cell proliferation and promote better chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) in vitro than both the FN modified surfaces and the negative control. Further, the Col II/rFN/Cad-11-MSCs composite stimulated cartilage formation in vivo; the chondrogenic effect of Col II alone was much less significant. These results suggested that the rFN/Cad-11-modified collagen type II biomimetic interface has dual biological functions of promoting adhesion and stimulating chondrogenic differentiation. This substance, thus, may serve as an ideal scaffold material for cartilage tissue engineering, enhancing repair of injured cartilage in vivo. PMID:23919505

  10. Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes

    SciTech Connect

    Lai, Peng-Yeh; Tsai, Chong-Bin; Tseng, Min-Jen

    2013-01-18

    Highlights: ► Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ► Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ► Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ► Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBPδ and PPARγ was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

  11. miR-429 Inhibits Differentiation and Promotes Proliferation in Porcine Preadipocytes

    PubMed Central

    Peng, Ying; Chen, Fen-Fen; Ge, Jing; Zhu, Jia-Yu; Shi, Xin-E; Li, Xiao; Yu, Tai-Yong; Chu, Gui-Yan; Yang, Gong-She

    2016-01-01

    MicroRNAs (miRNAs) are crucial regulatory molecules for adipogenesis. They contribute to the controlling of proliferation and differentiation of preadipocytes. Previous studies revealed an important role of miR-429 in cell invasion, migration, and apoptosis. Our previous work has shown that the expression of miR-429 in subcutaneous fat can be observed in newly born (3-day-old) Rongchang piglets rather than their adult counterparts (180-day-old). This expression pattern suggests that miR-429 might be functionally related to postnatal adipogenesis. However, we currently lack a mechanistic understanding of miR-429 within the context of preadipocyte differentiation. In this study, we investigated the function of miR-429 in porcine subcutaneous and intramuscular preadipocyte proliferation and differentiation. In our porcine preadipocyte differentiation model, miR-429 expression decreased remarkably upon adipogenic induction. Overexpression of miR-429 notably down-regulated the expression of adipogenic marker genes: PPARγ, aP2, FAS and impaired the triglyceride accumulation, while the expression of lipolytic gene ATGL was not affected. In addition, we observed that miR-429 significantly promoted the proliferation of porcine preadipocytes. We also found that miR-429 could directly bind to the 3′-UTRs of KLF9 and p27, which have been well documented to promote preadipocyte differentiation and repress cell cycle progression. Taken together, our data support a novel role of miR-429 in regulating porcine preadipocyte differentiation and proliferation, and KLF9 and p27 are potent targets of miR-429 during these processes. PMID:27941616

  12. Reciprocal role of vitamin D receptor on β-catenin regulated keratinocyte proliferation and differentiation.

    PubMed

    Hu, Lizhi; Bikle, Daniel D; Oda, Yuko

    2014-10-01

    The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), suppresses the proliferation while promoting the differentiation of keratinocytes through the vitamin D receptor (VDR). β-Catenin, on the other hand, promotes proliferation and blocks epidermal differentiation, although it stimulates hair follicle differentiation. In intestinal epithelia VDR binds β-catenin and blocks its proliferative effects. In this study we investigated the role of 1,25(OH)2D3/VDR on β-catenin regulated gene transcription during keratinocyte proliferation and differentiation. 1,25(OH)2D3 suppressed promoter reporter activity driven by synthetic and natural TCF/β-catenin response elements. Over-expression of VDR further suppressed these TCF/β-catenin promoter activities. 1,25(OH)2D3 also suppressed the mRNA expression of the β-catenin regulated gene Gli1 through VDR. These data were consistent with our previous observations that VDR silencing resulted in keratinocyte hyperproliferation with increased expression of Gli1 in vitro, whereas VDR null skin showed hyperproliferation in vivo. In contrast, 1,25(OH)2D3 induced expression of another β-catenin regulated gene, PADI1, important for both epidermal and hair follicle differentiation. Deletion of VDR resulted in defects in hair differentiation in vivo, with decreased expression of β-catenin regulated hair differentiation genes such as PADI1, hair keratin KRT31 and calcium binding protein S100a3. These genes possess vitamin D response elements (VDRE) adjacent to TCF/β-catenin response elements and are regulated by both VDR and β-catenin signaling. Therefore, we propose that VDR and β-catenin interact reciprocally to promote VDR stimulation of genes involved with differentiation that contain both VDR and β-catenin response elements while inhibiting β-catenin stimulation of genes involved with proliferation. Thus the major finding of this study is that while 1,25(OH)2D3/VDR inhibits the actions of β-catenin to

  13. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    SciTech Connect

    Brady, Robert T.; O'Brien, Fergal J.; Hoey, David A.

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  14. Differentiation and proliferation of periosteal osteoblast progenitors are differentially regulated by estrogens and intermittent parathyroid hormone administration.

    PubMed

    Ogita, Mami; Rached, Marie Therese; Dworakowski, Elzbieta; Bilezikian, John P; Kousteni, Stavroula

    2008-11-01

    The periosteum is now widely recognized as a homeostatic and therapeutic target for actions of sex steroids and intermittent PTH administration. The mechanisms by which estrogens suppress but PTH promotes periosteal expansion are not known. In this report, we show that intermittent PTH(1-34) promotes differentiation of periosteal osteoblast precursors as evidenced by the stimulation of the expression or activity of alkaline phosphatase as well as of targets of the bone morphogenetic protein 2 (BMP-2) and Wnt pathways. In contrast, 17beta-estradiol (E2) had no effect by itself. However, it attenuated PTH- or BMP-2-induced differentiation of primary periosteal osteoblast progenitors. Administration of intermittent PTH to ovariectomized mice induced rapid phosphorylation of the BMP-2 target Smad1/5/8 in the periosteum. A replacement dose of E2 had no effect by itself but suppressed PTH-induced phosphorylation of Smad1/5/8. In contrast to its effects to stimulate periosteal osteoblast differentiation, PTH promoted and subsequently suppressed proliferation of periosteal osteoblast progenitors in vitro and in vivo. E2 promoted proliferation and attenuated the antiproliferative effect of PTH. Both hormones protected periosteal osteoblasts from apoptosis induced by various proapoptotic agents. These observations suggest that the different effects of PTH and estrogens on the periosteum result from opposing actions on the recruitment of early periosteal osteoblast progenitors. Intermittent PTH promotes osteoblast differentiation from periosteum-derived mesenchymal progenitors through ERK-, BMP-, and Wnt-dependent signaling pathways. Estrogens promote proliferation of early osteoblast progenitors but inhibit their differentiation by osteogenic agents such as PTH or BMP-2.

  15. Effect of sertraline on proliferation and neurogenic differentiation of human adipose-derived stem cells

    PubMed Central

    Razavi, Shahnaz; Jahromi, Maliheh; Amirpour, Nushin; Khosravizadeh, Zahra

    2014-01-01

    Background: Antidepressant drugs are commonly employed for anxiety and mood disorders. Sertraline is extensively used as antidepressant in clinic. In addition, adipose tissue represents an abundant and accessible source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human adipose-derived stem cells (hADSCs) may be useful for autologous transplantation. Materials and Methods: In the present study, we assessed the effect of antidepressant drug Sertraline on the proliferation and neurogenic differentiation of hADSCs using MTT assay and immunofluorescence technique respectively. Results: MTT assay analysis showed that 0.5 μM Sertraline significantly increased the proliferation rate of hADSCs induced cells (P < 0.05), while immunofluorescent staining indicated that Sertraline treatment during neurogenic differentiation could be decreased the percentage of glial fibrillary acidic protein and Nestin-positive cells, but did not significantly effect on the percentage of MAP2 positive cells. Conclusion: Overall, our data show that Sertraline can be promoting proliferation rate during neurogenic differentiation of hADSCs after 6 days post-induction, while Sertraline inhibits gliogenesis of induced hADSCs. PMID:24800186

  16. C/EBPalpha and beta couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation.

    PubMed

    Lopez, Rodolphe G; Garcia-Silva, Susana; Moore, Susan J; Bereshchenko, Oksana; Martinez-Cruz, Ana B; Ermakova, Olga; Kurz, Elke; Paramio, Jesus M; Nerlov, Claus

    2009-10-01

    The transcriptional regulators that couple interfollicular basal keratinocyte proliferation arrest to commitment and differentiation are yet to be identified. Here we report that the basic region leucine zipper transcription factors C/EBPalpha and C/EBPbeta are co-expressed in basal keratinocytes, and are coordinately upregulated as keratinocytes exit the basal layer and undergo terminal differentiation. Mice lacking both C/EBPalpha and beta in the epidermis showed increased proliferation of basal keratinocytes and impaired commitment to differentiation. This led to ectopic expression of keratin 14 (K14) and DeltaNp63 in suprabasal cells, decreased expression of spinous and granular layer proteins, parakeratosis and defective epidermal water barrier function. Knock-in mutagenesis revealed that C/EBP-E2F interaction was required for control of interfollicular epidermis (IFE) keratinocyte proliferation, but not for induction of spinous and granular layer markers, whereas C/EBP DNA binding was required for DeltaNp63 downregulation and K1/K10 induction. Finally, loss of C/EBPalpha/beta induced stem cell gene expression signatures in the epidermis. C/EBPs, therefore, couple basal keratinocyte cell cycle exit to commitment to differentiation through E2F repression and DNA binding, respectively, and may act to restrict the epidermal stem cell compartment.

  17. MicroRNA-196b promotes cell proliferation and suppress cell differentiation in vitro

    SciTech Connect

    Cao, Donglin Hu, Liangshan; Lei, Da; Fang, Xiaolin; Zhang, Zhihong; Wang, Ting; Lin, Maorui; Huang, Jiwei; Yang, Huawen; Zhou, Xuan; Zhong, Limei

    2015-01-30

    Highlights: • miRNA-196b increases proliferation and blocks differentiation of progenitor cell. • miRNA-196b inhibits apoptosis and increases viability of cells lines. • Forced expression of miR-196b blocks the differentiation of THP1 induced by PMA. - Abstract: MicroRNA-196b (miR-196b) is frequently amplified and aberrantly overexpressed in acute leukemias. To investigate the role of miR-196b in acute leukemias, it has been observed that forced expression of this miRNA increases proliferation and inhibits apoptosis in human cell lines. More importantly, we show that this miRNA can significantly increase the colony-forming capacity of mouse normal bone marrow progenitor cells alone, as well as partially blocking the cells from differentiation. Taken together, our studies suggest that miRNA-196b may play an essential role in the development of MLL-associated leukemias through inhibiting cell differentiation and apoptosis, while promoting cell proliferation.

  18. The Effect on Proliferation and Differentiation of Cementoblast by Using Sclerostin as Inhibitor

    PubMed Central

    Bao, Xingfu; Liu, Yuyan; Han, Guanghong; Zuo, Zhigang; Hu, Min

    2013-01-01

    Cementogenesis is of great importance for normal teeth root development and is involved in the repair process of root resorption caused by orthodontic treatment. As highly differentiated mesenchymal cells, cementoblasts are responsible for this process under the regulation of many endogenous agents. Among these molecules, sclerostin has been much investigated recently for its distinct antagonism effect on bone metabolism. Encoded by the sost gene, sclerostin is expressed in osteocytes and cementocytes of cellular cementum. it is still unclear. In the current study, we investigated the effects of sclerostin on the processes of proliferation and differentiation; a series of experiments including MTT, apoptosis examination, alkaline phosphatase (ALP) activity, gene analysis, and alizarin red staining were carried out to evaluate the proliferation and differentiation of cementoblasts. Protein expression including osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) were also checked to analyze changes in osteoclastogenesis. Results show that sclerostin inhibits cementoblasts proliferation and differentiation, and promotes osteoclastogenesis. Interestingly, the monoclonal antibody for sclerostin has shown positive effects on osteoporosis, indicating that it may facilitate cementogenesis and benefit the treatment of cementum related diseases. PMID:24152444

  19. Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death

    SciTech Connect

    Curtis, Brandon M.; Leix, Kyle Alexander; Ji, Yajing; Glaves, Richard Samuel Elliot; Ash, David E.; Mohanty, Dillip K.

    2014-07-18

    Highlights: • Multipotent vascular stem cells (MVSCs) proliferate and differentiate. • Nitric oxide inhibits proliferation of MVSCs. • Nitric oxide inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs). • Smooth muscle cells (SMCs) neither de-differentiate nor proliferate. - Abstract: Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to such injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well.

  20. The effects of growth hormone on avian skeletal muscle satellite cell proliferation and differentiation.

    PubMed

    Halevy, O; Hodik, V; Mett, A

    1996-01-01

    Growth hormone receptor (GH-R) mRNA was expressed in avian skeletal muscle tissue and satellite cells in culture, and was capable of binding chicken growth hormone (cGH). In the satellite cells, GH-R gene expression was regulated by cGH in a biphasic manner which correlated with the GH effect on cell proliferation: 2-10 ng/ml of the hormone increased GH-R mRNA and DNA synthesis, whereas higher concentrations attenuated these effects. GH induced insulin-like growth factor I (IGF-I) mRNA, a potential factor for satellite cell proliferation and differentiation. However, GH inhibited the gene expression of myogenin and the expression of muscle-specific proteins in a dose-dependent manner. These results suggest a role of GH for inhibiting satellite cell differentiation in an IGF-I-independent manner. During satellite cell differentiation, both GH-R mRNA expression and cGH binding peaked when cells were still proliferating and beginning to fuse, and then declined as cells fully differentiated. GH-R mRNA expression in muscle tissue and the satellite cell fraction was evaluated during chicken growth. In both fractions, GH-R mRNA peaked at 4 days of age and then declined in correlation with the reduction of muscle regulatory gene expression. Our results are in contrast with previous studies on rat muscle satellite cells, suggesting a difference between mammalian and avian species in the mode of action of GH in these cells. Our notion is that GH, via its own receptor, promotes more satellite cells to proliferate by inhibiting their differentiation, leading to the addition of more nuclei to the growing muscle.

  1. MicroRNA93 Regulates Proliferation and Differentiation of Normal and Malignant Breast Stem Cells

    PubMed Central

    Liu, Suling; Patel, Shivani H.; Ginestier, Christophe; Ibarra, Ingrid; Martin-Trevino, Rachel; Bai, Shoumin; McDermott, Sean P.; Shang, Li; Ke, Jia; Ou, Sing J.; Heath, Amber; Zhang, Kevin J.; Korkaya, Hasan; Clouthier, Shawn G.; Charafe-Jauffret, Emmanuelle; Birnbaum, Daniel; Hannon, Gregory J.; Wicha, Max S.

    2012-01-01

    MicroRNAs (miRNAs) play important roles in normal cellular differentiation and oncogenesis. microRNA93 (mir-93), a member of the mir106b-25 cluster, located in intron 13 of the MCM7 gene, although frequently overexpressed in human malignancies may also function as a tumor suppressor gene. Using a series of breast cancer cell lines representing different stages of differentiation and mouse xenograft models, we demonstrate that mir-93 modulates the fate of breast cancer stem cells (BCSCs) by regulating their proliferation and differentiation states. In “claudinlow” SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFβ signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion. Enforced expression of mir-93 completely blocks tumor development in mammary fat pads and development of metastases following intracardiac injection in mouse xenografts. The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated “luminal” phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties. These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications. PMID:22685420

  2. Peroxisome Proliferator-Activated Receptor γ Activity is Required for Appropriate Cardiomyocyte Differentiation

    PubMed Central

    Peymani, Maryam; Ghaedi, Kamran; Irani, Shiva; Nasr-Esfahani, Mohammad Hossein

    2016-01-01

    Objective Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the PPAR nuclear receptor superfamily. Although PPARγ acts as a master transcription factor in adipocyte differentiation, it is also associated with a variety of cell functions including carbohydrate and lipid metabolism, glucose homeostasis, cell proliferation and cell differentiation. This study aimed to assess the expression level of PPARγ in order to address its role in cardiac cell differentiation of mouse embryonic stem cells (mESCs). Materials and Methods In this an intervening study, mESCs were subjected to cardiac differentiation. Total RNA was extracted from the cells and quantitative real time polymerase chain reaction (qPCR) was carried out to estimate level of gene expression. Furthermore, the requirement of PPARγ in cardiac differentiation of mESCs, during cardiac progenitor cells (CPCs) formation, was examined by applying the respective agonist and antagonist. Results The obtained data revealed an elevation in the expression level of PPARγ during spontaneous formation of CPCs and cardiomyocytes. Our results indicated that during CPC formation, PPARγ inactivation via treatment with GW9662 (GW) reduced expression of CPC and cardiac markers. Conclusion We conclude that PPARγ modulation has an effective role on cardiac differentiation of mESCs at the early stage of cardiomyogenesis. PMID:27540527

  3. Histamine is required during neural stem cell proliferation to increase neuron differentiation.

    PubMed

    Rodríguez-Martínez, G; Velasco, I; García-López, G; Solís, K H; Flores-Herrera, H; Díaz, N F; Molina-Hernández, A

    2012-08-02

    Histamine in the adult central nervous system (CNS) acts as a neurotransmitter. This amine is one of the first neurotransmitters to appear during development reaching its maximum concentration simultaneously with neuron differentiation peak. This suggests that HA plays an important role in neurogenesis. We have previously shown that HA is able to increase neuronal differentiation of neural stem cells (NSCs) in vitro, by activating the histamine type 1 receptor. However the mechanism(s) by which HA has a neurogenic effect on NSCs has not been explored. Here we explore how HA is able to increase neuron phenotype. Cortex neuroepithelium progenitors were cultured and at passage two treatments with 100 μM HA were given during cell proliferation and differentiation or only during differentiation. Immunocytochemistry was performed on differentiated cultures to detect mature neurons. To explore the expression of certain important transcriptional factors involved on asymmetric cell division and commitment, RT-PCR and qRT-PCR were performed. Results indicate that HA is required during cell proliferation in order to increase neuron differentiation and suggest that this amine increases neuron commitment during the proliferative phase probably by rising prospero1 and neurogenin1 expression.

  4. Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

    SciTech Connect

    Roura-Ferrer, Meritxell; Sole, Laura; Martinez-Marmol, Ramon; Villalonga, Nuria; Felipe, Antonio

    2008-05-16

    Voltage-dependent K{sup +} channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G{sub 1}-phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletal muscle cell proliferation.

  5. Low level light promotes the proliferation and differentiation of bone marrow derived mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Ahn, Jin-Chul; Rhee, Yun-Hee; Choi, Sun-Hyang; Kim, Dae Yu; Chung, Phil-Sang

    2015-03-01

    Low-level light irradiation (LLLI) reported to stimulate the proliferation or differentiation of a variety of cell types. However, very little is known about the effect of light therapy on stem cells. The aim of the present study was to evaluate the effect of LLLI on the molecular physiological change of human bone marrow derived stem cells (hBMSC) by wavelength (470, 630, 660, 740 and 850, 50mW). The laser diode was performed with different time interval (0, 7.5, 15, 30J/cm2, 50mW) on hBMSC. To determine the molecular physiological changes of cellular level of hBMSC, the clonogenic assay, ATP assay, reactive oxygen species (ROS) detection, mitochondria membrane potential (MMPΦ) staining and calcium efflux assay were assessed after irradiation. There was a difference between with and without irradiation on hBMSCs. An energy density up to 30 J/cm² improved the cell proliferation in comparison to the control group. Among these irradiated group, 630 and 660nm were significantly increased the cell proliferation. The cellular level of ATP and calcium influx was increased with energy dose-dependent in all LLLI groups. Meanwhile, ROS and MMPΦ were also increased after irradiation except 470nm. It can be concluded that LLLI using infrared light and an energy density up to 30 J/cm² has a positive stimulatory effect on the proliferation or differentiation of hBMSCs. Our results suggest that LLLI may influence to the mitochondrial membrane potential activity through ATP synthesis and increased cell metabolism which leads to cell proliferation and differentiation.

  6. Gene Expression upon Proliferation and Differentiation of Hematopoietic Cells with Ph Chromosome ex vivo

    PubMed Central

    Grineva, N.I.; Duchovenskay, E.A.; Timofeev, A.M.; Akhlynina, T.V.; Gerasimova, L.P.; Borovkova, T.V.; Schmarov, D.A.; Sarycheva, N.G.; Naydenova, N.M.; Gavrichkova, 
A.R.; Kolosova, L.Y.; Kolosheynova, T.I.; Kovaleva, L.G.

    2012-01-01

    The genesp53, mdm2, p21, c-myc,bcr/abl, bcr, bcl2, bax, and gapdh participate in the regulation of cell proliferation and differentiation, apoptosis and cell distribution for the cell cycle ex vivo in the Ph+cells of chronic myeloid leukemia containing the Ph chromosome andbcr/abloncogene. Expression of these genes correlates with regulation of cell proliferation and differentiation by alternating proliferation and maturation stages for three main Ph+cell types that occur under chronic myeloid leukemia. Thep53, p21, mdm2, and gapdh genes overexpress in active proliferating myeloid cells in the cell cycle S+ G2/M phases and when the phases are coincident with the proliferation stage. Expression of these genes decreases to a considerable level under alternation of the Ph+cell proliferation and maturation stages and whenever the expression is greatly diminished under significant neutrophil accumulation and especially under repeated alternation of the stages. In the course of neutrophil maturation, gene expression levels decrease in the range of gapdh > actin > c-myc, bcr/abl,p21 > p53 > bcl2 > bax.The expression levels of these genes in neutrophils are lower than those in myelocytes and lower by an order of magnitude than that in the cells with a prolonged proliferation stage. TheBcr/ablexpression gene under prolonged maturation and neutrophil accumulation is inhibited; however it is enhanced by 2–3 times for the proliferation stage with myelocyte accumulation. Minimalbcr/ablexpression is observed under overexpression ofp53, mdm2, p21, c-myc,as well as under cell maximum at the S and G2/M phases. Bcr/abloverexpression is observed under low expression of thep53, p21, mdm2genes. In the Ph+ cells with a high P/D efficiency index (5–20), overexpression of the genes in the range ofbcr> gapdh>bcr/abl, as well as a decreased expression of thep53, bcl2, mdm2, p21<< gapdh genes is observed for Ph+cells from the CML blast crisis and CML acceleration phase. Low control of

  7. Effects of nano tantalum implants on inducing osteoblast proliferation and differentiation

    PubMed Central

    Liu, Xinyu; Song, Xiaobin; Zhang, Peng; Zhu, Zhenkun; Xu, Xin

    2016-01-01

    In the present study, we examined the effects of nano tantalum (Ta) dental implants on inducing osteoblast proliferation and differentiation. The MG-63 osteoblasts were divided into 3 groups after recovery, passage and storage: i) Osteoblast culturing group (control group); ii) osteoblast and titanium (Ti) implant co-culturing group (Ti group); and iii) osteoblast and Ta implant co-culturing group (Ta group). After 7 days, a scanning electron microscope was used to observe the growth status, number and morphological changes of the cells on the surfaces of the materials. An MTT assay was used to detect cell proliferation after culturing for 1, 3 and 7 days. ELISA assay was used to detect the levels of alkaline phosphatase (ALP) after 1, 3 and 7 days. Western blot analysis was used to detect the expression levels of collagen type I (Col-1) and osteocalcin after 1, 3 and 7 days. There was significant cell spreading on the surfaces of Ti and of Ta after 7 days, flat and with many pseudopodia. Additionally, there were more cell components in the Ta group. Concurrently, cell proliferation in the Ti and Ta groups increased. There was also an increase in the level of ALP and the expression level of Col-1 over time. The indexes of the Ta group were more apparent than those of the Ti group at each time-point, and the differences were statistically significant (p<0.05). In conclusion, compared with Ti implants, Ta implants induced more osteoblast proliferation and differentiation. PMID:28101149

  8. Lowered Expression of Tumor Suppressor Candidate MYO1C Stimulates Cell Proliferation, Suppresses Cell Adhesion and Activates AKT

    PubMed Central

    Visuttijai, Kittichate; Pettersson, Jennifer; Mehrbani Azar, Yashar; van den Bout, Iman; Örndal, Charlotte; Marcickiewicz, Janusz; Nilsson, Staffan; Hörnquist, Michael; Olsson, Björn; Ejeskär, Katarina

    2016-01-01

    Myosin-1C (MYO1C) is a tumor suppressor candidate located in a region of recurrent losses distal to TP53. Myo1c can tightly and specifically bind to PIP2, the substrate of Phosphoinositide 3-kinase (PI3K), and to Rictor, suggesting a role for MYO1C in the PI3K pathway. This study was designed to examine MYO1C expression status in a panel of well-stratified endometrial carcinomas as well as to assess the biological significance of MYO1C as a tumor suppressor in vitro. We found a significant correlation between the tumor stage and lowered expression of MYO1C in endometrial carcinoma samples. In cell transfection experiments, we found a negative correlation between MYO1C expression and cell proliferation, and MYO1C silencing resulted in diminished cell migration and adhesion. Cells expressing excess of MYO1C had low basal level of phosphorylated protein kinase B (PKB, a.k.a. AKT) and cells with knocked down MYO1C expression showed a quicker phosphorylated AKT (pAKT) response in reaction to serum stimulation. Taken together the present study gives further evidence for tumor suppressor activity of MYO1C and suggests MYO1C mediates its tumor suppressor function through inhibition of PI3K pathway and its involvement in loss of contact inhibition. PMID:27716847

  9. Unsaturated Fatty Acids Drive Disintegrin and Metalloproteinase (ADAM)-dependent Cell Adhesion, Proliferation, and Migration by Modulating Membrane Fluidity*

    PubMed Central

    Reiss, Karina; Cornelsen, Isabell; Husmann, Matthias; Gimpl, Gerald; Bhakdi, Sucharit

    2011-01-01

    The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine changes in enzyme activity, but correlate with changes in membrane fluidity as revealed by measurement of 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and fluorescence recovery after photobleaching analyses. ELISA and immunoblot experiments conducted with granulocytes, endothelial cells, and keratinocytes revealed rapid increase of ectodomain shedding of ADAM10 and ADAM17 substrates upon membrane fluidization. Large amounts of unsaturated FFA may be liberated from cholesteryl esters in LDL that is entrapped in atherosclerotic lesions. Incubation of cells with thus modified LDL resulted in rapid cleavage of ADAM substrates with corresponding functional consequences on cell proliferation, cell migration, and endothelial permeability, events of high significance in atherogenesis. We propose that FFA represent critical regulators of ADAM function that may assume relevance in many biological settings through their influence on mobility of enzyme and substrate in lipid bilayers. PMID:21642425

  10. Unsaturated fatty acids drive disintegrin and metalloproteinase (ADAM)-dependent cell adhesion, proliferation, and migration by modulating membrane fluidity.

    PubMed

    Reiss, Karina; Cornelsen, Isabell; Husmann, Matthias; Gimpl, Gerald; Bhakdi, Sucharit

    2011-07-29

    The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine changes in enzyme activity, but correlate with changes in membrane fluidity as revealed by measurement of 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and fluorescence recovery after photobleaching analyses. ELISA and immunoblot experiments conducted with granulocytes, endothelial cells, and keratinocytes revealed rapid increase of ectodomain shedding of ADAM10 and ADAM17 substrates upon membrane fluidization. Large amounts of unsaturated FFA may be liberated from cholesteryl esters in LDL that is entrapped in atherosclerotic lesions. Incubation of cells with thus modified LDL resulted in rapid cleavage of ADAM substrates with corresponding functional consequences on cell proliferation, cell migration, and endothelial permeability, events of high significance in atherogenesis. We propose that FFA represent critical regulators of ADAM function that may assume relevance in many biological settings through their influence on mobility of enzyme and substrate in lipid bilayers.

  11. Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation.

    PubMed

    Tan, Si Hui; Senarath-Yapa, Kshemendra; Chung, Michael T; Longaker, Michael T; Wu, Joy Y; Nusse, Roeland

    2014-12-09

    Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated--to our knowledge for the first time--that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.

  12. Distinct human NUMB isoforms regulate differentiation vs. proliferation in the neuronal lineage.

    PubMed

    Verdi, J M; Bashirullah, A; Goldhawk, D E; Kubu, C J; Jamali, M; Meakin, S O; Lipshitz, H D

    1999-08-31

    Neuronal cell fate decisions are directed in Drosophila by NUMB, a signaling adapter protein with two protein-protein interaction domains: a phosphotyrosine-binding domain and a proline-rich region (PRR) that functions as an SH3-binding domain. Here we show that there are at least four human NUMB isoforms and that these serve two distinct developmental functions in the neuronal lineage: differentiation (but not proliferation) is promoted by human NUMB protein isoforms with a type I (short) PRR. In contrast, proliferation (but not differentiation) is directed by isoforms that have a type II (long) PRR. The two types of PRR may promote distinct intracellular signaling pathways downstream of the NOTCH receptor during mammalian neurogenesis.

  13. An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians

    PubMed Central

    Forsthoefel, David J.; James, Noelle P.; Escobar, David J.; Stary, Joel M.; Vieira, Ana P.; Waters, Forrest A.; Newmark, Phillip A.

    2012-01-01

    SUMMARY Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of post-mitotic tissues. Understanding how these processes are orchestrated requires characterizing cell type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis, and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling post-embryonic organogenesis. PMID:23079596

  14. Fbxw7β, E3 ubiquitin ligase, negative regulation of primary myoblast differentiation, proliferation and migration.

    PubMed

    Shin, Kyungshin; Hwang, Sang-Gu; Choi, Ik Joon; Ko, Young-Gyu; Jeong, Jaemin; Kwon, Heechung

    2017-04-01

    Satellite cells attached to skeletal muscle fibers play a crucial role in skeletal muscle regeneration. During regeneration, the satellite cells proliferate, migrate to the damaged region, and fuse to each other. Although it is important to determine the cellular mechanisms controlling myoblast behavior, their regulators are not well understood. In this study, we evaluated the roles of Fbxw7 in primary myoblasts and determined its potential as a therapeutic target for muscle disease. We originally found that Fbxw7β, one of the E3 ubiquitin ligase Fbxw7 subtypes, negatively regulates differentiation, proliferation and migration of myoblasts and satellite cells on muscle fiber. However, these phenomena were not observed in myoblasts expressing a dominant-negative, F-box deleted Fbxw7β, mutant. Our results suggest that myoblast differentiation potential and muscle regeneration can be regulated by Fbxw7β.

  15. An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians.

    PubMed

    Forsthoefel, David J; James, Noëlle P; Escobar, David J; Stary, Joel M; Vieira, Ana P; Waters, Forrest A; Newmark, Phillip A

    2012-10-16

    Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of postmitotic tissues. Understanding how these processes are orchestrated requires characterizing cell-type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling postembryonic organogenesis.

  16. Distinct human NUMB isoforms regulate differentiation vs. proliferation in the neuronal lineage

    PubMed Central

    Verdi, Joseph M.; Bashirullah, Arash; Goldhawk, Donna E.; Kubu, Chris J.; Jamali, Mina; Meakin, Susan O.; Lipshitz, Howard D.

    1999-01-01

    Neuronal cell fate decisions are directed in Drosophila by NUMB, a signaling adapter protein with two protein–protein interaction domains: a phosphotyrosine-binding domain and a proline-rich region (PRR) that functions as an SH3-binding domain. Here we show that there are at least four human NUMB isoforms and that these serve two distinct developmental functions in the neuronal lineage: differentiation (but not proliferation) is promoted by human NUMB protein isoforms with a type I (short) PRR. In contrast, proliferation (but not differentiation) is directed by isoforms that have a type II (long) PRR. The two types of PRR may promote distinct intracellular signaling pathways downstream of the NOTCH receptor during mammalian neurogenesis. PMID:10468633

  17. Functional cross-talk between the cellular prion protein and the neural cell adhesion molecule is critical for neuronal differentiation of neural stem/precursor cells.

    PubMed

    Prodromidou, Kanella; Papastefanaki, Florentia; Sklaviadis, Theodoros; Matsas, Rebecca

    2014-06-01

    Cellular prion protein (PrP) is prominently expressed in brain, in differentiated neurons but also in neural stem/precursor cells (NPCs). The misfolding of PrP is a central event in prion diseases, yet the physiological function of PrP is insufficiently understood. Although PrP has been reported to associate with the neural cell adhesion molecule (NCAM), the consequences of concerted PrP-NCAM action in NPC physiology are unknown. Here, we generated NPCs from the subventricular zone (SVZ) of postnatal day 5 wild-type and PrP null (-/-) mice and observed that PrP is essential for proper NPC proliferation and neuronal differentiation. Moreover, we found that PrP is required for the NPC response to NCAM-induced neuronal differentiation. In the absence of PrP, NCAM not only fails to promote neuronal differentiation but also induces an accumulation of doublecortin-positive neuronal progenitors at the proliferation stage. In agreement, we noted an increase in cycling neuronal progenitors in the SVZ of PrP-/- mice compared with PrP+/+ mice, as evidenced by double labeling for the proliferation marker Ki67 and doublecortin as well as by 5-bromo-2'-deoxyuridine incorporation experiments. Additionally, fewer newly born neurons were detected in the rostral migratory stream of PrP-/- mice. Analysis of the migration of SVZ cells in microexplant cultures from wild-type and PrP-/- mice revealed no differences between genotypes or a role for NCAM in this process. Our data demonstrate that PrP plays a critical role in neuronal differentiation of NPCs and suggest that this function is, at least in part, NCAM-dependent.

  18. The antimicrobial protein REG3A regulates keratinocyte proliferation and differentiation after skin injury.

    PubMed

    Lai, Yuping; Li, Dongqing; Li, Changwei; Muehleisen, Beda; Radek, Katherine A; Park, Hyun Jeong; Jiang, Ziwei; Li, Zhiheng; Lei, Hu; Quan, Yanchun; Zhang, Tian; Wu, Yelin; Kotol, Paul; Morizane, Shin; Hata, Tissa R; Iwatsuki, Keiji; Tang, Ce; Gallo, Richard L

    2012-07-27

    Epithelial keratinocyte proliferation is an essential element of wound repair, and abnormal epithelial proliferation is an intrinsic element in the skin disorder psoriasis. The factors that trigger epithelial proliferation in these inflammatory processes are incompletely understood. Here we have shown that regenerating islet-derived protein 3-alpha (REG3A) is highly expressed in keratinocytes during psoriasis and wound repair and in imiquimod-induced psoriatic skin lesions. The expression of REG3A by keratinocytes is induced by interleukin-17 (IL-17) via activation of keratinocyte-encoded IL-17 receptor A (IL-17RA) and feeds back on keratinocytes to inhibit terminal differentiation and increase cell proliferation by binding to exostosin-like 3 (EXTL3) followed by activation of phosphatidylinositol 3 kinase (PI3K) and the kinase AKT. These findings reveal that REG3A, a secreted intestinal antimicrobial protein, can promote skin keratinocyte proliferation and can be induced by IL-17. This observation suggests that REG3A may mediate the epidermal hyperproliferation observed in normal wound repair and in psoriasis.

  19. Differential adhesion between moving particles as a mechanism for the evolution of social groups.

    PubMed

    Garcia, Thomas; Brunnet, Leonardo Gregory; De Monte, Silvia

    2014-02-01

    The evolutionary stability of cooperative traits, that are beneficial to other individuals but costly to their carrier, is considered possible only through the establishment of a sufficient degree of assortment between cooperators. Chimeric microbial populations, characterized by simple interactions between unrelated individuals, restrain the applicability of standard mechanisms generating such assortment, in particular when cells disperse between successive reproductive events such as happens in Dicyostelids and Myxobacteria. In this paper, we address the evolutionary dynamics of a costly trait that enhances attachment to others as well as group cohesion. By modeling cells as self-propelled particles moving on a plane according to local interaction forces and undergoing cycles of aggregation, reproduction and dispersal, we show that blind differential adhesion provides a basis for assortment in the process of group formation. When reproductive performance depends on the social context of players, evolution by natural selection can lead to the success of the social trait, and to the concomitant emergence of sizeable groups. We point out the conditions on the microscopic properties of motion and interaction that make such evolutionary outcome possible, stressing that the advent of sociality by differential adhesion is restricted to specific ecological contexts. Moreover, we show that the aggregation process naturally implies the existence of non-aggregated particles, and highlight their crucial evolutionary role despite being largely neglected in theoretical models for the evolution of sociality.

  20. Synthesis of nanostructured porous silica coatings on titanium and their cell adhesive and osteogenic differentiation properties.

    PubMed

    Inzunza, Débora; Covarrubias, Cristian; Von Marttens, Alfredo; Leighton, Yerko; Carvajal, Juan Carlos; Valenzuela, Francisco; Díaz-Dosque, Mario; Méndez, Nicolás; Martínez, Constanza; Pino, Ana María; Rodríguez, Juan Pablo; Cáceres, Mónica; Smith, Patricio

    2014-01-01

    Nanostructured porous silica coatings were synthesized on titanium by the combined sol-gel and evaporation-induced self-assembly process. The silica-coating structures were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and nitrogen sorptometry. The effect of the nanoporous surface on apatite formation in simulated body fluid, protein adsorption, osteoblast cell adhesion behavior, and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) is reported. Silica coatings with highly ordered sub-10 nm porosity accelerate early osteoblast adhesive response, a favorable cell response that is attributed to an indirect effect due to the high protein adsorption observed on the large-specific surface area of the nanoporous coating but is also probably due to direct mechanical stimulus from the nanostructured topography. The nanoporous silica coatings, particularly those doped with calcium and phosphate, also promote the osteogenic differentiation of hBMSCs with spontaneous mineral nodule formation in basal conditions. The bioactive surface properties exhibited by the nanostructured porous silica coatings make these materials a promising alternative to improve the osseointegration properties of titanium dental implants and could have future impact on the nanoscale design of implant surfaces.

  1. Notch signaling is required for normal prostatic epithelial cell proliferation and differentiation.

    PubMed

    Wang, Xi-De; Leow, Ching Ching; Zha, Jiping; Tang, Zhijun; Modrusan, Zora; Radtke, Freddy; Aguet, Michel; de Sauvage, Frederic J; Gao, Wei-Qiang

    2006-02-01

    Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. Using a transgenic cell ablation approach, we found in our previous study that cells expressing Notch1 are crucial for prostate early development and re-growth. Here, we further define the role of Notch signaling in regulating prostatic epithelial cell growth and differentiation using biochemical and genetic approaches in ex vivo or in vivo systems. Treatment of developing prostate grown in culture with inhibitors of gamma-secretase/presenilin, which is required for Notch cleavage and activation, caused a robust increase in proliferation of epithelial cells co-expressing cytokeratin 8 and 14, lack of luminal/basal layer segregation and dramatically reduced branching morphogenesis. Using conditional Notch1 gene deletion mouse models, we found that inactivation of Notch1 signaling resulted in profound prostatic alterations, including increased tufting, bridging and enhanced epithelial proliferation. Cells within these lesions co-expressed both luminal and basal cell markers, a feature of prostatic epithelial cells in predifferentiation developmental stages. Microarray analysis revealed that the gene expression in a number of genetic networks was altered following Notch1 gene deletion in prostate. Furthermore, expression of Notch1 and its effector Hey-1 gene in human prostate adenocarcinomas were found significantly down-regulated compared to normal control tissues. Taken together, these data suggest that Notch signaling is critical for normal cell proliferation and differentiation in the prostate, and deregulation of this pathway may facilitate prostatic tumorigenesis.

  2. Lithium doped calcium phosphate cement maintains physical mechanical properties and promotes osteoblast proliferation and differentiation.

    PubMed

    Li, Li; Wang, Renchong; Li, Baichuan; Liang, Wei; Pan, Haobo; Cui, Xu; Tang, Jingli; Li, Bing

    2016-02-09

    Calcium phosphate cement (CPC) has been widely used in bone tissue repairing due to its physical mechanical properties and biocompatibility. Addition of trace element to CPC has shown promising evidence to improve the physical properties and biological activities of CPC. Lithium (Li) has effect on osteoblast proliferation and differentiation. In this study, we incorporated Li to CPC and examined the physical properties of Li/CPC and its effect on osteoblast proliferation and differentiation. We found that Li doped CPC maintained similar setting time, pore size distribution, compressive strength, composition, and morphology as CPC without Li. Additionally, Li doped CPC improved osteoblast proliferation and differentiation significantly compared to CPC without Li. To our knowledge, our results, for the first time, show that Li doped CPC has beneficial effect on osteoblast in cell culture while keeps the excellent physical-mechanical properties of CPC. This study will lead to potential application of Li doped CPC in bone tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

  3. Proliferation and Differentiation Potential of Human Adipose-Derived Stem Cells Grown on Chitosan Hydrogel

    PubMed Central

    Debnath, Tanya; Ghosh, Sutapa; Potlapuvu, Usha Shalini; Kona, Lakshmi; Kamaraju, Suguna Ratnakar; Sarkar, Suprabhat; Gaddam, Sumanlatha; Chelluri, Lakshmi Kiran

    2015-01-01

    Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate. PMID:25746846

  4. JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation

    PubMed Central

    Besancenot, Rodolphe; Roos-Weil, Damien; Tonetti, Carole; Abdelouahab, Hadjer; Lacout, Catherine; Pasquier, Florence; Willekens, Christophe; Rameau, Philippe; Lecluse, Yann; Micol, Jean-Baptiste; Constantinescu, Stefan N.; Vainchenker, William; Solary, Eric

    2014-01-01

    Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses. PMID:25143485

  5. Periodontitis promotes the proliferation and suppresses the differentiation potential of human periodontal ligament stem cells.

    PubMed

    Zheng, Wei; Wang, Shi; Wang, Jianguo; Jin, Fang

    2015-10-01

    The aim of the present study was to investigate the periodontitis-associated changes in the number, proliferation and differentiation potential of human periodontal ligament stem cells (PDLSCs). Cultures of human periodontal ligament cells (PDLCs) were established from healthy donors and donors with periodontitis. The numbers of stem cell were characterized using flow cytometry. PDLSCs were isolated from the PDLCs by immunomagnetic bead selection. Colony‑forming abilities, osteogenic and adipogenic potential, gene expression of cementoblast phenotype, alkaline phosphatase activity and in vivo differentiation capacities were then evaluated. Periodontitis caused an increase in the proliferation of PDLSCs and a decrease in the commitment to the osteoblast lineage. This is reflected by changes in the expression of osteoblast markers. When transplanted into immunocompromised mice, PDLSCs from the healthy donors exhibited the capacity to produce cementum PDL‑like structures, whereas, the inflammatory PDLSCs transplants predominantly formed connective tissues. In conclusion, the data from the present study suggest that periodontitis affects the proliferation and differentiation potential of human PDLSCs in vitro and in vivo.

  6. VEGF-mediated angiogenesis stimulates neural stem cell proliferation and differentiation in the premature brain

    SciTech Connect

    Sun, Jinqiao; Sha, Bin; Zhou, Wenhao; Yang, Yi

    2010-03-26

    This study investigated the effects of angiogenesis on the proliferation and differentiation of neural stem cells in the premature brain. We observed the changes in neurogenesis that followed the stimulation and inhibition of angiogenesis by altering vascular endothelial growth factor (VEGF) expression in a 3-day-old rat model. VEGF expression was overexpressed by adenovirus transfection and down-regulated by siRNA interference. Using immunofluorescence assays, Western blot analysis, and real-time PCR methods, we observed angiogenesis and the proliferation and differentiation of neural stem cells. Immunofluorescence assays showed that the number of vWF-positive areas peaked at day 7, and they were highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at every time point. The number of neural stem cells, neurons, astrocytes, and oligodendrocytes in the subventricular zone gradually increased over time in the VEGF up-regulation group. Among the three groups, the number of these cells was highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at the same time point. Western blot analysis and real-time PCR confirmed these results. These data suggest that angiogenesis may stimulate the proliferation of neural stem cells and differentiation into neurons, astrocytes, and oligodendrocytes in the premature brain.

  7. Epithelial Xbp1 Is Required for Cellular Proliferation and Differentiation during Mammary Gland Development

    PubMed Central

    Hasegawa, Daisuke; Calvo, Veronica; Avivar-Valderas, Alvaro; Lade, Abigale; Chou, Hsin-I; Lee, Youngmin A.; Farias, Eduardo F.; Aguirre-Ghiso, Julio A.

    2015-01-01

    Xbp1, a key mediator of the unfolded protein response (UPR), is activated by IRE1α-mediated splicing, which results in a frameshift to encode a protein with transcriptional activity. However, the direct function of Xbp1 in epithelial cells during mammary gland development is unknown. Here we report that the loss of Xbp1 in the mammary epithelium through targeted deletion leads to poor branching morphogenesis, impaired terminal end bud formation, and spontaneous stromal fibrosis during the adult virgin period. Additionally, epithelial Xbp1 deletion induces endoplasmic reticulum (ER) stress in the epithelium and dramatically inhibits epithelial proliferation and differentiation during lactation. The synthesis of milk and its major components, α/β-casein and whey acidic protein (WAP), is significantly reduced due to decreased prolactin receptor (Prlr) and ErbB4 expression in Xbp1-deficient mammary epithelium. Reduction of Prlr and ErbB4 expression and their diminished availability at the cell surface lead to reduced phosphorylated Stat5, an essential regulator of cell proliferation and differentiation during lactation. As a result, lactating mammary glands in these mice produce less milk protein, leading to poor pup growth and postnatal death. These findings suggest that the loss of Xbp1 induces a terminal UPR which blocks proliferation and differentiation during mammary gland development. PMID:25713103

  8. Vascular Cell Adhesion Molecule 1, Intercellular Adhesion Molecule 1, and Cluster of Differentiation 146 Levels in Patients with Type 2 Diabetes with Complications

    PubMed Central

    Saribal, Devrim; Yenmis, Guven; Guvenen, Guvenc

    2017-01-01

    Background Type 2 diabetes mellitus (T2DM) is a multisystemic, chronic disease accompanied by microvascular complications involving various complicated mechanisms. Intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and cluster of differentiation-146 (CD146) are mainly expressed by endothelial cells, and facilitate the adhesion and transmigration of immune cells, leading to inflammation. In the present study, we evaluated the levels of soluble adhesion molecules in patients with microvascular complications of T2DM. Methods Serum and whole blood samples were collected from 58 T2DM patients with microvascular complications and 20 age-matched healthy subjects. Levels of soluble ICAM-1 (sICAM-1) and soluble VCAM-1 (sVCAM-1) were assessed using enzyme-linked immunosorbent assay, while flow cytometry was used to determine CD146 levels. Results Serum sICAM-1 levels were lower in T2DM patients with microvascular complications than in healthy controls (P<0.05). No significant differences were found in sVCAM-1 and CD146 levels between the study and the control group. Although patients were subdivided into groups according to the type of microvascular complications that they experienced, cell adhesion molecule levels were not correlated with the complication type. Conclusion In the study group, most of the patients were on insulin therapy (76%), and 95% of them were receiving angiotensin-converting enzyme (ACE)-inhibitor agents. Insulin and ACE-inhibitors have been shown to decrease soluble adhesion molecule levels via various mechanisms, so we suggest that the decreased or unchanged levels of soluble forms of cellular adhesion molecules in our study group may have resulted from insulin and ACE-inhibitor therapy, as well as tissue-localized inflammation in patients with T2DM. PMID:28345319

  9. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration

    SciTech Connect

    Zhang, Lei; Wang, Huaxi; Yang, Yan; Liu, Hui; Zhang, Qihao; Xiang, Qi; Ge, Renshan; Su, Zhijian; Huang, Yadong

    2013-06-28

    Highlights: •Nerve growth factor has shown significant changes on mRNA levels during Adult Leydig cells regeneration. •We established the organ culture model of rat seminiferous tubules with ethane dimethyl sulphonate (EDS) treatment. •Nerve growth factor has shown proliferation and differentiation-promoting effects on Adult stem Leydig cells. •Nerve growth factor induces progenitor Leydig cells to proliferate and differentiate and immature Leydig cells to proliferate. -- Abstract: Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful

  10. Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells

    PubMed Central

    Murtaugh, Michael P.

    2017-01-01

    Immunological prevention of infectious disease, especially viral, is based on antigen-specific long-lived memory B cells. To test for cellular proliferation and differentiation factors in swine, an outbred model for humans, CD21+ B cells were activated in vitro with CD40L and stimulated with purported stimulatory cytokines to characterize functional responses. IL-21 induced a 3-fold expansion in total cell numbers with roughly 15% of all B cells differentiating to IgM or IgG antibody secreting cells (ASCs.) However, even with robust proliferation, cellular viability rapidly deteriorated. Therefore, a proliferation inducing ligand (APRIL) and B cell activating factor (BAFF) were evaluated as survival and maintenance factors. BAFF was effective at enhancing the viability of mature B cells as well as ASCs, while APRIL was only effective for ASCs. Both cytokines increased approximately two-fold the amount of IgM and IgG which was secreted by IL-21 differentiated ASCs. Mature B cells from porcine reproductive and respiratory virus (PRRSV) immune and naïve age-matched pigs were activated and treated with IL-21 and then tested for memory cell differentiation using a PRRSV non-structural protein 7 ELISPOT and ELISA. PRRSV immune pigs were positive on both ELISPOT and ELISA while naïve animals were negative on both assays. These results highlight the IL-21-driven expansion and differentiation of memory B cells in vitro without stimulation of the surface immunoglobulin receptor complex, as well as the establishment of a defined memory B cell culture system for characterization of vaccine responses in outbred animals. PMID:28125737

  11. Strategies to prepare TiO2 thin films, doped with transition metal ions, that exhibit specific physicochemical properties to support osteoblast cell adhesion and proliferation.

    PubMed

    Dhayal, Marshal; Kapoor, Renu; Sistla, Pavana Goury; Pandey, Ravi Ranjan; Kar, Satabisha; Saini, Krishan Kumar; Pande, Gopal

    2014-04-01

    Metal ion doped titanium oxide (TiO2) thin films, as bioactive coatings on metal or other implantable materials, can be used as surfaces for studying the cell biological properties of osteogenic and other cell types. Bulk crystallite phase distribution and surface carbon-oxygen constitution of thin films, play an important role in determining the biological responses of cells that come in their contact. Here we present a strategy to control the polarity of atomic interactions between the dopant metal and TiO2 molecules and obtain surfaces with smaller crystallite phases and optimal surface carbon-oxygen composition to support the maximum proliferation and adhesion of osteoblast cells. Our results suggest that surfaces, in which atomic interactions between the dopant metals and TiO2 were less polar, could support better adhesion, spreading and proliferation of cells.

  12. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds.

    PubMed

    Favi, Pelagie M; Benson, Roberto S; Neilsen, Nancy R; Hammonds, Ryan L; Bates, Cassandra C; Stephens, Christopher P; Dhar, Madhu S

    2013-05-01

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications.

  13. Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro.

    PubMed

    Ben-Dov, N; Shefer, G; Irintchev, A; Wernig, A; Oron, U; Halevy, O; Irinitchev, A

    1999-01-11

    Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rat's age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.

  14. Effects of celecoxib on proliferation and tenocytic differentiation of tendon-derived stem cells

    SciTech Connect

    Zhang, Kairui; Zhang, Sheng; Li, Qianqian; Yang, Jun; Dong, Weiqiang; Wang, Shengnan; Cheng, Yirong; Al-Qwbani, Mohammed; Wang, Qiang; Yu, Bin

    2014-07-18

    Highlights: • Celecoxib has no effects on TDSCs cell proliferation in various concentrations. • Celecoxib reduced mRNAs levels of tendon associated transcription factor. • Celecoxib reduced mRNAs levels of main tendon associated collagen. • Celecoxib reduced mRNAs levels of tendon associated molecules. - Abstract: NSAIDs are often ingested to reduce the pain and improve regeneration of tendon after tendon injury. Although the effects of NSAIDs in tendon healing have been reported, the data and conclusions are not consistent. Recently, tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and has been suggested involved in tendon repair. Our study aims to determine the effects of COX-2 inhibitor (celecoxib) on the proliferation and tenocytic differentiation of TDSCs. TDSCs were isolated from mice Achilles tendon and exposed to celecoxib. Cell proliferation rate was investigated at various concentrations (0.1, 1, 10 and 100 μg/ml) of celecoxib by using hemocytometer. The mRNA expression of tendon associated transcription factors, tendon associated collagens and tendon associated molecules were determined by reverse transcription-polymerase chain reaction. The protein expression of Collagen I, Collagen III, Scleraxis and Tenomodulin were determined by Western blotting. The results showed that celecoxib has no effects on TDSCs cell proliferation in various concentrations (p > 0.05). The levels of most tendon associated transcription factors, tendon associated collagens and tendon associated molecules genes expression were significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). Collagen I, Collagen III, Scleraxis and Tenomodulin protein expression were also significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). In conclusion, celecoxib inhibits tenocytic differentiation of tendon-derived stem cells but has no effects on cell proliferation.

  15. Measurement of generation-dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation.

    PubMed

    Akbarian, Vahe; Wang, Weijia; Audet, Julie

    2012-05-01

    Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture.

  16. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes

    SciTech Connect

    Li, Lei; Yang, Zheng; Zhang, Hai; Chen, Wenchuan; Chen, Mengshi; Zhu, Zhimin

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes inhibits proliferation of osteoblasts. Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes enhances differentiation of osteoblasts. Black-Right-Pointing-Pointer LIPUS stimulates MLO-Y4 cells to secrete PGE{sub 2} and NO. -- Abstract: Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE{sub 2} and NO assay showed that LIPUS could enhance PGE{sub 2} and NO secretion from MLO-Y4 cells at all time points within 24 h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE{sub 2} from osteocytes may play a role in this effect.

  17. α7 Nicotinic Acetylcholine Receptor Regulates Airway Epithelium Differentiation by Controlling Basal Cell Proliferation

    PubMed Central

    Maouche, Kamel; Polette, Myriam; Jolly, Thomas; Medjber, Kahina; Cloëz-Tayarani, Isabelle; Changeux, Jean-Pierre; Burlet, Henriette; Terryn, Christine; Coraux, Christelle; Zahm, Jean-Marie; Birembaut, Philippe; Tournier, Jean-Marie

    2009-01-01

    Airway epithelial basal cells are known to be critical for regenerating injured epithelium and maintaining tissue homeostasis. Recent evidence suggests that the α7 nicotinic acetylcholine receptor (nAChR), which is highly permeable to Ca2+, is involved in lung morphogenesis. Here, we have investigated the potential role of the α7 nAChR in the regulation of airway epithelial basal cell proliferation and the differentiation of the human airway epithelium. In vivo during fetal development and in vitro during the regeneration of the human airway epithelium, α7 nAChR expression coincides with epithelium differentiation. Inactivating α7 nAChR function in vitro increases cell proliferation during the initial steps of the epithelium regeneration, leading to epithelial alterations such as basal cell hyperplasia and squamous metaplasia, remodeling observed in many bronchopulmonary diseases. The regeneration of the airway epithelium after injury in α7−/− mice is delayed and characterized by a transient hyperplasia of basal cells. Moreover, 1-year-old α7−/− mice more frequently present basal cells hyperplasia. Modulating nAChR function or expression shows that only α7 nAChR, as opposed to heteropentameric αxβy nAChRs, controls the proliferation of human airway epithelial basal cells. These findings suggest that α7 nAChR is a key regulator of the plasticity of the human airway epithelium by controlling basal cell proliferation and differentiation pathway and is involved in airway remodeling during bronchopulmonary diseases. PMID:19808646

  18. Hepatocyte growth factor plays a dual role in regulating skeletal muscle satellite cell proliferation and differentiation.

    PubMed

    Gal-Levi, R; Leshem, Y; Aoki, S; Nakamura, T; Halevy, O

    1998-03-12

    The role of hepatocyte growth factor (HGF) and its receptor, c-met, in proliferation and differentiation of satellite cells was studied in primary cultures of chicken skeletal muscle satellite cells and a myogenic C2 cell line. HGF mRNA was expressed mainly in the myotubes of both cultures. The addition of conditioned medium derived from those cultures had a scattering effect on the canine kidney epithelial cell line, MDCK. In contrast, c-met mRNA levels decreased during cell differentiation of C2 and primary satellite cells. Application of exogenous HGF to chicken myoblasts resulted in their enhanced DNA synthesis. Among several growth factors, HGF was the first to induce DNA synthesis in quiescent satellite cells, thereby driving them into the cell cycle. Ectopic expression of chicken HGF in primary satellite cells suppressed the activation of muscle-regulatory gene reporter constructs MCK-CAT, MRF4-CAT, MEF2-CAT and 4Rtk-CAT, as well as the gene expression of MyoD and myogenin, and MHC protein expression. Ectopic MyoD reversed HGF's inhibitory effect on MCK transactivation. These data suggest that HGF inhibits cell differentiation by inhibiting the activity of basic helix-loop-helix (bHLH)/E protein heterodimers, thus inhibiting myogenic determination factor activity and subsequent muscle-specific protein expression. During muscle growth and regeneration, HGF plays a dual role in satellite-cell myogenesis, affecting both the proliferation and differentiation of these cells in a paracrine fashion.

  19. EGFR signaling regulates cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis.

    PubMed

    Fraguas, Susanna; Barberán, Sara; Cebrià, Francesc

    2011-06-01

    Similarly to development, the process of regeneration requires that cells accurately sense and respond to their external environment. Thus, intrinsic cues must be integrated with signals from the surrounding environment to ensure appropriate temporal and spatial regulation of tissue regeneration. Identifying the signaling pathways that control these events will not only provide insights into a fascinating biological phenomenon but may also yield new molecular targets for use in regenerative medicine. Among classical models to study regeneration, freshwater planarians represent an attractive system in which to investigate the signals that regulate cell proliferation and differentiation, as well as the proper patterning of the structures being regenerated. Recent studies in planarians have begun to define the role of conserved signaling pathways during regeneration. Here, we extend these analyses to the epidermal growth factor (EGF) receptor pathway. We report the characterization of three epidermal growth factor (EGF) receptors in the planarian Schmidtea mediterranea. Silencing of these genes by RNA interference (RNAi) yielded multiple defects in intact and regenerating planarians. Smed-egfr-1(RNAi) resulted in decreased differentiation of eye pigment cells, abnormal pharynx regeneration and maintenance, and the development of dorsal outgrowths. In contrast, Smed-egfr-3(RNAi) animals produced smaller blastemas associated with abnormal differentiation of certain cell types. Our results suggest important roles for the EGFR signaling in controlling cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis.

  20. Proliferation and differentiation of brown adipocytes from interstitial cells during cold acclimation

    SciTech Connect

    Bukowiecki, L.J.; Geloeen, A.; Collet, A.J.

    1986-06-01

    The mechanisms of brown adipocyte proliferation and differentiation during cold acclimation (and/or adaptation to hyperphagia) have been studied by quantitative photonic radioautography. (/sup 3/H)thymidine was injected to warm-acclimated (25/sup 0/C) rats and to animals exposed to 5/sup 0/C for 2 days. Samples of interscapular brown adipose tissue were collected for quantitative analysis of mitotic frequencies at various periods of time (4 h-15 days) after the injection of (/sup 3/H)thymidine, the rats being maintained at the temperatures to which they were initially exposed. It was found that cold exposure for 2 days markedly enhanced mitotic activity in endothelial cells, interstitial cells, and brown preadipocytes rather than in fully differentiated brown adipocytes. The total tissue labeling index (percent of labeled nuclei) increased approx.70 times over control values. The authors now report that cellular labeling progressively increased in mature brown adipocytes during cold acclimation, whereas it correspondingly decreased in interstitial cells and brown preadipocytes. This indicates that the sequence of events for cellular differentiation is interstitial cells ..-->.. brown preadipocytes ..-->.. mature brown adipocytes. Remarkable, labeling frequency did not change in endothelial cells during cold acclimation demonstrating that these cells cannot be considered as progenitors of brown adipocytes. It is suggested that brown adipocyte proliferation and differentiation from interstitial cells represent the fundamental phenomena explaining the enhanced capacity of cold-acclimated and/or hyperphagic rats to respond calorigenically to catecholamines.

  1. Oxidized Phospholipid Species Promote in Vivo Differential Cx43 Phosphorylation and Vascular Smooth Muscle Cell Proliferation

    PubMed Central

    Johnstone, Scott R.; Ross, Jeremy; Rizzo, Michael J.; Straub, Adam C.; Lampe, Paul D.; Leitinger, Norbert; Isakson, Brant E.

    2009-01-01

    Regulation of both the expression and function of connexins in the vascular wall is important during atherosclerosis. Progression of the disease state is marked by vascular smooth muscle cell (VSMC) proliferation, which coincides with the reduced expression levels of connexin 43 (Cx43). However, nothing is currently known about the factors that regulate post-translational modifications of Cx43 in atherogenesis, which could be of particular importance, due to the association between site-specific Cx43 phosphorylation and cellular proliferation. We compared the effects of direct carotid applications of two oxidized phospholipid derivatives, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), on Cx43 expression and phosphorylation, and on cell proliferation. Since both POVPC and PGPC have been shown to act through different intracellular pathways, we hypothesized that each oxidized phospholipid species could induce differential Cx43 phosphorylation events in the cytoplasmically located carboxyl-terminal region of the protein, which could potentially enhance cell proliferation. Application of POVPC caused a reduction in VSMC Cx43 levels, enhanced its phosphorylation at serine (pS) 279/282, and increased VSMC proliferation both in vivo and in vitro. Treatment with PGPC enhanced VSMC pS368 levels with no associated change in proliferation. These oxidized phospholipid-induced Cx43 post-translational changes in VSMCs were consistent with those identified in ApoE−/− mice. Taken together, these results demonstrate that post-translational phosphorylation of Cx43 could be a key factor in the pathogenesis of atherosclerosis. PMID:19608875

  2. Carbonated apatites obtained by the hydrolysis of monetite: influence of carbonate content on adhesion and proliferation of MC3T3-E1 osteoblastic cells.

    PubMed

    Pieters, Ilse Y; Van den Vreken, Natasja M F; Declercq, Heidi A; Cornelissen, Maria J; Verbeeck, Ronald M H

    2010-04-01

    The influence of the carbonate content in apatites on the adhesion and the proliferation of MC3T3-E1 osteoblastic cells was investigated. B-type carbonated apatites (DCAps) were prepared by the hydrolysis of monetite (CaHPO(4), DCP) in solutions with a carbonate concentration ranging from 0.001 to 0.075 mol l(-1). Stoichiometric hydroxyapatite (DCAp0) was synthesized in carbonate-free solution. MC3T3-E1 cells were seeded on the compacted DCAps and cell adhesion and proliferation were analysed after 24h and 7 days, respectively, using a MTS assay and fluorescence microscopy. Cell adhesion tends to increase with increasing carbonate content for carbonate contents between 0 and 6.9 wt.% and levels out to an acceptable value (+ or - 50% compared to the control) for carbonate contents between 6.9 and 16.1 wt.%. Only DCAps with a carbonate content equal to or higher than 11% support high cell proliferation comparable to the control. On the latter DCAps, the cells have a spread morphology and form a near-confluent layer. A decrease in charge density and crystallinity at the apatite surface, as well as the formation of more spheroidal crystals with increasing carbonate content, might attribute to changes in composition and three-dimensional structure of the protein adsorption layer and hence to the observed cell behaviour. Consequently, only DCAps with a high carbonate content, mimicking early in vivo mineralization, are possible candidates for bone regeneration.

  3. Computational modeling reveals that a combination of chemotaxis and differential adhesion leads to robust cell sorting during tissue patterning.

    PubMed

    Tan, Rui Zhen; Chiam, Keng-Hwee

    2014-01-01

    Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.

  4. Viral oncogenes, proto-oncogenes and homoeotic genes related to cell proliferation and differentiation.

    PubMed

    Antohi, S; Antohi-Talle, O

    1987-01-01

    Molecular studies on viral oncogenes and their products have led to the discovery of physiological proto-oncogenes, involved in the control of cell proliferation and gene activation. Other genetic and molecular investigations, initiated in Drosophila melanogaster and continued in different multicellular eukaryotes, have made evident the homoeotic genes, which are directly correlated with cell specialization, in the complex processes of differentiation and morphogenesis. Both gene classes are conserved to a high extent during evolution. They are involved in the eukaryotic mechanisms of differentiation control and proto-oncogenes, in particular, are related to malignant transformation. Some available data suggest a certain extent of relatedness between the gene products of both gene classes. A differentiation trigger model, including retroviral transposition, homoeotic genes and proto-oncogenes is discussed.

  5. Lithium Reversibly Inhibits Schwann Cell Proliferation and Differentiation Without Inducing Myelin Loss.

    PubMed

    Piñero, Gonzalo; Berg, Randall; Andersen, Natalia Denise; Setton-Avruj, Patricia; Monje, Paula Virginia

    2016-12-05

    This study was undertaken to examine the bioactivity, specificity, and reversibility of lithium's action on the growth, survival, proliferation, and differentiation of cultured Schwann cells (SCs). In isolated SCs, lithium promoted a state of cell cycle arrest that featured extensive cell enlargement and c-Jun downregulation in the absence of increased expression of myelin-associated markers. In addition, lithium effectively prevented mitogen-induced S-phase entry without impairing cell viability. When lithium was administered together with differentiating concentrations of cyclic adenosine monophosphate (cAMP) analogs, a dramatic inhibition of the expression of the master regulator of myelination Krox-20 was observed. Likewise, lithium antagonized the cAMP-dependent expression of various myelin markers such as protein zero, periaxin, and galactocerebroside and allowed SCs to maintain high levels of expression of immature SC markers even in the presence of high levels of cAMP and low levels of c-Jun. Most importantly, the inhibitory action of lithium on SC proliferation and differentiation was shown to be dose dependent, specific, and reversible upon removal of lithium compounds. In SC-neuron cultures, lithium suppressed myelin sheath formation while preserving axonal integrity, SC-axon contact, and basal lamina formation. Lithium was unique in its ability to prevent the onset of myelination without promoting myelin degradation or SC dedifferentiation. To conclude, our results underscored an unexpected antagonistic action of lithium on SC mitogenesis and myelin gene expression. We suggest that lithium represents an attractive pharmacological agent to safely and reversibly suppress the onset of SC proliferation, differentiation, and myelination while maintaining the integrity of pre-existing myelinated fibers.

  6. PU.1 regulates both cytokine-dependent proliferation and differentiation of granulocyte/macrophage progenitors.

    PubMed Central

    DeKoter, R P; Walsh, J C; Singh, H

    1998-01-01

    PU.1 is a unique regulatory protein required for the generation of both the innate and the adaptive immune system. It functions exclusively in a cell-intrinsic manner to control the development of granulocytes, macrophages, and B and T lymphocytes. We demonstrate that mutation of the PU.1 gene causes a severe reduction in myeloid (granulocyte/macrophage) progenitors. PU.1 -/- myeloid progenitors can proliferate in vitro in response to the multilineage cytokines interleukin-3 (IL-3), IL-6 and stem cell factor but are unresponsive to the myeloid-specific cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and M-CSF. The failure of PU.1 -/- progenitors to respond to G-CSF is bypassed by transient signaling with IL-3. In the presence of IL-3 and G-CSF, PU.1 -/- progenitors can differentiate into granulocytic precursors containing myeloperoxidase-positive granules. Thus PU.1 is not essential for specification of granulocytic precursors, but is required for their further differentiation. The failure of PU.1 -/- progenitors to respond to M-CSF is due to lack of c-fms gene transcription. Transduction of c-fms into PU.1 -/- myeloid progenitors bypasses the block to M-CSF-dependent proliferation but does not induce detectable macrophage differentiation. Therefore, PU. 1 appears to be essential for specification of monocytic precursors. Importantly, retroviral transduction of PU.1 into mutant progenitors restores responsiveness to myeloid-specific cytokines and development of mature granulocytes and macrophages. Thus PU.1 controls myelopoiesis by regulating both proliferation and differentiation pathways. PMID:9687512

  7. Temperature effect on proliferation and differentiation of satellite cells from turkeys with different growth rates.

    PubMed

    Clark, D L; Coy, C S; Strasburg, G M; Reed, K M; Velleman, S G

    2016-04-01

    Poultry selected for growth have an inefficient thermoregulatory system and are more sensitive to temperature extremes. Satellite cells are precursors to skeletal muscle and mediate all posthatch muscle growth. Their physiological functions are affected by temperature. The objective of the current study was to determine how temperature affects satellite cells isolated from the pectoralis major (p. major) muscle (breast muscle) of turkeys selected for increased 16 wk body weight (F line) in comparison to a randombred control line (RBC2) from which the F line originated. Pectoralis major muscle satellite cells were thermally challenged by culturing between 33°C and 43°C to analyze the effects of cold and heat on proliferation and differentiation as compared to control temperature of 38°C. Expression levels of myogenic regulatory factors: myogenic differentiation factor 1 (MYOD1) and myogenin (MYOG) were quantified by quantitative polymerase chain reaction (qPCR). At all sampling times, proliferation increased at a linear rate across temperature in both the RBC2 and F lines. Differentiation also increased at a linear rate across temperature from 33 to 41°C at all sampling times in both the F and RBC2 lines. Satellite cells isolated from F line turkeys were more sensitive to both hot and cold temperatures as proliferation and differentiation increased to a greater extent across temperature (33 to 43°C) when compared with the RBC2 line. Expression of MYOD1 and MYOG increased as temperatures increased from 33 to 41°C at all sampling times in both the F and RBC2 lines. These results demonstrate that satellite cell function is sensitive to both cold and hot temperatures and p. major muscle satellite cells from F line turkeys are more sensitive to temperature extremes than RBC2 satellite cells.

  8. Electrospun SF/PLCL nanofibrous membrane: a potential scaffold for retinal progenitor cell proliferation and differentiation

    PubMed Central

    Zhang, Dandan; Ni, Ni; Chen, Junzhao; Yao, Qinke; Shen, Bingqiao; Zhang, Yi; Zhu, Mengyu; Wang, Zi; Ruan, Jing; Wang, Jing; Mo, Xiumei; Shi, Wodong; Ji, Jing; Fan, Xianqun; Gu, Ping

    2015-01-01

    Biocompatible polymer scaffolds are promising as potential carriers for the delivery of retinal progenitor cells (RPCs) in cell replacement therapy for the repair of damaged or diseased retinas. The primary goal of the present study was to investigate the effects of blended electrospun nanofibrous membranes of silk fibroin (SF) and poly(L-lactic acid-co-ε-caprolactone) (PLCL), a novel scaffold, on the biological behaviour of RPCs in vitro. To assess the cell-scaffold interaction, RPCs were cultured on SF/PLCL scaffolds for indicated durations. Our data revealed that all the SF/PLCL scaffolds were thoroughly cytocompatible, and the SF:PLCL (1:1) scaffolds yielded the best RPC growth. The in vitro proliferation assays showed that RPCs proliferated more quickly on the SF:PLCL (1:1) than on the other scaffolds and the control. Quantitative polymerase chain reaction (qPCR) and immunocytochemistry analyses demonstrated that RPCs grown on the SF:PLCL (1:1) scaffolds preferentially differentiated toward retinal neurons, including, most interestingly, photoreceptors. In summary, we demonstrated that the SF:PLCL (1:1) scaffolds can not only markedly promote RPC proliferation with cytocompatibility for RPC growth but also robustly enhance RPCs’ differentiation toward specific retinal neurons of interest in vitro, suggesting that SF:PLCL (1:1) scaffolds may have potential applications in retinal cell replacement therapy in the future. PMID:26395224

  9. Role for WNT16B in human epidermal keratinocyte proliferation and differentiation.

    PubMed

    Teh, Muy-Teck; Blaydon, Diana; Ghali, Lucy R; Briggs, Victoria; Edmunds, Scott; Pantazi, Eleni; Barnes, Michael R; Leigh, Irene M; Kelsell, David P; Philpott, Michael P

    2007-01-15

    WNT signalling regulates a variety of cell functions including cell fate, polarity, and differentiation via the canonical or beta-catenin stabilisation pathway and/or the planar cell polarity or non-canonical pathway. We have previously demonstrated that two isoforms (A and B) from the WNT16 locus have differential expression in various adult human tissues. In this study we show that WNT16B but not WNT16A isoform was upregulated in basal cell carcinomas compared with normal skin. We further investigated the cellular and molecular functions of WNT16B in primary human epidermal keratinocytes and a keratinocyte cell line. Cellular expression of WNT16B neither stabilised beta-catenin nor activated the lymphoid enhancer factor or T-cell factor transcriptional reporter in primary keratinocytes. WNT16B activated the Jun-N-terminal kinase cascade suggesting the activation of a non-canonical WNT signalling pathway. Constitutive expression of WNT16B significantly enhanced the rate of cell proliferation and prolonged clonogenicity in primary keratinocytes. Silencing WNT16B by RNA interference reduced keratinocyte proliferation. Furthermore, overexpression of WNT16B induced a hyperproliferation phenotype in an organotypical culture system. This work presents the first evidence that WNT16B activates human keratinocyte proliferation possibly via a beta-catenin-independent non-canonical WNT transduction pathway.

  10. p53 and TAp63 promote keratinocyte proliferation and differentiation in breeding tubercles of the zebrafish.

    PubMed

    Fischer, Boris; Metzger, Manuel; Richardson, Rebecca; Knyphausen, Philipp; Ramezani, Thomas; Franzen, Rainer; Schmelzer, Elmon; Bloch, Wilhelm; Carney, Thomas J; Hammerschmidt, Matthias

    2014-01-01

    p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal appendages which according to our analyses display more advanced stratification and keratinization than regular epidermis, including continuous desquamation and renewal of superficial cells by derivatives of basal keratinocytes. Defects are further enhanced in TAp63/p53 double mutants, pointing to partially redundant roles of the two related factors. Molecular analyses, treatments with chemical inhibitors and epistasis studies further reveal the existence of a linear TAp63/p53->Notch->caspase 3 pathway required both for enhanced proliferation of keratinocytes at the base of the tubercles and their subsequent differentiation in upper layers. Together, these studies identify the zebrafish breeding tubercles as specific epidermal structures sharing crucial features with the cornified mammalian epidermis. In addition, they unravel essential roles of TAp63 and p53 to promote both keratinocyte proliferation and their terminal differentiation by promoting Notch signalling and caspase 3 activity, ensuring formation and proper homeostasis of this self-renewing stratified epithelium.

  11. Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells

    SciTech Connect

    Misu, Masayasu; Ouji, Yukiteru; Kawai, Norikazu; Nishimura, Fumihiko; Nakamura-Uchiyama, Fukumi; Yoshikawa, Masahide

    2015-08-07

    In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. - Highlights: • Wnt-10b inhibited proliferation and migration of melanoma cells. • Wnt-10b induced tyrosinase activity and senescence of melanoma cells. • Wnt-10b suppressed growth of cell aggregates in collagen 3D-gel cultures. • Wnt-10b represses tumor cell properties, driving them toward a more differentiated state along a melanocyte lineage.

  12. Notch regulates blastema proliferation and prevents differentiation during adult zebrafish fin regeneration.

    PubMed

    Münch, Juliane; González-Rajal, Alvaro; de la Pompa, José Luis

    2013-04-01

    Zebrafish have the capacity to regenerate several organs, including the heart and fins. Fin regeneration is epimorphic, involving the formation at the amputation plane of a mass of undifferentiated, proliferating mesenchymal progenitor-like cells, called blastema. This tissue provides all the cell types that form the fin, so that after damage or amputation the fin pattern and structure are fully restored. How blastema cells remain in this progenitor-like state is poorly understood. Here, we show that the Notch pathway plays an essential role during fin regeneration. Notch signalling is activated during blastema formation and remains active throughout the regeneration process. Chemical inhibition or morpholino-mediated knockdown of Notch signalling impairs fin regeneration via decreased proliferation accompanied by reduced expression of Notch target genes in the blastema. Conversely, overexpression of a constitutively active form of the Notch1 receptor (N1ICD) in the regenerating fin leads to increased proliferation and to the expansion of the blastema cell markers msxe and msxb, as well as increased expression of the proliferation regulator aldh1a2. This blastema expansion prevents regenerative fin outgrowth, as indicated by the reduction in differentiating osteoblasts and the inhibition of bone regeneration. We conclude that Notch signalling maintains blastema cells in a plastic, undifferentiated and proliferative state, an essential requirement for fin regeneration.

  13. Differential expression of alkaline phosphatase gene in proliferating primary lymphocytes and malignant lymphoid cell lines.

    PubMed

    Latheef, S A A; Devanabanda, Mallaiah; Sankati, Swetha; Madduri, Ramanadham

    2016-02-01

    Alkaline Phosphatase (APase) activity has been shown to be enhanced specifically in mitogen stimulated B lymphocytes committed to proliferation, but not in T lymphocytes. APase gene expression was analyzed in proliferating murine and human primary lymphocytes and human malignant cell lines using reverse transcriptase and real time PCR. In mitogen stimulated murine splenic lymphocytes, enhancement of APase activity correlated well with an increase in APase gene expression. However, in mitogen stimulated murine T lymphocytes and human PBL despite a vigorous proliferative response, no increase in APase enzyme activity or gene expression was observed. A constitutive expression of APase activity concomitant with APase gene expression was observed inhuman myeloma cell line, U266 B1. However, neither enzyme activity nor gene expression of APase were observed in human T cell lymphoma, SUPT-1. The results suggest a differential expression of APase activity and its gene in proliferating primary lymphocytes of mice and humans. The specific expression of APase activity and its gene only in human myeloma cells, but not in proliferating primary B cells can be exploited as a sensitive disease marker.

  14. Cell proliferation and differentiation during the three dimensional reconstitution of eccrine sweat glands.

    PubMed

    Li, Xuexue; Li, Haihong; Zhang, Mingjun; Chen, Liyun; Zhang, Bingna

    2017-04-01

    The aim of this study is to characterize the cell proliferation and proliferating cell types during three-dimensional reconstitution of eccrine sweat glands. Eccrine sweat gland cells suspended in Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 1, 2, 4, 6, 8, 14, 21, 28, 35 and 42 days post-implantation, Matrigel plugs were immunostained for Ki67, to detect cycling cells, and the Ki67 labeling index at different time points was calculated. Three pairs of antibodies, Ki67/K7, Ki67/K14 and Ki67/α-SMA, were used to identify proliferating cell types in the plugs, on days 28, 35 and 42, by immunofluorescence double staining. The Ki67 labeling index on the first day of implantation was 30.53%, rapidly reached a peak value of 81.43% at 2 days post-implantation, and then decreased gradually to a low of 2.87% at 42 days. Double immunofluorescence staining showed that K14/Ki67 double-stained cells accounted for 80% of the Ki67-positive cells, whereas K7/Ki67 and α-SMA/Ki67 double-stained cells each accounted for 10% of the Ki67-positive population on days 28, 35, or 42 post-implantation. We conclude that eccrine sweat gland cells rapidly enter the cell cycle after implantation, but quickly show decreased cell proliferation and increased cell differentiation.

  15. TGFβ2 Differentially Modulates Smooth Muscle Cell Proliferation and Migration in Electrospun Gelatin-Fibrinogen constructs

    PubMed Central

    Ardila, D. C.; Tamimi, E.; Danford, F.L.; Haskett, D. G.; Kellar, R. S.; Doetschman, T.; Vande Geest, J.P.

    2014-01-01

    A main goal of tissue engineering is the development of scaffolds that replace, restore and improve injured tissue. These scaffolds have to mimic natural tissue, constituted by an extracellular matrix (ECM) support, cells attached to the ECM, and signaling molecules such as growth factors that regulate cell function. In this study we created electrospun flat sheet scaffolds using different compositions of gelatin and fibrinogen. Smooth muscle cells (SMCs) were seeded on the scaffolds, and proliferation and infiltration were evaluated. Additionally, different concentrations of Transforming Growth Factor-beta2 (TGFβ2) were added to the medium with the aim of elucidating its effect on cell proliferation, migration and collagen production. Our results demostrated that a scafold with a composition of 80% gelatin-20% fibrinogen is suitable for tissue engineering applications since it promotes cell growth and migration. The addition of TGFβ2 at low concentrations (≤1ng/ml) to the culture medium resulted in an increase in SMC proliferation and scaffold infiltration, and in the reduction of collagen production. In contrast, TGFβ2 at concentrations >1ng/ml inhibited cell proliferation and migration while stimulating collagen production. According to our results TGFβ2 concentration has a differential effect on SMC function and thus can be used as a biochemical modulator that can be beneficial for tissue engineering applications. PMID:25453947

  16. The effect of temperature on proliferation and differentiation of chicken skeletal muscle satellite cells isolated from different muscle types.

    PubMed

    Harding, Rachel L; Halevy, Orna; Yahav, Shlomo; Velleman, Sandra G

    2016-04-01

    Skeletal muscle satellite cells are a muscle stem cell population that mediate posthatch muscle growth and repair. Satellite cells respond differentially to environmental stimuli based upon their fiber-type of origin. The objective of this study was to determine how temperatures below and above the in vitro control of 38°C affected the proliferation and differentiation of satellite cells isolated from the chicken anaerobic pectoralis major (p. major) or mixed fiber biceps femoris (b.femoris) muscles. The satellite cells isolated from the p. major muscle were more sensitive to both cold and hot temperatures compared to the b.femoris satellite cells during both proliferation and differentiation. The expressions of myogenic regulatory transcription factors were also different between satellite cells from different fiber types. MyoD expression, which partially regulates proliferation, was generally expressed at higher levels in p. major satellite cells compared to the b.femoris satellite cells from 33 to 43°C during proliferation and differentiation. Similarly, myogenin expression, which is required for differentiation, was also expressed at higher levels in p. major satellite cells in response to both cold and hot temperatures during proliferation and differentiation than b. femoris satellite cells. These data demonstrate that satellite cells from the anaerobic p. major muscle are more sensitive than satellite cells from the aerobic b. femoris muscle to both hot and cold thermal stress during myogenic proliferation and differentiation.

  17. Effects of cyclic stretch on proliferation of mesenchymal stem cells and their differentiation to smooth muscle cells

    SciTech Connect

    Ghazanfari, Samane; Tafazzoli-Shadpour, Mohammad; Shokrgozar, Mohammad Ali

    2009-10-23

    Bone marrow mesenchymal stem cells (MSCs) are capable of differentiating into a variety of cell types such as vascular smooth muscle cells (SMCs). In this study, we investigated influence of cyclic stretch on proliferation of hMSCs for different loading conditions, alignment of actin filaments, and consequent differentiation to SMCs. Isolated cells from bone marrow were exposed to cyclic stretch utilizing a customized device. Cell proliferation was examined by MTT assay, alignment of actin fibers by a designed image processing code, and cell differentiation by fluorescence staining. Results indicated promoted proliferation of hMSCs by cyclic strain, enhanced by elevated strain amplitude and number of cycles. Such loading regulated smooth muscle {alpha}-actin, and reoriented actin fibers. Cyclic stretch led to differentiation of hMSCs to SMCs without addition of growth factor. It was concluded that applying appropriate loading treatment on hMSCs could enhance proliferation capability, and produce functional SMCs for engineered tissues.

  18. p38 Mitogen-Activated Protein Kinase Pathway Regulates Genes during Proliferation and Differentiation in Oligodendrocytes

    PubMed Central

    Haines, Jeffery D.; Fulton, Debra L.; Richard, Stephane; Almazan, Guillermina

    2015-01-01

    We have previously shown that p38 mitogen-activated protein kinase (p38 MAPK) is important for oligodendrocyte (OLG) differentiation and myelination. However, the precise cellular mechanisms by which p38 regulates OLG differentiation remain largely unknown. To determine whether p38 functions in part through transcriptional events in regulating OLG identity, we performed microarray analysis on differentiating oligodendrocyte progenitors (OLPs) treated with a p38 inhibitor. Consistent with a role in OLG differentiation, pharmacological inhibition of p38 down-regulated the transcription of genes that are involved in myelin biogenesis, transcriptional control and cell cycle. Proliferation assays showed that OLPs treated with the p38 inhibitor retained a proliferative capacity which could be induced upon application of mitogens demonstrating that after two days of p38-inhibition OLGs remained poised to continue mitosis. Together, our results suggest that the p38 pathway regulates gene transcription which can coordinate OLG differentiation. Our microarray dataset will provide a useful resource for future studies investigating the molecular mechanisms by which p38 regulates oligodendrocyte differentiation and myelination. PMID:26714323

  19. Matriptase regulates proliferation and early, but not terminal, differentiation of human keratinocytes.

    PubMed

    Chen, Ya-Wen; Wang, Jehng-Kang; Chou, Fen-Pai; Wu, Bai-Yao; Hsiao, Hui-Chung; Chiu, Han; Xu, Zhonghong; Baksh, Adrienne N H; Shi, Galen; Kaul, Malvika; Barndt, Robert; Shanmugam, Victoria K; Johnson, Michael D; Lin, Chen-Yong

    2014-02-01

    Genetic defects in matriptase are linked to two congenital ichthyoses: autosomal recessive ichthyosis with hypotrichosis (ARIH, OMIM 610765) and ichthyosis, follicular atrophoderma, hypotrichosis, and hypohidrosis (IFAH, OMIM 602400). Mouse models with matriptase deficiency indicate an involvement of matriptase in suprabasal keratinocytes in the maintenance of the epidermal barrier. In contrast to what has been reported for mouse skin, we show that in human skin matriptase is primarily expressed in the basal and spinous keratinocytes, but not in the more differentiated keratinocytes of the granular layer. In addition, matriptase zymogen activation was predominantly detected in the basal cells. Furthermore, by using skin organotypic cultures as a model system to monitor the course of human epidermal differentiation, we found elevated matriptase zymogen activation during early stages of epidermal differentiation, coupled with a loss of matriptase expression in the late stages of this process. We also show here that matriptase deficiency in HaCaT cells modestly reduces cell proliferation and temporally affects calcium-induced expression of differentiation markers. These collective data suggest that, unlike mouse matriptase, human matriptase may be involved in the regulation of keratinocyte growth and early differentiation, rather than terminal differentiation, providing mechanistic insights into the pathology of the two congenital ichthyoses: ARIH and IFAH.

  20. Evaluation of early stage human bone marrow stromal proliferation, cell migration and osteogenic differentiation on μ-MIM structured stainless steel surfaces.

    PubMed

    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.

  1. Enhancement of growth and osteogenic differentiation of MC3T3-E1 cells via facile surface functionalization of polylactide membrane with chitooligosaccharide based on polydopamine adhesive coating

    NASA Astrophysics Data System (ADS)

    Li, Huihua; Luo, Chuang; Luo, Binghong; Wen, Wei; Wang, Xiaoying; Ding, Shan; Zhou, Changren

    2016-01-01

    To develop a chitooligosaccharide(COS)-functionalized poly(D,L-lactide) (PDLLA) membrane to enhance growth and osteogenic differentiation of MC3T3-E1 cells, firstly a thin polydopamine (PDOPA) layer was adhered to the PDLLA membrane via the self-polymerization and strong adhesion behavior of dopamine. Subsequently, COS was immobilized covalently on the resultant PDLLA/PDOPA composite membrane by coupling with PDOPA active coating. The successful immobilization of the PDOPA and COS was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) results indicated that the surface topography and roughness of the membranes were changed, and the root mean square increased from 0.613 nm to 6.96 and 7.12 nm, respectively after coating PDOPA and COS. Water contact angle and surface energy measurements revealed that the membrane hydrophilicity was remarkably improved by surface modification. In vitro cells culture results revealed that the PDOPA- and COS-functionalized surfaces showed a significant increase in MC3T3-E1 cells adhesion, proliferation, osteogenic differentiation and alkaline phosphate activity compared to the pristine PDLLA substrate. Furthermore the COS-functionalized PDLLA membrane was more effectively at enhancing osteoblast activity than the PDOPA-functionalized PDLLA membrane.

  2. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells.

    PubMed

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W Michael; Das, Mita

    2014-04-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.

  3. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells

    PubMed Central

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W. Michael

    2014-01-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases. PMID:24500281

  4. [The role of Piwi nuclear localization in the differentiation and proliferation of germline stem cells].

    PubMed

    Yakushev, E Y; Mikhaleva, E A; Abramov, Y A; Sokolova, O A; Zyrianova, I M; Gvozdev, V A; Klenov, M S

    2016-01-01

    The Piwi protein and its orthologs are considered as the key components of the piRNA machinery implicated in transcriptional silencing of transposons. Неre, we show that nuclear localization of the Piwi protein is required not only for transposon repression, but also for proper differentiation of germline stem cells (GSCs). piwi^(Nt) mutation that causes loss of nuclear Piwi and its retention in the cytoplasm leads to the accumulation of undifferentiated GSC-like cells. The analysis of piwi^(Nt) mutation in combination with a bam gene mutation blocking GSC differentiation shows that the loss of nuclear Piwi decreases GSC proliferation rate. This is accompanied by the accumulation of DNA double-strand breaks in GSCs that may be caused by transposition events. Here, for the first time a set of transposons repressed by Piwi in GSCs and surrounding niche cells has been identified. The present study together with our previous data show that nuclear and cytoplasmic Piwi can regulate different stages of the functioning of germinal cells: cytoplasmic Piwi is sufficient to maintain GSCs, while nuclear Piwi localization is necessary for their proper proliferation and differentiation.

  5. Nuclear EGFR characterize still controlled proliferation retained in better differentiated clear cell RCC.

    PubMed

    Ahel, J; Dordevic, G; Markic, D; Mozetic, V; Spanjol, J; Grahovac, B; Stifter, S

    2015-08-01

    Renal cell carcinoma (RCC) is the most common solid kidney tumor representing 2-3% of all cancers, with the highest frequency occurring in Western countries. There was a worldwide and European annual increase in incidence of approximately 2% although incidence has been stabilized in last few years. One third of the patients already have metastases in the time of the diagnosis with poor prognosis because RCC are radio and chemoresistant. The prognostic value of EGFR over-expression in RCC is a controversial issue that could be explained by different histological types of study tumors and non-standardized criteria for evaluation of expression. Recent evidences points to a new mode of EGFR signaling pathway in which activated EGFR undergoes nuclear translocalization and then, as transcription factor, mediates gene expression and other cellular events required for highly proliferating activities. According to our observations, the membranous expression of EGFR associates with high nuclear grade and poor differentiated tumors. On the other hand, nuclear EGFR expression was high in low nuclear graded and well differentiated tumors with good prognosis. We hypothesize that this mode of EGFR signaling characterizes still controlled proliferation retained in well differentiated RCC with Furhman nuclear grade I or II.

  6. Interleukin-7 co-ordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection

    PubMed Central

    Boudil, Amine; Matei, Irina R.; Shih, Han-Yu; Bogdanoski, Goce; Yuan, Julie S.; Chang, Stephen G.; Montpellier, Bertrand; Kowalski, Paul E.; Voisin, Veronique; Bashir, Shaheena; Bader, Gary D.; Krangel, Michael S.; Guidos, Cynthia J.

    2015-01-01

    pre-T cell receptor (TCR) and Notch signaling induce transient self-renewal or “β-selection” of TCRβ+ CD4 CD8 double-negative-3 (DN3) and DN4 progenitors that differentiate into CD4 CD8 double positive (DP) thymocytes which then rearrange Tcra. Interleukin-7 (IL-7) promotes Bcl2-dependent survival of TCRβ− DN thymocytes, but IL-7 functions during β-selection remain unclear. Here, we show that IL-7 signals TCRβ+ DN3 and DN4 thymocytes to upregulate genes involved in cell growth and represses Bcl6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate, but rearranged Tcra prematurely and differentiated rapidly. Bcl6 deletion, but not BCL2 over-expression, partially restored DN4 self-renewal in the absence of IL-7. Thus, IL-7 critically collaborates with pre-TCR and Notch signaling to coordinate proliferation, differentiation and Tcra recombination during β-selection. PMID:25729925

  7. Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation

    SciTech Connect

    Wang, Guang; Li, Yan; Wang, Xiao-yu; Han, Zhe; Chuai, Manli; Wang, Li-jing; Ho Lee, Kenneth Ka; Geng, Jian-guo; Yang, Xuesong

    2013-05-01

    development by tightly coordinating cell proliferation and differentiation during neurulation. - Highlights: ► The role of Slit/Robo1 signaling was investigated with chick and mouse models. ► Disturbance of Slit/Robo1 signaling resulted in neural tube defects. ► Slit/Robo1 signaling regulated the proliferation of neural tube cells. ► Slit/Robo1 signaling modulated the differentiation of neural tube cells. ► Slit/Robo1 signaling balanced the proliferation and differentiation of neural tube.

  8. Attachment, proliferation and differentiation of periodontal ligament cells on various guided tissue regeneration membranes.

    PubMed

    Takata, T; Wang, H L; Miyauchi, M

    2001-10-01

    The purpose of this study was to evaluate the biological effects of guided tissue regeneration (GTR) membrane materials, per se, on the periodontal tissue regeneration. Rat periodontal ligament (PDL)-derived cells were used to study the attachment, proliferation and differentiation, in vitro, on various GTR membranes. Five commercially available membranes bovine type I collagen (BioMend; BM), bovine type I atelocollagen (Tissue Guide; TG), polylactic acid (Epi-Guide; EG), co-polymer of polylactic acid and polyglycolic acid (Resolute; RL) and expanded polytetrafluoroethylene: e-PTFE (Gore Tex; GT)-were examined. A 3 x 3 mm section of the membrane was fixed to the bottom of a 35 x 10 mm style culture dish and plated with 2 ml of cell suspension at an initial density of 5 x 10(4) cells/ml in culture medium with 10% fetal bovine serum. For cell growth analysis, the specimens were fixed with 10% buffered formalin and stained with hematoxylin at 1.5 hours and 1, 3 and 5 days after cell seeding. The number of cells included in a unit area of 0.25 mm2 were counted under light microscopy. As a comparative scaffold of cell proliferation, a plastic cover for cell culture slip (Celldesk; CD) was used. For analysis of cell differentiation, activity of alkaline phosphatase (ALP) and calcification were histochemically revealed after 2-week cultivation. The initial number of PDL cells attached to the membrane at 1.5 hours after cell seeding was different among membranes. RL, TG and EG had the same level of attached cell numbers as that on CD, while the cell numbers on GT and BM were significantly lower than that on CD (p < 0.01). The rate of cell proliferation with time also differed among the membranes examined. RL and BM demonstrated a significantly higher number of cells at 5 days than at 1.5 hours (p < 0.01). TG had increased numbers of cells at 3 and 5 days after cell seeding. However, there was no statistical difference between the cell numbers at 1.5 hours and 5 days after

  9. Effects of lysophosphatidic acid on the in vitro proliferation and differentiation of a novel porcine preadipocyte cell line.

    PubMed

    Nobusue, Hiroyuki; Kondo, Daisuke; Yamamoto, Makiko; Kano, Koichiro

    2010-12-01

    We examined the effects of lysophosphatidic acid (LPA) on in vitro proliferation and differentiation of a porcine preadipocyte cell line, DFAT-P, and a mouse preadipocyte cell line, 3T3-L1. During the proliferation and differentiation phases, DFAT-P and 3T3-L1 cells expressed only the endothelial differentiation gene (EDG)-2 receptor and not EDG-4 and EDG-7 receptors. LPA promoted the proliferation of DFAT-P cells more extensively than that of 3T3-L1 cells. After adipogenic induction, LPA inhibited glycerol-3-phosphate dehydrogenase activity and lipid droplet accumulation, and suppressed peroxisome proliferator-activated receptor γ (PPARγ) protein expression, this inhibitory effect in DFAT-P cells was twice as high as that in 3T3-L1 cells. Furthermore, treatments with low LPA concentrations significantly inhibited adipocyte differentiation in DFAT-P cells but not in 3T3-L1 cells. We conclude that LPA promotes the proliferation of porcine preadipocytes through the EDG-2 receptor but inhibits their differentiation, and these effects depend on the down-regulation of PPARγ expression via the EDG-2 receptor. Furthermore, DFAT-P cells are more sensitive to LPA than 3T3-L1 cells. These findings in a porcine model will contribute to the understanding of LPA action mechanisms on in vitro proliferation and differentiation of preadipocytes in domestic animals and/or humans.

  10. Mitochondrial and lipogenic effects of vitamin D on differentiating and proliferating human keratinocytes.

    PubMed

    Consiglio, Marco; Viano, Marta; Casarin, Stefania; Castagnoli, Carlotta; Pescarmona, Gianpiero; Silvagno, Francesca

    2015-10-01

    Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin.

  11. Fsh Stimulates Spermatogonial Proliferation and Differentiation in Zebrafish via Igf3.

    PubMed

    Nóbrega, Rafael Henrique; Morais, Roberto Daltro Vidal de Souza; Crespo, Diego; de Waal, Paul P; de França, Luiz Renato; Schulz, Rüdiger W; Bogerd, Jan

    2015-10-01

    Growth factors modulate germ line stem cell self-renewal and differentiation behavior. We investigate the effects of Igf3, a fish-specific member of the igf family. Fsh increased in a steroid-independent manner the number and mitotic index of single type A undifferentiated spermatogonia and of clones of type A differentiating spermatogonia in adult zebrafish testis. All 4 igf gene family members in zebrafish are expressed in the testis but in tissue culture only igf3 transcript levels increased in response to recombinant zebrafish Fsh. This occurred in a cAMP/protein kinase A-dependent manner, in line with the results of studies on the igf3 gene promoter. Igf3 protein was detected in Sertoli cells. Recombinant zebrafish Igf3 increased the mitotic index of type A undifferentiated and type A differentiating spermatogonia and up-regulated the expression of genes related to spermatogonial differentiation and entry into meiosis, but Igf3 did not modulate testicular androgen release. An Igf receptor inhibitor blocked these effects of Igf3. Importantly, the Igf receptor inhibitor also blocked Fsh-induced spermatogonial proliferation. We conclude that Fsh stimulated Sertoli cell production of Igf3, which promoted via Igf receptor signaling spermatogonial proliferation and differentiation and their entry into meiosis. Because previous work showed that Fsh also released spermatogonia from an inhibitory signal by down-regulating anti-Müllerian hormone and by stimulating androgen production, we can now present a model, in which Fsh orchestrates the activity of stimulatory (Igf3, androgens) and inhibitory (anti-Müllerian hormone) signals to promote spermatogenesis.

  12. 14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization

    PubMed Central

    Sambandam, Sumitha A.T.; Kasetti, Ramesh Babu; Xue, Lei; Dean, Douglas C.; Lu, Qingxian; Li, Qiutang

    2015-01-01

    The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting DMBA/TPA induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In the present study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in DMBA/TPA-induced tumors from Er/+ skin. Furthermore, shRNA knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length WT or the mutant form found in Er/Er mice. However Er 14-3-3σ does not interact with Yap1, as demonstrated by co-immunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in Er/Er epidermis. PMID:25668240

  13. Differential effects of platelet rich plasma and washed platelets on the proliferation of mouse MSC cells.

    PubMed

    Duan, Jianmin; Kuang, Wei; Tan, Jiali; Li, Hongtao; Zhang, Yi; Hirotaka, Kikuchi; Tadashi, Katayama

    2011-04-01

    Multipotent mesenchymal stem cell (MSC) therapies are being tested clinically for a variety of disorders. However, despite the remarkable clinical advancements in this field, most applications still use traditional culture media containing fetal bovine serum. Platelet-rich plasma (PRP) appears as a novel application for tissue engineering and its effect on bone healing is thought to be mainly dependent on the proliferation promoting function, with the molecular mechanisms largely unknown. In this study, mouse osteogenic progenitor mesenchymal stem cells (MSCs) were cultured in PRP or washed platelet (WPLT)-treated wells or in untreated wells, and analyzed on cycloxygenase 2 (COX2) expression (qRT-PCR), cell growth (MTT assay) and cell differentiation (alkaline phosphatase activity). The results showed that PRP and WPLT stimulated cell growth similarly in the first 6 days, together with the steady induction of COX2 and PGE2. 10 μmol/l celecoxib (an inhibitor of COX2) significantly inhibited the pro-proliferation effects. Interestingly, WPLT had stronger effects than PRP in proliferation at the later time points (6-9 days). ALP activity assay and collagen 1a expression revealed PRP had a mild but statistically significant pro-differentiation effect, while no obvious effects observed in WLPT group. In summary, PRP stimulates initial growth of MSCs in a COX2 partially dependent manner and the less obvious osteogenic differentiation promoting effects of WPLT strongly indicates WPLT rather than the PRP should be the optional choice for expanding MSCs in vitro for clinical use.

  14. Effects of strontium on proliferation and differentiation of rat bone marrow mesenchymal stem cells

    SciTech Connect

    Li, Yunfeng; Li, Jihua; Zhu, Songsong; Luo, En; Feng, Ge; Chen, Qianming; Hu, Jing

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer Strontium ranelate (SrR) inhibits proliferation of BMMSCs. Black-Right-Pointing-Pointer SrR increases osteoblastic but decreases adipocytic differentiation of BMMSCs. Black-Right-Pointing-Pointer SrR increases expression of Runx2, BSP and OCN by BMMSCs in osteogenic medium. Black-Right-Pointing-Pointer SrR decreases expression of PPAR{gamma}, aP2/ALBP and LPL by BMMSCs in adipogenic medium. -- Abstract: Strontium ranelate (SrR) was an effective anti-osteoporotic drug to increase bone formation and decrease bone resorption. However, reports about the effect of SR on osteoblastic and adipocytic differentiation from bone marrow mesenchymal stem cells (BMMSCs) are limited. The purpose of this study is to evaluate whether SrR affects the ability of BMMSCs to differentiate into osteoblasts or adipocytes. Rat BMMSCs were identified by flow cytometry and exposed to SR (0.1 and 1.0 mM Sr{sup 2+}) under osteogenic or adipogenic medium for 1 and 2 weeks. The proliferation and differentiation of BMMSCs were analyzed by MTT, alkaline phosphatase (ALP), Oil red O staining, quantitative real-time RT-PCR and Western blot assays. SrR significantly inhibited the proliferation, increased osteoblastic but decreased adipocytic differentiation of rat BMMSCs dose-dependently. In osteogenic medium, SrR increased the expression of ALP, the mRNA levels of Cbfa1/Runx2, bone sialoprotein, and osteocalcin by RT-PCR, and the protein levels of Cbfa1/Runx2 by Western blot. In adipogenic medium, SrR decreased the mRNA levels of PPAR{gamma}2, adipocyte lipid-binding protein 2 (aP2/ALBP), and lipoprotein lipase (LPL) by RT-PCR, and the protein expression of PPAR{gamma} in Western blot analysis. These results indicated that the effects of SrR to promote osteoblastic but inhibit adipocytic differentiation of BMMSCs might contribute to its effect on osteoporosis treatment.

  15. The Transition from Proliferation to Differentiation in Colorectal Cancer Is Regulated by the Calcium Activated Chloride Channel A1

    PubMed Central

    Liu, Bin; McCaig, Colin D.; Pu, Jin

    2013-01-01

    Breaking the balance between proliferation and differentiation in animal cells can lead to cancer, but the mechanisms maintaining this balance remain largely undefined. The calcium activated chloride channel A1 (CLCA1) is a member of the calcium sensitive chloride conductance family of proteins and is expressed mainly in the colon, small intestine and appendix. We show that CLCA1 plays a functional role in differentiation and proliferation of Caco-2 cells and of intestinal tissue. Caco-2 cells spontaneously differentiate either in confluent culture or when treated with butyrate, a molecule present naturally in the diet. Here, we compared CLCA1 expressional levels between patients with and without colorectal cancer (CRC) and determined the functional role of CLCA1 in differentiation and proliferation of Caco-2 cells. We showed that: 1) CLCA1 and CLCA4 expression were down-regulated significantly in CRC patients; 2) CLCA1 expression was up-regulated in Caco-2 cells induced to differentiate by confluent culture or by treatment with sodium butyrate (NaBT); 3) Knockdown of CLCA1 with siRNA significantly inhibited cell differentiation and promoted cell proliferation in Caco-2 confluent cultures, and 4) In Caco-2 3D culture, suppression of CLCA1 significantly increased cell proliferation and compromised NaBT-induced inhibition of proliferation. In conclusion, CLCA1 may contribute to promoting spontaneous differentiation and reducing proliferation of Caco-2 cells and may be a target of NaBT-induced inhibition of proliferation and therefore a potential diagnostic marker for CRC prognosis. PMID:23593331

  16. Reference genes for gene expression analysis in proliferating and differentiating human keratinocytes.

    PubMed

    Lanzafame, Manuela; Botta, Elena; Teson, Massimo; Fortugno, Paola; Zambruno, Giovanna; Stefanini, Miria; Orioli, Donata

    2015-04-01

    Abnormalities in keratinocyte growth and differentiation have a pathogenic significance in many skin disorders and result in gene expression alterations detectable by quantitative real-time RT-PCR (qRT-PCR). Relative quantification based on endogenous control (EC) genes is the commonly adopted approach, and the use of multiple reference genes from independent pathways is considered a best practice guideline, unless fully validated EC genes are available. The literature on optimal reference genes during in vitro calcium-induced differentiation of normal human epidermal keratinocytes (NHEK) is inconsistent. In many studies, the expression of target genes is compared to that of housekeeping genes whose expression, however, significantly varies during keratinocyte differentiation. Here, we report the results of our investigations on the expression stability of 15 candidate EC genes, including those commonly used as reference in expression analysis by qRT-PCR, during NHEK calcium-induced differentiation. We demonstrate that YWHAZ and UBC are extremely stable genes, and therefore, they represent optimal EC genes for expression studies in proliferating and calcium-induced differentiating NHEK. Furthermore, we demonstrate that YWHAZ/14-3-3-zeta is a suitable reference for quantitative comparison of both transcript and protein levels.

  17. Inhibition of peroxisome proliferator-activated receptor (PPAR)-mediated keratinocyte differentiation by lipoxygenase inhibitors.

    PubMed Central

    Thuillier, Philippe; Brash, Alan R; Kehrer, James P; Stimmel, Julie B; Leesnitzer, Lisa M; Yang, Peiying; Newman, Robert A; Fischer, Susan M

    2002-01-01

    Lipoxygenase (LOX) metabolites from arachidonic acid and linoleic acid have been implicated in atherosclerosis, inflammation, keratinocyte differentiation and tumour progression. We previously showed that peroxisome proliferator-activated receptors (PPARs) play a role in keratinocyte differentiation and that the PPARalpha ligand 8S-hydroxyeicosatetraenoic acid is important in this process. We hypothesized that blocking LOX activity would block PPAR-mediated keratinocyte differentiation. Three LOX inhibitors, nordihydroguaiaretic acid, quercetin and morin, were studied for their effects on primary keratinocyte differentiation and PPAR activity. All three LOX inhibitors blocked calcium-induced expression of the differentiation marker keratin 1. In addition, activity of a PPAR-responsive element was inhibited in the presence of all three inhibitors, and this effect was mediated primarily through PPARalpha and PPARgamma. LOX inhibitors decreased the activity of a chimaeric PPAR-Gal4-ligand-binding domain reporter system and this effect was reversed by addition of PPAR ligands. Ligand-binding studies revealed that the LOX inhibitors bind directly to PPARs and demonstrate a novel mechanism for these inhibitors in altering PPAR-mediated gene expression. PMID:12069687

  18. MicroRNA-194 regulates keratinocyte proliferation and differentiation by targeting Grainyhead-like 2 in psoriasis.

    PubMed

    Yu, Xiaoyun; An, Jingang; Hua, Yunhui; Li, Zihai; Yan, Ning; Fan, Weixin; Su, Chuan

    2017-02-01

    MicroRNAs (miRNAs) are currently emerged as important regulators in psoriasis. Psoriasis is characterized by hyperproliferation and impaired differentiation of keratinocytes in skin lesions. miR-194 is a well-known regulator of cell proliferation and differentiation. However, the role of miR-194 in psoriasis pathogenesis remains unclear. In this study we aimed to investigate the role of miR-194 in keratinocyte hyperproliferation and differentiation. We found that miR-194 was significantly downregulated in psoriasis lesional skin. Overexpression of miR-194 inhibited the proliferation and promoted the differentiation of primary human keratinocytes, whereas miR-194 suppression promoted the proliferation and inhibited their differentiation. Bioinformatic analysis predicted that the Grainyhead-like 2 (GRHL2) was a target gene of miR-194, which we further validated with a dual-luciferase reporter assay, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot analysis. The effect of miR-194 on cell proliferation and differentiation was significantly reversed by overexpression of GRHL2. Moreover, the expression of miR-194 and GRHL2 was inversely correlated in psoriasis lesional skin. Taken together, our results suggest that miR-194 inhibits the proliferation and promotes the differentiation of keratinocytes through targeting GRHL2. The downregulation of miR-194 expression may contribute to the pathogenesis of psoriasis and targeting miR-194 may represent a novel and potential therapeutic strategy for psoriasis.

  19. Autophagy and modular restructuring of metabolism control germline tumor differentiation and proliferation in C. elegans.

    PubMed

    Gomes, Ligia C; Odedra, Devang; Dikic, Ivan; Pohl, Christian

    2016-01-01

    Autophagy can act either as a tumor suppressor or as a survival mechanism for established tumors. To understand how autophagy plays this dual role in cancer, in vivo models are required. By using a highly heterogeneous C. elegans germline tumor, we show that autophagy-related proteins are expressed in a specific subset of tumor cells, neurons. Inhibition of autophagy impairs neuronal differentiation and increases tumor cell number, resulting in a shorter life span of animals with tumors, while induction of autophagy extends their life span by impairing tumor proliferation. Fasting of animals with fully developed tumors leads to a doubling of their life span, which depends on modular changes in transcription including switches in transcription factor networks and mitochondrial metabolism. Hence, our results suggest that metabolic restructuring, cell-type specific regulation of autophagy and neuronal differentiation constitute central pathways preventing growth of heterogeneous tumors.

  20. Micro-RNA mediated regulation of proliferation, self-renewal and differentiation of mammalian stem cells

    PubMed Central

    Hime, Gary R

    2009-01-01

    Metazoan growth and development is maintained by populations of undifferentiated cells, commonly known as stem cells. Stem cells possess several characteristic properties, including dividing through self-renewing divisions and generating progeny that differentiate to have specialized cell fates. Multiple signaling pathways have been identified which coordinate stem cell proliferation with maintenance and differentiation. Relatively recently, the small, non-protein coding microRNAs (miRNAs) have been identified to function as important regulators in stem cell development. Individual miRNAs are capable of directing the translational repression of many mRNAs targets, generating widespread changes in gene expression. In addition, dysfunction of miRNA expression is commonly associated with cancer development. Cancer stem cells, which are likely responsible for initiating and maintaining tumorigenesis, share many similarities with stem cells and some mechanisms of miRNA function may be in common between these two cell types. PMID:19829062

  1. Low power laser and LED irradiation effect on proliferation and differentiation of Wistar rats mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Mancera, Diana; Solarte, Efrain; Fierro, Leonardo; Criollo, William

    2013-11-01

    It has been demonstrated that appropriately cultured and stimulated mesenchymal cells, can give rise to cells of all tissues of the body. We evaluate the cell proliferation and differentiation induced by low power light irradiation in cell cultures of mesenchymal cells, isolated and previously characterized, from Wistar rats. Roche® XTT and LDH tests were used to assess proliferation and cytotoxicity. Cellular differentiation was determined by optical microscopy and using specific fluorescent markers. We report laser cellular proliferation enhancement by 532 and 473 nm, and the best cell culture response by a dose of 2 Jcm-2. Although a three day irradiation protocol the cultures grown and no cytotoxicity was detected. Cellular differentiation occurred, and the production of cardiomyocytes was promoted by the cell proliferation stimulated by low power laser irradiation.

  2. Chronic treatment with the GLP1 analogue liraglutide increases cell proliferation and differentiation into neurons in an AD mouse model.

    PubMed

    Parthsarathy, Vadivel; Hölscher, Christian

    2013-01-01

    Neurogenesis is a life long process, but the rate of cell proliferation and differentiation decreases with age. In Alzheimer's patients, along with age, the presence of Aβ in the brain inhibits this process by reducing stem cell proliferation and cell differentiation. GLP-1 is a growth factor that has neuroprotective properties. GLP1 receptors are present on neuronal progenitor cells, and the GLP-1 analogue liraglutide has been shown to increase cell proliferation in an Alzheimer's disease (AD) mouse model. Here we investigated acute and chronic effects of liraglutide on progenitor cell proliferation, neuroblast differentiation and their subsequent differentiation into neurons in wild type and APP/PS-1 mice at different ages. APP/PS1 and their littermate controls, aged 3, 6, 12, 15 months were injected acutely or chronically with 25 nmol/kg liraglutide. Acute treatment with liraglutide showed an increase in cell proliferation in APP/PS1 mice, but not in controls whereas chronic treatment increased cell proliferation at all ages (BrdU and Ki67 markers). Moreover, numbers of immature neurons (DCX) were increased in both acute and chronic treated animals at all ages. Most newly generated cells differentiated into mature neurons (NeuN marker). A significant increase was observed with chronically treated 6, 12, 15 month APP/PS1 and WT groups. These results demonstrate that liraglutide, which is currently on the market as a treatment for type 2 diabetes (Victoza(TM)), increases neurogenesis, which may have beneficial effects in neurodegenerative disorders like AD.

  3. Differentiating plant cells switched to proliferation remodel the functional organization of nuclear domains.

    PubMed

    Testillano, P S; González-Melendi, P; Coronado, M J; Seguí-Simarro, J M; Moreno-Risueño, M A; Risueño, M C

    2005-01-01

    The immature pollen grain, the microspore, under stress conditions can switch its developmental program towards proliferation and embryogenesis. The comparison between the gametophytic and sporophytic pathways followed by the microspore permitted us to analyse the nuclear changes in plant differentiating cells when switched to proliferation. The nucleus is highly dynamic, the architecture of its well organised functional domains--condensed chromatin, interchromatin region, nuclear bodies and nucleolus--changing in response to DNA replication, RNA transcription, processing and transport. In the present work, the rearrangements of the nuclear domains during the switch to proliferation have been determined by in situ molecular identification methods for the subcellular localization of chromatin at different functional states, rDNA, elements of the nuclear machinery (PCNA, splicing factors), signalling and stress proteins. The study of the changes in the nuclear domains was determined by a correlative approach at confocal and electron microscopy levels. The results showed that the switch of the developmental program and the activation of the proliferative activity affected the functional organization of the nuclear domains, which accordingly changed their architecture and functional state. A redistribution of components, among them various signalling molecules which targeted structures within the interchromatin region upon translocation from the cytoplasm, was also observed.

  4. Parathyroid hormone mediates bone growth through the regulation of osteoblast proliferation and differentiation.

    PubMed

    Pettway, Glenda J; Meganck, Jeffrey A; Koh, Amy J; Keller, Evan T; Goldstein, Steven A; McCauley, Laurie K

    2008-04-01

    PTH (1-34) is the only FDA-approved anabolic agent for osteoporosis treatment in the U.S., but its mechanisms are not completely understood. This study investigated PTH effects on osteogenic cells at various stages of differentiation and proliferation using an engineered bone growth model in vivo. Ossicles were generated from bone marrow stromal cells (BMSCs) implanted in immunocompromised mice. Three weeks of PTH (40 microg/kg/day) or vehicle treatment initiated 1 day, 1, 2, or 3 weeks after BMSC implantation resulted in an anabolic response in PTH-treated implants (via histomorphometry and muCT) in all treatment groups. A novel in vivo tracking strategy with luciferase tagged BMSCs and weekly bioluminescent imaging of ossicles revealed increased donor cell proliferation in PTH-treated ossicles. The greatest increase occurred during the first week, and the activity remained elevated in PTH-treated implants over time. Zoledronic acid (ZA) was combined with PTH to delineate interactive mechanisms of these bone active agents. Combining ZA with PTH treatment reduced the PTH-mediated increase in luciferase BMSC activity, serum osteocalcin, and serum tartrate resistant acid phosphotase-5b (TRAP-5b) but ZA did not reduce the PTH-induced increase in total bone. Since zoledronic acid reduced PTH-induced proliferation without reducing bone volume, these data suggest that combining PTH and bisphosphonate therapy warrants further investigation in the treatment of bone disorders.

  5. Thyrotropin Stimulates Differentiation Not Proliferation of Normal Human Thyrocytes in Culture

    PubMed Central

    Morgan, Sarah J.; Neumann, Susanne; Marcus-Samuels, Bernice; Gershengorn, Marvin C.

    2016-01-01

    Although TSH has been suggested to be a proliferative agent for thyrocytes, the effect of TSH on human thyroid cells remains controversial. In particular, most of the reported studies relied primarily on changes in DNA synthesis but have not included measurement of the number of cells. We argue that only a direct count of cell number, demonstrating classical exponential expansion, serves as a valid measurement of proliferation. Thus, although some data support TSH as a proliferative agent, most do not provide conclusive evidence. To generate conclusive evidence with regard to a proliferative effect of TSH in human thyrocytes, we performed various experiments using primary cultures of human thyrocytes. In contrast to previous reports, TSH [±insulin-like growth factor 1 (IGF-1)] did not induce proliferation of thyrocytes under a variety of different conditions. However, TSH/IGF-1 cotreatment did upregulate thyroid-specific gene expression including thyroglobulin (TG) and TSHR in a manner consistent with cellular differentiation. Evidence for a proliferative effect of TSH has been used to inform the American Thyroid Association’s guidelines for the management of thyroid cancer patients, which include TSH suppression. While these recommendations are admittedly based on low- to moderate-quality evidence, TSH suppression is still widely used. We present data that question the consensus view that TSH promotes proliferation of human thyrocytes (upon which the American Thyroid Association’s guidelines are based) and suggest that additional studies, including randomized controlled trials, are warranted to address this important clinical question. PMID:28082948

  6. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    PubMed Central

    Bruno, Stefania; Grange, Cristina; Tapparo, Marta; Pasquino, Chiara; Romagnoli, Renato; Dametto, Ennia; Amoroso, Antonio; Tetta, Ciro; Camussi, Giovanni

    2016-01-01

    Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response. PMID:27127520

  7. Neural differentiation, NCAM-mediated adhesion, and gap junctional communication in neuroectoderm. A study in vitro

    PubMed Central

    1988-01-01

    We studied the development of NCAM and gap junctional communication, and their mutual relationship in chick neuroectoderm in vitro. Expression of NCAM, as detected by monoclonal and polyclonal antibodies, and development of junctional communication, as detected by extensive cell-to-cell transfer of 400-500-D fluorescent tracers, occurred in cultures from stage-2 embryos onward. Both expressions presumably required primary induction. The differentiating cells formed discrete fields of expression on the second to third day in culture, with the NCAM fields coinciding with the junctional communication fields delineated by the tracers. Other neural differentiations developed in the following order: tetanus toxin receptors, neurofilament protein, and neurite outgrowth. Chronic treatment with antibody Fab fragments against NCAM interfered with the development of communication, suggesting that NCAM-mediated adhesion promotes formation of cell-to-cell channels. Temperature-sensitive mutant Rous sarcoma virus blocked (reversibly) communication and the subsequent development of neurofilament protein and neurites, but expression of NCAM continued. PMID:2834404

  8. RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation.

    PubMed

    Morales, Mònica; Arenas, Enrique J; Urosevic, Jelena; Guiu, Marc; Fernández, Esther; Planet, Evarist; Fenwick, Robert Bryn; Fernández-Ruiz, Sonia; Salvatella, Xavier; Reverter, David; Carracedo, Arkaitz; Massagué, Joan; Gomis, Roger R

    2014-07-01

    In estrogen receptor-negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease. Thus, a better understanding of the biology of metastasis is needed. In particular, biomarkers to identify patients that are at risk of lung metastasis could open the avenue for new therapeutic opportunities. Here we characterize the biological activity of RARRES3, a new metastasis suppressor gene whose reduced expression in the primary breast tumors identifies a subgroup of patients more likely to develop lung metastasis. We show that RARRES3 downregulation engages metastasis-initiating capabilities by facilitating adhesion of the tumor cells to the lung parenchyma. In addition, impaired tumor cell differentiation due to the loss of RARRES3 phospholipase A1/A2 activity also contributes to lung metastasis. Our results establish RARRES3 downregulation as a potential biomarker to identify patients at high risk of lung metastasis who might benefit from a differentiation treatment in the adjuvant programme.

  9. Adhesion, vitality and osteogenic differentiation capacity of adipose derived stem cells seeded on nitinol nanoparticle coatings.

    PubMed

    Strauss, Sarah; Neumeister, Anne; Barcikowski, Stephan; Kracht, Dietmar; Kuhbier, Jörn W; Radtke, Christine; Reimers, Kerstin; Vogt, Peter M

    2013-01-01

    Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells.

  10. Adhesion, Vitality and Osteogenic Differentiation Capacity of Adipose Derived Stem Cells Seeded on Nitinol Nanoparticle Coatings

    PubMed Central

    Strauß, Sarah; Neumeister, Anne; Barcikowski, Stephan; Kracht, Dietmar; Kuhbier, Jörn W.; Radtke, Christine; Reimers, Kerstin; Vogt, Peter M.

    2013-01-01

    Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells. PMID:23308190

  11. RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation

    PubMed Central

    Morales, Mònica; Arenas, Enrique J; Urosevic, Jelena; Guiu, Marc; Fernández, Esther; Planet, Evarist; Fenwick, Robert Bryn; Fernández-Ruiz, Sonia; Salvatella, Xavier; Reverter, David; Carracedo, Arkaitz; Massagué, Joan; Gomis, Roger R

    2014-01-01

    In estrogen receptor-negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease. Thus, a better understanding of the biology of metastasis is needed. In particular, biomarkers to identify patients that are at risk of lung metastasis could open the avenue for new therapeutic opportunities. Here we characterize the biological activity of RARRES3, a new metastasis suppressor gene whose reduced expression in the primary breast tumors identifies a subgroup of patients more likely to develop lung metastasis. We show that RARRES3 downregulation engages metastasis-initiating capabilities by facilitating adhesion of the tumor cells to the lung parenchyma. In addition, impaired tumor cell differentiation due to the loss of RARRES3 phospholipase A1/A2 activity also contributes to lung metastasis. Our results establish RARRES3 downregulation as a potential biomarker to identify patients at high risk of lung metastasis who might benefit from a differentiation treatment in the adjuvant programme. PMID:24867881

  12. Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

    PubMed

    Sanfelice, Raquel Arruda; da Silva, Suelen Santos; Bosqui, Larissa Rodrigues; Miranda-Sapla, Milena Menegazzo; Barbosa, Bellisa Freitas; Silva, Rafaela José; Ferro, Eloísa A Vieira; Panagio, Luciano Aparecido; Navarro, Italmar Teodorico; Bordignon, Juliano; Conchon-Costa, Ivete; Pavanelli, Wander Rogerio; Almeida, Ricardo Sergio; Costa, Idessania Nazareth

    2017-03-01

    The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×10(5)) were infected with T. gondii tachyzoites (5×10(5)) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×10(5)) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms

  13. Electrical stimulation by enzymatic biofuel cell to promote proliferation, migration and differentiation of muscle precursor cells.

    PubMed

    Lee, Jae Ho; Jeon, Won-Yong; Kim, Hyug-Han; Lee, Eun-Jung; Kim, Hae-Won

    2015-01-01

    Electrical stimulation is a very important biophysical cue for skeletal muscle maintenance and myotube formation. The absence of electrical signals from motor neurons causes denervated muscles to atrophy. Herein, we investigate for the first time the utility of an enzymatic biofuel cell (EBFC) as a promising means for mimicking native electrical stimulation. EBFC was set up using two different enzymes: one was glucose oxidase (GOX) used for the generation of anodic current followed by the oxidation of glucose; the other was Bilirubin oxidase (BOD) for the generation of cathodic current followed by the reduction of oxygen. We studied the behaviors of muscle precursor cells (MPCs) in terms of proliferation, migration and differentiation under different electrical conditions. The EBFC electrical stimulations significantly increased cell proliferation and migration. Furthermore, the electrical stimulations promoted the differentiation of cells into myotube formation based on expressions at the gene and protein levels. The EBFC set up, with its free forms adjustable to any implant design, was subsequently applied to the nanofiber scaffolding system. The MPCs were demonstrated to be stimulated in a similar manner as the 2D culture conditions, suggesting potential applications of the EBFC system for muscle repair and regeneration.

  14. Tenascin-W inhibits proliferation and differentiation of preosteoblasts during endochondral bone formation

    SciTech Connect

    Kimura, Hiroaki; Akiyama, Haruhiko . E-mail: hakiyama@kuhp.kyoto-u.ac.jp; Nakamura, Takashi; Crombrugghe, Benoit de

    2007-05-18

    We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterix-null embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblastic cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TN-W suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.

  15. MRI reporter genes: applications for imaging of cell survival, proliferation, migration and differentiation.

    PubMed

    Vandsburger, Moriel H; Radoul, Marina; Cohen, Batya; Neeman, Michal

    2013-07-01

    Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly or with targeted contrast media. However many genes and proteins and particularly those located in intracellular compartments are not accessible for targeted agents. The transcriptional regulation of these genes can nevertheless be detected, although indirectly, using reporter gene encoding for readily detectable proteins. Such reporter proteins can be expressed in the tissue of interest by genetically introducing the reporter gene in the target cells. Imaging of reporter genes has become a powerful tool in modern biomedical research. Typically, expression of fluorescent and bioluminescent proteins and the reaction product of expressed enzymes and exogenous substrates were examined using in vitro histological methods and in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raised the possibility that MRI could become a powerful tool for concomitant high-resolution anatomical and functional imaging and for imaging of reporter gene activity. An immediate application of MRI reporter gene methods was by monitoring gene expression patterns in gene therapy and in vivo imaging of the survival, proliferation, migration and differentiation of pluripotent and multipotent cells used in cell-based regenerative therapies for cancer, myocardial infarction and neural degeneration. In this review, we characterized a variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, migration and differentiation. In particular, we discussed which methods were best suited for translation to clinical use in regenerative therapies.

  16. TMPRSS2:ERG blocks neuroendocrine and luminal cell differentiation to maintain prostate cancer proliferation.

    PubMed

    Mounir, Z; Lin, F; Lin, V G; Korn, J M; Yu, Y; Valdez, R; Aina, O H; Buchwalter, G; Jaffe, A B; Korpal, M; Zhu, P; Brown, M; Cardiff, R D; Rocnik, J L; Yang, Y; Pagliarini, R

    2015-07-01

    The biological outcome of TMPRSS2:ERG chromosomal translocations in prostate cancer (PC) remains poorly understood. To address this, we compared the transcriptional effects of TMPRSS2:ERG expression in a transgenic mouse model with those of ERG knockdown in a TMPRSS2:ERG-positive PC cell line. This reveals that ERG represses the expression of a previously unreported set of androgen receptor (AR)-independent neuronal genes that are indicative of neuroendocrine (NE) cell differentiation-in addition to previously reported AR-regulated luminal genes. Cell sorting and proliferation assays performed after sustained ERG knockdown indicate that ERG drives proliferation and blocks the differentiation of prostate cells to both NE and luminal cell types. Inhibition of ERG expression in TMPRSS2:ERG-positive PC cells through blockade of AR signaling is tracked with increased NE gene expression. We also provide evidence that these NE cells are resistant to pharmacological AR inhibition and can revert to the phenotype of parental cells upon restoration of AR/ERG signaling. Our findings highlight an ERG-regulated mechanism capable of repopulating the parent tumor through the transient generation of an anti-androgen therapy-resistant cell population, suggesting that ERG may have a direct role in preventing resistance to anti-androgen therapy.

  17. Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation

    PubMed Central

    Tan, Si Hui; Senarath-Yapa, Kshemendra; Chung, Michael T.; Longaker, Michael T.; Wu, Joy Y.; Nusse, Roeland

    2014-01-01

    Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated—to our knowledge for the first time—that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation. PMID:25422448

  18. Abnormal proliferation and differentiation of osteoblasts from girls with adolescent idiopathic scoliosis to melatonin.

    PubMed

    Man, Gene Chi-Wai; Wang, William Wei-Jun; Yeung, Benson Hiu-Yan; Lee, Simon Kwong-Man; Ng, Bobby Kin-Ah; Hung, Wing-Yin; Wong, Jack Ho; Ng, Tzi-Bun; Qiu, Yong; Cheng, Jack Chun-Yiu

    2010-08-01

    Melatonin deficiency has been postulated as an etiologic factors in adolescent idiopathic scoliosis (AIS). In previous studies, melatonin was shown to regulate skeletal growth and bone formation in both humans and rats. Although it remains controversial whether there are differences in serum melatonin level between AIS and control subjects, melatonin signaling pathway dysfunction in osteoblasts has been reported in patients with AIS. Recently, our group found that melatonin receptor 1B (MT2) gene polymorphism was associated with the occurrence of AIS. Hence, the present study investigated the effect of melatonin on AIS osteoblasts. In vitro assays were performed with osteoblasts isolated from 17 severe AIS girls and nine control subjects. The osteoblasts were exposed to different concentrations of melatonin for 3 days. The effects of melatonin on cell proliferation (as evidenced by MTT assay) and differentiation (demonstrated by alkaline phosphatase activity) were determined. In the control group, melatonin significantly stimulated osteoblasts to proliferate and differentiate. However, in the AIS group, the stimulatory effects of melatonin were not discernible. Importantly, this finding demonstrated that there is a significant difference between AIS and control osteoblasts in functional response toward melatonin. Melatonin-stimulated proliferation of control osteoblasts was inhibited by the MT2 antagonist, 4-phenyl-2-propionamidotetraline, as well as by luzindole, a nonselective melatonin receptor antagonist, suggesting that MT2 is associated with the proliferative action of melatonin. The lack of response in AIS osteoblasts might be because of dysfunction of the melatonin signaling pathway, which may contribute to the low bone mineral density and abnormal skeletal growth observed in patients with AIS.

  19. Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway.

    PubMed

    Dai, Jie-Min; Yu, Mu-Xue; Shen, Zhen-Yu; Guo, Chu-Yi; Zhuang, Si-Qi; Qiu, Xiao-Shan

    2015-05-08

    Signaling through the mammalian target of rapamycin (mTOR) in response to leucine modulates many cellular and developmental processes. However, in the context of satellite cell proliferation and differentiation, the role of leucine and mTORC1 is less known. This study investigates the role of leucine in the process of proliferation and differentiation of primary preterm rat satellite cells, and the relationship with mammalian target of rapamycin complex 1 (mTORC1) activation. Dissociation of primary satellite cells occurred with type I collagenase and trypsin, and purification, via different speed adherence methods. Satellite cells with positive expression of Desmin were treated with leucine and rapamycin. We observed that leucine promoted proliferation and differentiation of primary satellite cells and increased the phosphorylation of mTOR. Rapamycin inhibited proliferation and differentiation, as well as decreased the phosphorylation level of mTOR. Furthermore, leucine increased the expression of MyoD and myogenin while the protein level of MyoD decreased due to rapamycin. However, myogenin expressed no affect by rapamycin. In conclusion, leucine may up-regulate the activation of mTORC1 to promote proliferation and differentiation of primary preterm rat satellite cells. We have shown that leucine promoted the differentiation of myotubes in part through the mTORC1-MyoD signal pathway.

  20. SDF-1 and CXCR4 play an important role in adult SVZ lineage cell proliferation and differentiation.

    PubMed

    Zhu, Chang; Yao, Wen-Long; Tan, Wei; Zhang, Chuan-Han

    2017-02-15

    Evidence has shown that stromal cell-derived factor (SDF-1/CXCL12) plays an important role in maintaining adult neural progenitor cells (NPCs). SDF-1 is also known to enhance recovery by recruiting NPCs to damaged regions and recent studies have revealed that SDF-1α exhibits pleiotropism, thereby differentially affecting NPC subpopulations. In this study, we investigated the role of SDF-1 in in vitro NPC self-renewal, proliferation and differentiation, following treatment with different concentrations of SDF-1 or a CXCR4 antagonist, AMD3100. We observed that AMD3100 inhibited the formation of primary neurospheres. However, SDF-1 and AMD3100 exhibited no effect on proliferation upon inclusion of growth factors in the media. Following growth factor withdrawal, AMD3100 and SDF-1 treatment resulted in differential effects on NPC proliferation. SDF-1, at a concentration of 500ng/ml, resulted in an increase in the relative proportion of oligodendrocytes following growth factor withdrawal-induced differentiation. Using CXCR4 knockout mice, we observed that SDF-1 affected NPC proliferation in the sub-ventricular zone (SVZ). We also investigated the occurrence of differential CXCR4 expression at different stages during lineage progression. These results clearly indicate that signaling interactions between SDF-1 and CXCR4 play an important role in adult SVZ lineage cell proliferation and differentiation.

  1. NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

    PubMed

    Hu, Xiu-Xiu; Zhong, Liang; Zhang, Xi; Gao, Yuan-Mei; Liu, Bei-Zhong

    2014-01-01

    A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The

  2. Surfactant functionalization induces robust, differential adhesion of tumor cells and blood cells to charged nanotube-coated biomaterials under flow.

    PubMed

    Mitchell, Michael J; Castellanos, Carlos A; King, Michael R

    2015-07-01

    The metastatic spread of cancer cells from the primary tumor to distant sites leads to a poor prognosis in cancers originating from multiple organs. Increasing evidence has linked selectin-based adhesion between circulating tumor cells (CTCs) and endothelial cells of the microvasculature to metastatic dissemination, in a manner similar to leukocyte adhesion during inflammation. Functionalized biomaterial surfaces hold promise as a diagnostic tool to separate CTCs and potentially treat metastasis, utilizing antibody and selectin-mediated interactions for cell capture under flow. However, capture at high purity levels is challenged by the fact that CTCs and leukocytes both possess selectin ligands. Here, a straightforward technique to functionalize and alter the charge of naturally occurring halloysite nanotubes using surfactants is reported to induce robust, differential adhesion of tumor cells and blood cells to nanotube-coated surfaces under flow. Negatively charged sodium dodecanoate-functionalized nanotubes simultaneously enhanced tumor cell capture while negating leukocyte adhesion, both in the presence and absence of adhesion proteins, and can be utilized to isolate circulating tumor cells regardless of biomarker expression. Conversely, diminishing nanotube charge via functionalization with decyltrimethylammonium bromide both abolished tumor cell capture while promoting leukocyte adhesion.

  3. Surfactant Functionalization Induces Robust, Differential Adhesion of Tumor Cells and Blood Cells to Charged Nanotube-Coated Biomaterials Under Flow

    PubMed Central

    Mitchell, Michael J.; Castellanos, Carlos A.; King, Michael R.

    2015-01-01

    The metastatic spread of cancer cells from the primary tumor to distant sites leads to a poor prognosis in cancers originating from multiple organs. Increasing evidence has linked selectin-based adhesion between circulating tumor cells (CTCs) and endothelial cells of the microvasculature to metastatic dissemination, in a manner similar to leukocyte adhesion during inflammation. Functionalized biomaterial surfaces hold promise as a diagnostic tool to separate CTCs and potentially treat metastasis, utilizing antibody and selectin-mediated interactions for cell capture under flow. However, capture at high purity levels is challenged by the fact that CTCs and leukocytes both possess selectin ligands. Here, a straightforward technique to functionalize and alter the charge of naturally occurring halloysite nanotubes using surfactants is reported to induce robust, differential adhesion of tumor cells and blood cells to nanotube-coated surfaces under flow. Negatively charged sodium dodecanoate-functionalized nanotubes simultaneously enhanced tumor cell capture while negating leukocyte adhesion, both in the presence and absence of adhesion proteins, and can be utilized to isolate circulating tumor cells regardless of biomarker expression. Conversely, diminishing nanotube charge via functionalization with decyltrimethylammonium bromide both abolished tumor cell capture while promoting leukocyte adhesion. PMID:25934290

  4. β1 integrin adhesion enhances IL-6 mediated STAT3 signaling in Myeloma cells: Implications for Microenvironment Influence on Tumor Survival and Proliferation

    PubMed Central

    Shain, Kenneth H.; Yarde, Danielle N.; Meads, Mark B.; Huang, Mei; Jove, Richard; Hazlehurst, Lori A.; Dalton, William S.

    2009-01-01

    The bone marrow microenvironmental components interleukin (IL)-6 and fibronectin (FN) individually influence the proliferation and survival of Multiple Myeloma (MM) cells; however, in vivo these effectors most likely work together. We examined signaling events, cell cycle progression, and levels of drug response in MM cells either adhered to FN via β1 integrins, stimulated with IL-6, or treated with the two combined. While G1/S cell cycle arrest associated with FN adhesion was overcome when IL-6 as added, the cell adhesion mediated drug resistance (CAM-DR) was maintained in the presence of IL-6. Concomitant exposure of MM cells to IL-6 and FN adhesion revealed a dramatic increase in STAT3 phosphorylation, nuclear translocation and DNA-binding, as compared to either IL-6 or FN adhesion alone in four MM cell lines. Importantly, this increase in STAT3 activation correlated with a novel association between STAT3 and gp130 in cells adhered to FN prior to stimulation with IL-6, relative to non-adherent cells. Taken together, these results suggest a mechanism by which collaborative signaling by β1 integrin and gp130 confers an increased survival advantage to MM cells. PMID:19155309

  5. Ectopic TBX1 suppresses thymic epithelial cell differentiation and proliferation during thymus organogenesis.

    PubMed

    Reeh, Kaitlin A G; Cardenas, Kim T; Bain, Virginia E; Liu, Zhijie; Laurent, Micheline; Manley, Nancy R; Richie, Ellen R

    2014-08-01

    The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis.

  6. 10e12z CLA alters adipocyte differentiation and adipocyte cytokine expression and induces macrophage proliferation.

    PubMed

    Belda, Benjamin J; Thompson, Jerry T; Eser, Pinar O; Vanden Heuvel, John P

    2012-05-01

    The trans-10, cis-12 (10e12z) conjugated linoleic acid (CLA) isomer of CLA is responsible for loss of lipid storage or adipose tissue in vitro or in vivo. This isomer also induces inflammatory signaling in both mouse and human adipocytes in vitro. However, when these events occur and whether they are significant enough to affect other cell types are unclear. In these experiments, the 3T3-L1 cell line has been used to examine the interaction between inflammatory signaling and decreased differentiation or lipid storage induced by 10e12z CLA. In assays measuring both lipid accumulation and gene expression, differentiating 3T3-L1 cells exhibit concurrent induction of inflammatory signaling, as measured by cyclooxygenase-2 expression, and a decrease in adipocyte marker gene expression. Furthermore, in fully differentiated adipocytes, as identified in microarray assays and confirmed with real-time polymerase chain reaction, 10e12z CLA also significantly affected expression of both matrix metalloprotein-3 (MMP-3), collagen VI α 3 ColVI alpha 3 (VIα3) and the cytokine epiregulin, demonstrating that the effects of 10e12z broadly impact adipocyte function. In agreement with other experimental systems, 10e12z CLA inhibited RAW 264.7 cell proliferation; however, in response to adipocyte-conditioned media, 10e12z-CLA-treated adipocytes induced proliferation of this cell line, suggesting that the effect of 10e12z CLA is context dependent. These results are largely consistent with the known activation of the inflammatory mediator nuclear factor-κB in adipocytes in vitro and in vivo by 10e12z CLA treatment and demonstrate that adipose is an important target tissue of this isomer that impacts other cell types.

  7. Kv3.1 channels stimulate adult neural precursor cell proliferation and neuronal differentiation.

    PubMed

    Yasuda, Takahiro; Cuny, Hartmut; Adams, David J

    2013-05-15

    Adult neural stem/precursor cells (NPCs) play a pivotal role in neuronal plasticity throughout life. Among ion channels identified in adult NPCs, voltage-gated delayed rectifier K(+) (KDR) channels are dominantly expressed. However, the KDR channel subtype and its physiological role are still undefined. We used real-time quantitative RT-PCR and gene knockdown techniques to identify a major functional KDR channel subtype in adult NPCs. Dominant mRNA expression of Kv3.1, a high voltage-gated KDR channel, was quantitatively confirmed. Kv3.1 gene knockdown with specific small interfering RNAs (siRNA) for Kv3.1 significantly inhibited Kv3.1 mRNA expression by 63.9% (P < 0.001) and KDR channel currents by 52.2% (P < 0.001). This indicates that Kv3.1 is the subtype responsible for producing KDR channel outward currents. Resting membrane properties, such as resting membrane potential, of NPCs were not affected by Kv3.1 expression. Kv3.1 knockdown with 300 nm siRNA inhibited NPC growth (increase in cell numbers) by 52.9% (P < 0.01). This inhibition was attributed to decreased cell proliferation, not increased cell apoptosis. We also established a convenient in vitro imaging assay system to evaluate NPC differentiation using NPCs from doublecortin-green fluorescent protein transgenic mice. Kv3.1 knockdown also significantly reduced neuronal differentiation by 31.4% (P < 0.01). We have demonstrated that Kv3.1 is a dominant functional KDR channel subtype expressed in adult NPCs and plays key roles in NPC proliferation and neuronal lineage commitment during differentiation.

  8. Repeated morphine treatment alters polysialylated neural cell adhesion molecule, glutamate decarboxylase-67 expression and cell proliferation in the adult rat hippocampus.

    PubMed

    Kahn, Laëtitia; Alonso, Gérard; Normand, Elisabeth; Manzoni, Olivier J

    2005-01-01

    Altered synaptic transmission and plasticity in brain areas involved in reward and learning are thought to underlie the long-lasting effects of addictive drugs. In support of this idea, opiates reduce neurogenesis [A.J. Eisch et al. (2000) Proceedings of the National Academy of Sciences USA, 97, 7579-7584] and enhance long-term potentiation in adult rodent hippocampus [J.M. Harrison et al. (2002) Journal of Neurophysiology, 87, 2464-2470], a key structure of learning and memory processes. Here we studied how repeated morphine treatment and withdrawal affect cell proliferation and neuronal phenotypes in the dentate gyrus-CA3 region of the adult rat hippocampus. Our data showed a strong reduction of cellular proliferation in morphine-dependent animals (54% of control) that was followed by a rebound increase after 1 week withdrawal and a return to normal after 2 weeks withdrawal. Morphine dependence was also associated with a drastic reduction in the expression levels of the polysialylated form of neural cell adhesion molecule (68% of control), an adhesion molecule expressed by newly generated neurons and involved in cell migration and structural plasticity. Polysialylated neural cell adhesion molecule levels quickly returned to normal following withdrawal. In morphine-dependent rats, we found a significant increase of glutamate decarboxylase-67 mRNA transcription (170% of control) in dentate gyrus granular cells which was followed by a marked rebound decrease after 1 week withdrawal and a return to normal after 4 weeks withdrawal. Together, the results show, for the first time, that, in addition to reducing cell proliferation and neurogenesis, chronic exposure to morphine dramatically alters neuronal phenotypes in the dentate gyrus-CA3 region of the adult rat hippocampus.

  9. BMP4-induced differentiation of a rat spermatogonial stem cell line causes changes in its cell adhesion properties.

    PubMed

    Carlomagno, Gianfranco; van Bragt, Maaike P A; Korver, Cindy M; Repping, Sjoerd; de Rooij, Dirk G; van Pelt, Ans M M

    2010-11-01

    Spermatogonial stem cells (SSCs) are at the basis of the spermatogenic process and are essential for the continuous lifelong production of spermatozoa. Although several factors that govern SSC self-renewal and differentiation have been investigated, the direct effect of such factors on SSCs has not yet been studied, mainly because of the absence of markers to identify SSCs and the lack of effective methods to obtain and culture a pure population of SSCs. We now have used a previously established rat SSC cell line (GC-6spg) to elucidate the role of BMP4 in SSC differentiation. We found that GC-6spg cells cultured in the presence of BMP4 upregulate KIT expression, which is an early marker for differentiating spermatogonia. GC-6spg cells were found to express three BMP4 receptors and the downstream SMAD1/5/8 proteins were phosphorylated during BMP4-induced differentiation. A time-course DNA micro-array analysis revealed a total of 529 differentially regulated transcripts (≥2-fold), including several known downstream targets of BMP4 such as Id2 and Gata2. Pathway analysis revealed that the most affected pathways were those involved in adherens junctions, focal junctions, gap junctions, cell adhesion molecules, and regulation of actin cytoskeleton. Interestingly, among the genes belonging to the most strongly affected adhesion pathways was Cdh1 (known as E-cadherin), an adhesion molecule known to be expressed by a subpopulation of spermatogonia including SSCs. Overall, our results suggest that BMP4 induces early differentiation of SSCs in a direct manner by affecting cell adhesion pathways.

  10. Exosome complex orchestrates developmental signaling to balance proliferation and differentiation during erythropoiesis

    PubMed Central

    McIver, Skye C; Katsumura, Koichi R; Davids, Elsa; Liu, Peng; Kang, Yoon-A; Yang, David; Bresnick, Emery H

    2016-01-01

    Since the highly conserved exosome complex mediates the degradation and processing of multiple classes of RNAs, it almost certainly controls diverse biological processes. How this post-transcriptional RNA-regulatory machine impacts cell fate decisions and differentiation is poorly understood. Previously, we demonstrated that exosome complex subunits confer an erythroid maturation barricade, and the erythroid transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the cognate genes. While dissecting requirements for the maturation barricade in Mus musculus, we discovered that the exosome complex is a vital determinant of a developmental signaling transition that dictates proliferation/amplification versus differentiation. Exosome complex integrity in erythroid precursor cells ensures Kit receptor tyrosine kinase expression and stem cell factor/Kit signaling, while preventing responsiveness to erythropoietin-instigated signals that promote differentiation. Functioning as a gatekeeper of this developmental signaling transition, the exosome complex controls the massive production of erythroid cells that ensures organismal survival in homeostatic and stress contexts. DOI: http://dx.doi.org/10.7554/eLife.17877.001 PMID:27543448

  11. Invariant NKT cells require autophagy to coordinate proliferation and survival signals during differentiation.

    PubMed

    Pei, Bo; Zhao, Meng; Miller, Brian C; Véla, Jose Luis; Bruinsma, Monique W; Virgin, Herbert W; Kronenberg, Mitchell

    2015-06-15

    Autophagy regulates cell differentiation, proliferation, and survival in multiple cell types, including cells of the immune system. In this study, we examined the effects of a disruption of autophagy on the differentiation of invariant NKT (iNKT) cells. Using mice with a T lymphocyte-specific deletion of Atg5 or Atg7, two members of the macroautophagic pathway, we observed a profound decrease in the iNKT cell population. The deficit is cell-autonomous, and it acts predominantly to reduce the number of mature cells, as well as the function of peripheral iNKT cells. In the absence of autophagy, there is reduced progression of iNKT cells in the thymus through the cell cycle, as well as increased apoptosis of these cells. Importantly, the reduction in Th1-biased iNKT cells is most pronounced, leading to a selective reduction in iNKT cell-derived IFN-γ. Our findings highlight the unique metabolic and genetic requirements for the differentiation of iNKT cells.

  12. Heterogeneous activation of a slow myosin gene in proliferating myoblasts and differentiated single myofibers

    PubMed Central

    Wang, Jing-hua; Wang, Qiao-jing; Wang, Chao; Reinholt, Brad; Grant, Alan L; Gerrard, David E; Kuang, Shihuan

    2015-01-01

    Each skeletal muscle contains a fixed ratio of fast and slow myofibers that are distributed in a stereotyped pattern to achieve a specific motor function. How myofibers are specified during development and regeneration is poorly understood. Here we address this question using transgenic reporter mice that indelibly mark the myofiber lineages based on activation of fast or slow myosin. Lineage tracing indicates that during development all muscles have activated the fast myosin gene Myl1, but not the slow myosin gene Myh7, which is activated in all slow but a subset of fast myofibers. Similarly, most nascent myofibers do not activate Myh7 during fast muscle regeneration, but the ratio and pattern of fast and slow myofibers are restored at the completion of regeneration. At the single myofiber level, most mature fast myofibers are heterogeneous in nuclear composition, manifested by mosaic activation of Myh7. Strikingly, Myh7 is activated in a subpopulation of proliferating myoblasts that co-express the myogenic progenitor marker Pax7. When induced to differentiate, the Myh7-activated myoblasts differentiate more readily than the non-activated myoblasts, and have a higher tendency, but not restricted, to become slow myotubes. Together, our data reveal significant nuclear heterogeneity within a single myofiber, and challenge the conventional view that myosin genes are only expressed after myogenic differentiation. These results provide novel insights into the regulation of muscle fiber type specification. PMID:25794679

  13. Different Culture Media Affect Proliferation, Surface Epitope Expression, and Differentiation of Ovine MSC

    PubMed Central

    Tolba, René; Jahnen-Dechent, Wilhelm; Lethaus, Bernd; Neuss, Sabine

    2013-01-01

    Orthopedic implants including engineered bone tissue are commonly tested in sheep. To avoid rejection of heterologous or xenogeneic cells, autologous cells are preferably used, that is, ovine mesenchymal stem cells (oMSC). Unlike human MSC, ovine MSC are not well studied regarding isolation, expansion, and characterization. Here we investigated the impact of culture media composition on growth characteristics, differentiation, and surface antigen expression of oMSC. The culture media varied in fetal calf serum (FCS) content and in the addition of supplements and/or additional epidermal growth factor (EGF). We found that FCS strongly influenced oMSC proliferation and that specific combinations of supplemental factors (MCDB-201, ITS-plus, dexamethasone, and L-ascorbic acid) determined the expression of surface epitopes. We compared two published protocols for oMSC differentiation towards the osteogenic, adipogenic, and chondrogenic fate and found (i) considerable donor to donor variations, (ii) protocol-dependent variations, and (iii) variations resulting from the preculture medium composition. Our results indicate that the isolation and culture of oMSC in different growth media are highly variable regarding oMSC phenotype and behaviour. Furthermore, variations from donor to donor critically influence growth rate, surface marker expression, and differentiation. PMID:24228035

  14. Adhesion protein VSIG1 is required for the proper differentiation of glandular gastric epithelia.

    PubMed

    Oidovsambuu, Odgerel; Nyamsuren, Gunsmaa; Liu, Shuai; Göring, Wolfgang; Engel, Wolfgang; Adham, Ibrahim M

    2011-01-01

    VSIG1, a cell adhesion protein of the immunoglobulin superfamily, is preferentially expressed in stomach, testis, and certain gastric, esophageal and ovarian cancers. Here, we describe the expression patterns of three alternatively spliced isoforms of mouse Vsig1 during pre- and postnatal development of stomach and potential function of Vsig1 in differentiation of gastric epithelia. We show that isoforms Vsig1A and Vsig1B, which differ in the 3'untranslated region, are expressed in the early stages of stomach development. Immunohistochemical analysis revealed that VSIG1 is restricted to the adherens junction of the glandular epithelium. The shorter transcript Vsig1C is restricted to the testis, encodes an N-terminal truncated protein and is presumably regulated by an internal promoter, which is located upstream of exon 1b. To determine whether the 5' flanking region of exon 1a specifically targets the expression of Vsig1 to stomach epithelia, we generated and analyzed transgenic mice. The 4.8-kb fragment located upstream of exon 1a was sufficient to direct the expression of the reporter gene to the glandular epithelia of transgenic stomach. To determine the role of VSIG1 during the development of stomach epithelia, an X-linked Vsig1 was inactivated in embryonic stem cells (ESCs). Although Vsig1(-/Y) ESCs were only able to generate low coat color chimeric mice, no male chimeras transmitted the targeted allele to their progeny suggesting that the high contribution of Vsig1(-/Y) cells leads to the lethality of chimeric embryos. Analysis of chimeric stomachs revealed the differentiation of VSIG1-null cells into squamous epithelia inside the glandular region. These results suggest that VSIG1 is required for the establishment of glandular versus squamous epithelia in the stomach.

  15. Meclozine facilitates proliferation and differentiation of chondrocytes by attenuating abnormally activated FGFR3 signaling in achondroplasia.

    PubMed

    Matsushita, Masaki; Kitoh, Hiroshi; Ohkawara, Bisei; Mishima, Kenichi; Kaneko, Hiroshi; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

    2013-01-01

    Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.

  16. Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells.

    PubMed

    Yi, Lan; Shan, Jian; Chen, Xin; Li, Guoqing; Li, Linwei; Tan, Hui; Su, Qi

    2016-09-01

    Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, calreticulin (CRT) was found to be downregulated in differentiated HL-60 cells treated with DADS. The present study investigated the role of CRT proteins in DADS-induced proliferation, invasion and differentiation in HL-60 cells. The present study demonstrated that DADS treatment significantly changed the morphology of HL-60 cells and caused the significant time-dependent downregulation of CRT. Small interfering RNA (siRNA)-mediated knockdown of CRT expression significantly inhibited proliferation, decreased invasion ability, increased the expression of cluster of differentiation (CD)11b and reduced the expression of CD33 in DADS-treated HL-60 cells. DADS also significantly affected cell proliferation, invasion and differentiation in CRT-overexpressed HL-60 cells. Nitroblue tetrazolium (NBT) reduction assays showed decreased NBT reduction activity in the CRT overexpression group and increased NBT reduction in the CRT siRNA group. Following treatment with DADS, the NBT reduction abilities in all groups were increased. In conclusion, the present study clearly demonstrates the downregulation of CRT during DADS-induced differentiation in HL-60 cells and indicates that CRT is involved in cell proliferation, invasion and differentiation in DADS-treated HL-60 cells.

  17. Proinflammatory cytokines differentially influence adult hippocampal cell proliferation depending upon the route and chronicity of administration.

    PubMed

    Seguin, Julie Anne; Brennan, Jordan; Mangano, Emily; Hayley, Shawn

    2009-01-01

    Disturbances of hippocampal plasticity, including impaired dendritic branching and reductions of neurogenesis, are provoked by stressful insults and may occur in depression. Although corticoids likely contribute to stressor-induced reductions of neurogenesis, other signaling messengers, including pro-inflammatory cytokines might also be involved. Accordingly, the present investigation assessed whether three proinflammatory cytokines, namely interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) (associated with depression) influenced cellular proliferation within the hippocampus. In this regard, systemic administration of TNF-alpha reduced 5-bromo-2-deoxyuridine (BrdU) labeling within the hippocampus, whereas IL-1beta and IL-6 had no such effect. However, repeated but not a single intra-hippocampal infusion of IL-6 and IL-1beta actually increased cellular proliferation and IL-6 infusion also enhanced microglial staining within the hippocampus. Yet, no changes in doublecortin expression were apparent, suggesting that the cytokine did not influence the birth of cells destined to become neurons. Essentially, the route of administration and chronicity of cytokine administration had a marked influence upon the nature of hippocampal alterations provoked, suggesting that cytokines may differentially regulate hippocampal plasticity in neuropsychiatric conditions.

  18. Normal proliferation and differentiation of Hoxc-8 transgenic chondrocytes in vitro

    PubMed Central

    Cormier, Stephania A; Mello, Maria Alice; Kappen, Claudia

    2003-01-01

    Background Hox genes encode transcription factors that are involved in pattern formation in the skeleton, and recent evidence suggests that they also play a role in the regulation of endochondral ossification. To analyze the role of Hoxc-8 in this process in more detail, we applied in vitro culture systems, using high density cultures of primary chondrocytes from neonatal mouse ribs. Results Cultured cells were characterized on the basis of morphology (light microscopy) and production of cartilage-specific extracellular matrix (sulfated proteoglycans and type II Collagen). Hypertrophy was demonstrated by increase in cell size, alkaline phosphatase activity and type X Collagen immunohistochemistry. Proliferation was assessed by BrdU uptake and flow cytometry. Unexpectedly, chondrocytes from Hoxc-8 transgenic mice, which exhibit delayed cartilage maturation in vivo [1], were able to proliferate and differentiate normally in our culture systems. This was the case even though freshly isolated Hoxc-8 transgenic chondrocytes exhibited significant molecular differences as measured by real-time quantitative PCR. Conclusions The results demonstrate that primary rib chondrocytes behave similar to published reports for chondrocytes from other sources, validating in vitro approaches for studies of Hox genes in the regulation of endochondral ossification. Our analysis of cartilage-producing cells from Hoxc-8 transgenic mice provides evidence that the cellular phenotype induced by Hoxc-8 overexpression in vivo is reversible in vitro. PMID:12713673

  19. Early-age heat exposure affects skeletal muscle satellite cell proliferation and differentiation in chicks.

    PubMed

    Halevy, O; Krispin, A; Leshem, Y; McMurtry, J P; Yahav, S

    2001-07-01

    Exposure of young chicks to thermal conditioning (TC; i.e., 37 degrees C for 24 h) resulted in significantly improved body and muscle growth at a later age. We hypothesized that TC causes an increase in satellite cell proliferation, necessary for further muscle hypertrophy. An immediate increase was observed in satellite cell DNA synthesis in culture and in vivo in response to TC of 3-day-old chicks to levels that were significantly higher than those of control chicks. This was accompanied by a marked induction of insulin-like growth factor-I (IFG-I), but not hepatocyte growth factor in the breast muscle. No significant difference between treatments in plasma IGF-I levels was observed. A marked elevation in muscle regulatory factors on day 5, followed by a decline in cell proliferation on day 6 together with continuous high levels of IGF-I in the TC chick muscle may indicate accelerated cell differentiation. These data suggest a central role for IGF-I in the immediate stimulation of satellite cell myogenic processes in response to heat exposure.

  20. Mesenchymal stem cell proliferation and differentiation on load-bearing trabecular Nitinol scaffolds.

    PubMed

    Gotman, Irena; Ben-David, Dror; Unger, Ronald E; Böse, Thomas; Gutmanas, Elazar Y; Kirkpatrick, C James

    2013-09-01

    Bone tissue regeneration in load-bearing regions of the body requires high-strength porous scaffolds capable of supporting angiogenesis and osteogenesis. 70% porous Nitinol (NiTi) scaffolds with a regular 3-D architecture resembling trabecular bone were produced from Ni foams using an original reactive vapor infiltration technique. The "trabecular Nitinol" scaffolds possessed a high compressive strength of 79 MPa and high permeability of 6.9×10(-6) cm2. The scaffolds were further modified to produce a near Ni-free surface layer and evaluated in terms of Ni ion release and human mesenchymal stem cell (hMSC) proliferation (AlamarBlue), differentiation (alkaline phosphatase activity, ALP) and mineralization (Alizarin Red S staining). Scanning electron microscopy was employed to qualitatively corroborate the results. hMSCs were able to adhere and proliferate on both as-produced and surface-modified trabecular NiTi scaffolds, to acquire an osteoblastic phenotype and produce a mineralized extracellular matrix. Both ALP activity and mineralization were increased on porous scaffolds compared to control polystyrene plates. Experiments in a model coculture system of microvascular endothelial cells and hMSCs demonstrated the formation of prevascular structures in trabecular NiTi scaffolds. These data suggest that load-bearing trabecular Nitinol scaffolds could be effective in regenerating damaged or lost bone tissue.

  1. Atf4 regulates chondrocyte proliferation and differentiation during endochondral ossification by activating Ihh transcription

    PubMed Central

    Wang, Weiguang; Lian, Na; Li, Lingzhen; Moss, Heather E.; Wang, Weixi; Perrien, Daniel S.; Elefteriou, Florent; Yang, Xiangli

    2009-01-01

    Activating transcription factor 4 (Atf4) is a leucine-zipper-containing protein of the cAMP response element-binding protein (CREB) family. Ablation of Atf4 (Atf4−/−) in mice leads to severe skeletal defects, including delayed ossification and low bone mass, short stature and short limbs. Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. In Atf4−/− growth plate, the typical columnar structure of proliferative chondrocytes is disturbed. The proliferative zone is shortened, whereas the hypertrophic zone is transiently expanded. The expression of Indian hedgehog (Ihh) is markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), is normal. Furthermore, forced expression of Atf4 in chondrocytes induces endogenous Ihh mRNA, and Atf4 directly binds to the Ihh promoter and activates its transcription. Supporting these findings, reactivation of Hh signaling pharmacologically in mouse limb explants corrects the Atf4−/− chondrocyte proliferation and short limb phenotypes. This study thus identifies Atf4 as a novel transcriptional activator of Ihh in chondrocytes that paces longitudinal bone growth by controlling growth plate chondrocyte proliferation and differentiation. PMID:19906842

  2. PROX1 Gene is Differentially Expressed in Oral Cancer and Reduces Cellular Proliferation

    PubMed Central

    Rodrigues, Maria F.S.D.; de Oliveira Rodini, Camila; de Aquino Xavier, Flávia C.; Paiva, Katiúcia B.; Severino, Patrícia; Moyses, Raquel A.; López, Rossana M.; DeCicco, Rafael; Rocha, Lília A.; Carvalho, Marcos B.; Tajara, Eloiza H.; Nunes, Fabio D.

    2014-01-01

    Abstract Homeobox genes are a family of transcription factors that play a pivotal role in embryogenesis. Prospero homeobox 1 (PROX1) has been shown to function as a tumor suppressor gene or oncogene in various types of cancer, including oral squamous cell carcinoma (OSCC). We have previously identified PROX1 as a downregulated gene in OSCC. The aim of this study is to clarify the underlying mechanism by which PROX1 regulates tumorigenicity of OSCC cells. PROX1 mRNA and protein expression levels were first investigated in 40 samples of OSCC and in nontumor margins. Methylation and amplification analysis was also performed to assess the epigenetic and genetic mechanisms involved in controlling PROX1 expression. OSCC cell line SCC9 was also transfected to stably express the PROX1 gene. Next, SCC9-PROX1-overexpressing cells and controls were subjected to proliferation, differentiation, apoptosis, migration, and invasion assays in vitro. OSCC samples showed reduced PROX1 expression levels compared with nontumor margins. PROX1 amplification was associated with better overall survival. PROX1 overexpression reduces cell proliferation and downregulates cyclin D1. PROX1-overexpressing cells also exhibited reduced CK18 and CK19 expression and transcriptionally altered the expression of WISP3, GATA3, NOTCH1, and E2F1. Our results suggest that PROX1 functions as a tumor suppressor gene in oral carcinogenesis. PMID:25526434

  3. p120-catenin and β-catenin differentially regulate cadherin adhesive function.

    PubMed

    Oas, Rebecca G; Nanes, Benjamin A; Esimai, Chimdimnma C; Vincent, Peter A; García, Andrés J; Kowalczyk, Andrew P

    2013-03-01

    Vascular endothelial (VE)-cadherin, the major adherens junction adhesion molecule in endothelial cells, interacts with p120-catenin and β-catenin through its cytoplasmic tail. However, the specific functional contributions of the catenins to the establishment of strong adhesion are not fully understood. Here we use bioengineering approaches to identify the roles of cadherin-catenin interactions in promoting strong cellular adhesion and the ability of the cells to spread on an adhesive surface. Our results demonstrate that the domain of VE-cadherin that binds to β-catenin is required for the establishment of strong steady-state adhesion strength. Surprisingly, p120 binding to the cadherin tail had no effect on the strength of adhesion when the available adhesive area was limited. Instead, the binding of VE-cadherin to p120 regulates adhesive contact area in a Rac1-dependent manner. These findings reveal that p120 and β-catenin have distinct but complementary roles in strengthening cadherin-mediated adhesion.

  4. Human Olfactory Mucosa Multipotent Mesenchymal Stromal Cells Promote Survival, Proliferation, and Differentiation of Human Hematopoietic Cells

    PubMed Central

    Diaz-Solano, Dylana; Wittig, Olga; Ayala-Grosso, Carlos; Pieruzzini, Rosalinda

    2012-01-01

    Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit–granulocyte/macrophage (CFU-GM) progenitors and CD34+ cells were found, at 43 days of co-culture. Reverse transcriptase–polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines. PMID:22471939

  5. Altered proliferation and differentiation properties of primary mammary epithelial cells from BRCA1 mutation carriers.

    PubMed

    Burga, Laura N; Tung, Nadine M; Troyan, Susan L; Bostina, Mihnea; Konstantinopoulos, Panagiotis A; Fountzilas, Helena; Spentzos, Dimitrios; Miron, Alexander; Yassin, Yosuf A; Lee, Bernard T; Wulf, Gerburg M

    2009-02-15

    Female BRCA1 mutation carriers have a nearly 80% probability of developing breast cancer during their life-time. We hypothesized that the breast epithelium at risk in BRCA1 mutation carriers harbors mammary epithelial cells (MEC) with altered proliferation and differentiation properties. Using a three-dimensional culture technique to grow MECs ex vivo, we found that the ability to form colonies, an indication of clonality, was restricted to the aldehyde dehydrogenase 1-positive fraction in MECs but not in HCC1937 BRCA1-mutant cancer cells. Primary MECs from BRCA1 mutation carriers (n = 9) had a 28% greater ability for clonal growth compared with normal controls (n = 6; P = 0.006), and their colonies were significantly larger. Colonies in controls and BRCA1 mutation carriers stained positive for BRCA1 by immunohistochemistry, and 79% of the examined single colonies from BRCA1 carriers retained heterozygosity for BRCA1 (ROH). Colonies from BRCA1 mutation carriers frequently showed high epidermal growth factor receptor (EGFR) expression (71% EGFR positive versus 44% in controls) and were negative for estrogen receptor (ERalpha; 32% ER negative, 44% mixed, 24% ER positive versus 90% ER positive in controls). Expression of CK14 and p63 were not significantly different. Microarray studies revealed that colonies from BRCA1-mutant PMECs anticipate expression profiles found in BRCA1-related tumors, and that the EGFR pathway is up-regulated. We conclude that BRCA1 haploinsufficiency leads to an increased ability for clonal growth and proliferation in the PMECs of BRCA1 mutation carriers, possibly as a result of EGFR pathway activation. These altered growth and differentiation properties may render BRCA1-mutant PMECs vulnerable to transformation and predispose to the development of ER-negative, EGFR-positive breast cancers.

  6. Altered Proliferation and Differentiation Properties of Primary Mammary Epithelial Cells from BRCA1 Mutation Carriers

    PubMed Central

    Burga, Laura N.; Tung, Nadine M.; Troyan, Susan L.; Bostina, Mihnea; Konstantinopoulos, Panagiotis A.; Fountzilas, Helena; Spentzos, Dimitrios; Miron, Alexander; Yassin, Yosuf A.; Lee, Bernard T.; Wulf, Gerburg M.

    2011-01-01

    Female BRCA1 mutation carriers have a nearly 80% probability of developing breast cancer during their life-time. We hypothesized that the breast epithelium at risk in BRCA1 mutation carriers harbors mammary epithelial cells (MEC) with altered proliferation and differentiation properties. Using a three-dimensional culture technique to grow MECs ex vivo, we found that the ability to form colonies, an indication of clonality, was restricted to the aldehyde dehydrogenase 1–positive fraction in MECs but not in HCC1937 BRCA1-mutant cancer cells. Primary MECs from BRCA1 mutation carriers (n = 9) had a 28% greater ability for clonal growth compared with normal controls (n= 6; P = 0.006), and their colonies were significantly larger. Colonies in controls and BRCA1 mutation carriers stained positive for BRCA1 by immunohistochemistry, and 79% of the examined single colonies from BRCA1 carriers retained heterozygosity for BRCA1 (ROH). Colonies from BRCA1 mutation carriers frequently showed high epidermal growth factor receptor (EGFR) expression (71% EGFR positive versus 44% in controls) and were negative for estrogen receptor (ERα; 32% ER negative, 44% mixed, 24% ER positive versus 90% ER positive in controls). Expression of CK14 and p63 were not significantly different. Microarray studies revealed that colonies from BRCA1-mutant PMECs anticipate expression profiles found in BRCA1-related tumors, and that the EGFR pathway is up-regulated. We conclude that BRCA1 haploin-sufficiency leads to an increased ability for clonal growth and proliferation in the PMECs of BRCA1 mutation carriers, possibly as a result of EGFR pathway activation. These altered growth and differentiation properties may render BRCA1-mutant PMECs vulnerable to transformation and predispose to the development of ER-negative, EGFR-positive breast cancers. PMID:19190334

  7. Differential Effects of Polyphenols on Proliferation and Apoptosis in Human Myeloid and Lymphoid Leukemia Cell Lines

    PubMed Central

    Mahbub, Amani A; Le Maitre, Christine L.; Haywood-Small, Sarah L.; McDougall, Gordon J.; Cross, Neil A.; Jordan-Mahy, Nicola

    2013-01-01

    Background: Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors, our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin, chrysin, apigenin, emodin, aloe-emodin, rhein, cis-stilbene and trans-stilbene) were studied on cell proliferation, cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines, together with normal haematopoietic control cells. Methods: Cellular proliferation was measured by CellTiter-Glo® luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. Results: Emodin, quercetin, and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 µM, 8-33 µM, and 25-85 µM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 µM, 19-50 µM, and 8-50 µM respectively). Generally, lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines, however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. Conclusions: These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly, the differential sensitivity of emodin, quercetin, and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia. PMID:23796248

  8. MRI Reporter Genes: Application to Imaging of Cell Survival, Proliferation, Migration, and Differentiation

    PubMed Central

    Vandsburger, Moriel H; Radoul, Marina; Cohen, Batya; Neeman, Michal

    2013-01-01

    Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly, or through the use of targeted contrast media. However many genes and proteins, and particularly those located in intracellular compartments, are not accessible for targeted agents. The transcriptional regulation of these genes can never the less be detected, though indirectly, through the use of reporter genes encoding for readily detectable proteins. Such reporter proteins can be expressed in the tissue of interest by genetically introducing the reporter gene in the target cells. Imaging of reporter genes has become a powerful tool in modern biomedical research. Typically, expression of fluorescent or bioluminescent proteins, or the reaction product of expressed enzymes and exogenous substrates, are examined using in vitro histological methods, or in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raise the possibility that MRI could become a powerful tool for concomitant high resolution anatomical and functional imaging and for imaging of reporter gene activity. An immediate application of MRI reporter gene methods is for monitoring gene expression patterns in gene therapy and for in vivo imaging of the survival, proliferation, migration, and differentiation of pluripotent or multipotent cells used in cell based regenerative therapies for cancer, myocardial infarction, and neural degeneration. In this review, we characterize the variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, cell migration, and cell differentiation. In particular, we discuss which methods are best suited for translation to clinical use in regenerative therapies. PMID:23225197

  9. Differential regulation of proliferation and neuronal differentiation in adult rat spinal cord neural stem/progenitors by ERK1/2, Akt, and PLCγ

    PubMed Central

    Chan, Wai Si; Sideris, Alexandra; Sutachan, Jhon J.; Montoya G, Jose V.; Blanck, Thomas J. J.; Recio-Pinto, Esperanza

    2013-01-01

    Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2). We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2) over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2) or the phosphoinositide 3-kinase (PI3K)/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase Cγ (PLCγ) pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCγ signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs. PMID:23986655

  10. miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells

    PubMed Central

    Tomé, M; López-Romero, P; Albo, C; Sepúlveda, J C; Fernández-Gutiérrez, B; Dopazo, A; Bernad, A; González, M A

    2011-01-01

    In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses revealed that miR-335 is significantly downregulated upon hMSC differentiation. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, a positive regulator of MSC self-renewal, and downregulated by interferon-γ (IFN-γ), a pro-inflammatory cytokine that has an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3′UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 downregulation is critical for the acquisition of reparative MSC phenotypes. PMID:21164520

  11. Effects of FGFR Signaling on Cell Proliferation and Differentiation of Apert Dental Cells.

    PubMed

    Lu, Changming; Huguley, Samuel; Cui, Chun; Cabaniss, Lauren B; Waite, Peter D; Sarver, David M; Mamaeva, Olga A; MacDougall, Mary

    2016-01-01

    The Apert syndrome is a rare congenital disorder most often arising from S252W or P253R mutations in fibroblast growth factor receptor (FGFR2). Numerous studies have focused on the regulatory role of Apert FGFR2 signaling in bone formation, whereas its functional role in tooth development is largely unknown. To investigate the role of FGFR signaling in cell proliferation and odontogenic differentiation of human dental cells in vitro, we isolated dental pulp and enamel organ epithelia (EOE) tissues from an Apert patient carrying the S252W FGFR2 mutation. Apert primary pulp and EOE cells were established and shown to exhibit normal morphology and express alkaline phosphatase under differentiation conditions. Similar to control cells, Apert dental pulp and EOE cells expressed all FGFRs, with highest levels of FGFR1 followed by FGFR2 and low levels of FGFR3 and FGFR4. However, Apert cells had increased cell growth compared with control cells. Distinct from previous findings in osteoblast cells, gain-of-function S252W FGFR2 mutation did not upregulate the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFRα), but elevated extracellular signal-regulated kinase (ERK) signaling in cells after EGF stimulation. Unexpectedly, there was little effect of the S252W mutation on odontogenic gene expression in dental pulp and EOE cells. However, after inhibition of total FGFR signaling or ERK signaling, the expression of odontogenic genes was upregulated in both dental cell types, indicating the negative effect of whole FGFR signaling on odontogenic differentiation. This study provides novel insights on FGFR signaling and a common Apert FGFR2 mutation in the regulation of odontogenic differentiation of dental mesenchymal and epithelial cells.

  12. Clonal Characterization of Rat Muscle Satellite Cells: Proliferation, Metabolism and Differentiation Define an Intrinsic Heterogeneity

    PubMed Central

    Rossi, Carlo A.; Pozzobon, Michela; Ditadi, Andrea; Archacka, Karolina; Gastaldello, Annalisa; Sanna, Marta; Franzin, Chiara; Malerba, Alberto; Milan, Gabriella; Cananzi, Mara; Schiaffino, Stefano; Campanella, Michelangelo; Vettor, Roberto; De Coppi, Paolo

    2010-01-01

    Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨm), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described. PMID:20049087

  13. Differential modulation of mitogen driven proliferation and homeostasis driven proliferation of T cells by rapamycin, Ly294002 and chlorophyllin.

    PubMed

    Sharma, Deepak; Kumar, Sandur Santosh; Raghu, Rashmi; Khanam, Shazia; Sainis, Krishna Balaji

    2007-04-01

    Homeostasis driven proliferation (HDP) of naïve CD4+ T cells depends upon T cell receptor ligation with self-MHC II along with availability of interleukin-7. But the exact nature of downstream signaling events involved in HDP of helper T cells remains elusive. To identify the specific involvement of signaling molecules in HDP, purified CD4+ T cells were treated with either mTOR inhibitor rapamycin or PI3kinase inhibitor Ly294002 or with an antioxidant chlorophyllin (CHL) in vitro. Rapamycin treated cells failed to proliferate, expressed anergic T cell specific transcription factor genes egr-2 and egr-3 and showed diminished IFN-gamma production in response to Con A stimulation in vitro. Although CHL treated cells also failed to proliferate, they showed a normal IFN-gamma production during primary stimulation and did not upregulate egr-2 and egr-3 genes following restimulation in vitro. Ly294002 treated cells failed to express IL-2 and IFN-gamma and did not divide in response to Con A stimulation in vitro. While all these inhibitors significantly inhibited CD4+ T cell proliferation in response to the mitogen in vitro, only CHL treatment could inhibit their HDP in lymphopenic mice. Our results also demonstrate that combined treatment with rapamycin and Ly294002 did not inhibit HDP of CD4+ T cells. Thus, the present study, for the first time, shows a non-essential role of mTOR and PI3kinase during HDP of CD4+ T cells and also describes its possible regulation by an antioxidant.

  14. The effects of caffeic, coumaric and ferulic acids on proliferation, superoxide production, adhesion and migration of human tumor cells in vitro.

    PubMed

    Nasr Bouzaiene, Nouha; Kilani Jaziri, Soumaya; Kovacic, Hervé; Chekir-Ghedira, Leila; Ghedira, Kamel; Luis, José

    2015-11-05

    Reactive oxygen species are well-known mediators of various biological responses. In this study, we examined the effect of three phenolic acids, caffeic, coumaric and ferulic acids, on superoxide anion production, adhesion and migration of human lung (A549) and colon adenocarcinoma (HT29-D4) cancer cell lines. Proliferation of both tumor cells was inhibited by phenolic acids. Caffeic, coumaric and ferulic acids also significantly inhibited superoxide production in A549 and HT29-D4 cells. Superoxide anion production decreased by 92% and 77% at the highest tested concentration (200 µM) of caffeic acid in A549 and HT29-D4 cell lines respectively. Furthermore, A549 and HT29-D4 cell adhesion was reduced by 77.9% and 79.8% respectively at the higher tested concentration of ferulic acid (200 µM). Migration assay performed towards A549 cell line, revealed that tested compounds reduced significantly cell migration. At the highest concentration tested (200 µM), the covered surface was 7.7%, 9.5% and 35% for caffeic, coumaric or ferulic acids, respectively. These results demonstrate that caffeic, coumaric and ferulic acids may participate as active ingredients in anticancer agents against lung and colon cancer development, at adhesion and migration steps of tumor progression.

  15. Enhanced proliferation and osteogenic differentiation of mesenchymal stem cells on graphene oxide-incorporated electrospun poly(lactic-co-glycolic acid) nanofibrous mats.

    PubMed

    Luo, Yu; Shen, He; Fang, Yongxiang; Cao, Yuhua; Huang, Jie; Zhang, Mengxin; Dai, Jianwu; Shi, Xiangyang; Zhang, Zhijun

    2015-03-25

    Currently, combining biomaterial scaffolds with living stem cells for tissue regeneration is a main approach for tissue engineering. Mesenchymal stem cells (MSCs) are promising candidates for musculoskeletal tissue repair through differentiating into specific tissues, such as bone, muscle, and cartilage. Thus, successfully directing the fate of MSCs through factors and inducers would improve regeneration efficiency. Here, we report the fabrication of graphene oxide (GO)-doped poly(lactic-co-glycolic acid) (PLGA) nanofiber scaffolds via electrospinning technique for the enhancement of osteogenic differentiation of MSCs. GO-PLGA nanofibrous mats with three-dimensional porous structure and smooth surface can be readily produced via an electrospinning technique. GO plays two roles in the nanofibrous mats: first, it enhances the hydrophilic performance, and protein- and inducer-adsorption ability of the nanofibers. Second, the incorporated GO accelerates the human MSCs (hMSCs) adhesion and proliferation versus pure PLGA nanofiber and induces the osteogenic differentiation. The incorporating GO scaffold materials may find applications in tissue engineering and other fields.

  16. ADAMTS-10 and -6 differentially regulate cell-cell junctions and focal adhesions

    PubMed Central

    Cain, Stuart A.; Mularczyk, Ewa J.; Singh, Mukti; Massam-Wu, Teresa; Kielty, Cay M.

    2016-01-01

    ADAMTS10 and ADAMTS6 are homologous metalloproteinases with ill-defined roles. ADAMTS10 mutations cause Weill-Marchesani syndrome (WMS), implicating it in fibrillin microfibril biology since some fibrillin-1 mutations also cause WMS. However little is known about ADAMTS6 function. ADAMTS10 is resistant to furin cleavage, however we show that ADAMTS6 is effectively processed and active. Using siRNA, over-expression and mutagenesis, it was found ADAMTS6 inhibits and ADAMTS10 is required for focal adhesions, epithelial cell-cell junction formation, and microfibril deposition. Either knockdown of ADAMTS6, or disruption of its furin processing or catalytic sites restores focal adhesions, implicating its enzyme activity acts on targets in the focal adhesion complex. In ADAMTS10-depleted cultures, expression of syndecan-4 rescues focal adhesions and cell-cell junctions. Recombinant C-termini of ADAMTS10 and ADAMTS6, both of which induce focal adhesions, bind heparin and syndecan-4. However, cells overexpressing full-length ADAMTS6 lack heparan sulphate and focal adhesions, whilst depletion of ADAMTS6 induces a prominent glycocalyx. Thus ADAMTS10 and ADAMTS6 oppositely affect heparan sulphate-rich interfaces including focal adhesions. We previously showed that microfibril deposition requires fibronectin-induced focal adhesions, and cell-cell junctions in epithelial cultures. Here we reveal that ADAMTS6 causes a reduction in heparan sulphate-rich interfaces, and its expression is regulated by ADAMTS10. PMID:27779234

  17. Effects of Cerium Oxide Nanoparticles on the Proliferation, Osteogenic Differentiation and Adipogenic Differentiation of Primary Mouse Bone Marrow Stromal Cells In Vitro.

    PubMed

    Zhang, Qun; Ge, Kun; Ren, Huihui; Zhang, Cuimiao; Zhang, Jinchao

    2015-09-01

    The effects of cerium oxide nanoparticles (nanoceria) on the proliferation, osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells (BMSCs) were studied by employing 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny tetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, collagen production, alizarin red-S (ARS) and oil red o stain assays. The results indicated that nanoceria increased the viability of BMSCs at all tested concentrations with evident dose dependence for 24 and 72 h. On day 14, nanoceria inhibited the osteogenic differentiation of BMSCs at all tested concentrations. On day 19 and 24, nanoceria inhibited the formation of mineralized matrix nodules of BMSCs at all tested concentrations. On day 17, nanoceria inhibited the adipogenic differentiation of BMSCs at all tested concentrations. This suggests that the effects of nanoceria on the proliferation, osteogenic differentiation and adipogenic differentiation of BMSCs are very complicated. Both the concentration and culture time have significant influence on the proliferation, osteogenic differentiation and adipogenic differentiation of BMSCs. These results will be helpful for rational applications of nanoceria in the future.

  18. MicroRNA-128 regulates the proliferation and differentiation of bovine skeletal muscle satellite cells by repressing Sp1.

    PubMed

    Dai, Yang; Zhang, Wei Ran; Wang, Yi Min; Liu, Xin Feng; Li, Xin; Ding, Xiang Bin; Guo, Hong

    2016-03-01

    MicroRNAs (miRNAs) play essential roles in muscle cell proliferation and differentiation. The muscle-specific miRNAs miR-1 and miR-206 have been shown to regulate muscle development and promote myogenic differentiation; however, it is likely that a number of other miRNAs play important roles in regulating myogenesis as well. microRNA-128 (miR-128) has been reported to be highly expressed in brain and skeletal muscle, and we found that miR-128 is also up-regulated during bovine skeletal muscle satellite cell differentiation using microarray analysis and qRT-PCR. However, little is known about the functions of miR-128 in bovine skeletal muscle satellite cell development. In this study, we investigated the biological functions of miR-128 in bovine skeletal muscle cell development. Using a dual-luciferase reporter assay, we confirmed that miR-128 regulates the Sp1 gene. Over-expression of miR-128 reduced Sp1 protein levels and inhibited muscle satellite cell proliferation and differentiation. Inhibition of miR-128 increased Sp1 protein levels and promoted muscle satellite cell differentiation but also suppressed proliferation. Changes in miR-128 and Sp1 expression levels also affected the protein levels of MyoD and CDKN1A. Sp1, an activator of MyoD and a suppressor of CDKN1A, plays an important role in bovine muscle cell proliferation and differentiation. The results of our study reveal a mechanism by which miR-128 regulates bovine skeletal muscle satellite cell proliferation and myogenic differentiation via Sp1.

  19. Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation.

    PubMed

    Herzmann, Nicole; Salamon, Achim; Fiedler, Tomas; Peters, Kirsten

    2017-01-01

    Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.

  20. Impact of low oxygen tension on stemness, proliferation and differentiation potential of human adipose-derived stem cells

    SciTech Connect

    Choi, Jane Ru; Pingguan-Murphy, Belinda; Wan Abas, Wan Abu Bakar; Noor Azmi, Mat Adenan; Omar, Siti Zawiah; Chua, Kien Hui; Wan Safwani, Wan Kamarul Zaman

    2014-05-30

    Highlights: • Hypoxia maintains the stemness of adipose-derived stem cells (ASCs). • ASCs show an increased proliferation rate under low oxygen tension. • Oxygen level as low as 2% enhances the chondrogenic differentiation potential of ASCs. • HIF-1α may regulate the proliferation and differentiation activities of ASCs under hypoxia. - Abstract: Adipose-derived stem cells (ASCs) have been found adapted to a specific niche with low oxygen tension (hypoxia) in the body. As an important component of this niche, oxygen tension has been known to play a critical role in the maintenance of stem cell characteristics. However, the effect of O{sub 2} tension on their functional properties has not been well determined. In this study, we investigated the effects of O{sub 2} tension on ASCs stemness, differentiation and proliferation ability. Human ASCs were cultured under normoxia (21% O{sub 2}) and hypoxia (2% O{sub 2}). We found that hypoxia increased ASC stemness marker expression and proliferation rate without altering their morphology and surface markers. Low oxygen tension further enhances the chondrogenic differentiation ability, but reduces both adipogenic and osteogenic differentiation potential. These results might be correlated with the increased expression of HIF-1α under hypoxia. Taken together, we suggest that growing ASCs under 2% O{sub 2} tension may be important in expanding ASCs effectively while maintaining their functional properties for clinical therapy, particularly for the treatment of cartilage defects.

  1. Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression

    PubMed Central

    Ruijtenberg, Suzan; van den Heuvel, Sander

    2016-01-01

    ABSTRACT Cell proliferation and differentiation show a remarkable inverse relationship. Precursor cells continue division before acquiring a fully differentiated state, while terminal differentiation usually coincides with proliferation arrest and permanent exit from the division cycle. Mechanistic insight in the temporal coordination between cell cycle exit and differentiation has come from studies of cells in culture and genetic animal models. As initially described for skeletal muscle differentiation, temporal coordination involves mutual antagonism between cyclin-dependent kinases that promote cell cycle entry and transcription factors that induce tissue-specific gene expression. Recent insights highlight the contribution of chromatin-regulating complexes that act in conjunction with the transcription factors and determine their activity. In particular SWI/SNF chromatin remodelers contribute to dual regulation of cell cycle and tissue-specific gene expression during terminal differentiation. We review the concerted regulation of the cell cycle and cell type-specific transcription, and discuss common mutations in human cancer that emphasize the clinical importance of proliferation versus differentiation control. PMID:26825227

  2. Periostin differentially induces proliferation, contraction and apoptosis of primary Dupuytren's disease and adjacent palmar fascia cells

    SciTech Connect

    Vi, Linda; Feng, Lucy; Zhu, Rebecca D.; Wu, Yan; Satish, Latha; Gan, Bing Siang; O'Gorman, David B.

    2009-12-10

    Dupuytren's disease, (DD), is a fibroproliferative condition of the palmar fascia in the hand, typically resulting in permanent contracture of one or more fingers. This fibromatosis is similar to scarring and other fibroses in displaying excess collagen secretion and contractile myofibroblast differentiation. In this report we expand on previous data demonstrating that POSTN mRNA, which encodes the extra-cellular matrix protein periostin, is up-regulated in Dupuytren's disease cord tissue relative to phenotypically normal palmar fascia. We demonstrate that the protein product of POSTN, periostin, is abundant in Dupuytren's disease cord tissue while little or no periostin immunoreactivity is evident in patient-matched control tissues. The relevance of periostin up-regulation in DD was assessed in primary cultures of cells derived from diseased and phenotypically unaffected palmar fascia from the same patients. These cells were grown in type-1 collagen-enriched culture conditions with or without periostin addition to more closely replicate the in vivo environment. Periostin was found to differentially regulate the apoptosis, proliferation, {alpha} smooth muscle actin expression and stressed Fibroblast Populated Collagen Lattice contraction of these cell types. We hypothesize that periostin, secreted by disease cord myofibroblasts into the extra-cellular matrix, promotes the transition of resident fibroblasts in the palmar fascia toward a myofibroblast phenotype, thereby promoting disease progression.

  3. Proliferation and differentiation of osteoblast-like cells on apatite-wollastonite/polyethylene composites.

    PubMed

    Rea, S M; Brooks, R A; Best, S M; Kokubo, T; Bonfield, W

    2004-08-01

    Glass-ceramic apatite-wollastonite (A-W)/high-density polyethylene composite (AWPEX) materials have been designed to match the mechanical strength of human cortical bone and to provide favourable bioactivity, with potential use in many orthopaedic applications. To better understand AWPEX properties, the effects of surface finish and ceramic filler size and content on osteoblast-like cell attachment, proliferation, and differentiation were examined. Glass-ceramic content was tested at 30 and 50 vol% and median particle size at 4.5 and 7.7 microm. Samples were prepared as 1 x 10 x 10 mm(3) tiles with polished or rough surfaces, sterilized by gamma irradiation (2.5 Mrad), and characterized by scanning electron microscopy (SEM) and surface profilometry. Saos-2 human osteoblast-like cells were cultured on each surface at an initial concentration of 4500 cells/cm(2) for 1, 3, or 7 days. At each time point, adenosine triphosphate and alkaline phosphatase levels were measured to assess cell number and osteoblast differentiation. SEM imaging of cells on the composite surfaces showed preferential cell attachment to filler particles within the polymer matrix. Significant biochemical assay differences were found at 7 days, confirmed by ANOVA post-hoc testing using Bonferroni's correction. Overall, increased exposure of the glass-ceramic A-W phase in AWPEX through surface polishing, higher volume fraction and/or larger particle size was found to lead to an improved cell response.

  4. Microcephaly gene links Trithorax and REST/NRSF to control neural stem cell proliferation and differentiation

    PubMed Central

    Yang, Yawei J.; Baltus, Andrew E.; Mathew, Rebecca S.; Murphy, Elisabeth A.; Evrony, Gilad D.; Gonzalez, Dilenny M.; Wang, Estee P.; Marshall-Walker, Christine A.; Barry, Brenda J.; Murn, Jernej; Tatarakis, Antonis; Mahajan, Muktar A.; Samuels, Herbert H.; Shi, Yang; Golden, Jeffrey A.; Mahajnah, Muhammad; Shenhav, Ruthie; Walsh, Christopher A.

    2013-01-01

    SUMMARY Microcephaly is a neurodevelopmental disorder causing significantly reduced cerebral cortex size. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation. Here, we identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335-null mice are embryonically lethal and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that Znf335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF, and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation. PMID:23178126

  5. Enhancement of Dopaminergic Differentiation in Proliferating Midbrain Neuroblasts by Sonic Hedgehog and Ascorbic Acid

    PubMed Central

    Volpicelli, Floriana; Consales, Claudia; Caiazzo, Massimiliano; Colucci-D'Amato, Luca; Perrone-Capano, Carla; di Porzio, Umberto

    2004-01-01

    We analyzed the molecular mechanisms involved in the acquisition and maturation of dopaminergic (DA) neurons generated in vitro from rat ventral mesencephalon (MES) cells in the presence of mitogens or specific signaling molecules. The addition of basic fibroblast growth factor (bFGF) to MES cells in serum-free medium stimulates the proliferation of neuroblasts but delays DA differentiation. Recombinant Sonic hedgehog (SHH) protein increases up to three fold the number of tyrosine hydroxylase (TH)-positive cells and their differentiation, an effect abolished by anti-SHH antibodies. The expanded cultures are rich in nestin-positive neurons, glial cells are rare, all TH+ neurons are DA, and all DA and GABAergic markers analyzed are expressed. Adding ascorbic acid to bFGF/SHH-treated cultures resulted in a further five- to seven-fold enhancement of viable DA neurons. This experimental system also provides a powerful tool to generate DA neurons from single embryos. Our strategy provides an enriched source of MES DA neurons that are useful for analyzing molecular mechanisms controlling their function and for experimental regenerative approaches in DA dysfunction. PMID:15303305

  6. Eltrombopag inhibits the proliferation of leukemia cells via reduction of intracellular iron and induction of differentiation.

    PubMed

    Roth, Michael; Will, Britta; Simkin, Guillermo; Narayanagari, Swathi; Barreyro, Laura; Bartholdy, Boris; Tamari, Roni; Mitsiades, Constantine S; Verma, Amit; Steidl, Ulrich

    2012-07-12

    Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist that has been approved recently for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Prior studies have shown that EP stimulates megakaryopoiesis in BM cells from patients with acute myeloid leukemia and myelodysplastic syndrome, and the results also suggested that it may inhibit leukemia cell growth. In the present study, we studied the effects of EP on leukemia cell proliferation and the mechanism of its antiproliferative effects. We found that EP leads to a decreased cell division rate, a block in G(1) phase of cell cycle, and increased differentiation in human and murine leukemia cells. Because EP is species specific in that it can only bind TPO-R in human and primate cells, these findings further suggested that the antileukemic effect is independent of TPO-R. We found that treatment with EP leads to a reduction in free intracellular iron in leukemic cells in a dose-dependent manner. Experimental increase of intracellular iron abrogated the antiproliferative and differentiation-inducing effects of EP, demonstrating that its antileukemic effects are mediated through modulation of intracellular iron content. Finally, determination of EP's antileukemic activity in vivo demonstrated its ability to prolong survival in 2 mouse models of leukemia.

  7. Exendin-4 promotes proliferation and differentiation of MC3T3-E1 osteoblasts by MAPKs activation.

    PubMed

    Feng, Yingyu; Su, Lei; Zhong, Xing; Guohong, Wei; Xiao, Haipeng; Li, Yanbing; Xiu, Lingling

    2016-04-01

    Glucagon-like peptide-1 (GLP1) and its receptor agonist have been previously reported to play a positive role in bone metabolism in aged ovariectomized rats and insulin-resistant models. However, whether GLP1 has a direct effect on the proliferation and differentiation of osteoblasts or any cellular mechanism for this potential role is unknown. We examined the effects of the GLP1 receptor agonist exendin-4 on the proliferation, differentiation, and mineralization of mouse osteoblastic MC3T3-E1 cells. GLP1 receptor was detected in MC3T3-E1 cells by polymerase chain reaction (PCR) and Western blot assay. Cell proliferation was assessed using MTT assay, revealing that exendin-4 increased cell proliferation at effective concentrations between 10(-10) and 10(-5) M. Quantitative PCR analysis showed that exendin-4 increased the mRNA expression of the differentiation markers alkaline phosphatase (ALP), collagen-1 (COL1), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2) under osteogenic conditions. Alizarin red staining confirmed that 10(-7) M exendin-4 increased osteoblast mineralization by 18.7%. Exendin-4 upregulated the phosphorylation of ERK1/2, p38, and JNK, with the peak effect at 1.5 h in the Western blot analysis. The use of selective MAPK inhibitors, namely PD98059, SB203580, and SP600125, blocked the effects of exendin-4 on kinase activation (ERK1/2, p38, and JNK), as well as cell proliferation and differentiation in MC3T3-E1 cells. These findings demonstrate that exendin-4 promotes both the proliferation and differentiation of preosteoblasts MC3T3-E1 via activation of the MAPK pathway.

  8. Short Term Morphine Exposure In Vitro Alters Proliferation and Differentiation of Neural Progenitor Cells and Promotes Apoptosis via Mu Receptors

    PubMed Central

    Willner, Dafna; Cohen-Yeshurun, Ayelet; Avidan, Alexander; Ozersky, Vladislav; Shohami, Esther; Leker, Ronen R.

    2014-01-01

    Background Chronic morphine treatment inhibits neural progenitor cell (NPC) progression and negatively effects hippocampal neurogenesis. However, the effect of acute opioid treatment on cell development and its influence on NPC differentiation and proliferation in vitro is unknown. We aim to investigate the effect of a single, short term exposure of morphine on the proliferation, differentiation and apoptosis of NPCs and the mechanism involved. Methods Cell cultures from 14-day mouse embryos were exposed to different concentrations of morphine and its antagonist naloxone for 24 hours and proliferation, differentiation and apoptosis were studied. Proliferating cells were labeled with bromodeoxyuridine (BrdU) and cell fate was studied with immunocytochemistry. Results Cells treated with morphine demonstrated decreased BrdU expression with increased morphine concentrations. Analysis of double-labeled cells showed a decrease in cells co-stained for BrdU with nestin and an increase in cells co-stained with BrdU and neuron-specific class III β-tubuline (TUJ1) in a dose dependent manner. Furthermore, a significant increase in caspase-3 activity was observed in the nestin- positive cells. Addition of naloxone to morphine-treated NPCs reversed the anti-proliferative and pro-apoptotic effects of morphine. Conclusions Short term morphine exposure induced inhibition of NPC proliferation and increased active caspase-3 expression in a dose dependent manner. Morphine induces neuronal and glial differentiation and decreases the expression of nestin- positive cells. These effects were reversed with the addition of the opioid antagonist naloxone. Our results demonstrate the effects of short term morphine administration on the proliferation and differentiation of NPCs and imply a mu-receptor mechanism in the regulation of NPC survival. PMID:25072277

  9. Effects of surface molecular chirality on adhesion and differentiation of stem cells.

    PubMed

    Yao, Xiang; Hu, Yiwen; Cao, Bin; Peng, Rong; Ding, Jiandong

    2013-12-01

    Chirality is one of the most fascinating and ubiquitous cues in nature, especially in life. The effects of chiral surfaces on stem cells have, however, not yet been revealed. Herein we examined the molecular chirality effect on stem cell behaviors. Self assembly monolayers of L- or D-cysteine (Cys) were formed on a glass surface coated with gold. Mesenchymal stem cells (MSCs) derived from bone marrow of rats exhibited more adhering preference and thus less cell spreading on the L surface than on the d one at the confluent condition. More protein adsorption was observed on the L surface after immersed in cell culture medium with fetal bovine serum. After osteogenic and adipogenic co-induction at the confluent condition, a larger proportion of cells became osteoblasts on the d surface, while the adipogenic fraction on the L surface was found to be higher than on the D surface. In order to interpret how this chirality effect worked, we fabricated Cys microislands of two sizes on the non-fouling poly(ethylene glycol) hydrogel to pre-define the spreading areas of single cells. Then the differentiation extents did not exhibit a significant difference between L and D surfaces under a given area of microislands, yet very significant differences of osteogenesis and adipogenesis were found between different areas. So, the molecular chirality influenced stem cells, probably via favored adsorption of natural proteins on the L surface, which led to more cell adhesion; and the larger cell spreading area with higher cell tension in turn favored osteogenesis rather than adipogenesis. As a result, this study reveals the molecular chirality on material surfaces as an indirect regulator of stem cells.

  10. Differential arrest and adhesion of tumor cells and microbeads in the microvasculature.

    PubMed

    Guo, Peng; Cai, Bin; Lei, Ming; Liu, Yang; Fu, Bingmei M

    2014-06-01

    To investigate the mechanical mechanisms behind tumor cell arrest in the microvasculature, we injected fluorescently labeled human breast carcinoma cells or similarly sized rigid beads into the systemic circulation of a rat. Their arrest patterns in the microvasculature of mesentery were recorded and quantified. We found that 93% of rigid beads were arrested either at arteriole-capillary intersections or in capillaries. Only 3% were at the capillary-postcapillary venule intersections and in postcapillary venules. In contrast, most of the flexible tumor cells were either entrapped in capillaries or arrested at capillary or postcapillary venule-postcapillary venule intersections and in postcapillary venules. Only 12% of tumor cells were arrested at the arteriole-capillary intersections. The differential arrest and adhesion of tumor cells and microbeads in the microvasculature was confirmed by a χ(2) test (p < 0.001). These results demonstrate that mechanical trapping was responsible for almost all the arrest of beads and half the arrest of tumor cells. Based on the measured geometry and blood flow velocities at the intersections, we also performed a numerical simulation using commercial software (ANSYS CFX 12.01) to depict the detailed distribution profiles of the velocity, shear rate, and vorticity at the intersections where tumor cells preferred to arrest and adhere. Simulation results reveal the presence of localized vorticity and shear rate regions at the turning points of the microvessel intersections, implying that hemodynamic factors play an important role in tumor cell arrest in the microcirculation. Our study helps elucidate long-debated issues related to the dominant factors in early-stage tumor hematogenous metastasis.

  11. Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

    PubMed

    Lohmann, Michael; Walenda, Gudrun; Hemeda, Hatim; Joussen, Sylvia; Drescher, Wolf; Jockenhoevel, Stefan; Hutschenreuter, Gabriele; Zenke, Martin; Wagner, Wolfgang

    2012-01-01

    The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

  12. Adhesion and differentiation of Saos-2 osteoblast-like cells on chromium-doped diamond-like carbon coatings.

    PubMed

    Filova, Elena; Vandrovcova, Marta; Jelinek, Miroslav; Zemek, Josef; Houdkova, Jana; Jan Remsa; Kocourek, Tomas; Stankova, Lubica; Bacakova, Lucie

    2017-01-01

    Diamond-like carbon (DLC) thin films are promising for use in coating orthopaedic, dental and cardiovascular implants. The problem of DLC layers lies in their weak layer adhesion to metal implants. Chromium is used as a dopant for improving the adhesion of DLC films. Cr-DLC layers were prepared by a hybrid technology, using a combination of pulsed laser deposition (PLD) from a graphite target and magnetron sputtering. Depending on the deposition conditions, the concentration of Cr in the DLC layers moved from zero to 10.0 at.%. The effect of DLC layers with 0.0, 0.9, 1.8, 7.3, 7.7 and 10.0 at.% Cr content on the adhesion and osteogenic differentiation of human osteoblast-like Saos-2 cells was assessed in vitro. The DLC samples that contained 7.7 and 10.0 at.% of Cr supported cell spreading on day 1 after seeding. On day three after seeding, the most apparent vinculin-containing focal adhesion plaques were also found on samples with higher concentrations of chromium. On the other hand, the expression of type I collagen and alkaline phosphatase at the mRNA and protein level was the highest on Cr-DLC samples with a lower concentration of Cr (0-1.8 at.%). We can conclude that higher concentrations of chromium supported cell adhesion; however DLC and DLC doped with a lower concentration of chromium supported osteogenic cell differentiation.

  13. Why are enteric ganglia so small? Role of differential adhesion of enteric neurons and enteric neural crest cells.

    PubMed Central

    Rollo, Benjamin N.; Zhang, Dongcheng; Simkin, Johanna E.; Menheniott, Trevelyan R.; Newgreen, Donald F.

    2015-01-01

    The avian enteric nervous system (ENS) consists of a vast number of unusually small ganglia compared to other peripheral ganglia. Each ENS ganglion at mid-gestation has a core of neurons and a shell of mesenchymal precursor/glia-like enteric neural crest (ENC) cells. To study ENS cell ganglionation we isolated midgut ENS cells by HNK-1 fluorescence-activated cell sorting (FACS) from E5 and E8 quail embryos, and from E9 chick embryos. We performed cell-cell aggregation assays which revealed a developmentally regulated functional increase in ENS cell adhesive function, requiring both Ca 2+ -dependent and independent adhesion. This was consistent with N-cadherin and NCAM labelling. Neurons sorted to the core of aggregates, surrounded by outer ENC cells, showing that neurons had higher adhesion than ENC cells. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM and N-cadherin on this surface of the outer non-neuronal ENC cells. Aggregation assays showed that ENS cells FACS selected for NCAM-high and enriched for enteric neurons formed larger and more coherent aggregates than unsorted ENS cells. In contrast, ENS cells of the NCAM-low FACS fraction formed small, disorganised aggregates.  This suggests a novel mechanism for control of ENS ganglion morphogenesis where i) differential adhesion of ENS neurons and ENC cells controls the core/shell ganglionic structure and ii) the ratio of neurons to ENC cells dictates the equilibrium ganglion size by generation of an outer non-adhesive surface. PMID:26064478

  14. A novel role for junctional adhesion molecule-A in tumor proliferation: modulation by an anti-JAM-A monoclonal antibody.

    PubMed

    Goetsch, Liliane; Haeuw, Jean-François; Beau-Larvor, Charlotte; Gonzalez, Alexandra; Zanna, Laurence; Malissard, Martine; Lepecquet, Anne-Marie; Robert, Alain; Bailly, Christian; Broussas, Matthieu; Corvaia, Nathalie

    2013-03-15

    To identify new potential targets in oncology, functional approaches were developed using tumor cells as immunogens to select monoclonal antibodies targeting membrane receptors involved in cell proliferation. For that purpose cancer cells were injected into mice and resulting hybridomas were screened for their ability to inhibit cell proliferation in vitro. Based on this functional approach coupled to proteomic analysis, a monoclonal antibody specifically recognizing the human junctional adhesion molecule-A (JAM-A) was defined. Interestingly, compared to both normal and tumor tissues, we observed that JAM-A was mainly overexpressed on breast, lung and kidney tumor tissues. In vivo experiments demonstrated that injections of anti-JAM-A antibody resulted in a significant tumor growth inhibition of xenograft human tumors. Treatment with monoclonal antibody induced a decrease of the Ki67 expression and downregulated JAM-A levels. All together, our results show for the first time that JAM-A can interfere with tumor proliferation and suggest that JAM-A is a potential novel target in oncology. The results also demonstrate that a functional approach coupled to a robust proteomic analysis can be successful to identify new antibody target molecules that lead to promising new antibody-based therapies against cancers.

  15. Polycomb Group Protein Ezh2 Regulates Hepatic Progenitor Cell Proliferation and Differentiation in Murine Embryonic Liver

    PubMed Central

    Ueno, Yasuharu; Nakata, Susumu; Obana, Yuta; Sekine, Keisuke; Zheng, Yun-Wen; Takebe, Takanori; Isono, Kyoichi; Koseki, Haruhiko; Taniguchi, Hideki

    2014-01-01

    In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. Enhancer of zeste homolog 2 (Ezh2), a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3), which trigger the gene suppression. In the present study, we investigated a role of Ezh2 in the regulation of the expanding hepatic progenitor population in vivo. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of Ezh2, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from in vivo fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion in vivo, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3. PMID:25153170

  16. Retrovirus delivered neurotrophin-3 promotes survival, proliferation and neuronal differentiation of human fetal neural stem cells in vitro.

    PubMed

    Lu, Haixia; Li, Minjie; Song, Tusheng; Qian, Yihua; Xiao, Xinli; Chen, Xinlin; Zhang, Pengbo; Feng, Xinshun; Parker, Terence; Liu, Yong

    2008-10-22

    Poor survival and insufficient neuronal differentiation are the main obstacles to neural stem cell (NSC) transplantation therapy. Genetic modification of NSCs with neurotrophins is considered a promising approach to overcome these difficulties. In this study, the effects on survival, proliferation and neuronal differentiation of human fetal NSCs (hfNSCs) were observed after infection by a neurotrophin-3 (NT-3) recombinant retrovirus. The hfNSCs, from 12-week human fetal brains formed neurospheres, expressed the stem cell marker nestin and differentiated into the three main cell types of the nervous system. NT-3 recombinant retrovirus (Retro-NT-3) infected hfNSCs efficiently expressed NT-3 gene for at least 8 weeks, presented an accelerated proliferation, and therefore produced an increased number of neurospheres and after differentiation in vitro, contained a higher percentage of neuronal cells. Eight weeks after infection, 37.9+/-4.2% of hfNSCs in the Retro-NT-3 infection group expressed the neuronal marker, this was significantly higher than the control and mock infection groups. NT-3 transduced hfNSCs also displayed longer protruding neurites compared with other groups. Combined these results demonstrate that NT-3 modification promote the survival/proliferation, neuronal differentiation and growth of neurites of hfNSCs in vitro. This study proposes recombinant retrovirus mediated NT-3 modification may provide a promising means to resolve the poor survival and insufficient neuronal differentiation of NSCs.

  17. Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells

    PubMed Central

    Jha, Rajneesh; Wu, Qingling; Singh, Monalisa; Preininger, Marcela K.; Han, Pengcheng; Ding, Gouliang; Cho, Hee Cheol; Jo, Hanjoong; Maher, Kevin O.; Wagner, Mary B.; Xu, Chunhui

    2016-01-01

    Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes. PMID:27492371

  18. Differential Role of β1C and β1A Integrin Cytoplasmic Variants in Modulating Focal Adhesion Kinase, Protein Kinase B/AKT, and Ras/Mitogen-activated Protein Kinase Pathways

    PubMed Central

    Fornaro, Mara; Steger, Craig A.; Bennett, Anton M.; Wu, J. Julie; Languino, Lucia R.

    2000-01-01

    The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The β1 integrin subunits, β1C and β1A, that contain variant cytoplasmic domains differentially affect cell proliferation; β1C inhibits proliferation, whereas β1A promotes it. We investigated the ability of β1C and β1A to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human β1C or human β1A. The different cytodomains of either β1C or β1A did not affect either association with the endogenous α2, αV, and α5 subunits or cell adhesion to fibronectin or TS2/16, a mAb to human β1. Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing β1C showed significantly inhibited extracellular signal–regulated kinase (ERK) 2 activation compared with β1A stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed β1C by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, β1A engagement induced activation of both proteins. We show that Ras activation was strongly reduced in β1C stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued β1C-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in β1C stable cell lines was attributable to an inhibitory effect of β1C on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued β1C antiproliferative effect. These findings show that the β1C variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings

  19. Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation.

    PubMed

    Wang, Guang; Li, Yan; Wang, Xiao-yu; Han, Zhe; Chuai, Manli; Wang, Li-jing; Ho Lee, Kenneth Ka; Geng, Jian-guo; Yang, Xuesong

    2013-05-01

    coordinating cell proliferation and differentiation during neurulation.

  20. Sitagliptin inhibit human lymphocytes proliferation and Th1/Th17 differentiation in vitro.

    PubMed

    Pinheiro, Marcelo Maia; Stoppa, Caroline Lais; Valduga, Claudete Justina; Okuyama, Cristina Eunice; Gorjão, Renata; Pereira, Regina Mara Silva; Diniz, Susana Nogueira

    2017-03-30

    Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new class of anti-diabetic agents that are widely used in clinical practice to improve glycemic control in patients with type 2 diabetes. DPP-4 is also known as lymphocyte cell surface protein, CD26, and plays an important role in T-cell immunity. Recent studies suggest that DPP-4 inhibitors improve beta-cell function and attenuate autoimmunity in type 1 diabetic mouse models. To investigate the direct effect of DPP4 in immune response, human peripheral blood mononuclear cells (PBMC) from healthy volunteers were obtained by Ficoll gradient and cultivated in the absence (control) or presence of phytohemagglutinin (PHA), or stimulated with PHA and treated with sitagliptin. The immune modulation mechanisms analyzed were: cell proliferation, by MTT assay; cytokine quantification by ELISA or cytometric bead array (CBA), Th1/Th2/Th17 phenotyping by flow cytometric analysis and CD26 gene expression by real time PCR. The results showed that sitagliptin treatment inhibited the proliferation of PBMC-PHA stimulated cells in a dose dependent manner and decreased CD26 expression by these cells, suggesting that sitagliptin may interfere in CD26 expression, dimerization and cell signaling. Sitagliptin treatment not only inhibited IL-10 (p<0.05) and IFN-gamma (p=0.07) cytokines, but also completely abolish IL-6 expression by PBMCs (p<0.001). On the other hand, IL-4 were secreted in culture supernatants from sitagliptin treated cells. A statistically significant increase (p<0.05) in the ratio of TGF-beta/proliferation index after sitagliptin treatment (2627.97±1351.65), when comparing to untreated cells (646.28±376.94), was also demonstrated, indicating higher TGF-beta1 production by viable cells in cultures. Sitagliptin treatment induced a significantly (p<0.05) decrease in IL-17 and IFN-gamma intracellular expression compared with PHA alone. Also, the percentage of T CD4(+)IL-17(+), T CD4(+)IFNgamma(+) and T CD4(+)IL-4(+) cells

  1. Differentiation of Crohn’s Disease-Associated Isolates from Other Pathogenic Escherichia coli by Fimbrial Adhesion under Shear Force

    PubMed Central

    Szunerits, Sabine; Zagorodko, Oleksandr; Cogez, Virginie; Dumych, Tetiana; Chalopin, Thibaut; Alvarez Dorta, Dimitri; Sivignon, Adeline; Barnich, Nicolas; Harduin-Lepers, Anne; Larroulet, Iban; Yanguas Serrano, Aritz; Siriwardena, Aloysius; Pesquera, Amaia; Zurutuza, Amaia; Gouin, Sébastien G.; Boukherroub, Rabah; Bouckaert, Julie

    2016-01-01

    Shear force exerted on uropathogenic Escherichia coli adhering to surfaces makes type-1 fimbriae stretch out like springs to catch on to mannosidic receptors. This mechanism is initiated by a disruption of the quaternary interactions between the lectin and the pilin of the two-domain FimH adhesin and transduces allosterically to the mannose-binding pocket of FimH to increase its affinity. Mannose-specific adhesion of 14 E. coli pathovars was measured under flow, using surface plasmon resonance detection on functionalized graphene-coated gold interfaces. Increasing the shear had important differential consequences on bacterial adhesion. Adherent-invasive E. coli, isolated from the feces and biopsies of Crohn’s disease patients, consistently changed their adhesion behavior less under shear and displayed lower SPR signals, compared to E. coli opportunistically infecting the urinary tract, intestines or loci of knee and hip prostheses. We exemplified this further with the extreme behaviors of the reference strains UTI89 and LF82. Whereas their FimA major pilins have identical sequences, FimH of LF82 E. coli is marked by the Thr158Pro mutation. Positioned in the inter-domain region known to carry hot spots of mutations in E. coli pathotypes, residue 158 is indicated to play a structural role in the allosteric regulation of type-1 fimbriae-mediated bacterial adhesion. PMID:27043645

  2. Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate

    PubMed Central

    Wang, Weiguang; Song, Buer; Anbarchian, Teni; Shirazyan, Anna

    2016-01-01

    TGFβs act through canonical and non-canonical pathways, and canonical signals are transduced via Smad2 and Smad3. However, the contribution of canonical vs. non-canonical pathways in cartilage is unknown because the role of Smad2 in chondrogenesis has not been investigated in vivo. Therefore, we analyzed mice in which Smad2 is deleted in cartilage (Smad2CKO), global Smad3-/- mutants, and crosses of these strains. Growth plates at birth from all mutant strains exhibited expanded columnar and hypertrophic zones, linked to increased proliferation in resting chondrocytes. Defects were more severe in Smad2CKO and Smad2CKO;Smad3-/- (Smad2/3) mutant mice than in Smad3-/- mice, demonstrating that Smad2 plays a role in chondrogenesis. Increased levels of Ihh RNA, a key regulator of chondrocyte proliferation and differentiation, were seen in prehypertrophic chondrocytes in the three mutant strains at birth. In accordance, TGFβ treatment decreased Ihh RNA levels in primary chondrocytes from control (Smad2fx/fx) mice, but inhibition was impaired in cells from mutants. Consistent with the skeletal phenotype, the impact on TGFβ-mediated inhibition of Ihh RNA expression was more severe in Smad2CKO than in Smad3-/- cells. Putative Smad2/3 binding elements (SBEs) were identified in the proximal Ihh promoter. Mutagenesis demonstrated a role for three of them. ChIP analysis suggested that Smad2 and Smad3 have different affinities for these SBEs, and that the repressors SnoN and Ski were differentially recruited by Smad2 and Smad3, respectively. Furthermore, nuclear localization of the repressor Hdac4 was decreased in growth plates of Smad2CKO and double mutant mice. TGFβ induced association of Hdac4 with Smad2, but not with Smad3, on the Ihh promoter. Overall, these studies revealed that Smad2 plays an essential role in the development of the growth plate, that both Smads 2 and 3 inhibit Ihh expression in the neonatal growth plate, and suggested they accomplish this by binding to

  3. Effects of oncostatin M on cell proliferation and osteogenic differentiation in C3H10T1/2

    PubMed Central

    Zou, F.; Xu, J-C.; Wu, G-H.; Zhou, L-L.; Wa, Q-D.; Peng, J-Q.; Zou, X-N.

    2016-01-01

    Objective: To explore the effects of protein factor Oncostatin M (OSM), a member of the Interleukin-6 (IL-6) family on cell proliferation, osteogenic differentiation and mineralization. Materials and methods: Basal nutrient solutions of different concentrations of OSM (0, 5, 10, 20, 40, 80 ng/ml) were used. In order to divide embryonic origin between mesenchymal stem cells C3H10T1/2 of in vitro cultured mice, and the effects of in vitro proliferation efficiencies of C3H10T1/2 cells of different concentrations of OSM, the C3H10T1/2 cells were divided into four groups: (1) Basal nutrient solution group (negative control); (2) Osteogenesis induced liquid group (positive control); (3) OSM (20 ng/ml) group; (4) Experimental group (osteogenesis induced liquid + OSM (20 ng/ml)). The expressions levels of relevant osteogenesis and mineralization genes were detected. Results: OSM had several effects on promoting the proliferation of embryonic origin mesenchymal stem cells C3H10T1/2 with respect to time of exposure as well as concentrations. In the present study, it has been shown that when the concentration of OSM is 20 ng/ml, the effects of promoting proliferation are most obvious. OSM can induce osteogenic differentiation of C3H10T1/2, make the process of osteogenic differentiation in advance, and promote the formation of end-stage calcium deposits and mineralized nodule, and osteogenic differentiation of C3H10T1/2 is finally achieved. Conclusion: OSM can promote the proliferation of C3H10T1/2, and induce its osteogenic differentiation and end-stage mineralization. PMID:27973390

  4. Ethacrynic acid and 1 alpha,25-dihydroxyvitamin D3 cooperatively inhibit proliferation and induce differentiation of human myeloid leukemia cells.

    PubMed

    Makishima, M; Honma, Y

    1996-09-01

    The active form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 (VD3), inhibits proliferation and induces differentiation of leukemia cells, but its clinical use is limited by the adverse effect of hypercalcemia. In this study we found that the loop diuretic ethacrynic acid, which is used to treat hypercalcemia, enhanced the differentiation of human leukemia cells induced by VD3. Ethacrynic acid alone inhibited the proliferation of human promyelocytic HL-60 cells while only slightly increasing differentiation markers such as nitroblue tetrazolium (NBT)-reducing and lysozyme activities. Ethacrynic acid effectively enhanced the growth-inhibiting action of VD3. In the presence of ethacrynic acid, VD3 increased the NBT-reducing and lysozyme activities and the CD11b expression of HL-60 cells more effectively than VD3 alone. Other loop diuretics, furosemide and bumetanide, also enhanced the differentiation of HL-60 cells induced by VD3, but to a lesser extent than ethacrynic acid. The differentiation of HL-60 cells induced by all-trans retinoic acid, dimethyl sulfoxide or phorbol-12-myristate 13-acetate was also enhanced by ethacrynic acid with increasing NBT-reducing and lysozyme activities and the expression of CD11b or CD14 surface antigen. Morphologically, ethacrynic acid enhanced the monocytic differentiation of HL-60 cells induced by VD3 and phorbol ester and the granulocytic differentiation by retinoic acid and dimethyl sulfoxide. Other human myelomonocytic leukemia ML-1, U937, P39/TSU and P31/FUJ cells were induced to differentiate by VD3 and this was also enhanced by ethacrynic acid. The long-term culture of HL-60 cells showed that ethacrynic acid plus VD3 induced the complete growth arrest of HL-60 cells. Therefore ethacrynic acid, which is used to treat hypercalcemia, enhanced the proliferation-inhibiting and differentiation-inducing activities of VD3 and the combination of ethacrynic acid and VD3 may be useful in therapy for myeloid leukemia.

  5. Ampakine CX546 increases proliferation and neuronal differentiation in subventricular zone stem/progenitor cell cultures.

    PubMed

    Schitine, Clarissa; Xapelli, Sara; Agasse, Fabienne; Sardà-Arroyo, Laura; Silva, Ana P; De Melo Reis, Ricardo A; de Mello, Fernando G; Malva, João O

    2012-06-01

    Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK), and dendritogenesis (MAP2-positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies.

  6. Meningioma 1 is required for appropriate osteoblast proliferation, motility, differentiation, and function.

    PubMed

    Zhang, Xiaoxue; Dowd, Diane R; Moore, Meika C; Kranenburg, Tanya A; Meester-Smoor, Magda A; Zwarthoff, Ellen C; MacDonald, Paul N

    2009-07-03

    The vitamin D endocrine system is essential for calcium and phosphate homeostasis and skeletal mineralization. The 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) hormone binds to the vitamin D receptor (VDR) to regulate gene expression. These gene products in turn mediate the actions of 1,25(OH)(2)D(3) in mineral-regulating target cells such as the osteoblast. We showed previously that meningioma 1 (MN1) is a novel target of 1,25(OH)(2)D(3) in MG-63 osteoblastic cells and that it is a coactivator for VDR-mediated transcription (Sutton, A. L., Zhang, X., Ellison, T. I., and MacDonald, P. N. (2005) Mol. Endocrinol. 19, 2234-2244). However, the functional significance of MN1 in osteoblastic cell biology is largely unknown. Here, we demonstrate that MN1 expression is increased dramatically during differentiation of primary osteoblastic cells. Using calvarial osteoblasts derived from wild-type and MN1 knock-out mice, we provide data supporting an essential role of MN1 in maintaining appropriate osteoblast proliferation, differentiation, and function. MN1 knock-out osteoblasts displayed altered morphology, decreased growth rate, impaired motility, and attenuated 1,25(OH)(2)D(3)/VDR-mediated transcription as well as reduced alkaline phosphatase activity and mineralized nodule formation. MN1 null osteoblasts were also impaired in supporting osteoclastogenesis in co-culture studies presumably because of marked reduction in the RANKL:OPG ratio in the MN1 null cells. Mechanistic studies supported a transcriptional role for MN1 in controlling RANKL gene expression through activation of the RANKL promoter. Cumulatively, these studies indicate an important role for MN1 in maintaining the appropriate maturation and function of calvarial osteoblasts.

  7. miR-143 regulates proliferation and differentiation of bovine skeletal muscle satellite cells by targeting IGFBP5.

    PubMed

    Zhang, Wei Ran; Zhang, Hui Na; Wang, Yi Min; Dai, Yang; Liu, Xin Feng; Li, Xin; Ding, Xiang Bin; Guo, Hong

    2017-03-01

    Development of skeletal muscle is a complicated biological process regulated by various regulation factors and signal pathways. MicroRNAs (miRNAs) are novel gene regulators that control muscle cell development. microRNA-143 (miR-143) is highly expressed in skeletal muscle, and we found that miR-143 level is significantly increased during bovine skeletal muscle satellite cells (MSCs) differentiation process through microarray analysis and qRT-PCR detection. However, the function of miR-143 in bovine muscle development remained unclear. In our work, the functions of miR-143 in bovine MSCs myogenic differentiation were investigated. We discovered that IGFBP5 is directly regulated by miR-143 using a dual-luciferase reporter assay. Overexpression of miR-143 led to decreased level of IGFBP5 protein and restrained cell proliferation and differentiation, while downregulation of miR-143 resulted in increased levels of IGFBP5 protein and restrained cell proliferation but improved differentiation. IGFBP5, an important component of IGF signaling pathway, contributes greatly to bovine muscle cell development. A mechanism that miR-143 can regulate the proliferation and differentiation of bovine MSCs through changing expression of IGFBP5 was elucidated by our study.

  8. Comparison of human mesenchymal stem cells proliferation and differentiation on poly(methyl methacrylate) bone cements with and without mineralized collagen incorporation.

    PubMed

    Wu, Jingjing; Xu, Suju; Qiu, Zhiye; Liu, Peng; Liu, Huiying; Yu, Xiang; Cui, Fu-Zhai; Chunhua, Zhao Robert

    2016-01-01

    Poly(methyl methacrylate) bone cement is widely used in vertebroplasty, joint replacement surgery, and other orthopaedic surgeries, while it also exposed many problems on mechanical property and biocompatibility. Better performance in mechanical match and bone integration is highly desirable. Recently, there reported that incorporation of mineralized collagen into poly(methyl methacrylate) showed positive results in mechanical property and osteointegration ability in vivo. In the present study, we focused on the comparison of osteogenic behavior between mineralized collagen incorporated in poly(methyl methacrylate) and poly(methyl methacrylate). Human marrow mesenchymal stem cells are used in this experiment. Adhesion and proliferation were used to characterize biocompatibility. Activity of alkaline phosphatase was used to assess the differentiation of human marrow mesenchymal stem cells into osteoblasts. Real-time PCR was performed to detect the expression of osteoblast-related markers at messenger RNA level. The results show that osteogenic differentiation on mineralized collagen incorporated in poly(methyl methacrylate) bone cement is more than two times higher than that of poly(methyl methacrylate) after culturing for 21 days. Thus, important mechanism on mineralized collagen incorporation increasing the osteogenetic ability of poly(methyl methacrylate) bone cement may be understood in this concern.

  9. Regulation of Articular Chondrocyte Proliferation and Differentiation by Indian Hedgehog and Parathyroid Hormone-related Protein

    PubMed Central

    Chen, Xuesong; Macica, Carolyn; Nasiri, Ali; Broadus, Arthur E.

    2008-01-01

    Objective The chondrocytes of the epiphyseal growth zone are regulated by the Indian hedgehog (Ihh)-parathyroid hormone-related protein (PTHrP) axis. In weight-bearing joints, this growth zone comes to be subdivided by the secondary ossification center into distinct articular and growth cartilage structures. Here, we explored the cells of origin, localization, regulation of expression, and putative functions of Ihh and PTHrP in articular cartilage in the mouse. Methods We assessed Ihh and PTHrP expression in an allelic PTHrP-lacZ knockin mouse and several versions of PTHrP-null mice. Selected joints were unloaded surgically to examine load-induction of PTHrP and Ihh. Results The embryonic growth zone appears to serve as the source of PTHrP-expressing proliferative chondrocytes that populate both the forming articular cartilage and growth plate structures. In articular cartilage, these cells take the form of articular chondrocytes in the mid-zone. In PTHrP-knockout mice, mineralizing chondrocytes encroach upon developing articular cartilage but appear to be prevented from mineralizing the joint space by Ihh-driven surface chondrocyte proliferation. In growing and adult mice, PTHrP expression in articular chondrocytes is load-induced, and unloading is associated with rapid changes in PTHrP expression and articular chondrocyte differentiation. Conclusion We conclude that the PTHrP-Ihh axis participates in the maintenance of articular cartilage. Dysregulation of this system might contribute to the pathogenesis of arthritis. PMID:19035497

  10. HB-EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins.

    PubMed

    Puschmann, Till B; Zandén, Carl; Lebkuechner, Isabell; Philippot, Camille; de Pablo, Yolanda; Liu, Johan; Pekny, Milos

    2014-03-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a vascular-derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia-inducible proteins released by astrocytes in CNS injuries. It was suggested that HB-EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB-EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB-EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia-like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT-qPCR analysis demonstrated that HB-EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB-EGF-mediated astrocyte response. HB-EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de-differentiation of astrocytes.

  11. Electrospun tilapia collagen nanofibers accelerating wound healing via inducing keratinocytes proliferation and differentiation.

    PubMed

    Zhou, Tian; Wang, Nanping; Xue, Yang; Ding, Tingting; Liu, Xin; Mo, Xiumei; Sun, Jiao

    2016-07-01

    The development of biomaterials with the ability to induce skin wound healing is a great challenge in biomedicine. In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4(+)/CD8(+) lymphocytes, and the level of IgG or IgM in Sprague-Dawley rats. The tensile strength and contact angle of collagen nanofibers were 6.72±0.44MPa and 26.71±4.88°, respectively. They also had good thermal stability and swelling property. Furthermore, the nanofibers could significantly promote the proliferation of human keratinocytes (HaCaTs) and stimulate epidermal differentiation through the up-regulated gene expression of involucrin, filaggrin, and type I transglutaminase in HaCaTs. The collagen nanofibers could also facilitate rat skin regeneration. In the present study, electrospun biomimetic tilapia skin collagen nanofibers were succesfully prepared, were proved to have good bioactivity and could accelerate rat wound healing rapidly and effectively. These biological effects might be attributed to the biomimic extracellular matrix structure and the multiple amino acids of the collagen nanofibers. Therefore, the cost-efficient tilapia collagen nanofibers could be used as novel wound dressing, meanwhile effectively avoiding the risk of transmitting animal disease in the future clinical apllication.

  12. Microfabricated discontinuous-edge surface topographies influence osteoblast adhesion, migration, cytoskeletal organization, and proliferation and enhance matrix and mineral deposition in vitro.

    PubMed

    Hamilton, D W; Wong, K S; Brunette, D M

    2006-05-01

    The fabrication of surfaces that stimulate increased adhesion, migration, and differentiated function of osteoblasts has been viewed as being desirable for many orthopedic applications. Previous studies have shown that microfabricated pits and grooves alter adhesion, spreading, matrix secretion, and production of mineral by rat calvarial osteoblasts (RCOs). The mechanisms underlying these effects are unknown, although microenvironment and cell alignment are considered to play a role. The aim of this work was to investigate the behavior of RCOs on microfabricated discontinuous-edge surfaces (DESs), which could provide an alternative means to control both the microenvironment and cellular alignment. Two types of discontinuous-type structures were employed, gap-cornered boxes and micron scale pillars. DES gap-cornered boxes and the pillars influenced the arrangement of F-actin, microtubules, and vinculin. Osteoblasts were guided in their direction of migration on both types of substrata. Both box DESs and pillars altered the staining intensity and localization pattern of phosphotyrosine and src-activated FAK localization. Cell multilayering, matrix deposition, and mineralization were enhanced on both discontinuous topographies when compared with smooth controls. This study shows that DESs alter adhesion, migration, and proliferative responses from osteoblasts at early time points (<1 week) and promote multilayering, matrix deposition, and mineral deposition at later times (2-6 weeks). Such topographical patterns could potentially be employed as effective surface features on bone-contacting implants or in membrane-based periodontal applications.

  13. HO-1 inhibits preadipocyte proliferation and differentiation at the onset of obesity via ROS dependent activation of Akt2

    PubMed Central

    Wagner, Gabriel; Lindroos-Christensen, Josefine; Einwallner, Elisa; Husa, Julia; Zapf, Thea-Christin; Lipp, Katharina; Rauscher, Sabine; Gröger, Marion; Spittler, Andreas; Loewe, Robert; Gruber, Florian; Duvigneau, J. Catharina; Mohr, Thomas; Sutterlüty-Fall, Hedwig; Klinglmüller, Florian; Prager, Gerhard; Huppertz, Berthold; Yun, Jeanho; Wagner, Oswald; Esterbauer, Harald; Bilban, Martin

    2017-01-01

    Excessive accumulation of white adipose tissue (WAT) is a hallmark of obesity. The expansion of WAT in obesity involves proliferation and differentiation of adipose precursors, however, the underlying molecular mechanisms remain unclear. Here, we used an unbiased transcriptomics approach to identify the earliest molecular underpinnings occuring in adipose precursors following a brief HFD in mice. Our analysis identifies Heme Oxygenase-1 (HO-1) as strongly and selectively being upregulated in the adipose precursor fraction of WAT, upon high-fat diet (HFD) feeding. Specific deletion of HO-1 in adipose precursors of Hmox1fl/flPdgfraCre mice enhanced HFD-dependent visceral adipose precursor proliferation and differentiation. Mechanistically, HO-1 reduces HFD-induced AKT2 phosphorylation via ROS thresholding in mitochondria to reduce visceral adipose precursor proliferation. HO-1 influences adipogenesis in a cell-autonomous way by regulating events early in adipogenesis, during the process of mitotic clonal expansion, upstream of Cebpα and PPARγ. Similar effects on human preadipocyte proliferation and differentiation in vitro were observed upon modulation of HO-1 expression. This collectively renders HO-1 as an essential factor linking extrinsic factors (HFD) with inhibition of specific downstream molecular mediators (ROS & AKT2), resulting in diminished adipogenesis that may contribute to hyperplastic adipose tissue expansion. PMID:28102348

  14. Effect of recombinant human tumour necrosis factor beta (TNF beta) on activation, proliferation and differentiation of human B lymphocytes.

    PubMed Central

    Zola, H; Nikoloutsopoulos, A

    1989-01-01

    Activation, proliferation and differentiation of B lymphocytes are processes controlled by T cells, and the control is mediated in part by the action of lymphokines derived from T cells. In this study we have examined the ability of tumour necrosis factor-beta (TNF beta), a T-cell product, to induce a state of activation in resting B cells, to induce proliferation of already activated B cells, and to stimulate differentiation. Recombinant tumour necrosis factor beta (rTNF beta) was used alone and in conjunction with known stimulators. As judged by several markers of activation (CD23, CDw40, LFA-1, 4F2, MHC class I and class II), rTNF beta did not contribute to the activation of resting B cells, either alone or in conjunction with anti-IgM and IL-4. However, the activation marker detected by the monoclonal antibody Leu 21 did show a greater degree of up-regulation by anti-IgM + IL-4 + rTNF beta when compared with anti-IgM + IL-4. rTNF beta induced proliferation of B cells, but only if activating stimuli were also present. Two other factors which induce proliferation of activated B cells, low molecular weight B-cell growth factor (LMW-BCGF) and IL-2, showed additive effects with rTNF beta. No evidence of changes in differentiation status of the B cells was seen. PMID:2787779

  15. Identification of small-molecule modulators of mouse SVZ progenitor cell proliferation and differentiation through high-throughput screening.

    PubMed

    Liu, Yaping; Lacson, Raul; Cassaday, Jason; Ross, David A; Kreamer, Anthony; Hudak, Edward; Peltier, Richard; McLaren, Donna; Muñoz-Sanjuan, Ignacio; Santini, Francesca; Strulovici, Berta; Ferrer, Marc

    2009-04-01

    Adult mouse subventricular zone (SVZ) neural stem/progenitor cells are multipotent self-renewing cells that retain the capacity to generate the major cell types of the central nervous system in vitro and in vivo. The relative ease of expanding SVZ cells in culture as neurospheres makes them an ideal model for carrying out large-scale screening to identify compounds that regulate neural progenitor cell proliferation and differentiation. The authors have developed an adenosine triphosphate-based cell proliferation assay using adult SVZ cells to identify small molecules that activate or inhibit progenitor cell proliferation. This assay was miniaturized to a 1536-well format for high-throughput screening (HTS) of >1 million small-molecule compounds, and 325 and 581 compounds were confirmed as potential inducers of SVZ cell proliferation and differentiation, respectively. A number of these compounds were identified as having a selective proliferative and differentiation effect on SVZ cells versus mouse Neuro2a neuroblastoma cells. These compounds can potentially be useful pharmacological tools to modulate resident stem cells and neurogenesis in the adult brain. This study represents a novel application of primary somatic stem cells in the HTS of a large-scale compound library.

  16. Glucagon-like Peptide-1 improves proliferation and differentiation of endothelial progenitor cells via upregulating VEGF generation

    PubMed Central

    Xie, Xiao-Yun; Mo, Zhao-Hui; Chen, Ke; He, Hong-Hui; Xie, Yan-Hong

    2011-01-01

    Summary Background Glucagon-like peptide-1(GLP-1), released from enteroendocrine cells of the intestine, exerted cardiovascular protective effect. Circulating endothelial progenitor cells (EPCs) play an important role in maintaining endothelial integrity regulating neovascularization and reendothelialization after endothelial injury. Vascular endothelial growth factor (VEGF) is an important cytokine in the process of EPCs vascular differentiation and proliferation. Material/Methods This study was designed to investigate the association between VEGF changes and the proliferation/differentiation function of EPCs in the presence of GLP-1. Results We demonstrated that GLP-1 markedly enhanced the EPCs proliferation and expression of EC-specific markers, and simultaneously upregulated VEGF secretion in EPCs. Exogenous VEGF augmented EPCs proliferation/differentiation abilities in a dose-dependent manner. However, all of the beneficial effects of GLP-1 were suppressed by anti-VEGFmAb or the KDR-specific tyrosine kinase inhibitor SU1498. Conclusions These findings suggest that GLP-1 improves VEGF generation, which contributed to improvement of EPCs biological function, partly by tyrosine kinase KDR. VEGF is a necessary intermediate, mediating the effects of GLP-1 on EPCs. These changes offer a novel explanation that upregulation EPCs bioactivities may be one of the mechanisms of GLP-1 cardiovascular protective effect. PMID:21278683

  17. Tetraploid cells produced by absence of substrate adhesion during cytokinesis are limited in their proliferation and enter senescence after DNA replication.

    PubMed

    De Santis Puzzonia, Marco; Gonzalez, Laetitia; Ascenzi, Sonia; Cundari, Enrico; Degrassi, Francesca

    2016-01-01

    Tetraploidy has been proposed as an intermediate state in neoplastic transformation due to the intrinsic chromosome instability of tetraploid cells. Despite the identification of p53 as a major factor in growth arrest of tetraploid cells, it is still unclear whether the p53-dependent mechanism for proliferation restriction is intrinsic to the tetraploid status or dependent on the origin of tetraploidy. Substrate adherence is fundamental for cytokinesis completion in adherent untransformed cells. Here we show that untransformed fibroblast cells undergoing mitosis in suspension produce binucleated tetraploid cells due to defective cleavage furrow constriction that leads to incomplete cell abscission. Binucleated cells obtained after loss of substrate adhesion maintain an inactive p53 status and are able to progress into G1 and S phase. However, binucleated cells arrest in G2, accumulate p53 and are not able to enter mitosis as no tetraploid metaphases were recorded after one cell cycle time. In contrast, tetraploid metaphases were found following pharmacological inhibition of Chk1 kinase, suggesting the involvement of the ATR/Chk1 pathway in the G2 arrest of binucleated cells. Interestingly, after persistence in the G2 phase of the cell cycle, a large fraction of binucleated cells become senescent. These findings identify a new pathway of proliferation restriction for tetraploid untransformed cells that seems to be specific for loss of adhesion-dependent cytokinesis failure. This involves Chk1 and p53 activation during G2. Inhibition of growth and entrance into senescence after cytokinesis in suspension may represent an important mechanism to control tumor growth. In fact, anchorage independent growth is a hallmark of cancer and it has been demonstrated that binucleated transformed cells can enter a cycle of anchorage independent growth.

  18. PED/PEA-15 interacts with the 67 kD laminin receptor and regulates cell adhesion, migration, proliferation and apoptosis

    PubMed Central

    Formisano, Pietro; Ragno, Pia; Pesapane, Ada; Alfano, Daniela; Alberobello, Anna Teresa; Rea, Vincenza Elena Anna; Giusto, Raffaella; Rossi, Francesca W; Beguinot, Francesco; Rossi, Guido; Montuori, Nunzia

    2012-01-01

    Abstract Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is an anti-apoptotic protein whose expression is increased in several human cancers. In addition to apoptosis, PED/PEA-15 is involved in the regulation of other major cellular functions, including cell adhesion, migration, proliferation and glucose metabolism. To further understand the functions of this protein, we performed a yeast two-hybrid screening using PED/PEA-15 as a bait and identified the 67 kD high-affinity laminin receptor (67LR) as an interacting partner. 67 kD laminin receptor is a non-integrin cell-surface receptor for the extracellular matrix (ECM), derived from the dimerization of a 37 kD cytosolic precursor (37LRP). The 67LR is highly expressed in human cancers and widely recognized as a molecular marker of metastatic aggressiveness. The molecular interaction of PED/PEA-15 with 67LR was confirmed by pull-down experiments with recombinant His-tagged 37LRP on lysates of PED/PEA-15 transfected HEK-293 cells. Further, overexpressed or endogenous PED/PEA-15 was co-immunoprecipitated with 67LR in PED/PEA-15-transfected HEK-293 cells and in U-373 glioblastoma cells, respectively. PED/PEA-15 overexpression significantly increased 67LR-mediated HEK-293 cell adhesion and migration to laminin that, in turn, determined PED/PEA-15 phosphorylation both in Ser-104 and Ser-116, thus enabling cell proliferation and resistance to apoptosis. PED/PEA-15 ability to induce cell responses to ECM-derived signals through interaction with 67LR may be of crucial importance for tumour cell survival in a poor microenvironment, thus favouring the metastatic spread and colonization. PMID:21895963

  19. Photo-initiated grafting of gelatin/N-maleic acyl-chitosan to enhance endothelial cell adhesion, proliferation and function on PLA surface.

    PubMed

    Zhu, Aiping; Zhao, Feng; Ma, Teng

    2009-07-01

    Vascular graft surface properties significantly affect adhesion, growth and function of endothelial cells (ECs). The bulk degradation property of poly(lactic acid) (PLA) makes it possible for it to be replaced by cellular materials and PLA is desirable as a scaffold material for vascular grafts. However, PLA has an unfavorable surface property for EC adhesion and proliferation due to the lack of a selective cell adhesion motif. Photo-initiated surface-grafting polymerization is a promising method for immobilizing certain biomacromolecules on material surfaces without compromising bulk properties. N-Maleic acyl-chitosan (NMCS) is a novel biocompatible amphiphilic derivative of chitosan with double bonds and can be initiated by ultraviolet light. In this study, gelatin was complexed with NMCS via hydrophobic interaction, and gel/NMCS complex thus formed was then grafted on the PLA surface to improve EC biocompatibility. X-ray photoelectron and Fourier transform infrared spectroscopy, and water contact angle measurement confirmed immobilization of the gel/NMCS complex on PLA surface. Moreover, the gel/NMCS modified PLA enhanced human umbilical vein endothelial cell (HUVEC) spreading and flattening, and promoted the expression of more structured CD31 and vWF compared to unmodified PLA film. Compared to the unmodified PLA surface, the HUVECs on the modified PLA surface had elevated uptake of acetylated low-density lipoprotein, and maintained the ability to modulate metabolic activity upon exposure to shear stress at 5dyncm(-2) by up-regulating nitric oxide and prostacyclin production. Cell retention was 1.6 times higher on the gel/NMCS-PLA surface, demonstrating its improved potential for hemocompatibility. These results indicate that photo-initiated surface-grafting of the biomimetic gel/NMCS complex is an effective method to modify material surfaces as vascular grafts.

  20. The effect of mechanical stress on the proliferation, adipogenic differentiation and gene expression of human adipose-derived stem cells.

    PubMed

    Paul, Nora E; Denecke, Bernd; Kim, Bong-Sung; Dreser, Alice; Bernhagen, Jürgen; Pallua, Norbert

    2017-01-17

    To allow for a better implementation of external volume expansion to clinical applications for soft tissue regeneration, it is necessary to comprehensively understand the underlying mechanisms. Since human adipose-derived stem cells (hASCs) play a crucial role in soft tissue enlargement, we investigated the impact of cyclic stretch on gene expression, proliferation rate, and adipogenic differentiation of these cells. After cyclic stretching, RNA was extracted and subjected to DNA microarray analysis and RT-qPCR. Also, the expression of FABP4 mRNA was analysed by RT-qPCR to test whether mechanical stretch affected adipogenic differentiation of hASCs. Proliferation rate was assessed by alamarBlue assay and Ki-67 staining. Cell cycle analysis was performed with flow cytometry and Western blot. We found that cyclic stretch significantly induced the expression of CYP1B1 mRNA. Further, the adipogenic differentiation of hASCs was impaired as was the proliferation. This was partly due to a decrease in extracellular signal-regulated kinase (ERK) 1/2 and histone H3 phosphorylation, suggesting a growth arrest in the G2 /M phase of the cell cycle. Enrichment analyses demonstrated that stretch-regulated genes were over-represented in pathways and biological processes involved in extracellular matrix organisation, vascular remodelling, and responses to cell stress. Taken together, mechanical stress impaired both the proliferation and adipogenic differentiation, but led to a tissue-remodelling phenotype of hASCs. These data suggest that extracellular matrix remodelling and neoangiogenesis may play a more important role in external volume expansion than proliferation and adipogenesis of hASCs.

  1. Blood-compatible poly(2-methoxyethyl acrylate) for the adhesion and proliferation of endothelial and smooth muscle cells.

    PubMed

    Sato, Chikako; Aoki, Makiko; Tanaka, Masaru

    2016-09-01

    Thrombus formation presents a serious hindrance in the development of functional artificial blood vessels, especially those with a small diameter. Endothelialization can prevent thrombus formation; however, the adhesion of endothelial cells to existing polymer materials is generally weak. Therefore, polymers that have both anti-thrombotic and endothelialization properties do not currently exist. We previously reported that platelets do not adhere to poly(2-methoxyethyl acrylate) (PMEA) or poly(tetrahydrofurfuryl acrylate)(PTHFA). Here, we investigated whether endothelial cells and smooth muscle cells, both of which are blood vessel components, could adhere to these synthetic polymers. Polyethylene terephthalate films were coated with PMEA and PTHFA using a spin-coater. Human umbilical vein endothelial cells or aorta smooth muscle cells were seeded on the polymer surfaces, after which we analyzed the number of adherent cells, their morphologies and vinculin expression. We found that both endothelial and smooth muscle cells adhered to PMEA and PTHFA, while platelets did not. We propose that, by using PMEA and PTHFA with no modifications, it should be possible to develop artificial blood vessels with both anti-thrombotic and endothelialization properties. In addition, we discuss the mechanism of selective cell adhesion in PMEA and PTHFA.

  2. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activates promyogenic signaling pathways, thereby promoting myoblast differentiation.

    PubMed

    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee; Seo, Dong-Wan; Kang, Jong-Sun; Bae, Gyu-Un

    2016-01-29

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) regulates postnatal myogenesis by alleviating myostatin activity, but the molecular mechanisms by which it regulates myogenesis are not fully understood. In this study, we investigate molecular mechanisms of PPARβ/δ in myoblast differentiation. C2C12 myoblasts treated with a PPARβ/δ agonist, GW0742 exhibit enhanced myotube formation and muscle-specific gene expression. GW0742 treatment dramatically activates promyogenic kinases, p38MAPK and Akt, in a dose-dependent manner. GW0742-stimulated myoblast differentiation is mediated by p38MAPK and Akt, since it failed to restore myoblast differentiation repressed by inhibition of p38MAPK and Akt. In addition, GW0742 treatment enhances MyoD-reporter activities. Consistently, overexpression of PPARβ/δ enhances myoblast differentiation accompanied by elevated activation of p38MAPK and Akt. Collectively, these results suggest that PPARβ/δ enhances myoblast differentiation through activation of promyogenic signaling pathways.

  3. Differential expression of cell adhesion molecules in an ionizing radiation-induced breast cancer model system.

    PubMed

    Calaf, Gloria M; Roy, Debasish; Narayan, Gopeshwar; Balajee, Adayabalam S

    2013-07-01

    Cell-cell adhesion is mediated by members of the cadherin-catenin system and among them E-cadherin and β-catenin are important adhesion molecules for epithelial cell function and preservation of tissue integrity. To investigate the importance of cell adhesion molecules in breast carcinogenesis, we developed an in vitro breast cancer model system wherein immortalized human breast epithelial cell line, MCF-10F, was malignantly transformed by exposure to low doses of high linear energy transfer (LET) α particle radiation (150 keV/µm) and subsequent growth in the presence or absence of 17β-estradiol. This model consisted of human breast epithelial cells in different stages of transformation: i) parental cell line MCF-10F; ii) MCF-l0F continuously grown with estradiol at 10(-8) (Estrogen); iii) a non-malignant cell line (Alpha3); and iv) a malignant and tumorigenic cell line (Alpha5) and the Tumor2 cell line derived from the nude mouse xenograft of the Alpha5 cell line. Expression levels of important cell adhesion molecules such as α-catenin, β-catenin, γ-catenin, E-cadherin and integrin were found to be higher at the protein level in the Alpha5 and Tumor2 cell lines relative to these levels in the non-tumorigenic MCF-10F, Estrogen and Alpha3 cell lines. In corroboration, cDNA expression analysis revealed elevated levels of genes involved in the cell adhesion function [E-cadherin, integrin β6 and desmocollin3 (DSc3)] in the Alpha5 and Tumor2 cell lines relative to the levels in the MCF-10F, Estrogen and Alpha3 cell lines. Collectively, our results suggest that cell adhesion molecules are expressed at higher levels in malignantly transformed breast epithelial cells relative to levels in non-malignant cells. However, reduced levels of adhesion molecules observed in the mouse xenograft-derived Tumor 2 cell line compared to the pre-tumorigenic Alpha5 cell line suggests that the altered expression levels of adhesion molecules depend on the tumor tissue

  4. Wnt/{beta}-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

    SciTech Connect

    Zhang, Long; Shi, Songting; Zhang, Juan; Zhou, Fangfang; Dijke, Peter ten

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. Black-Right-Pointing-Pointer Wnt3a induces Id3 expression via canonical Wnt/{beta}-catenin pathway. Black-Right-Pointing-Pointer Wnt3a-induced Id3 expression does not depend on BMP signaling activation. Black-Right-Pointing-Pointer Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a {beta}-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/{beta}-catenin induced gene in myoblast cell fate determination.

  5. Nuclear Factor I-C promotes proliferation and differentiation of apical papilla-derived human stem cells in vitro

    SciTech Connect

    Zhang, Jing; Wang, Zhihua; Jiang, Yong; Niu, Zhongying; Fu, Lei; Luo, Zhirong; Cooper, Paul R.; Smith, Anthony J.; He, Wenxi

    2015-03-15

    The transcription factor Nuclear Factor I-C (NFIC) has been implicated in the regulation of tooth root development, where it may be anticipated to impact on the behavior of stem cells from the apical papilla (SCAPs) and root odontoblast activity. We hypothesized that NFIC may provide an important target for promoting dentin/root regeneration. In the present study, the effects of NFIC on the proliferation and differentiation of SCAPs were investigated. Over-expression of NFIC increased cell proliferation, mineralization nodule formation and alkaline phosphatase (ALP) activity in SCAPs. Furthermore, NFIC up-regulated the mRNA levels of odontogenic-related markers, ALP, osteocalcin and collagen type I as well as dentin sialoprotein protein levels. In contrast, knockdown of NFIC by si-RNA inhibited the mineralization capacity of SCAPs and down-regulated the expression of odontogenic-related markers. In conclusion, the results indicated that upregulation of NFIC activity in SCAPs may promote osteo/odontoblastic differentiation of SCAPs. - Highlights: • NFIC promotes the proliferation of SCAPs in vitro. • NFIC promotes osteo/odontogenic differentiation of SCAPs in vitro. • Knockdown of NFIC inhibits odontogenic differentiation in SCAPs.

  6. Molecular targeting regulation of proliferation and differentiation of the bone marrow-derived mesenchymal stem cells or mesenchymal stromal cells.

    PubMed

    Chen, Bei-Yu; Wang, Xi; Chen, Liang-Wei; Luo, Zhuo-Jing

    2012-04-01

    The bone marrow-derived mesenchymal stem cells or mesenchymal stromal cells (MSCs), with pluripotent differentiation capacity, present an ideal source for cell transplantation or tissue engineering therapies, but exact understanding of regulating mechanism underling MSC proliferation and differentiation remains a critical issue in securing their safe and efficient clinical application. This review outlines current knowledge regarding MSC cell surface biomarkers and molecular mechanisms of MSC differentiation and proliferation with emphasis on Wnt/β-catenin signaling, Notch signaling pathway, bone morphogenesis proteins and various growth factors functioning in regulation of differentiation and proliferation of MSCs. Possible relation of oncogene and immunosuppressive activities of MSCs with tumorigenicity or tumor generation is also addressed for safe translational clinical application. Fast increase of MSC knowledge and techniques has led to some successful clinical trials and helped devising new tissue engineering therapies for bone and cartilage diseases that severely afflict human health. Production of adult MSC-derived functional neurons can further extend their therapeutic application in nerve injury and neurodegenerative diseases. It is promising that MSCs shall overcome ethical and immunorejection problems appeared in human embryonic stem cells, and specific molecular targeting manipulation may result in practical MSC therapy for personalized treatment of various diseases in the regeneration medicine.

  7. Metamorphosis of summer flounder, Paralichthys dentatus: cell proliferation and differentiation of the gastric mucosa and developmental effects of altered thyroidal status.

    PubMed

    Soffientino, B; Specker, J L

    2001-06-15

    Summer flounder, like most marine fishes studied to date, are stomachless at first feeding, and subsequently acquire gastric function during the process of metamorphosis. Stomach formation is controlled largely by thyroxine (T4). In the present work we sought to understand gastric organogenesis in terms of cell proliferation and its relationship to histological differentiation. The objectives of the study were (1) to obtain a developmental pattern of cell proliferation in the gastric mucosa and to relate that pattern to the progress of gastric differentiation; and (2) to understand the regulatory role of T4 on cell proliferation and histological differentiation by altering the thyroidal status of the developing larvae. We observed that (1) in normally developing larvae, cell proliferation increased by early metamorphic climax (MC), remained high until mid-MC, and decreased to basal levels by late MC; concomitantly, the gastric glands appeared and differentiated in the fundic mucosa, and were complete by late MC; (2) T4 accelerated the differentiation of gastric glands and mucus neck cells, while inhibiting the concomitant increase in cell proliferation observed in controls; and (3) the goitrogen thiourea inhibited both cell proliferation and gastric differentiation compared to controls. These results indicate that T(4) is necessary for the three-fold increase in cell proliferation that occurs in early metamorphic climax, but that high T4 levels promote differentiation at the expense of proliferation. The observed effects would be consistent with the normal, metamorphosis-related increase in whole body T4.

  8. Conditioned media from differentiating craniofacial bone marrow stromal cells influence mineralization and proliferation in periodontal ligament stem cells.

    PubMed

    Jin, Zhenyu; Feng, Yuan; Liu, Hongwei

    2016-10-01

    Previous reports have mainly focused on the behavioral responses of human periodontal ligament stem cells (hPDLSCs) in interaction with tibia bone marrow stromal cells (BMSCs). However, there is little study on the biologic features of hPDLSCs under the induction of maxilla BMSCs (M-BMSCs) at different phases of osteogenic differentiation. We hypothesized that M-BMSCs undergoing osteogenic differentiation acted on the proliferation, differentiation, and bone-forming capacity of hPDLSCs. In this paper, primary hPDLSCs and human M-BMSCs (hM-BMSCs) were expanded in vitro. After screening of surface markers for characterization, hPDLSCs were cocultured with different phases of differentiating hM-BMSCs. Cell proliferation and alkaline phosphatase activity were examined, and mineralization-associated markers such as osteocalcin and runt-related transcription factor 2 of hPDLSCs in coculture with uninduced/osteoinduced hM-BMSCs were evaluated. hPDLSCs in hM-BMSCs-conditioned medium (hM-BMSCs-CM) group showed a reduction in proliferation compared with untreated hPDLSCs, while osteoinduced hM-BMSCs for 10 day-conditioned medium (hM-BMSCs-CM-10ds) and osteoinduced hM-BMSCs for 15 day-conditioned medium (hM-BMSCs-CM-15ds) enhance the proliferation of hPDLSCs. hM-BMSCs of separate differentiation stages temporarily inhibited osteogenesis of hPDLSCs in the early days. Upon extending time periods, uninduced/osteoinduced hM-BMSCs markedly enhanced osteogenesis of hPDLSCs to different degrees. The transplantation results showed hM-BMSCs-CM-15ds treatment promoted tissue regeneration to generate cementum/periodontal ligament-like structure characterized by hard-tissue formation. This research supported the notion that hM-BMSCs triggered osteogenesis of hPDLSCs suggesting important implications for periodontal engineering.

  9. Silicon-hydroxyapatite bioactive coatings (Si-HA) from diatomaceous earth and silica. Study of adhesion and proliferation of osteoblast-like cells.

    PubMed

    López-Alvarez, M; Solla, E L; González, P; Serra, J; León, B; Marques, A P; Reis, R L

    2009-05-01

    The aim of this study consisted on investigating the influence of silicon substituted hydroxyapatite (Si-HA) coatings over the human osteoblast-like cell line (SaOS-2) behaviour. Diatomaceous earth and silica, together with commercial hydroxyapatite were respectively the silicon and HA sources used to produce the Si-HA coatings. HA coatings with 0 wt% of silicon were used as control of the experiment. Pulsed laser deposition (PLD) was the selected technique to deposit the coatings. The Si-HA thin films were characterized by Fourier Transformed Infrared Spectroscopy (FTIR) demonstrating the efficient transfer of Si to the HA structure. The in vitro cell culture was established to assess the cell attachment, proliferation and osteoblastic activity respectively by, Scanning Electron Microscopy (SEM), DNA and alkaline phosphatase (ALP) quantification. The SEM analysis demonstrated a similar adhesion behaviour of the cells on the tested materials and the maintenance of the typical osteoblastic morphology along the time of culture. The Si-HA coatings did not evidence any type of cytotoxic behaviour when compared with HA coatings. Moreover, both the proliferation rate and osteoblastic activity results showed a slightly better performance on the Si-HA coatings from diatoms than on the Si-HA from silica.

  10. Influence of sodium hypochlorite treatment of electropolished and magnetoelectropolished nitinol surfaces on adhesion and proliferation of MC3T3 pre-osteoblast cells.

    PubMed

    Rokicki, Ryszard; Haider, Waseem; Hryniewicz, Tadeusz

    2012-09-01

    The influence of 6 % sodium hypochlorite (NaClO) treatment on adhesion and proliferation of MC3T3 pre-osteoblast cells seeded on electropolished (EP) and magnetoelectropolished (MEP) nitinol surfaces were investigated. The chemistry, topography, roughness, surface energy, wettability of EP and MEP nitinol surfaces before and after NaClO treatment were studied with X-ray photoelectron spectroscopy (XPS), profilometry, and contact angle meter. In vitro interaction of osteoblast cell and NaClO treated EP and MEP nitinol surfaces were assessed after 3 days of incubation by scanning electron microscopy. The XPS analysis shows that NaClO treatment increases oxygen content especially in subsurface oxide layer of EP and MEP nitinol. The changes of both basic components of nitinol, namely nickel and titanium in oxide layer, were negligible. The NaClO treatment did not influence physico-morphological surface properties of EP and MEP nitinol to a big extent. The osteoblast cells show remarkable adherence and proliferation improvement on NaClO treated EP and MEP nitinol surfaces. After 3 days of incubation they show almost total confluence on both NaClO treated surfaces. The present study shows that NaClO treatment of EP and MEP nitinol surfaces alters oxide layer by enriching it in oxygen and by this improves bone cell-nitinol interaction.

  11. Effects of cerium oxide nanoparticles on the proliferation, differentiation, and mineralization function of primary osteoblasts in vitro.

    PubMed

    Zhou, Guoqiang; Gu, Guangqi; Li, Yang; Zhang, Qun; Wang, Wenying; Wang, Shuxiang; Zhang, Jinchao

    2013-06-01

    The effects of cerium oxide nanoparticles on the proliferation, differentiation, and mineralization function of primary osteoblasts in vitro were evaluated. The results showed that the cell biological effects of cerium oxide nanoparticles varied with different diameters. The cytotoxicity of cerium oxide nanoparticles on primary osteoblasts varies with the size and incubation time. Sixty-nanometer cerium oxide nanoparticles show significant cytotoxicity on primary osteoblasts at 48 h exposure. Cerium oxide nanoparticles with diameters of 40 nm promoted the differentiation of osteoblasts and the promotion rate was enhanced with increasing concentration. Cerium oxide nanoparticles with diameters of 60 nm promoted the differentiation of osteoblasts at lower concentrations, but turned to inhibit the differentiation at higher concentrations. Cerium oxide nanoparticles promoted the adipogenic transdifferentiation of osteoblasts at all tested concentrations. Moreover, the effects of 60-nm cerium oxide nanoparticles were stronger than that of 40-nm cerium oxide nanoparticles. Cerium oxide nanoparticles promoted the formation of mineralized matrix nodules of osteoblasts at all tested concentrations in a dose-dependent manner and the promotion rate increased with decreasing size. The results showed that cerium oxide nanoparticles had no acute cytotoxic effects on osteoblasts and could promote the osteogenic differentiation and mineralization of osteoblasts. Moreover, the size, concentration, and culture time of nanoparticles have significant influence on the proliferation, differentiation, and mineralization of osteoblasts.

  12. Effects of tacrolimus on morphology, proliferation and differentiation of mesenchymal stem cells derived from gingiva tissue

    PubMed Central

    HA, DONG-HO; YONG, CHUL SOON; KIM, JONG OH; JEONG, JEE-HEON; PARK, JUN-BEOM

    2016-01-01

    Tacrolimus is a 23-membered macrolide lactone with potent immunosuppressive activity that is effective in the prophylaxis of organ rejection following kidney, heart and liver transplantation. Tacrolimus also exerts a variety of actions on bone metabolism. The aim of the present study was to evaluate the effects of different concentrations of tacrolimus on the morphology and viability of human stem cells derived from the gingiva. Gingival-derived stem cells were grown in the presence of tacrolimus at final concentrations ranging from 0.001 to 100 µg/ml. The morphology of the cells was viewed under an inverted microscope and the cell viability was analyzed using Cell Counting kit-8 (CCK-8) on days 1, 3, 5 and 7. Alizarin Red S staining was used to assess mineralization of treated cells. The control group showed spindle-shaped, fibroblast-like morphology and the shapes of the cells in 0.001, 0.01, 0.1, 1 and 10 µg/ml tacrolimus were similar to those of the control group. All groups except the 100 µg/ml group showed increased cell proliferation over time. Cultures grown in the presence of tacrolimus at 0.001, 0.01, 0.1, 1 and 10 µg/ml were not identified to be significantly different compared with the control at days 1, 3 and 5 using the CCK-8 assays. Increased mineralized deposits were noted with increased incubation time. Treatment with tacrolimus from 0.001 to 1 µg/ml led to an increase in mineralization compared with the control group. Within the limits of this study, tacrolimus at the tested concentrations (ranging from 0.001 to 10 µg/ml) did not result in differences in the viability of stem cells derived from gingiva; however it did enhance osteogenic differentiation of the stem cells. PMID:27177273

  13. Habitat-specific shaping of proliferation and neuronal differentiation in adult hippocampal neurogenesis of wild rodents.

    PubMed

    Cavegn, Nicole; van Dijk, R Maarten; Menges, Dominik; Brettschneider, Helene; Phalanndwa, Mashudu; Chimimba, Christian T; Isler, Karin; Lipp, Hans-Peter; Slomianka, Lutz; Amrein, Irmgard

    2013-01-01

    Daily life of wild mammals is characterized by a multitude of attractive and aversive stimuli. The hippocampus processes complex polymodal information associated with such stimuli and mediates adequate behavioral responses. How newly generated hippocampal neurons in wild animals contribute to hippocampal function is still a subject of debate. Here, we test the relationship between adult hippocampal neurogenesis (AHN) and habitat types. To this end, we compare wild Muridae species of southern Africa [Namaqua rock mouse (Micaelamys namaquensis), red veld rat (Aethomys chrysophilus), highveld gerbil (Tatera brantsii), and spiny mouse (Acomys spinosissimus)] with data from wild European Muridae [long-tailed wood mice (Apodemus sylvaticus), pygmy field mice (Apodemus microps), yellow-necked wood mice (Apodemus flavicollis), and house mice (Mus musculus domesticus)] from previous studies. The pattern of neurogenesis, expressed in normalized numbers of Ki67- and Doublecortin(DCX)-positive cells to total granule cells (GCs), is similar for the species from a southern African habitat. However, we found low proliferation, but high neuronal differentiation in rodents from the southern African habitat compared to rodents from the European environment. Within the African rodents, we observe additional regulatory and morphological traits in the hippocampus. Namaqua rock mice with previous pregnancies showed lower AHN compared to males and nulliparous females. The phylogenetically closely related species (Namaqua rock mouse and red veld rat) show a CA4, which is not usually observed in murine rodents. The specific features of the southern environment that may be associated with the high number of young neurons in African rodents still remain to be elucidated. This study provides the first evidence that a habitat can shape adult neurogenesis in rodents across phylogenetic groups.

  14. Habitat-specific shaping of proliferation and neuronal differentiation in adult hippocampal neurogenesis of wild rodents

    PubMed Central

    Cavegn, Nicole; van Dijk, R. Maarten; Menges, Dominik; Brettschneider, Helene; Phalanndwa, Mashudu; Chimimba, Christian T.; Isler, Karin; Lipp, Hans-Peter; Slomianka, Lutz; Amrein, Irmgard

    2013-01-01

    Daily life of wild mammals is characterized by a multitude of attractive and aversive stimuli. The hippocampus processes complex polymodal information associated with such stimuli and mediates adequate behavioral responses. How newly generated hippocampal neurons in wild animals contribute to hippocampal function is still a subject of debate. Here, we test the relationship between adult hippocampal neurogenesis (AHN) and habitat types. To this end, we compare wild Muridae species of southern Africa [Namaqua rock mouse (Micaelamys namaquensis), red veld rat (Aethomys chrysophilus), highveld gerbil (Tatera brantsii), and spiny mouse (Acomys spinosissimus)] with data from wild European Muridae [long-tailed wood mice (Apodemus sylvaticus), pygmy field mice (Apodemus microps), yellow-necked wood mice (Apodemus flavicollis), and house mice (Mus musculus domesticus)] from previous studies. The pattern of neurogenesis, expressed in normalized numbers of Ki67- and Doublecortin(DCX)-positive cells to total granule cells (GCs), is similar for the species from a southern African habitat. However, we found low proliferation, but high neuronal differentiation in rodents from the southern African habitat compared to rodents from the European environment. Within the African rodents, we observe additional regulatory and morphological traits in the hippocampus. Namaqua rock mice with previous pregnancies showed lower AHN compared to males and nulliparous females. The phylogenetically closely related species (Namaqua rock mouse and red veld rat) show a CA4, which is not usually observed in murine rodents. The specific features of the southern environment that may be associated with the high number of young neurons in African rodents still remain to be elucidated. This study provides the first evidence that a habitat can shape adult neurogenesis in rodents across phylogenetic groups. PMID:23616743

  15. Clonal Tests of Conventional Kinesin Function during Cell Proliferation and Differentiation

    PubMed Central

    Brendza, Robert P.; Sheehan, Kathy B.; Turner, F.R.; Saxton, William M.

    2000-01-01

    Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks. PMID:10749933

  16. Cholinesterases in development: AChE as a firewall to inhibit cell proliferation and support differentiation.

    PubMed

    Layer, Paul G; Klaczinski, Janine; Salfelder, Anika; Sperling, Laura E; Thangaraj, Gopenath; Tuschl, Corina; Vogel-Höpker, Astrid

    2013-03-25

    Acetylcholinesterase (AChE) is a most remarkable protein, not only because it is one of the fastest enzymes in nature, but also since it appears in many molecular forms and is regulated by elaborate genetic networks. AChE is expressed in many tissues during development and in mature organisms, as well as in healthy and diseased states. In search for alternative, "non-classical" functions of cholinesterases (ChEs), AChE could either work within the frame of classic cholinergic systems, but in non-neural tissues ("non-synaptic function"), or act non-enzymatically. Here, we review briefly some of the major ideas and advances of this field, and report on some recent progress from our own experimental work, e.g. that (i) non-neural ChEs have pronounced, predominantly enzymatic effects on early embryonic (limb) development in chick and mouse, that (ii) retinal R28 cells of the rat overexpressing synaptic AChE present a significantly decreased cell proliferation, and that (iii) in developing chick retina ACh-synthesizing and ACh-degrading cells originate from the same postmitotic precursor cells, which later form two locally opposing cell populations. We suggest that such distinct distributions of ChAT(+) vs. AChE(+) cells in the inner half retina provide graded distributions of ACh, which can direct cell differentiation and network formation. Thus, as corroborated by works from many labs, AChE can be considered a highly co-opting protein, which can combine enzymatic and non-enzymatic functions within one molecule.

  17. EGCG Inhibits Proliferation, Invasiveness and Tumor Growth by Up-Regulation of Adhesion Molecules, Suppression of Gelatinases Activity, and Induction of Apoptosis in Nasopharyngeal Carcinoma Cells

    PubMed Central

    Fang, Chih-Yeu; Wu, Chung-Chun; Hsu, Hui-Yu; Chuang, Hsin-Ying; Huang, Sheng-Yen; Tsai, Ching-Hwa; Chang, Yao; Tsao, George Sai-Wah; Chen, Chi-Long; Chen, Jen-Yang

    2015-01-01

    (−)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, has been shown to inhibit the proliferation of a variety of tumor cells. Epidemiological studies have shown that drinking green tea can reduce the incidence of nasopharyngeal carcinoma (NPC), yet the underlying mechanism is not well understood. In this study, the inhibitory effect of EGCG was tested on a set of Epstein Barr virus-negative and -positive NPC cell lines. Treatment with EGCG inhibited the proliferation of NPC cells but did not affect the growth of a non-malignant nasopharyngeal cell line, NP460hTert. Moreover, EGCG treated cells had reduced migration and invasive properties. The expression of the cell adhesion molecules E-cadherin and β-catenin was found to be up-regulated by EGCG treatment, while the down-regulation of matrix metalloproteinases (MMP)-2 and MMP-9 were found to be mediated by suppression of extracellular signal-regulated kinase (ERK) phosphorylation and AP-1 and Sp1 transactivation. Spheroid formation by NPC cells in suspension was significantly inhibited by EGCG. Oral administration of EGCG was capable of suppressing tumor growth in xenografted mice bearing NPC tumors. Treatment with EGCG was found to elevate the expression of p53 and p21, and eventually led to apoptosis of NPC cells via caspase 3 activation. The nuclear translocation of NF-κB and β-catenin was also suppressed by EGCG treatment. These results indicate that EGCG can inhibit the proliferation and invasiveness, and induce apoptosis, of NPC cells, making it a promising agent for chemoprevention or adjuvant therapy of NPC. PMID:25625511

  18. Clodronate acts on human osteoclastic cell proliferation, differentiation and function in a bioreversible manner

    PubMed Central

    Recenti, Raffaella; Leone, Giuseppe; Simi, Lisa; Orfei, Marco; Pinzani, Pamela; Pieraccini, Giuseppe; Moneti, Gloriano; Carossino, Anna Maria; Franchi, Alessandro; Bartolucci, Gianluca; Carbonell Sala, Silvia; Ginanneschi, Mauro; Tanini, Annalisa; Brandi, Maria Luisa

    2007-01-01

    clodronate was evaluated by luciferin-luciferase assay. The Mann-Whitney “U” test was conducted for statistical analysis. Results. We found that clodronate inhibited both proliferation and differentiative features of cells of the osteoclastic lineage. Furthermore, treatment of both cell types with clodronate caused apoptosis, generation of measurable levels of AppCCl2p, and reduction of intracellular ATP levels. Addition of ATP to the culture medium caused an inhibition of the biological actions of clodronate on the human osteoclastic cell lineage. Conclusions. These data indicate that intracellular accumulation of the metabolite AppCCl2p is the likely route by which clodronate inhibits osteoclastic function and this effect is reversed by ATP. PMID:22461215

  19. Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

    NASA Astrophysics Data System (ADS)

    Zheng, Dong; Neoh, Koon Gee; Kang, En-Tang

    2016-01-01

    In this work, alkaline phosphatase (ALP) was covalently immobilized on carboxymethyl chitosan (CMCS)-coated polydopamine (PDA)-functionalized Ti to achieve a bifunctional surface. Our results showed ∼89% reduction in Staphylococcus epidermidis adhesion on this surface compared to that on pristine Ti. The ALP-modified Ti supported cell proliferation, and significantly enhanced cellular ALP activity and calcium deposition of osteoblasts, human mesenchymal stem cells (hMSCs) and human adipose-derived stem cells (hADSCs). The extent of enhancement in the functions of these cells is dependent on the surface density of immobilized ALP. The substrate prepared using an ALP solution of 50 μg/cm2 resulted in 44%, 54% and 129% increase in calcium deposited by osteoblasts, hMSCs and hADSCs, respectively, compared to those cultured on pristine Ti. The ALP-modified substrates also promoted the osteogenic differentiation of hMSCs and hADSCs by up-regulating gene expressions of runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteocalcin (OC) in the two types of stem cells. The surface-immobilized ALP was stable after being subjected to 1 h immersion in 70% ethanol and autoclaving at 121 °C for 20 min. However, the enzymatic bioactivity of the surface-immobilized ALP was reduced by about 50% after these substrates were immersed in phosphate buffered saline (PBS) or PBS containing lysozyme for 14 days.

  20. Influence of biomechanical and biochemical stimulation on the proliferation and differentiation of bone marrow stromal cells seeded on polyurethane scaffolds

    PubMed Central

    TENG, SONGSONG; LIU, CHAOXU; GUENTHER, DANIEL; OMAR, MOHAMED; NEUNABER, CLAUDIA; KRETTEK, CHRISTIAN; JAGODZINSKI, MICHAEL

    2016-01-01

    The aim of the present investigation was to compare the effects of cyclic compression, perfusion, dexamethasone (DEX) and bone morphogenetic protein-7 (BMP-7) on the proliferation and differentiation of human bone marrow stromal cells (hBMSCs) in polyurethane scaffolds in a perfusion bioreactor. Polyurethane scaffolds seeded with hBMSCs were cultured under six different conditions, as follows: 10% Cyclic compression at 0.5 and 5 Hz; 10 ml/min perfusion; 100 nM DEX; 100 ng/ml BMP-7; and 1 ml/min perfusion without mechanical and biochemical stimulation (control). On days 7 and 14, samples were tested for the following data: Cell proliferation; mRNA expression of Runx2, COL1A1 and osteocalcin; osteocalcin content; calcium deposition; and the equilibrium modulus of the tissue specimen. The results indicated that BMP-7 and 10 ml/min perfusion promoted cell proliferation, which was inhibited by 5 Hz cyclic compression and DEX. On day 7, the 5 Hz cyclic compression inhibited Runx2 expression, whereas the 0.5 Hz cyclic compression and BMP-7 upregulated the COL1A1 mRNA levels on day 7 and enhanced the osteocalcin expression on day 14. The DEX-treated hBMSCs exhibited downregulated osteocalcin expression. After 14 days, the BMP-7 group exhibited the highest calcium deposition, followed by the 0.5 Hz cyclic compression and the DEX groups. The equilibrium modulus of the engineered constructs significantly increased in the BMP-7, 0.5 Hz cyclic compression and DEX groups. In conclusion, the present results suggest that BMP-7 and perfusion enhance cell proliferation, whereas high frequency cyclic compression inhibits the proliferation and osteogenic differentiation of hBMSCs. Low frequency cyclic compression is more effective than DEX, but less effective compared with BMP-7 on the osteogenic differentiation of hBMSCs seeded on polyurethane scaffolds. PMID:27284290

  1. Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

    SciTech Connect

    Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun; Choi, Yun Jin; Kim, Yong Sang; Ko, Kinarm; Koh, Yong-Gon

    2014-08-08

    Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.

  2. Stochastic cellular automata model of cell migration, proliferation and differentiation: validation with in vitro cultures of muscle satellite cells.

    PubMed

    Garijo, N; Manzano, R; Osta, R; Perez, M A

    2012-12-07

    Cell migration and proliferation has been modelled in the literature as a process similar to diffusion. However, using diffusion models to simulate the proliferation and migration of cells tends to create a homogeneous distribution in the cell density that does not correlate to empirical observations. In fact, the mechanism of cell dispersal is not diffusion. Cells disperse by crawling or proliferation, or are transported in a moving fluid. The use of cellular automata, particle models or cell-based models can overcome this limitation. This paper presents a stochastic cellular automata model to simulate the proliferation, migration and differentiation of cells. These processes are considered as completely stochastic as well as discrete. The model developed was applied to predict the behaviour of in vitro cell cultures performed with adult muscle satellite cells. Moreover, non homogeneous distribution of cells has been observed inside the culture well and, using the above mentioned stochastic cellular automata model, we have been able to predict this heterogeneous cell distribution and compute accurate quantitative results. Differentiation was also incorporated into the computational simulation. The results predicted the myotube formation that typically occurs with adult muscle satellite cells. In conclusion, we have shown how a stochastic cellular automata model can be implemented and is capable of reproducing the in vitro behaviour of adult muscle satellite cells.

  3. Attachment, proliferation and differentiation of BMSCs on gas-jet/electrospun nHAP/PHB fibrous scaffolds

    NASA Astrophysics Data System (ADS)

    Guan, Donghua; Chen, Zhiqing; Huang, Chunpeng; Lin, Yinghe

    2008-11-01

    In this study, poly(3-hydroxybutyrate) (PHB)-based scaffolds containing nanosized hydroxyapatite (nHAP) were manufactured by gas-jet/electrospinning. The morphologies of the scaffolds were characterized. The effect of the scaffolds on attachment, proliferation and differentiation of the bone marrow stroma cells (BMSCs) were accessed by using scanning electron microscopy (SEM), methylthiazol tetrazolium (MTT) assay and alkaline phosphatase (ALP) activity. The results show that the gas-jet/electrospun scaffolds possess an extracellular matrix-like topography. In vitro studies describe that the scaffolds have positive effects on attachment, proliferation and differentiation of BMSCs in vitro. It can be concluded that the scaffolds combing the unique structural features generated by gas-jet/electrospinning with functional factors, have the potential to be used in bone tissue engineering.

  4. ErbB3 binding protein-1 (Ebp1) controls proliferation and myogenic differentiation of muscle stem cells.

    PubMed

    Figeac, Nicolas; Serralbo, Olivier; Marcelle, Christophe; Zammit, Peter S

    2014-02-01

    Satellite cells are resident stem cells of skeletal muscle, supplying myoblasts for post-natal muscle growth, hypertrophy and repair. Many regulatory networks control satellite cell function, which includes EGF signalling via the ErbB family of receptors. Here we investigated the role of ErbB3 binding protein-1 (Ebp1) in regulation of myogenic stem cell proliferation and differentiation. Ebp1 is a well-conserved DNA/RNA binding protein that is implicated in cell growth, apoptosis and differentiation in many cell types. Of the two main Ebp1 isoforms, only p48 was expressed in satellite cells and C2C12 myoblasts. Although not present in quiescent satellite cells, p48 was strongly induced during activation, remaining at high levels during proliferation and differentiation. While retroviral-mediated over-expression of Ebp1 had only minor effects, siRNA-mediated Ebp1 knockdown inhibited both proliferation and differentiation of satellite cells and C2C12 myoblasts, with a clear failure of myotube formation. Ebp1-knockdown significantly reduced ErbB3 receptor levels, yet over-expression of ErbB3 in Ebp1 knockdown cells did not rescue differentiation. Ebp1 was also expressed by muscle cells during developmental myogenesis in mouse. Since Ebp1 is well-conserved between mouse and chick, we switched to chick to examine its role in muscle formation. In chick embryo, Ebp1 was expressed in the dermomyotome, and myogenic differentiation of muscle progenitors was inhibited by specific Ebp1 down-regulation using shRNA electroporation. These observations demonstrate a conserved function of Ebp1 in the regulation of embryonic muscle progenitors and adult muscle stem cells, which likely operates independently of ErbB3 signaling.

  5. ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential

    SciTech Connect

    Carcamo-Orive, Ivan; Tejados, Naiara; Delgado, Jesus; Gaztelumendi, Ainhoa; Otaegui, David; Lang, Valerie; Trigueros, Cesar

    2008-05-01

    Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes.

  6. Effects of non-steroidal anti-inflammatory drugs on proliferation, differentiation and migration in equine mesenchymal stem cells.

    PubMed

    Müller, Maike; Raabe, Oksana; Addicks, Klaus; Wenisch, Sabine; Arnhold, Stefan

    2011-03-01

    In equine medicine, stem cell therapies for orthopaedic diseases are routinely accompanied by application of NSAIDs (non-steroidal anti-inflammatory drugs). Thus, it has to be analysed how NSAIDs actually affect the growth and differentiation potential of MSCs (mesenchymal stem cells) in vitro in order to predict the influence of NSAIDs such as phenylbutazone, meloxicam, celecoxib and flunixin on MSCs after grafting in vivo. The effects of NSAIDs were evaluated regarding cell viability and proliferation. Additionally, the multilineage differentiation capacity and cell migration was analysed. NSAIDs at lower concentrations (0.1-1 μM for celecoxib and meloxicam and 10-50 μM for flunixin) exert a positive effect on cell proliferation and migration, while at higher concentrations (10-200 μM for celecoxib and meloxicam and 100-1000 μM for flunixin and phenylbutazone), there is rather a negative influence. While there is hardly any influence on the adipogenic as well as on the chondrogenic MSC differentiation, the osteogenic differentiation potential, as demonstrated with the von Kossa staining, is significantly disturbed. Thus, it can be concluded that the effects of NSAIDs on MSCs are largely dependent on the concentrations used. Additionally, for some differentiation lineages, also the choice of NSAID is critical.

  7. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    SciTech Connect

    Xin, Ying; Lu, Qingxian; Li, Qiutang

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  8. Impact of the putative differentiating agents sodium phenylbutyrate and sodium phenylacetate on proliferation, differentiation, and apoptosis of primary neoplastic myeloid cells.

    PubMed

    Gore, S D; Samid, D; Weng, L J

    1997-10-01

    Sodium phenylacetate (PA) and sodium phenylbutyrate (PB) are aromatic fatty acids that can effect differentiation in a variety of cell lines at doses that may be clinically attainable. We have studied the impact of these two agents on lineage- and differentiation stage-specific antigen expression, proliferation, apoptosis, and clonogenic cell survival in primary cultures of bone marrow samples from patients with myeloid neoplasms at presentation and in remission and from normal volunteers. PB inhibited the proliferation of primary acute myeloid leukemia cells in suspension culture with an ID50 of 6.6 mM, similar to its ED50 in cell lines. At higher doses (>/=5 mM), PB also induced apoptosis. PB inhibited clonogenic leukemia cell growth with a median ID50 of less than 2 mM; however, colony-forming units-granulocyte/macrophage from patients with myelodysplasia and normal volunteers were inhibited with a similar ID50. In contrast to PB, its metabolite PA had no significant effect on either acute myeloid leukemia proliferation or apoptosis. Expression of the monocytic marker CD14 was increased in monocytic and myelomonocytic leukemias in response to PB, and to a lesser extent, PA. Surprisingly, both agents appeared to increase expression of the progenitor cell antigen CD34, as well as the DR locus of the human leukocyte antigen. These data indicate that PB, but not its metabolite PA, has significant cytostatic and differentiating activity against primary neoplastic myeloid cells at doses that may be achievable clinically.

  9. Alternating Current Electric Fields of Varying Frequencies: Effects on Proliferation and Differentiation of Porcine Neural Progenitor Cells

    PubMed Central

    Lim, Ji-Hey; McCullen, Seth D.; Piedrahita, Jorge A.

    2013-01-01

    Abstract Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes. PMID:23961767

  10. Alternating current electric fields of varying frequencies: effects on proliferation and differentiation of porcine neural progenitor cells.

    PubMed

    Lim, Ji-Hey; McCullen, Seth D; Piedrahita, Jorge A; Loboa, Elizabeth G; Olby, Natasha J

    2013-10-01

    Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes.

  11. Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1.

    PubMed

    Keohane, Aoife; Ryan, Sinead; Maloney, Eimer; Sullivan, Aideen M; Nolan, Yvonne M

    2010-01-01

    Tumour necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine, which influences neuronal survival and function yet there is limited information available on its effects on hippocampal neural precursor cells (NPCs). We show that TNFalpha treatment during proliferation had no effect on the percentage of proliferating cells prepared from embryonic rat hippocampal neurosphere cultures, nor did it affect cell fate towards either an astrocytic or neuronal lineage when cells were then allowed to differentiate. However, when cells were differentiated in the presence of TNFalpha, significantly reduced percentages of newly born and post-mitotic neurons, significantly increased percentages of astrocytes and increased expression of TNFalpha receptors, TNF-R1 and TNF-R2, as well as expression of the anti-neurogenic Hes1 gene, were observed. These data indicate that exposure of hippocampal NPCs to TNFalpha when they are undergoing differentiation but not proliferation has a detrimental effect on their neuronal lineage fate, which may be mediated through increased expression of Hes1.

  12. Extremely low-frequency electromagnetic fields enhance the proliferation and differentiation of neural progenitor cells cultured from ischemic brains.

    PubMed

    Cheng, Yannan; Dai, Yiqin; Zhu, Ximin; Xu, Haochen; Cai, Ping; Xia, Ruohong; Mao, Lizhen; Zhao, Bing-Qiao; Fan, Wenying

    2015-10-21

    In the mammalian brain, neurogenesis persists throughout the embryonic period and adulthood in the subventricular zone of the lateral ventricle and the granular zone (dentate gyrus) of the hippocampus. Newborn neural progenitor cells (NPCs) in the two regions play a critical role in structural and functional plasticity and neural regeneration after brain injury. Previous studies have reported that extremely low-frequency electromagnetic fields (ELF-EMF) could promote osteogenesis, angiogenesis, and cardiac stem cells' differentiation, which indicates that ELF-EMF might be an effective tool for regenerative therapy. The present studies were carried out to examine the effects of ELF-EMF on hippocampal NPCs cultured from embryonic and adult ischemic brains. We found that exposure to ELF-EMF (50 Hz, 0.4 mT) significantly enhanced the proliferation capability both in embryonic NPCs and in ischemic NPCs. Neuronal differentiation was also enhanced after 7 days of cumulative ELF-EMF exposure, whereas glial differentiation was not influenced markedly. The expression of phosphorylated Akt increased during the proliferation process when ischemic NPCs were exposed to ELF-EMF. However, blockage of the Akt pathway abolished the ELF-EMF-induced proliferation of ischemic NPCs. These data show that ELF-EMF promotes neurogenesis of ischemic NPCs and suggest that this effect may occur through the Akt pathway.Video abstract, Supplemental Digital Content 1, http://links.lww.com/WNR/A347.

  13. Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

    PubMed Central

    Chen, Yongjuan; Roohani-Esfahani, Seyed-Iman; Lu, ZuFu; Zreiqat, Hala; Dunstan, Colin R.

    2015-01-01

    Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling. PMID:25602473

  14. Acupuncture Induces the Proliferation and Differentiation of Endogenous Neural Stem Cells in Rats with Traumatic Brain Injury

    PubMed Central

    Jiang, Shuting; Chen, Weihao; Zhang, Yimin; Zhang, Yujuan; Chen, Ailian; Dai, Qiufu; Lin, Shujun; Lin, Hanyu

    2016-01-01

    Purpose. To investigate whether acupuncture induced the proliferation and differentiation of endogenous neural stem cells (NSCs) in a rat model of traumatic brain injury (TBI). Methods. 104 Sprague-Dawley rats were randomly divided into normal, model, and acupuncture groups. Each group was subdivided into three-day (3 d), seven-day (7 d), and fourteen-day (14 d) groups. The rat TBI model was established using Feeney's freefall epidural impact method. The rats in the acupuncture group were treated at acupoints (Baihui, Shuigou, Fengfu, Yamen, and bilateral Hegu). The normal and model groups did not receive acupuncture. The establishment of the rat TBI model and the therapeutic effect of acupuncture were assessed using neurobehavioral scoring and hematoxylin-eosin staining. The proliferation and differentiation of NSCs in TBI rats were analyzed using immunofluorescence microscopy. Results. The levels of nestin-expressing cells and bromodeoxyuridine/glial fibrillary acidic protein- (BrdU/GFAP-) and BrdU/S100 calcium-binding protein B-positive and BrdU/microtubule-associated protein 2- and BrdU/galactocerebrosidase-positive cells were more significantly increased at various time points in the acupuncture group than in the model group (P < 0.01), except for a decreased level of BrdU/GFAP-positive cells at 7 d and 14 d. Conclusion. Acupuncture induced the proliferation and differentiation of NSCs, thereby promoting neural repair in the TBI rats. PMID:27313641

  15. A role of KIT receptor signaling for proliferation and differentiation of rat stem Leydig cells in vitro.

    PubMed

    Liu, Shiwen; Chen, Xiaomin; Wang, Yiyan; Li, Linxi; Wang, Guimin; Li, Xiaoheng; Chen, Haolin; Guo, Jingjing; Lin, Han; Lian, Qing-Quan; Ge, Ren-Shan

    2017-03-15

    In the testis, KIT ligand (KITL, also called stem cell factor) is expressed by Sertoli cells and its receptor (c-kit, KIT) is expressed by spermatogonia and Leydig cells. Although KITL-KIT signaling is critical for the spermatogenesis, its roles in Leydig cell development during puberty are not clear. In the present study, we investigated effects of KITL on stem Leydig cell proliferation and differentiation. Using an in vitro culture system of seminiferous tubules from Leydig cell-depleted testis, we found that KITL increased the proliferation activity of putative stem Leydig cells at higher concentration (10 and 100 ng/ml). Low concentration (1 ng/ml) of KITL significantly induced the differentiation of stem Leydig cells via increasing the expression level of steroidogenic acute regulatory protein (Star). In contrast, higher concentration (100 ng/ml) of KITL inhibited the differentiation of stem Leydig cells via inhibiting the steroidogenic enzyme (Cyp11a1, Cyp17a1, and Hsd17b3) expression levels. We cultured rat progenitor Leydig cells with KITL for 48 h and did not find any influence of KITL on the proliferation and androgen production of these cells. In conclusion, KITL is a growth factor that regulates the development of the stem Leydig cell.

  16. The MMP-1/PAR-1 Axis Enhances Proliferation and Neuronal Differentiation of Adult Hippocampal Neural Progenitor Cells

    PubMed Central

    Valente, Maria Maddalena; Allen, Megan; Bortolotto, Valeria; Lim, Seung T.; Conant, Katherine; Grilli, Mariagrazia

    2015-01-01

    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play a role in varied forms of developmental and postnatal neuroplasticity. MMP substrates include protease-activated receptor-1 (PAR-1), a G-protein coupled receptor expressed in hippocampus. We examined proliferation and differentiation of adult neural progenitor cells (aNPCs) from hippocampi of mice that overexpress the potent PAR-1 agonist MMP-1. We found that, as compared to aNPCs from littermate controls, MMP-1 tg aNPCs display enhanced proliferation. Under differentiating conditions, these cells give rise to a higher percentage of MAP-2+ neurons and a reduced number of oligodendrocyte precursors, and no change in the number of astrocytes. The fact that these results are MMP and PAR-1 dependent is supported by studies with distinct antagonists. Moreover, JSH-23, an inhibitor of NF-κB p65 nuclear translocation, counteracted both the proliferation and differentiation changes seen in MMP-1 tg-derived NPCs. In complementary studies, we found that the percentage of Sox2+ undifferentiated progenitor cells is increased in hippocampi of MMP-1 tg animals, compared to wt mice. Together, these results add to a growing body of data suggesting that