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Sample records for cell colony formation

  1. Study of budding yeast colony formation and its characterizations by using circular granular cell

    NASA Astrophysics Data System (ADS)

    Aprianti, D.; Haryanto, F.; Purqon, A.; Khotimah, S. N.; Viridi, S.

    2016-03-01

    Budding yeast can exhibit colony formation in solid substrate. The colony of pathogenic budding yeast can colonize various surfaces of the human body and medical devices. Furthermore, it can form biofilm that resists drug effective therapy. The formation of the colony is affected by the interaction between cells and with its growth media. The cell budding pattern holds an important role in colony expansion. To study this colony growth, the molecular dynamic method was chosen to simulate the interaction between budding yeast cells. Every cell was modelled by circular granular cells, which can grow and produce buds. Cohesion force, contact force, and Stokes force govern this model to mimic the interaction between cells and with the growth substrate. Characterization was determined by the maximum (L max) and minimum (L min) distances between two cells within the colony and whether two lines that connect the two cells in the maximum and minimum distances intersect each other. Therefore, it can be recognized the colony shape in circular, oval, and irregular shapes. Simulation resulted that colony formation are mostly in oval shape with little branch. It also shows that greater cohesion strength obtains more compact colony formation.

  2. Effect of aspirin on tumour cell colony formation and evolution.

    PubMed

    Wodarz, Dominik; Goel, Ajay; Boland, C Richard; Komarova, Natalia L

    2017-09-01

    Aspirin is known to reduce the risk of colorectal cancer (CRC) incidence, but the underlying mechanisms are not fully understood. In a previous study, we quantified the in vitro growth kinetics of different CRC tumour cell lines treated with varying doses of aspirin, measuring the rate of cell division and cell death. Here, we use these measured parameters to calculate the chances of successful clonal expansion and to determine the evolutionary potential of the tumour cell lines in the presence and absence of aspirin. The calculations indicate that aspirin increases the probability that a single tumour cell fails to clonally expand. Further, calculations suggest that aspirin increases the evolutionary potential of an expanding tumour cell colony. An aspirin-treated tumour cell population is predicted to result in the accumulation of more mutations (and is thus more virulent and more difficult to treat) than a cell population of the same size that grew without aspirin. This indicates a potential trade-off between delaying the onset of cancer and increasing its evolutionary potential through chemoprevention. Further work needs to investigate to what extent these findings apply to in vivo settings, and to what degree they contribute to the epidemiologically documented aspirin-mediated protection. © 2017 The Author(s).

  3. Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture.

    PubMed

    Farcet, J P; Gourdin, M F; Testa, U; Andre, C; Jouault, H; Reyes, F

    1983-01-01

    Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal T lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes.

  4. Studies on T-cell colony formation in chronic renal failure (CRF) patients.

    PubMed

    Wakabayashi, Y; Sugimoto, M; Ishiyama, T; Horie, S; Abe, S; Hirose, S; Okuda, T

    1989-12-01

    In order to study the possibility of abnormal differentiation and proliferation of T-cell precursors in chronic renal failure (CRF), we studied T-cell colony formation in CRF patients. The two-step monolayer method, with phytohemagglutinin-P as the inducer, was used for T-cell colony formation. In our results, colony formation was markedly reduced in CRF patients in comparison with normal controls, with about half of the former showing no colony growth. All cases showed a significant increase in colony numbers with in vitro plasmapheresis (the replacement of autologous plasma in the culture system with normal AB plasma). A significant increase in colony numbers was also seen with the addition of exogenous interleukin-2 (IL-2). The addition of IL-2 in the presence of normal plasma, in particular, induced an increase in colony numbers to near the levels in normal subjects. These results suggest that T-cell precursors exist in near normal numbers in CRF patients and that there are uremic inhibitors in the plasma. A reduced production of IL-2 is also indicated. These factors may be involved in the pathogenesis of immunodeficiency in CRF patients.

  5. Periodic Colony Formation of Bacteria Due to their Cell Reproduction and Movement

    NASA Astrophysics Data System (ADS)

    Itoh, H.; Wakita, J.; Watanabe, K.; Matsuyama, T.; Matsushita, M.

    We have experimentally investigated periodic pattern formation produced by bacterial species Proteus mirabilis, which forms concentric-ring-like colonies by repeating migration and rest alternately on the surface of a solid agar medium. We distinguish three phases (initial lag phase, the following migration and consolidation phases that appear alternately) for the colony growth. Here we mainly used physical approaches in order to try to understand the formation of concentric-ring-like colonies, such as cutting the part of a colony during its growth. Global chemical signals governing the colony formation from the center were not found. We also checked phase entrainment quantitatively by letting two colonies collide with each other and confirmed that it does not take place in macroscopic scales. When we cut a colony just behind the migrating front shortly after the migration started, the migration ended earlier and the following consolidation lasted longer. However, the following cycles were not influenced by the cut, i.e., the following migration and consolidation phases were both found to return normal. The cut results in the stop of supply of cell population to the migrating front by internal waves. In fact the cell population on the new terrace during the first migration after the cut was less than that without cut. Furthermore, the cell population density was found to be recovered to the ordinary value by the end of the consolidation. All these experimental results suggest that the most important factor for the repetition of migration and consolidation phases is the cell population density.

  6. Colony formation and interleukin 2 production by leukaemic human T cells.

    PubMed Central

    Krajewski, A S; Dewar, A E; Seidelin, P H; Murray, R

    1983-01-01

    PHA-induced colony formation and interleukin 2 (IL-2) production were studied in four patients with T cell leukaemia (three cases OKT4+/T helper and one case OKT8+/T cytotoxic suppressor). Cases of T helper cell leukaemia showed colony formation that was comparable to normal purified blood T cells and was not dependent on the addition of conditioned medium, containing IL-2 activity, to cultures. In contrast the T suppressor cell leukaemia formed colonies only when cultures were supplemented with IL-2 containing medium. When IL-2 production by PHA stimulated cells was measured culture supernatants from the three T helper cell leukaemias all showed normal or high levels of activity, when compared to normal blood mononuclear cells, whereas the T suppressor cell leukaemia showed no activity. PMID:6604606

  7. [CFU-HPP colony formation of bone marrow hematopoietic proginitor cells in psoriatic patients and methylation of p16 gene promotor in CFU-HPP colony cells].

    PubMed

    Zhang, Rui-Li; Niu, Xu-Ping; Li, Xin-Hua; Zhang, Kai-Ming; Yin, Guo-Hua

    2007-08-01

    This study was purposed to investigate the colony formation of high-proliferative potential colony-forming units (CFU-HPP) from bone marrow-derived hematopoietic cells of psoriatic patients and p16 gene promotor methylation in CFU-HPP cells, and to explore the relationship between the colony formation and the methylation status of p16 gene promoter. Bone marrow-derived mononuclear cells from psoriatic patients and normal controls were separated by density gradient centrifugation, and were cultured in methycellulose semi-solid culture medium with SCF, GM-CSF, IL-3 and IL-6 for 14 days to measure the colonies of CFU-HPP. The CFU-HPP colony cells were collected and methylation status of p16 gene promoter of CFU-HPP cell DNA modified with sodium bisulfite was detected by the methylation-specific polymerase chain reaction (MSP). The results showed that in methycellulose semi-solid culture system, the number and the size of CFU-HPP colonies of bone marrow of psoriatic patients were all significantly less than that of normal controls, the positive frequency of p16 gene promoter methylation in CFU-HPP cells was lower than that in CFU-HPP colony cells of normal controls. It is concluded that the colony formation capability of CFU-HPP from bone marrow hematopoietic progenitor cells in psoriatic patients is lower than that in normal controls, and the lower positive frequency of P16 gene promoter methylation in CFU-HPP cells perhaps closely correlated with lower CFU-HPP colony-forming capability.

  8. Arsenic promotes centrosome abnormalities and cell colony formation in p53 compromised human lung cells

    SciTech Connect

    Liao Weiting; Lin Pinpin; Cheng, T.-S.; Yu, H.-S.; Chang, Louis W.

    2007-12-01

    Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus, an interaction between arsenic and cigarette smoking in lung carcinogenesis was suspected. p53 dysfunction or mutation in lung epithelial cells was frequently observed in cigarette smokers. Our present study was to explore the differential effects by arsenic on H1355 cells (human lung adenocarcinoma cell line with mutation in p53), BEAS-2B (immortalized lung epithelial cell with functional p53) and pifithrin-{alpha}-treated BEAS-2B cells (p53-inhibited cells). These cells were treated with different doses of sodium arsenite (0, 0.1, 1, 5 and 10 {mu}M) for 48 h. A greater reduction in cell viability was observed in the BEAS-2B cells vs. p53 compromised cells (H1355 or p53-inhibited BEAS-2B). Similar observation was also made on 7-day cell survival (growth) study. TUNEL analysis confirmed that there was indeed a significantly reduced arsenite-induced apoptosis found in p53-compromised cells. Centrosomal abnormality has been attributed to eventual chromosomal missegregation, aneuploidy and tumorigenesis. In our present study, reduced p21 and Gadd45a expressions and increased centrosomal abnormality (atopic and multiple centrosomes) were observed in both arsenite-treated H1355 and p53-inhibited BEAS-2B cells as compared with similarly treated BEAS-2B cells. Increased anchorage-independent growth (colony formation) of BEAS-2B cells co-treated with pifithrin-{alpha} and 5 {mu}M sodium arsenite was also observed in soft agar. Our present investigation demonstrated that arsenic would act specifically on p53 compromised cells (either with p53 dysfunction or inhibited) to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenic, especially under the condition of p53 dysfunction.

  9. Comparison of colony formation of human spermatogonial stem cells (SSCs) with and without collagen.

    PubMed

    Shiva, Razi; Ghasem, Saki; Masoud, Hemadi; Sadat, Khorsandi Laya; Ali, Khodadadi; Reza, Dadfar Mohammad

    2016-03-01

    To investigate the effects of collagen and growth factors on in vitro proliferation of human spermatogonial stem cells obtained from patients with non-obstructive azoospermia. The experimental cross-sectional study was conducted from February 2013 to April 2015 after obtaining approval from the ethics committee of Ahvaz Jundishapur University of Medical Sciences, Iran. Testicular sperm extractions of non-obstructive azoospermic patients were obtained from the Clinical Urology and Embryology, In Vitro Fertilization Department of Imam Khomeini Hospital. Spermatogonial stem cells and Sertoli cells, obtained from human testis biopsies by a two-step enzymatic digestion method, were purified using fluorescence- activated cell-sorting and daturastramonium-lectin, and were cultured separately. To investigate a more direct influential factor on colony formation, one control and two experimental groups were formed. Group 1 acted as the control in which spermatogonial stem cells were co-cultured with Sertoli cells alone. In group 2 they were co-cultured with Sertoli cells and growth factors such as leukaemia inhibitory factor, epidermal growth factor and glial cell-derived neurotrophic factor, and in group 3 with Sertoli cells along with growth factors in the presence of collagen-coated dishes. Number and diameter of the colonies were evaluated after 7 weeks. Specimens obtained related to 21 patients. Number and diameter of the colonies in group 3 (18±2.6 and 276.6±45.5) were significantly more than both groups 1 (3.5±1 and D1:81.6±12) and group 2(11±2.2 and 165.2±32.5) (p<0.05 each). Also, the number and diameter of colony in group 2 were significantly better than the control group (p<0.05).Expression profile of the VASA, promyelocytic leukaemia zinc-finger (PLZF), Octamer-binding transcription factor 4 (OCT4) and integrin a6 (INTGa6) were detected in all groups. Based on cytochemical findings, OCT4 was expressed in the colonies of all three groups. According to

  10. Nanofibrous substrates support colony formation and maintain stemness of human embryonic stem cells

    PubMed Central

    Gauthaman, Kalamegam; Venugopal, Jayarama Reddy; Yee, Fong Chui; Peh, Gary Swee Lim; Ramakrishna, Seeram; Bongso, Ariff

    2009-01-01

    Inadequate cell numbers in culture is one of the hurdles currently delaying the application of human embryonic stem cells (hESCs) for transplantation therapy. Nanofibrous scaffolds have been effectively used to expand and differentiate non-colony forming multipotent mesenchymal stem cells (MSC) for the repair of tissues or organs. In the present study, we evaluated the influence of nanofibrous scaffolds for hESC proliferation, increase in colony formation, self-renewal properties, undifferentiation and retention of ‘stemness’. Polycaprolactone/collagen (PCL/collagen) and PCL/gelatin nanofibrous scaffolds were fabricated using electrospinning technology. The hESCs were seeded on the nanofibrous scaffolds in the presence or absence of mitomycin-C treated mouse embryonic fibroblasts (MEFs). The hESCs grown on both scaffolds in the presence of the MEFs produced an increase in cell growth of 47.58% (P≤ 0.006) and 40.18% (P≤ 0.005), respectively, over conventional controls of hESCs on MEFs alone. The hESC colonies were also larger in diameter on the scaffolds compared to controls (PCL/collagen, 156.25 ± 7 μM and PCL/gelatin, 135.42 ± 5 μM). Immunohistochemistry of the hESCs grown on the nanofibrous scaffolds with MEFs, demonstrated positive staining for the various stemness-related markers (octamer 4 [OCT-4], tumour rejection antigen-1–60, GCTM-2 and TG-30), and semi-quantitative RT-PCR for the pluripotent stemness genomic markers (NANOG, SOX-2, OCT-4) showed that they were also highly expressed. Continued successful propagation of hESC colonies from nanofibrous scaffolds back to conventional culture on MEFs was also possible. Nanofibrous scaffolds support hESC expansion in an undifferentiated state with retention of stemness characteristics thus having tremendous potential in scaling up cell numbers for transplantation therapy. PMID:19228268

  11. Biophysical Properties of Scaffolds Modulate Human Blood Vessel Formation from Circulating Endothelial Colony-Forming Cells

    NASA Astrophysics Data System (ADS)

    Critser, Paul J.; Yoder, Mervin C.

    A functional vascular system forms early in development and is continually remodeled throughout the life of the organism. Impairment to the regeneration or repair of this system leads to tissue ischemia, dysfunction, and disease. The process of vascular formation and remodeling is complex, relying on local microenvironmental cues, cytokine signaling, and multiple cell types to function properly. Tissue engineering strategies have attempted to exploit these mechanisms to develop functional vascular networks for the generation of artificial tissues and therapeutic strategies to restore tissue homeostasis. The success of these strategies requires the isolation of appropriate progenitor cell sources which are straightforward to obtain, display high proliferative potential, and demonstrate an ability to form functional vessels. Several populations are of interest including endothelial colony-forming cells, a subpopulation of endothelial progenitor cells. Additionally, the development of scaffolds to deliver and support progenitor cell survival and function is crucial for the formation of functional vascular networks. The composition and biophysical properties of these scaffolds have been shown to modulate endothelial cell behavior and vessel formation. However, further investigation is needed to better understand how these mechanical properties and biophysical properties impact vessel formation. Additionally, several other cell populations are involved in neoangiogenesis and formation of tissue parenchyma and an understanding of the potential impact of these cell populations on the biophysical properties of scaffolds will also be needed to advance these strategies. This chapter examines how the biophysical properties of matrix scaffolds can influence vessel formation and remodeling and, in particular, the impact on in vivo human endothelial progenitor cell vessel formation.

  12. Formation and dissolution of bacterial colonies

    NASA Astrophysics Data System (ADS)

    Weber, Christoph A.; Lin, Yen Ting; Biais, Nicolas; Zaburdaev, Vasily

    2015-09-01

    Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation.

  13. Formation and dissolution of bacterial colonies.

    PubMed

    Weber, Christoph A; Lin, Yen Ting; Biais, Nicolas; Zaburdaev, Vasily

    2015-09-01

    Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation.

  14. Enhancement of committed hematopoietic stem cell colony formation by nandrolone decanoate after sublethal whole body irradiation

    SciTech Connect

    Gallicchio, V.S.; Chen, M.G.; Watts, T.D.

    1984-11-01

    The ability of an anabolic steroid, nandrolone decanoate, to increase committed topoietic stem cell (CFU-gm, CFU-e, and BFU-e) colony formation after sublethal irradiation was evaluated. Immediately after receiving whole body irradiation and on the next two days, each mouse was injected intraperitoneally with nandrolone decanoate (1.25 mg) in propylene glycol. Irradiated control mice received only propylene glycol. Compared to controls, drug-treated mice showed marked peripheral blood leukocytosis and more stable packed red cell volume. Drug-treated mice also demonstrated increased erythropoiesis, as CFU-e/BFU-e concentrations from both marrow (9% to 581%) and spleen (15% to 797%) were elevated. Granulopoiesis was increased similarly, as CFU-gm concentrations from marrow (38% to 685%) and spleen (9% to 373%) were elevated. These results demonstrate that nandrolone decanoate enhances hematopoietic stem cell recovery after sublethal whole body irradiation. This suggests that following hematopoietic suppression, nandrolone decanoate may stimulate the recovery of hematopoiesis at the stem cell level and in peripheral blood.

  15. Abiotic factors in colony formation: effects of nutrition and light on extracellular polysaccharide production and cell aggregates of Microcystis aeruginosa

    NASA Astrophysics Data System (ADS)

    Yang, Zhen; Kong, Fanxiang

    2013-07-01

    Colony morphology is important for Microcystis to sustain a competitive advantage in eutrophic lakes. The mechanism of colony formation in Microcystis is currently unclear. Extracellular polysaccharide (EPS) has been reported to play an important role in cell aggregate formation of some phytoplankton. Microcystis aeruginosa was cultivated under varied abiotic conditions, including different nutrient, light, and temperature conditions, to investigate their effects on EPS production and morphological change. The results show that nutrient concentration and light intensity have great effects on EPS productionin M. aeruginosa. There was a considerable increase in EPS production after M. aeruginosa was cultivated in adjusted culture conditions similar to those present in the field (28.9 mg C/L, 1.98 mg N/L, 0.65 mg P/L, light intensity: 100 μmol/(m2 · s)). These results indicate that abiotic factors might be one of the triggers for colony formation in Microcystis.

  16. Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

    SciTech Connect

    Jiang Fangxu . E-mail: jiang@wehi.edu.au; Harrison, Leonard C.

    2005-08-01

    We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its {alpha}{sub 6} integrin and {alpha}-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells.

  17. Substrate Stiffness Affects Human Keratinocyte Colony Formation

    PubMed Central

    Zarkoob, Hoda; Bodduluri, Sandeep; Ponnaluri, Sailahari V.; Selby, John C.; Sander, Edward A.

    2015-01-01

    Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned

  18. Freeze-Drying of Mononuclear Cells Derived from Umbilical Cord Blood Followed by Colony Formation

    PubMed Central

    Natan, Dity; Nagler, Arnon; Arav, Amir

    2009-01-01

    Background We recently showed that freeze-dried cells stored for 3 years at room temperature can direct embryonic development following cloning. However, viability, as evaluated by membrane integrity of the cells after freeze-drying, was very low; and it was mainly the DNA integrity that was preserved. In the present study, we improved the cells' viability and functionality after freeze-drying. Methodology/Principal Findings We optimized the conditions of directional freezing, i.e. interface velocity and cell concentration, and we added the antioxidant EGCG to the freezing solution. The study was performed on mononuclear cells (MNCs) derived from human umbilical cord blood. After freeze-drying, we tested the viability, number of CD34+-presenting cells and ability of the rehydrated hematopoietic stem cells to differentiate into different blood cells in culture. The viability of the MNCs after freeze-drying and rehydration with pure water was 88%–91%. The total number of CD34+-presenting cells and the number of colonies did not change significantly when evaluated before freezing, after freeze-thawing, and after freeze-drying (5.4×104±4.7, 3.49×104±6 and 6.31×104±12.27 cells, respectively, and 31±25.15, 47±45.8 and 23.44±13.3 colonies, respectively). Conclusions This is the first report of nucleated cells which have been dried and then rehydrated with double-distilled water remaining viable, and of hematopoietic stem cells retaining their ability to differentiate into different blood cells. PMID:19381290

  19. Periodic Pattern Formation of Bacterial Colonies

    NASA Astrophysics Data System (ADS)

    Itoh, Hiroto; Wakita, Jun-ichi; Matsuyama, Tohey; Matsushita, Mitsugu

    1999-04-01

    We have experimentally investigated pattern formation of colonies ofbacterial species Proteus mirabilis, which is famous forforming concentric-ring-like colonies.The colony grows cyclically with the interface repeating an advance anda stop alternately on a surface of a solid agar medium.We distinguish three phases (initial lag phase, the followingmigration and consolidation phases that appear alternately) for the colony growth.When we cut a colony just behind a migrating front shortly after the migrationstarted, the migration ended earlier and the following consolidationlasted longer.However, the following cycles were not influenced by the cut, i.e., thephases of the migration and consolidation were not affected.Global chemical signals governing the colony formation from thecenter were not found to exist.We also quantitatively checked phase entrainment by letting two coloniescollide with each other and found that it does not take place in macroscopic scales.All these experimental results suggest that the most important factorfor the migration is the cell population density.

  20. Interleukin 18 inhibits osteoclast formation via T cell production of granulocyte macrophage colony-stimulating factor.

    PubMed Central

    Horwood, N J; Udagawa, N; Elliott, J; Grail, D; Okamura, H; Kurimoto, M; Dunn, A R; Martin, T; Gillespie, M T

    1998-01-01

    IL-18 inhibits osteoclast (OCL) formation in vitro independent of IFN-gamma production, and this was abolished by the addition of neutralizing antibodies to GM-CSF. We now establish that IL-18 was unable to inhibit OCL formation in cocultures using GM-CSF-deficient mice (GM-CSF -/-). Reciprocal cocultures using either wild-type osteoblasts with GM-CSF -/- spleen cells or GM-CSF -/- osteoblasts with wild-type spleen cells were examined. Wild-type spleen cells were required to elicit a response to IL-18 indicating that cells of splenic origin were the IL-18 target. As T cells comprise a large proportion of the spleen cell population, the role of T cells in osteoclastogenesis was examined. Total T cells were removed and repleted in various combinations. Addition of wild-type T cells to a GM-CSF -/- coculture restored IL-18 inhibition of osteoclastogenesis. Major subsets of T cells, CD4+ and CD8+, were also individually depleted. Addition of either CD4+ or CD8+ wild-type T cells restored IL-18 action in a GM-CSF -/- background, while IL-18 was ineffective when either CD4+ or CD8+ GM-CSF -/- T cells were added to a wild-type coculture. These results highlight the involvement of T cells in IL-18-induced OCL inhibition and provide evidence for a new OCL inhibitory pathway whereby IL-18 inhibits OCL formation due to action upon T cells promoting the release of GM-CSF, which in turn acts upon OCL precursors. PMID:9449693

  1. Knockdown of interferon-induced transmembrane protein 3 expression suppresses breast cancer cell growth and colony formation and affects the cell cycle.

    PubMed

    Yang, Mei; Gao, Hongwen; Chen, Peng; Jia, Jiaoyuan; Wu, Shan

    2013-07-01

    Interferon-induced transmembrane protein 3 (IFITM3) is an important anti-virus protein and has been recently shown to play a role in human cancer development. Thus, the present study aimed to assess the expression of the IFITM3 protein in breast cancer tissues and to investigate the in vitro effects of IFITM3 knockdown in the regulation of breast cancer cell growth and cell cycle distributions. A total of 64 patients of breast cancer and the matched normal tissue specimens were obtained for immunohistochemical analysis of IFITM3 expression. Lentivirus-carrying IFITM3 shRNA was used to knock down IFITM3 expression in breast cancer cell lines. Phenotypic changes of cell viability, growth, colony formation and cell cycle distribution was also assayed using flow cytometry, MTT, BrdU incorporation and colony formation assays. The IFITM3 protein was highly expressed in invasive breast cancer compared to normal tissues and was significantly associated with estrogen receptor and progesterone receptor status. The lentivirus-carried IFITM3 shRNA significantly reduced the expression of IFITM3 mRNA and protein in breast cancer cells, inhibiting tumor cell viability, growth and colony formation, arrested tumor cells at the G0/G1 phase of the cell cycle and reduced the number of cells in the S phase of the cell cycle. Expression of IFITM3 protein could be a potential therapeutic target in future treatment of breast cancer.

  2. Murine embryonic stem cells secrete cytokines/growth modulators that enhance cell survival/anti-apoptosis and stimulate colony formation of murine hematopoietic progenitor cells.

    PubMed

    Guo, Ying; Graham-Evans, Barbara; Broxmeyer, Hal E

    2006-04-01

    Stromal cell-derived factor (SDF)-1/CXCL12, released by murine embryonic stem (ES) cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells. Conditioned medium (CM) from murine ES cells growing in the presence of leukemia inhibitory factor (LIF) was generated while the ES cells were in an undifferentiated Oct-4 expressing state. ES cell-CM enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased apoptosis of murine bone marrow c-kit(+)lin- cells. ES CM contained interleukin (IL)-1alpha, IL-10, IL-11, macrophage-colony stimulating factor (CSF), oncostatin M, stem cell factor, vascular endothelial growth factor, as well as a number of chemokines and other proteins, some of which are known to enhance survival/anti-apoptosis of progenitors. Irradiation of ES cells enhanced release of some proteins and decreased release of others. IL-6, FGF-9, and TNF-alpha, not detected prior to irradiation was found after ES cells were irradiated. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins, results which may be of physiological and/or practical significance.

  3. Downregulation of ROS-FIG inhibits cell proliferation, colony-formation, cell cycle progression, migration and invasion, while inducing apoptosis in intrahepatic cholangiocarcinoma cells

    PubMed Central

    DENG, GANG; HU, CHENGHUAN; ZHU, LEI; HUANG, FEIZHOU; HUANG, WEI; XU, HONGBO; NIE, WANPIN

    2014-01-01

    Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer with poor responsiveness to existing drug therapies. Therefore, novel treatment strategies against ICC are required to improve survival. The aim of this study was to demonstrate the role of fused-in-glioblastoma-c-ros-oncogene1 (FIG-ROS) fusion gene in ICC. ROS was positively expressed in ICC tissues and HUCCT1 cells. Plasmids expressing ROS- and FIG-specific shRNAs were constructed and transfected into HUCCT1 cells. The results showed that single transfection of ROS- or FIG-specific shRNA inhibited HUCCT1 cell proliferation, colony formation, cell cycle progression, migration and invasion, while inducing apoptosis. Moreover, the co-inhibition of ROS- and FIG-specific shRNA exhibited stronger effects on HUCCT1 cell proliferation, apoptosis, colony formation, cell cycle progression, migration and invasion, when compared to single inhibition of ROS and FIG. Furthermore, findings of this study suggested that the AKT signaling pathway was involved in the ROS-FIG-mediated biological processes of HUCCT1 cells. In summary, the results suggest that FIG-ROS plays an oncogenic role in ICC. Additionally, ROS1-6290 and FIG-363 segments may become effective therapeutic targets for ICC harboring ROS-FIG fusion protein. PMID:24968753

  4. Ferulic acid decreases cell viability and colony formation while inhibiting migration of MIA PaCa-2 human pancreatic cancer cells in vitro.

    PubMed

    Fahrioğlu, Umut; Dodurga, Yavuz; Elmas, Levent; Seçme, Mücahit

    2016-01-15

    Novel and combinatorial treatment methods are becoming sought after entities in cancer treatment and these treatments are even more valuable for pancreatic cancer. The scientists are always on the lookout for new chemicals to help them in their fight against cancer. In this study, we examine the effects of ferulic acid (FA), a phenolic compound, on gene expression, viability, colony formation and migration/invasion in the cultured MIA PaCa-2 human pancreatic cancer cell. Cytotoxic effects of FA were determined by using trypan blue dye exclusion test and Cell TiterGlo (CTG) assay. IC50 dose in MIA PaCa-2 cells was detected as 500μM/ml at the 72nd hour. Expression profiles of certain cell cycle and apoptosis genes such as CCND1 (cyclin D1),CDK4, CDK6, RB, p21, p16, p53, caspase-3, caspase-9, caspase-8, caspase-10, Bcl-2, BCL-XL,BID, DR4,DR5,FADD,TRADD,PARP, APAF, Bax, Akt, PTEN, PUMA, NOXA, MMP2, MMP9, TIMP1 and TIMP2 were determined by real-time PCR. The effect of FA on cell viability was determined by CellTiter-Glo® Luminescent Cell Viability Assay. Additionally, effects of FA on colony formation and invasion were also investigated. It was observed that FA caused a significant decrease in the expression of CCND1, CDK 4/6, Bcl2 and caspase 8 and 10 in the MIA PaCa-2 cells while causing an increase in the expression of p53, Bax, PTEN caspase 3 and 9. FA was observed to decrease colony formation while inhibiting cell invasion and migration as observed by the BioCoat Matrigel Invasion Chamber guide and colony formation assays. In conclusion, FA is thought to behave as an anti-cancer agent by affecting cell cycle, apoptotic, invasion and colony formation behavior of MIA PaCa-2 cells. Therefore, FA is placed as a strong candidate for further studies aimed at finding a better, more effective treatment approach for pancreatic cancer.

  5. Mesenchymal stromal cells, colony-forming unit fibroblasts, from bone marrow of untreated advanced breast and lung cancer patients suppress fibroblast colony formation from healthy marrow.

    PubMed

    Hofer, Erica Leonor; Labovsky, Vivian; La Russa, Vincent; Vallone, Valeria Fernández; Honegger, Alba Elizabeth; Belloc, Carlos Gabriel; Wen, Huei Chi; Bordenave, Raúl Horacio; Bullorsky, Eduardo Oscar; Feldman, Leonardo; Chasseing, Norma Alejandra

    2010-03-01

    We have shown that bone marrow (BM) from untreated advanced lung and breast cancer patients (LCP and BCP) have a reduced number of colony-forming unit fibroblasts (CFU-Fs) or mesenchymal stem cells (MSCs). Factors that regulate the proliferation and differentiation of CFU-F are produced by the patients' BM microenvironment. We have now examined whether conditioned media (CM) from patients' CFU-F-derived stromal cells also inhibits the colony-forming efficiency (CFE) of CFU-F in primary cultures from healthy volunteers (HV)-BM. Thus the number and proliferation potential of HV-CFU-F were also found to be decreased and similar to colony numbers and colony size of patients' CFU-F. Stromal cells from both of these types of colonies appeared relatively larger and lacked the characteristic spindle morphology typically seen in healthy stromal cells. We developed an arbitrary mesenchymal stromal cell maturational index by taking three measures consisting of stromal cell surface area, longitudinal and horizontal axis. All stromal indices derived from HV-CFU-F grown in patients' CM were similar to those from stromal elements derived from patients' CFU-F. These indices were markedly higher than stromal indices typical of HV-CFU-F cultured in healthy CM or standard medium [alpha-medium plus 20% heat-inactivated fetal bovine serum (FBS)]. Patients' CM had increased concentrations of the CFU-F inhibitor, GM-CSF, and low levels of bFGF and Dkk-1, strong promoters of self-renewal of MSCs, compared to the levels quantified in CM from HV-CFU-F. Moreover, the majority of patients' MSCs were unresponsive in standard medium and healthy CM to give CFU-F, indicating that the majority of mesenchymal stromal cells from patients' CFU-F are locked in maturational arrest. These results show that alterations of GM-CSF, bFGF, and Dkk-1 are associated with deficient cloning and maturation arrest of CFU-F. Defective autocrine and paracrine mechanisms may be involved in the BM microenvironments of

  6. Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay

    PubMed Central

    WANG, FENGCHAO; SCOVILLE, DAVID; HE, XI C.; MAHE, MAXIME M.; BOX, ANDREW; PERRY, JOHN M.; SMITH, NICHOLAS R.; LEI, NAN YE; DAVIES, PAIGE S.; FULLER, MEGAN K.; HAUG, JEFFREY S.; MCCLAIN, MELAINIA; GRACZ, ADAM D.; DING, SHENG; STELZNER, MATTHIAS; DUNN, JAMES C. Y.; MAGNESS, SCOTT T.; WONG, MELISSA H.; MARTIN, MARTIN G.; HELMRATH, MICHAEL; LI, LINHENG

    2013-01-01

    BACKGROUND & AIMS Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. METHODS We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. RESULTS CD44+CD24loCD166+ cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78lo/− putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. CONCLUSIONS We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44+CD24loCD166+, GRP78lo/−, and c-Kit− facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44+CD24−/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs. PMID:23644405

  7. Robust conversion of marrow cells to skeletal muscle with formation of marrow-derived muscle cell colonies: A multifactorial process

    SciTech Connect

    Abedi, Mehrdad; Greer, Deborah A.; Colvin, Gerald A.; Demers, Delia A.; Dooner, Mark S.; Harpel, Jasha A.; Weier, Heinz-Ulrich G.; Lambert, Jean-Francois; Quesenberry, Peter J.

    2004-01-10

    Murine marrow cells are capable of repopulating skeletal muscle fibers. A point of concern has been the robustness of such conversions. We have investigated the impact of type of cell delivery, muscle injury, nature of delivered cell, and stem cell mobilizations on marrow to muscle conversion. We transplanted GFP transgenic marrow into irradiated C57BL/6 mice and then injured anterior tibialis muscle by cardiotoxin. One month after injury, sections were analyzed by standard and deconvolutional microscopy for expression of muscle and hematopietic markers. Irradiation was essential to conversion although whether by injury or induction of chimerism is not clear. Cardiotoxin and to a lesser extent PBS injected muscles showed significant number of GFP+ muscle fibers while uninjected muscles showed only rare GFP+ cells. Marrow conversion to muscle was increased by two cycles of G-CSF mobilization and to a lesser extent with G-CSF and steel or GM-CSF. Transplantation of female GFP to male C57 BL/6 and GFP to Rosa26 mice showed fusion of donor cells to recipient muscle. High numbers of donor derived muscle colonies and up to12 percent GFP positive muscle cells were seen after mobilization or direct injection. These levels of donor muscle chimerism approach levels which could be clinically significant in developing strategies for the treatment of muscular dystrophies. In summary, the conversion of marrow to skeletal muscle cells is based on cell fusion and is critically dependent on injury. This conversion is also numerically significant and increases with mobilization.

  8. Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells

    PubMed Central

    Miyashita, Tomoyuki; Higuchi, Youichi; Kojima, Motohiro; Ochiai, Atsushi; Ishii, Genichiro

    2017-01-01

    Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas. PMID:28059107

  9. Comparison of colony-formation efficiency of bovine fetal fibroblast cell lines cultured with low oxygen, hydrocortisone, L-carnosine, bFGF, or different levels of FBS.

    PubMed

    Talbot, Neil C; Powell, Anne M; Caperna, Thomas J

    2004-01-01

    A comparison of colony-formation efficiency (CFE) was made between six independent bovine fetal fibroblast (BFF) cell lines used in somatic cell nuclear transfer. Variation in CFE was assessed under different culture conditions. The conditions examined were ambient atmosphere (approximately 20% oxygen) culture versus 5% oxygen culture, three levels of fetal bovine serum (FBS) in the medium (5%, 10% or 20%), and the amendment of 10% FBS medium with basic fibroblast growth factor (1 ng/mL), L-carnosine (20 mM), or hydrocortisone (1 microM). The six BFF cell lines showed significant differences from one another in CFE. No significant difference in CFE was found with reduced oxygen culture. L-Carnosine also had no significant effect on CFE. A FBS concentration of 10% was found to produce the best overall CFE. Hydrocortisone treatment reduced the size of colonies although the number of colonies formed was not affected. Basic FGF increased the size of colonies but the number of colonies formed was not affected. The results showed that different BFF cell lines varied significantly in their CFE. Also, some medium supplements or culture conditions that have shown positive CFE effects on the fibroblasts of other species failed to show significant positive CFE effects on the BFF cell lines tested.

  10. A new preclinical 3-dimensional agarose colony formation assay.

    PubMed

    Kajiwara, Yoshinori; Panchabhai, Sonali; Levin, Victor A

    2008-08-01

    The evaluation of new drug treatments and combination treatments for gliomas and other cancers requires a robust means to interrogate wide dose ranges and varying times of drug exposure without stain-inactivation of the cells (colonies). To this end, we developed a 3-dimensional (3D) colony formation assay that makes use of GelCount technology, a new cell colony counter for gels and soft agars. We used U251MG, SNB19, and LNZ308 glioma cell lines and MiaPaCa pancreas adenocarcinoma and SW480 colon adenocarcinoma cell lines. Colonies were grown in a two-tiered agarose that had 0.7% agarose on the bottom and 0.3% agarose on top. We then studied the effects of DFMO, carboplatin, and SAHA over a 3-log dose range and over multiple days of drug exposure. Using GelCount we approximated the area under the curve (AUC) of colony volumes as the sum of colony volumes (microm2xOD) in each plate to calculate IC50 values. Adenocarcinoma colonies were recognized by GelCount scanning at 3-4 days, while it took 6-7 days to detect glioma colonies. The growth rate of MiaPaCa and SW480 cells was rapid, with 100 colonies counted in 5-6 days; glioma cells grew more slowly, with 100 colonies counted in 9-10 days. Reliable log dose versus AUC curves were observed for all drugs studied. In conclusion, the GelCount method that we describe is more quantitative than traditional colony assays and allows precise study of drug effects with respect to both dose and time of exposure using fewer culture plates.

  11. A novel small molecule STAT3 inhibitor, LY5, inhibits cell viability, colony formation, and migration of colon and liver cancer cells

    PubMed Central

    Yu, Wenying; Jou, David; Wang, Yina; Ma, Haiyan; Xiao, Hui; Qin, Hua; Zhang, Cuntai; Lü, Jiagao; Li, Sheng; Li, Chenglong; Lin, Jiayuh; Lin, Li

    2016-01-01

    Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated in human liver and colon cancer cells and is required for cancer cell viability, survival and migration. Therefore, inhibition of STAT3 signaling may be a viable therapeutic approach for these two cancers. We recently designed a non-peptide small molecule STAT3 inhibitor, LY5, using in silico site-directed Fragment-based drug design (FBDD). The inhibitory effect on STAT3 phosphorylation, cell viability, migration and colony forming ability by LY5 were examined in human liver and colon cancer cells. We demonstrated that LY5 inhibited constitutive Interleukin-6 (IL-6)-induced STAT3 phosphorylation, STAT3 nuclear translocation, decreased STAT3 downstream targeted gene expression and induced apoptosis in liver and colon cancer cells. LY5 had little effect on STAT1 phosphorylation mediated by IFN-γ. Inhibition of persistent STAT3 phosphorylation by LY5 also inhibited colony formation, cell migration, and decreased the viability of liver cancer and colon cancer cells. Furthermore, LY5 inhibited STAT3 phosphorylation and suppressed colon tumor growth in a mouse model in vivo. Our results suggest that LY5 is a potent STAT3 inhibitor and may be a potential drug candidate for liver and colon cancer therapy. PMID:26883202

  12. Interleukin-6 from Ovarian Mesenchymal Stem Cells Promotes Proliferation, Sphere and Colony Formation and Tumorigenesis of an Ovarian Cancer Cell Line SKOV3

    PubMed Central

    Ding, Dah-Ching; Liu, Hwan-Wun; Chu, Tang-Yuan

    2016-01-01

    The origin of the majority of epithelial ovarian cancers (EOC) is regarded as extraovarian, with the ovary being the secondary site. The aim of this study was to explore the possible role of ovarian mesenchymal stem cells (OvMSCs) and secreted IL-6 in the development of EOC. OvMSCs were derived from normal ovarian stroma. Cell surface markers and differentiation capability were determined. The effects of IL-6 and conditioned medium of OvMSCs on the malignant phenotype of SKOV3 ovarian cancer cells were tested, and the status of STAT3 and ERK phosphorylation was investigated. OvMSCs had similar surface marker profiles as bone marrow mesenchymal stem cells, i.e., CD44 (+), CD90 (+) and CD45 (-), and was readily inducible to osteogenic, adipogenic and chondrogenic differentiation. OvMSCs secreted an extremely high level (>2500 pg/ml) of IL-6. Treatment of SKOV3 cells with conditioned media from OvMSCs increased cell proliferation, tumor sphere formation and anchorage independent growth, and resulted in activation of STAT3 but not ERK. Coinjection of OvMSCs with SKOV3 cell enhanced tumorigenesis in NOD-SCID mice. All of these behaviors were blocked by IL-6 receptor blocking antibody administered in vitro or in vivo. The OvMSCs alone injected into mice had no tumor growth after 3 months. By secreting high levels of IL-6, OvMSCs enhance the proliferation, sphere and colony formation and tumorigenesis of SKOV3 cells. PMID:27698921

  13. Inhibition of MEK and GSK3 supports ES cell-like domed colony formation from avian and reptile embryos.

    PubMed

    Nakanoh, Shota; Okazaki, Kenji; Agata, Kiyokazu

    2013-07-01

    As amniotes diversified, mammals may have modified mechanisms of cellular pluripotency along with the acquisition of a placenta. What then defined pluripotent states in the ancestral amniotes? To study the evolutionary background of pluripotency in amniotes, we tested the effects of extracellular effectors on primary culture cells from avian and reptile embryos in serum-free medium. When treated with a combination of a MEK inhibitor and a GSK3 inhibitor (2i condition), chicken early embryos formed domed colonies (DCs), which were morphologically indistinguishable from the colonies formed by mouse and rat naïve embryonic stem cells. However, no DCs formed when cells from further-developed embryos were cultured in the 2i condition, indicating that there is a clear boundary of DC-forming ability at around the stage of primitive streak elongation. Quail embryos at the blastoderm and cleavage stages also formed DCs in the 2i condition, which is consistent with the notion that the appearance of DCs corresponds with the presence of pluripotent cells in embryos. Gecko blastoderms also formed DCs in the 2i condition, but gastrulas did not. ERK activation by bFGF caused an effect opposite to that of the 2i condition, namely, it dispersed colonies of cells even from early embryos in all species examined. These results suggest that the regulation of pluripotency by FGF/ERK signaling may date back at least to the common ancestor of mammals, birds, and reptiles. However, gene expression analysis indicated the possibility that mammalian pluripotency transcription factors function differently in non-mammalian amniotes.

  14. Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation.

    PubMed

    Kusakawa, Shinji; Yasuda, Satoshi; Kuroda, Takuya; Kawamata, Shin; Sato, Yoji

    2015-12-08

    Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process.

  15. High throughput and automatic colony formation assay based on impedance measurement technique.

    PubMed

    Lei, Kin Fong; Kao, Chich-Hao; Tsang, Ngan-Ming

    2017-03-02

    To predict the response of in vivo tumors, in vitro culture of cell colonies was suggested to be a standard assay to achieve high clinical relevance. To describe the responses of cell colonies, the most widely used quantification method is to count the number and size of cell colonies under microscope. That makes the colony formation assay infeasible to be high throughput and automated. In this work, in situ analysis of cell colonies suspended in soft hydrogel was developed based on impedance measurement technique. Cell colonies cultured between a pair of parallel plate electrodes were successfully analyzed by coating a layer of base hydrogel on one side of electrode. Real-time and label-free monitoring of cell colonies was realized during the culture course. Impedance magnitude and phase angle respectively represented the summation effect of colony responses and size of colonies. In addition, dynamic response of drug-treated colonies was demonstrated. High throughput and automatic colony formation assay was realized to facilitate more objective assessments in cancer research. Graphical Abstract High throughput and automatic colony formation assay was realized by in situ impedimetric analysis across a pair of parallel plate electrodes in a culture chamber. Cell colonies suspended in soft hydrogel were cultured under the tested substance and their dynamic response was represented by impedance data.

  16. Impaired growth, hematopoietic colony formation, and ribosome maturation in human cells depleted of Shwachman-Diamond syndrome protein SBDS.

    PubMed

    Sezgin, Gulay; Henson, Adrianna L; Nihrane, Abdallah; Singh, Sharon; Wattenberg, Max; Alard, Pascale; Ellis, Steven R; Liu, Johnson M

    2013-02-01

    Shwachman-Diamond syndrome (SDS), associated with SBDS mutations, is characterized by pancreatic exocrine dysfunction and marrow failure. Sdo1, the yeast ortholog of SBDS, is implicated in maturation of the 60S ribosomal subunit, with delayed export of 60S-like particles from the nucleoplasm when depleted. Sdo1 is needed for release of the anti-subunit association factor Tif6 from 60S subunits, and Tif6 may not be recycled to the nucleus when Sdo1 is absent. To clarify the role of SBDS in human ribosome function, TF-1 erythroleukemia and A549 lung carcinoma cells were transfected with vectors expressing RNAi against SBDS. Growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to controls. To analyze the effect of SBDS on 60S subunit maturation in A549 cells, subunit localization was assessed by transfection with a vector expressing a fusion between human RPL29 and GFP: we found a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits. Polysome analysis of TF-1 knockdown cells showed a decrease in free 60S and 80S subunits. We also analyzed the levels of eIF6 (human ortholog of Tif6) following near-complete knockdown of SBDS in TF-1 cells and found an approximately 20% increase in the amount of eIF6 associated with the 60S subunit. We conclude that knockdown of SBDS leads to growth inhibition and defects in ribosome maturation, suggesting a role for wild-type SBDS in nuclear export of pre-60S subunits. Furthermore, knockdown of SBDS may interfere with eIF6 recycling. Copyright © 2012 Wiley Periodicals, Inc.

  17. A generalized discrete model linking rippling pattern formation and individual cell reversal statistics in colonies of myxobacteria

    NASA Astrophysics Data System (ADS)

    Börner, Uwe; Deutsch, Andreas; Bär, Markus

    2006-06-01

    Self-organization processes in multicellular aggregates of bacteria and amoebae offer fascinating insights into the evolution of cooperation and differentiation of cells. During myxobacterial development a variety of spatio-temporal patterns emerges such as counterpropagating waves of cell density that are known as rippling. Recently, several models have been introduced that qualitatively reproduce these patterns. All models include active motion and a collision-triggered reversal of individual bacteria. Here, we present a systematic study of a generalized discrete model that is based on similar assumptions as the continuous model by Igoshin et al (2001 Proc. Natl Acad. Sci. USA 98 14913). We find counterpropagating as well as unidirectional rippling waves in extended regions of the parameter space. If the interaction strength and the degree of cooperativity are large enough, rippling patterns appear even in the absence of a refractory period. We show for the first time that the experimentally observed double peak in the reversal statistics of bacteria in rippling colonies (Welch and Kaiser 2001 Proc. Natl Acad. Sci. USA 98 14907) can be reproduced in simulations of counterpropagating rippling waves which are dominant in experiments. In addition, the reversal statistics in the pre-rippling phase is correctly reproduced.

  18. Macrophage colony-stimulating factor gene transduction into human lung cancer cells differentially regulates metastasis formations in various organ microenvironments of natural killer cell-depleted SCID mice.

    PubMed

    Yano, S; Nishioka, Y; Nokihara, H; Sone, S

    1997-02-15

    We investigated whether local production of macrophage colony-stimulating factor (M-CSF), responsible for migration and activation of monocytes/macrophages at a tumor growth site, affected the metastatic pattern of lung cancer. For this, highly metastatic human squamous (RERF-LC-AI) or small (H69/VP) cell lung carcinoma cells were transduced with the human M-CSF gene inserted into pRc/CMV-MCSF to establish M-CSF-producing clones (MCSF-AI-9-18, MCSF-AI-9-24, and MCSF-VP-5). M-CSF gene transduction had no effect on the expression of surface antigen or on in vitro proliferation. After s.c. injection into SCID mice, the growth rates of M-CSF-producing cells were slower than those of parent or mock-transduced cells. In the metastatic model in SCID mice depleted of natural killer cells, RERF-LC-AI cells formed metastases mainly in the liver and kidneys, whereas H69/VP cells metastasized mainly to the liver and systemic lymph nodes. The numbers of metastatic colonies of MCSF-AI-9-18 and MCSF-AI-9-24 cells in the liver but not the kidneys were significantly reduced. The development of lymph node metastases of MCSF-VP-5 cells was also less than that of parent or mock-transduced cells. Treatment of SCID mice with anti-human M-CSF antibody resulted in a significant increase in liver metastases of their M-CSF gene transfectants. No significant differences were observed in the distributions in mice or in the in vitro invasive potentials of MCSF-AI-9-18 cells and Neo-AI-3 cells. These findings indicate that the antimetastatic effect of M-CSF may be specific to particular organs, suggesting the influence of heterogeneity of organ microenvironments on the metastasis of lung cancer.

  19. NS-018, a selective JAK2 inhibitor, preferentially inhibits CFU-GM colony formation by bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients.

    PubMed

    Kuroda, Junya; Kodama, Ayumi; Chinen, Yoshiaki; Shimura, Yuji; Mizutani, Shinsuke; Nagoshi, Hisao; Kobayashi, Tsutomu; Matsumoto, Yosuke; Nakaya, Yohei; Tamura, Ayako; Kobayashi, Yutaka; Naito, Haruna; Taniwaki, Masafumi

    2014-05-01

    JAK2/STAT signaling promotes survival and expansion of myelodysplastic syndrome (MDS) clones, but little is known about the potential of JAK2/STAT as a therapeutic target in MDS. We investigated the effect of NS-018, a novel antagonist for JAK2, on the colony-forming ability of bone marrow mononuclear cells (BMMNCs) from high-risk MDS patients. NS-018 decreased colony-forming unit-granulocyte/macrophage (CFU-GM) colony numbers from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent than against normal BMMNCs. In addition, NS-018 suppressed the phosphorylation of STAT3 in colony-forming cells from MDS patients. Collectively, NS-018 could be a new therapeutic option for high-risk MDS.

  20. Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

    NASA Astrophysics Data System (ADS)

    Meng, Qing-Yuan; Akaike, Toshihiro

    2013-03-01

    Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

  1. Budding yeast colony growth study based on circular granular cell

    NASA Astrophysics Data System (ADS)

    Aprianti, Devi; Khotimah, S. N.; Viridi, S.

    2016-08-01

    Yeast colony growth can be modelled by using circular granular cells, which can grow and produce buds. The bud growth angle can be set to regulate cell budding pattern. Cohesion force, contact force and Stokes force were adopted to accommodate the behaviour and interactions among cells. Simulation steps are divided into two steps, the explicit step is due to cell growing and implicit step for the cell rearrangement. Only in explicit step that time change was performed. In this study, we examine the influence of cell diameter growth time and reproduction time combination toward the growth of cell number and colony formation. We find a commutative relation between the cell diameter growth time and reproduction time to the specific growth rate. The greater value of the multiplication of the parameters, the smaller specific growth rate is obtained. It also shows a linear correlation between the specific growth rate and colony diameter growth rate.

  2. Time-Lapse Analysis of Human Embryonic Stem Cells Reveals Multiple Bottlenecks Restricting Colony Formation and Their Relief upon Culture Adaptation

    PubMed Central

    Barbaric, Ivana; Biga, Veronica; Gokhale, Paul J.; Jones, Mark; Stavish, Dylan; Glen, Adam; Coca, Daniel; Andrews, Peter W.

    2014-01-01

    Summary Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns. PMID:25068128

  3. Formation of complex bacterial colonies via self-generated vortices

    NASA Astrophysics Data System (ADS)

    Czirók, András; Ben-Jacob, Eshel; Cohen, Inon; Vicsek, Tamás

    1996-08-01

    Depending on the environmental conditions bacterial colonies growing on agar surfaces can exhibit complex colony formation and various types of collective motion. Experimental results are presented concerning the hydrodynamics (vortices, migration of bacteria in clusters) and colony formation of a morphotype of Bacillus subtilis. Some of these features are not specific to this morphotype but also have been observed in several other bacterial strains, suggesting the presence of universal effects. A simple model of self-propelled particles is proposed, which is capable of describing the hydrodynamics on the intermediate level, including the experimentally observed rotating disks of bacteria. The colony formation is captured by a complex generic model taking into account nutrient diffusion, reproduction, and sporulation of bacteria, extracellular slime deposition, chemoregulation, and inhomogeneous population. Our model also sheds light on some possible biological benefits of this ``multicellular behavior.''

  4. Diffusion-limited growth in bacterial colony formation

    NASA Astrophysics Data System (ADS)

    Matsushita, Mitsugu; Fujikawa, Hiroshi

    1990-09-01

    Colonies of bacterial species called Bacillus subtilis have been found to grow two-dimensionally and self-similarly on agar plates through diffusion-limited processes in a nutrient concentration field. We obtained a fractal dimension of the colony patterns of D=1.73±0.02, very close to that of the two-dimensional DLA model, and confirmed the existence of the screening effect of protruding main branches against inner ones in a colony, the repulsion between two neighboring colonies and the tendency to grow toward nutrient. These effects are all characteristic of the pattern formation in a Laplacian field. This finding implies the importance of physical properties of the environment for the morphology of bacterial colonies in general.

  5. Improved microbioassay for plasma erythropoietin based on CFU-E colony formation.

    PubMed

    Sakata, S; Enoki, Y

    1992-05-01

    We examined the conditions necessary for performing a reliable erythropoietin (EPO) assay based on CFU-E colony formation in fetal mouse liver cell (FMLC) microcultures using 96-well microtiter plates. Both linearity of colony numbers with the number of cells plated and comparison among the colony ratios at various densities of seeding cells indicated that the colonies originated from a single progenitor cell when 7500 or fewer cells were plated into individual microtiter wells. About a twofold CFU-E enrichment in 12- to 13-day FMLC was achieved by Ficoll-Paque centrifugation. Plasma treated with acid-boiling stimulated the colony formation most and contained no colony inhibitor. Dose-response curve for the plasma was parallel to the EPO standard curve. The "erythroid colony-stimulating activity" in the plasma was additive to that in the standard EPO, and was completely neutralized by a monoclonal antibody against recombinant human EPO. Using the assay procedure thus established, plasma EPO titer was determined in normal subjects, in patients with nonuremic anemia and polycythemia vera, and in dialysis patients with chronic renal failure. The use of different preparations of standard EPO resulted in a significant difference in the titers because their dose-response curves differed from one another. An inverse relationship was found between EPO titers and hemoglobin concentrations in the nonuremic anemic patients, but not in the dialysis patients with about one half the normal EPO level.

  6. Observations on colony formation by the cosmopolitan phytoplankton genus Phaeocystis

    NASA Astrophysics Data System (ADS)

    Verity, Peter G.; Medlin, Linda K.

    2003-12-01

    Few marine phytoplankton have heteromorphic life cycles and also often dominate the ecosystems in which they occur. The class Prymnesiophyceae contains a notable exception: the genus Phaeocystis includes three species that form gelatinous colonies but also occur within their ranges as solitary cells. Phaeocystis antarctica and P. pouchetii are exclusively high latitude taxa, and are notable for regionally tremendous blooms of the colony stage. P. globosa occurs circumglobally, yet its colony blooms primarily are confined to colder waters within its range. Three additional species are warm water forms that have been reported only as solitary cells or loose aggregations that bear little resemblance to the organized colonies of the other taxa. Interpretation of existing data indicates that resource availability (light, temperature and nutrients) by itself is not sufficient to explain this distinction between cold-water colony-forming taxa and warm water solitary cell taxa, nor why colony development in P. globosa is essentially a spatially restricted phenomenon within a much broader geographic range. Colony development by P. globosa in situ has been observed at temperatures ≥20 °C, but only rarely and generally under conditions of seasonally or anthropogenically elevated nutrient supply. Data presented here demonstrate colony development at 20-22 °C in natural plankton communities from oligotrophic waters that were pre-screened through 63 μm mesh (i.e. lacking mesozooplankton and large microzooplankton), but not in unscreened communities containing microzooplankton and >63 μm zooplankton. Reduction of colony proliferation at higher temperatures by mesozooplankton grazing remains as an intriguing possibility that is consistent with available evidence to help explain differences in latitudinal extent of in situ colony development. These data are interpreted within a theoretical framework regarding the potential advantages and disadvantages of the two life cycle

  7. Mixed colony formation in vitro by the heterogeneous compartment of multipotential progenitors in human bone marrow.

    PubMed

    Holyoake, T L; Freshney, M G; Konwalinka, G; Haun, M; Petzer, A; Fitzsimons, E; Lucie, N P; Wright, E G; Pragnell, I B

    1993-02-01

    The physiology of the human haemopoietic primitive progenitor populations can be studied in normal and disease states by clonal in vitro cultures in which the primitive progenitor cells proliferate and differentiate to form mixed colonies. For many applications it is essential that such assays detect a high proportion of primitive progenitor cells. We describe an in vitro assay which detects a high incidence of human CD34+ multipotential progenitor cells. Bone marrow mononuclear cells (MNC) or selected CD34+ cells were plated at low cell concentrations in semisolid agar cultures with synergizing growth factor combinations. The optimum growth factor combination of conditioned medium from Mia PaCa-2 cells (Mia-CM), recombinant granulocyte-macrophage colony-stimulating factor and recombinant stem cell factor (SCF) supported the formation of macroscopic (> or = mm) colonies (97% of which were multilineage), at an average incidence of 250/10(5) MNC. The colony-forming cells (human colony-forming unit, type A) detected, showed a low cycling status (7.3%) and the macroscopic colonies had a high replating efficiency (46%), reflecting their probable primitive nature. This assay should prove invaluable, for studies on the regulation of proliferation of the multipotential compartment and in studies involving the assessment of these cells in transplantation and neoplastic disease.

  8. 17β-Estradiol regulates cell proliferation, colony formation, migration, invasion and promotes apoptosis by upregulating miR-9 and thus degrades MALAT-1 in osteosarcoma cell MG-63 in an estrogen receptor-independent manner

    SciTech Connect

    Fang, Dengfeng; Yang, Hui; Lin, Jing; Teng, Yi; Jiang, Yingying; Chen, Jiao; Li, Yu

    2015-02-20

    In bone, different concentration of estrogen leads to various of physiological processes in osteoblast, such as the proliferation, migration, and apoptosis in an estrogen receptor-dependent manner. But little was known about the estrogen effects on osteosarcoma (OS). In this study, OS cell MG-63 was treated with low (1 nM) or high (100 nM) dose of 17β-Estradiol (E2) with the presence or absence of estrogen receptor α (ERα), for evaluating the E2 effects on proliferation, migration, invasion, colony formation and apoptosis. Consistent with a previous study, high dose of E2 treatment dramatically downregulated expressing level of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT-1). The observation of upregulation of miR-9 after a high dose of E2 treatment indicated the cause of MALAT-1 reduction. Downregulation of MALAT-1 promoted the combination of SFPQ/PTBP2 complex. It was also observed that the proliferation, migration, invasion, colony formation and apoptosis of OS cells were remarkably affected by high dose of E2 treatment, but not by low dose, in an ERα independent manner. Furthermore, the abolishment of the effects on these physiological processes caused by ectopic expression of miR-9 ASOs suggested the necessity of miR-9 in MALAT-1 regulation. Here we found that the high dose of E2 treatment upregulated miR-9 thus posttranscriptionally regulated MALAT-1 RNA level in OS cells, and then the downregulation of MALAT-1 inhibited cell proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) processes in the E2-dose dependent and ER-independent ways. - Highlights: • E2 affects osteosarcoma cell MG-63 in an Estrogen receptor-independent way. • High dose of E2 treatment upregulates miR-9 which target to MALAT-1 RNA. • Upregulated miR-9 degrades MALAT-1 and thus affects combination of SFPQ/PTBP2. • E2 treatment block cell proliferation, colony formation, mobility, and enhance apoptosis.

  9. Stage-dependent reduction in T colony formation in Hodgkin's disease. Coincidence with monocyte synthesis of prostaglandins.

    PubMed Central

    Bockman, R S

    1980-01-01

    Prostaglandin synthesis and T lymphocyte colony formation have been examined in previously untreated patients with Hodgkin's disease. Mononuclear cells have been isolated from peripheral blood and spleens of these patients. Significant augmentation in prostaglandin E levels were noted in the mononuclear cell cutures from Hodgkin's disease patients compared with controls (1.64 +/- 0.29 vs. 0.39 +/- 0.09 ng/10(6) cells, P < 0.005). Measured prostaglandin E levels increased with advancing stage of disease. Virtually all of the prostaglandins were synthesized by the adherent monocyte cell population. Prostaglandin E was the major product. Clonal expansion of a T lymphocyte precursor cell, which gives rise to colonies > 50 cells, was determined by a layered soft agar method. T colony formation was significantly reduced in patients with stage II, III, and IV disease. There were progressively reduced colony numbers seen with advancing stage of disease (609 +/- 209, 416 +/- 158, 207 +/- 58 compared with normals 2,274 +/- 360 colonies/10(6) cells plated; P < 0.005). The addition of inhibitors of endogenous prostaglandin synthesis resulted in significant augmentation of T colony number. However, a consistent relative decrease in T colony number was seen even when endogenous prostaglandin E synthesis was blocked. It would appear that both the prostaglandin-dependent and independent T colony precursor cells are lost with progressive stage of disease. A causative role of augmented prostaglandin synthesis in this stage-dependent reduction of T colony formation could not be established. PMID:6967491

  10. Growth dynamics of cancer cell colonies and their comparison with noncancerous cells

    NASA Astrophysics Data System (ADS)

    Huergo, M. A. C.; Pasquale, M. A.; González, P. H.; Bolzán, A. E.; Arvia, A. J.

    2012-01-01

    The two-dimensional (2D) growth dynamics of HeLa (cervix cancer) cell colonies was studied following both their growth front and the pattern morphology evolutions utilizing large population colonies exhibiting linearly and radially spreading fronts. In both cases, the colony profile fractal dimension was df=1.20±0.05 and the growth fronts displaced at the constant velocity 0.90±0.05 μm min-1. Colonies showed changes in both cell morphology and average size. As time increased, the formation of large cells at the colony front was observed. Accordingly, the heterogeneity of the colony increased and local driving forces that set in began to influence the dynamics of the colony front. The dynamic scaling analysis of rough colony fronts resulted in a roughness exponent α = 0.50±0.05, a growth exponent β = 0.32±0.04, and a dynamic exponent z=1.5±0.2. The validity of this set of scaling exponents extended from a lower cutoff lc≈60 μm upward, and the exponents agreed with those predicted by the standard Kardar-Parisi-Zhang continuous equation. HeLa data were compared with those previously reported for Vero cell colonies. The value of df and the Kardar-Parisi-Zhang-type 2D front growth dynamics were similar for colonies of both cell lines. This indicates that the cell colony growth dynamics is independent of the genetic background and the tumorigenic nature of the cells. However, one can distinguish some differences between both cell lines during the growth of colonies that may result from specific cooperative effects and the nature of each biosystem.

  11. Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation

    PubMed Central

    Taguchi, Kazumi; Kitamura, Hiroshi; Yasui, Takahiro; Naiki, Taku; Hamamoto, Shuzo; Ando, Ryosuke; Mizuno, Kentaro; Kawai, Noriyasu; Tozawa, Keiichi; Asano, Kenichi; Tanaka, Masato; Miyoshi, Ichiro; Kohri, Kenjiro

    2014-01-01

    We recently reported evidence suggesting that migrating macrophages (Mϕs) eliminate renal crystals in hyperoxaluric mice. Mϕs can be inflammatory (M1) or anti-inflammatory (M2), and colony-stimulating factor-1 (CSF-1) mediates polarization to the M2Mϕ phenotype. M2Mϕs promote renal tissue repair and regeneration, but it is not clear whether these cells are involved in suppressing renal crystal formation. We investigated the role of M2Mϕs in renal crystal formation during hyperoxaluria using CSF-1–deficient mice, which lack M2Mϕs. Compared with wild-type mice, CSF-1–deficient mice had significantly higher amounts of renal calcium oxalate crystal deposition. Treatment with recombinant human CSF-1 increased the expression of M2-related genes and markedly decreased the number of renal crystals in both CSF-1–deficient and wild-type mice. Flow cytometry of sorted renal Mϕs showed that CSF-1 deficiency resulted in a smaller population of CD11b+F4/80+CD163+CD206hi cells, which represent M2-like Mϕs. Additionally, transfusion of M2Mϕs into CSF-1–deficient mice suppressed renal crystal deposition. In vitro phagocytosis assays with calcium oxalate monohydrate crystals showed a higher rate of crystal phagocytosis by M2-polarized Mϕs than M1-polarized Mϕs or renal tubular cells. Gene array profiling showed that CSF-1 deficiency resulted in disordered M2- and stone-related gene expressions. Collectively, our results provide compelling evidence for a suppressive role of CSF-1 signaling in renal crystal formation. PMID:24578130

  12. The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apoptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and inhibit colony formation of primary MPN cells

    PubMed Central

    Mazzacurati, Lucia; Lambert, Que T.; Pradhan, Anuradha; Griner, Lori N.; Huszar, Dennis; Reuther, Gary W.

    2015-01-01

    Classical myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that exhibit excess mature myeloid cells, bone marrow fibrosis, and risk of leukemic transformation. Aberrant JAK2 signaling plays an etiological role in MPN formation. Because neoplastic cells in patients are largely insensitive to current anti-JAK2 therapies, effective therapies remain needed. Members of the PIM family of serine/threonine kinases are induced by JAK/STAT signaling, regulate hematopoietic stem cell growth, protect hematopoietic cells from apoptosis, and exhibit hematopoietic cell transforming properties. We hypothesized that PIM kinases may offer a therapeutic target for MPNs. We treated JAK2-V617F-dependent MPN model cells as well as primary MPN patient cells with the PIM kinase inhibitors SGI-1776 and AZD1208 and the JAK2 inhibitor ruxolitinib. While MPN model cells were rather insensitive to PIM inhibitors, combination of PIM inhibitors with ruxolitinib led to a synergistic effect on MPN cell growth due to enhanced apoptosis. Importantly, PIM inhibitor mono-therapy inhibited, and AZD1208/ruxolitinib combination therapy synergistically suppressed, colony formation of primary MPN cells. Enhanced apoptosis by combination therapy was associated with activation of BAD, inhibition of downstream components of the mTOR pathway, including p70S6K and S6 protein, and activation of 4EBP1. Importantly, PIM inhibitors re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib by inducing apoptosis. Finally, exogenous expression of PIM1 induced ruxolitinib resistance in MPN model cells. These data indicate that PIMs may play a role in MPNs and that combining PIM and JAK2 kinase inhibitors may offer a more efficacious therapeutic approach for MPNs over JAK2 inhibitor mono-therapy. PMID:26472029

  13. Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

    PubMed

    Galbraith, R M; Goust, J M; Fudenberg, H H

    1977-12-01

    The presence of phytohemagglutinin or pokeweed mitogen in cultures of human peripheral blood mononuclear cells in agar is known to stimulate the formation of lymphoid colonies. We now report that similar colonies can be induced in the absence of plant lectins upon addition of filtered and ultracentrifuged conditioned medium (CM) obtained from certain human lymphoblastoid cell lines. Colony formation required at least 6 X 10(5) mononuclear cells per milliliter, and optimum results were obtained at concentrations of 1 X 10(6) cells/ml in the presence of 20% CM (50-500 colonies per 10(6) cells cultured). Individual cells within colonies displayed uniform morphological characteristics of lymphoid cells, and the majority formed rosettes with sheep erythrocytes, suggesting that they were of T-cell type.

  14. Effect of Corynebacterium parvum on colony-stimulating factor and granulocyte-macrophage colony formation.

    PubMed

    Foster, R S; MacPherson, B R; Browdie, D A

    1977-05-01

    Because Corynebacterium parvum has tumor-inhibitory properties and stimulates granulocyte-macrophage production, it may have clinical value in combination with chemotherapy. The leukopoietic effect of killed suspensions of C. parvum was studied in mice using the technique of in vitro clonal culture of hematopoietic cells. After C. parvum injection, there was a prompt, sustained elevation of serum colony-stimulating factor followed by an increase in granulocyte-macrophage precursor cells in the spleen and increases in blood mononuclear and granulocyte cells. Colony-stimulating factor production is suggested as a major mechanism of stimulation of granulocyte-macrophage proliferation by C. parvum. Since rapidly proliferating hematopoietic cells may have increased sensititity to cytotoxic agents, the details of hematopoietic stimulation by C. parvum may be critical in the sequential timing of combined C. parvum and chemotherapy treatment to obtain maximal tumor inhibition and minimal hematopoietic toxicity.

  15. Breast cancer cells induce osteoclast formation by stimulating host IL-11 production and downregulating granulocyte/macrophage colony-stimulating factor.

    PubMed

    Morgan, Hayley; Tumber, Anthony; Hill, Peter A

    2004-05-01

    Breast cancer cells frequently metastasize to the skeleton, where they induce OCL formation and activity, resulting in extensive bone destruction. However, the mechanisms by which breast cancer cells mediate increased osteolysis remain unclear. To elucidate this point, we investigated how 3 human breast cancer cell lines, MDA-MB-231, MDA-MB-435 and MCF-7, induce OCL formation using a murine osteoblast-spleen cell coculture system and compared their effects with a human colorectal cancer cell line, HCT-15; a human lung cancer cell line, HT-1080; and a normal human breast cell line, HME. The breast cancer cell lines supported OCL formation only when osteoblasts were present in spleen cell cocultures, whilst the non-breast cancer cell lines and the normal breast cell line, HME, had no effect. Fractionation of BCCM by ultrafiltration established that osteoclastogenic activity was associated with factors having m.w. >3 kDa. Breast cancer cell lines produced primarily PTHrP, with lesser amounts of IL-6, IL-11 and TNF-alpha. The effect of BCCM on OCL formation in osteoblast-spleen cell cocultures was partially prevented by a neutralising antibody to human PTHrP and completely prevented by a neutralising antibody to either murine IL-11 or the murine IL-11 receptor; neutralising antibodies to human IL-6, IL-11 or TNF-alpha were without effect. BCCM or human PTHrP induced an increase in murine osteoblast IL-11 mRNA and protein production, effects that were prevented in the presence of a neutralising antibody to human PTHrP. The osteoclastogenic activity of IL-11 was mediated by enhancing osteoblast production of PGE(2) effects, which were abrogated by an inhibitor of cyclooxygenase. PGE(2) apparently enhanced OCL formation by downregulating GM-CSF production by spleen cells since recombinant murine GM-CSF inhibited OCL formation and a neutralising antibody to murine GM-CSF blocked these inhibitory effects. We conclude that breast cancer cells induce OCL formation by

  16. Periodic Colony Formation by Bacterial Species Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Wakita, Jun-ichi; Shimada, Hirotoshi; Itoh, Hiroto; Matsuyama, Tohey; Matsushita, Mitsugu

    2001-03-01

    We have investigated the periodic colony growth of bacterial species Bacillus subtilis. A colony grows cyclically with the interface repeating an advance (migration phase) and a rest (consolidation phase) alternately on a surface of semi-solid agar plate under appropriate environmental conditions, resulting in a concentric ring-like colony. It was found from macroscopic observations that the characteristic quantities for the periodic growth such as the migration time, the consolidation time and the terrace spacing do not depend so much on nutrient concentration Cn, but do on agar concentration Ca. The consolidation time was a weakly increasing function of Ca, while the migration time and the terrace spacing were, respectively, weakly and strongly decreasing function of Ca. Overall, the cycle (migration-plus-consolidation) time seems to be constant, and does not depend so much on both Cn and Ca. Microscopically, bacterial cells inside the growing front of a colony keep increasing their population during both migration and consolidation phases. It was also confirmed that their secreting surfactant called surfactin does not affect their periodic growth qualitatively, i.e., mutant cells which cannot secrete surfactin produce a concentric ring-like colony. All these results suggest that the diffusion of the nutrient and the surfactin are irrelevant to their periodic growth.

  17. Monoclonal origin of B lymphocyte colony-forming cells in spleen colonies formed by multipotential hemopoietic stem cells

    PubMed Central

    Lala, PK; Johnson, GR

    1978-01-01

    Spleen colonies produced by transplanting lethally irradiated mice with either 12 day fetal liver or adult bone marrow cells were found to contain B- lymphocyte colony-forming cells (BL-CFC) . The proportion of BL-CFC positive spleen colonies did not increase substantially between 8 and 14 days after transplantation, the range being 18-45 percent. However, the absolute number of BL-CFC per spleen colony varied considerably (between 1 and 10,318), although the majority of colonies contained less than 200 BL-CFC. Irrespective of the time after transplantation, smaller spleen colonies were found to have a higher frequency of BL-CFC than larger spleen colonies. To determine the possible clonal origin of BL-CFC from spleen colony- forming unit (CFU-S), CBA mice were injected with equal numbers of CBA and CBA T(6)/T(6) fetal liver or adult bone marrow cells. Analysis of 7-15-day spleen colonies demonstrated that 90 percent were either exclusively T(6) positive or T(6) negative and approximately equal numbers ofboth colony types were observed. B-lymphocyte colonies were grown and successfully karyotyped from 19 spleen colonies. When compared with the original spleen colony karyotype the B-lymphocyte colony cells karyotype was identical in all 19 cases. In 3 of the 19 colonies analyzed a mixture of T(6) positive and T(6) negative karyotypes was present and identical proportions of the karyotypes were present in the pooled B-lymphocyte colony cells and spleen colony cells. The data indicate that the B-lymphocyte colony-forming cells detected in spleen colonies are genuine members of the hemopoietic clone derived from the initiating hemopoietic stem cell (CFU-S). PMID:309918

  18. [Antigen-binding clone cells in hematopoietic spleen colonies].

    PubMed

    Khazanova, I V; Van'ko, L V; Malaĭtsev, V V; Khamitova, N S; Zhuravel', G M

    1976-05-01

    The antigen-binding cell clones and the Ig-positive cells were found and quantitatively assessed in the primary hemopoietic splenic colonies. The data ogtained were analyzed assuming that the ratio of clone of specialized B-cells should reflect the quantitative ration in the corresponding V-genes in the given lymphocyte populations at definite stages of its development. The colonies differed from one another markedly by the curves of inhibition by DNP-lysine of rosette-formation with DNP-erythrocytes, i.e. by the avidity of B-cells of the given specificity. The colonies differed significantly by the ratio of the volumes of the two clone groups (cell with anti-DNP and anti-BE Ig-receptors) between themselves and the combination of the Ig-positive cells. These quantitaive regularities were incompatible with the view that each B-cell had any conceivable set of V-genes, i.e. with the germ-line hypothesis of the antibody and receptor diversity.

  19. Active mechanics and geometry of adherent cells and cell colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2014-03-01

    Measurements of traction stresses exerted by adherent cells or cell colonies on elastic substrates have yielded new insight on how the mechanical and geometrical properties of the substrate regulate cellular force distribution, mechanical energy, spreading, morphology or stress ber architecture. We have developed a generic mechanical model of adherent cells as an active contractile gel mechanically coupled to an elastic substrate and to neighboring cells in a tissue. The contractile gel model accurately predicts the distribution of cellular and traction stresses as observed in single cell experiments, and captures the dependence of cell shape, traction stresses and stress ber polarization on the substrate's mechanical and geometrical properties. The model further predicts that the total strain energy of an adherent cell is solely regulated by its spread area, in agreement with recent experiments on micropatterned substrates with controlled geometry. When used to describe the behavior of colonies of adherent epithelial cells, the model demonstrates the crucial role of the mechanical cross-talk between intercellular and extracellular adhesion in regulating traction force distribution. Strong intercellular mechanical coupling organizes traction forces to the colony periphery, whereas weaker intercellular coupling leads to the build up of traction stresses at intercellular junctions. Furthermore, in agreement with experiments on large cohesive keratinocyte colonies, the model predicts a linear scaling of traction forces with the colony size. This scaling suggests the emergence of an effective surface tension as a scale-free material property of the adherent tissue, originating from actomyosin contractility.

  20. Two-dimensionality of yeast colony expansion accompanied by pattern formation.

    PubMed

    Chen, Lin; Noorbakhsh, Javad; Adams, Rhys M; Samaniego-Evans, Joseph; Agollah, Germaine; Nevozhay, Dmitry; Kuzdzal-Fick, Jennie; Mehta, Pankaj; Balázsi, Gábor

    2014-12-01

    Yeasts can form multicellular patterns as they expand on agar plates, a phenotype that requires a functional copy of the FLO11 gene. Although the biochemical and molecular requirements for such patterns have been examined, the mechanisms underlying their formation are not entirely clear. Here we develop quantitative methods to accurately characterize the size, shape, and surface patterns of yeast colonies for various combinations of agar and sugar concentrations. We combine these measurements with mathematical and physical models and find that FLO11 gene constrains cells to grow near the agar surface, causing the formation of larger and more irregular colonies that undergo hierarchical wrinkling. Head-to-head competition assays on agar plates indicate that two-dimensional constraint on the expansion of FLO11 wild type (FLO11) cells confers a fitness advantage over FLO11 knockout (flo11Δ) cells on the agar surface.

  1. Two-Dimensionality of Yeast Colony Expansion Accompanied by Pattern Formation

    PubMed Central

    Chen, Lin; Noorbakhsh, Javad; Adams, Rhys M.; Samaniego-Evans, Joseph; Agollah, Germaine; Nevozhay, Dmitry; Kuzdzal-Fick, Jennie; Mehta, Pankaj; Balázsi, Gábor

    2014-01-01

    Yeasts can form multicellular patterns as they expand on agar plates, a phenotype that requires a functional copy of the FLO11 gene. Although the biochemical and molecular requirements for such patterns have been examined, the mechanisms underlying their formation are not entirely clear. Here we develop quantitative methods to accurately characterize the size, shape, and surface patterns of yeast colonies for various combinations of agar and sugar concentrations. We combine these measurements with mathematical and physical models and find that FLO11 gene constrains cells to grow near the agar surface, causing the formation of larger and more irregular colonies that undergo hierarchical wrinkling. Head-to-head competition assays on agar plates indicate that two-dimensional constraint on the expansion of FLO11 wild type (FLO11) cells confers a fitness advantage over FLO11 knockout (flo11Δ) cells on the agar surface. PMID:25504059

  2. The induction of human peripheral blood lymphoid colonies by conditioned media from human tumour cell lines.

    PubMed Central

    Vesole, D H; Moore, G E

    1980-01-01

    Conditioned medium (CM) from 29 human tumour cell lines and 3 malignant pleural fluids were tested for their ability to stimulate lymphoid colony formation in semi-solid agar; 9 of 14 malignant melanomas, 3 of 6 colonic carcinomas, 2 of 5 ovarian carcinomas, 3 of 4 breast carcinomas and 1 of 3 pleural fluids from breast cancer patients contained colony-stimulating activity (CSA) for human peripheral blood lymphoid cells (PBL) in semi-solid agar. Conditioned media also stimulated PBL proliferation in liquid medium; these effects were dose dependent. With the exception of one pleural fluid, extensive dialysis of CM did not significantly increase colony formation; CM from two tumour cell lines demonstrated a significant decrease in the induction of colony formation after dialysis. PMID:6970165

  3. Modeling cell-matrix traction forces in Keratinocyte colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2013-03-01

    Crosstalk between cell-cell and cell-matrix adhesions plays an essential role in the mechanical function of tissues. The traction forces exerted by cohesive keratinocyte colonies with strong cell-cell adhesions are mostly concentrated at the colony periphery. In contrast, for weak cadherin-based intercellular adhesions, individual cells in a colony interact with their matrix independently, with a disorganized distribution of traction forces extending throughout the colony. In this talk I will present a minimal physical model of the colony as contractile elastic media linked by springs and coupled to an elastic substrate. The model captures the spatial distribution of traction forces seen in experiments. For cell colonies with strong cell-cell adhesions, the total traction force of the colony measured in experiments is found to scale with the colony's geometrical size. This scaling suggests the emergence of an effective surface tension of magnitude comparable to that measured for non-adherent, three-dimensional cell aggregates. The physical model supports the scaling and indicates that the surface tension may be controlled by acto-myosin contractility. Supported by the NSF through grant DMR-1004789. This work was done in collaboration with Aaron F. Mertz, Eric R. Dufresne and Valerie Horsley (Yale University) and M. Cristina Marchetti (Syracuse University).

  4. Colony forming cell (CFC) assay for human hematopoietic cells.

    PubMed

    Sarma, Nayan J; Takeda, Akiko; Yaseen, Nabeel R

    2010-12-18

    Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages. We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and

  5. Dynamics and morphology characteristics of cell colonies with radially spreading growth fronts

    NASA Astrophysics Data System (ADS)

    Huergo, M. A. C.; Pasquale, M. A.; González, P. H.; Bolzán, A. E.; Arvia, A. J.

    2011-08-01

    The dynamics of two-dimensional (2D) radially spreading growth fronts of Vero cell colonies was investigated utilizing two types of colonies, namely type I starting from clusters with a small number of cells, which initially exhibited arbitrary-shaped rough growth fronts and progressively approached quasicircular ones as the cell population increased; and type II colonies, starting from a relatively large circular three-dimensional (3D) cell cluster. For large cell population colonies, the fractal dimension of the fronts was DF=1.20±0.05. For low cell populations, the mean colony radius increased exponentially with time, but for large ones the constant radial front velocity 0.20±0.02 μm min-1 was reached. Colony spreading was accompanied by changes in both cell morphology and average size, and by the formation of very large cells, some of them multinuclear. Therefore the heterogeneity of colonies increased and local driving forces that set in began to influence the 2D growth front kinetics. The retardation effect related to the exponential to constant radial front velocity transition was assigned to a number of possible interferences including the cell duplication and 3D growth in the bulk of the colony. The dynamic scaling analysis of overhang-corrected rough colony fronts, after arc-radius coordinate system transformation, resulted in roughness exponent α = 0.50±0.05 and growth exponent β = 0.32±0.04, for arc lengths greater than 100 μm. This set of scaling exponents agreed with that predicted by the Kardar, Parisi, and Zhang continuous equation. For arc lengths shorter than 2-3 cell diameters, the value α = 0.85±0.05 would be related to a cell front roughening caused by temporarily membrane deformations occasionally interfered by cell proliferation.

  6. A Novel Type of Colony Formation in Marine Planktonic Diatoms Revealed by Atomic Force Microscopy

    PubMed Central

    Bosak, Sunčica; Pletikapić, Galja; Hozić, Amela; Svetličić, Vesna; Sarno, Diana; Viličić, Damir

    2012-01-01

    Diatoms have evolved a variety of colonial life forms in which cells are connected by organic threads, mucilage pads or silicate structures. In this study, we provide the first description of a novel strategy of colony formation among marine planktonic diatoms. Bacteriastrum jadranum forms loose but regular chains with distinct heterovalvate terminal cells. The colonial cells and their siliceous projections, the setae, are not in direct contact; instead, they are enclosed within the optically transparent organic matrix. This cell jacket structure was detected by staining procedure with Alcian Blue, which showed that the polysaccharides are predominant matrix constituents and revealed that the jacket reaches the span of the setae. The scanning electron microscopy (SEM) observations showed distinguishable fibrillar network firmly associated with cells. Using atomic force microscopy (AFM), we were able to visualise and characterise the cell jacket structure at molecular resolution. At nanoscale resolution, the cell jacket appears as a cross-linked fibrillar network organised into a recognisable structure. The circular patches of self-repeating pattern (hexagonal pores with openings of 8–100 nm) are connected through thicker surrounding fibrils and reinforced by branching fibrils. The pore-forming fibrils within the patches are only 0.6–1.6 nm high, the surrounding fibrils connecting patches are 2.0–2.8 nm high, and the branching fibrils are considerably wider but not higher than 4.0 nm. The discovered polysaccharide fibrillar network is highly organised and delicately structured with a monomolecular fibril height of 0.6 nm. We conclude that the Bacteriastrum polysaccharide jacket represents an essential part of the cell, as the conjunction of the polymer network with the frustule appears to be extremely tight and such specific and unique patterns have never been found in self-assembled polysaccharide gel networks, which are usually encountered in the marine

  7. A novel type of colony formation in marine planktonic diatoms revealed by atomic force microscopy.

    PubMed

    Bosak, Sunčica; Pletikapić, Galja; Hozić, Amela; Svetličić, Vesna; Sarno, Diana; Viličić, Damir

    2012-01-01

    Diatoms have evolved a variety of colonial life forms in which cells are connected by organic threads, mucilage pads or silicate structures. In this study, we provide the first description of a novel strategy of colony formation among marine planktonic diatoms. Bacteriastrum jadranum forms loose but regular chains with distinct heterovalvate terminal cells. The colonial cells and their siliceous projections, the setae, are not in direct contact; instead, they are enclosed within the optically transparent organic matrix. This cell jacket structure was detected by staining procedure with Alcian Blue, which showed that the polysaccharides are predominant matrix constituents and revealed that the jacket reaches the span of the setae. The scanning electron microscopy (SEM) observations showed distinguishable fibrillar network firmly associated with cells. Using atomic force microscopy (AFM), we were able to visualise and characterise the cell jacket structure at molecular resolution. At nanoscale resolution, the cell jacket appears as a cross-linked fibrillar network organised into a recognisable structure. The circular patches of self-repeating pattern (hexagonal pores with openings of 8-100 nm) are connected through thicker surrounding fibrils and reinforced by branching fibrils. The pore-forming fibrils within the patches are only 0.6-1.6 nm high, the surrounding fibrils connecting patches are 2.0-2.8 nm high, and the branching fibrils are considerably wider but not higher than 4.0 nm. The discovered polysaccharide fibrillar network is highly organised and delicately structured with a monomolecular fibril height of 0.6 nm. We conclude that the Bacteriastrum polysaccharide jacket represents an essential part of the cell, as the conjunction of the polymer network with the frustule appears to be extremely tight and such specific and unique patterns have never been found in self-assembled polysaccharide gel networks, which are usually encountered in the marine

  8. Erythroid colony formation and effect of hemin in vitro in hereditary sideroblastic anemias.

    PubMed

    Partanen, S; Pasanen, A; Juvonen, E; Tenhunen, R; Ruutu, T

    1988-05-01

    Colony formation by erythroid burst-forming units (BFU-E) and erythroid colony-forming units (CFU-E) and the effect of hemin on colony growth was studied in vitro in three Finnish families with hereditary sideroblastic anemia (HSA). Defective activity of heme synthase has been demonstrated in family A and that of delta-aminolevulinic acid synthase in family B. No biochemical defect has been recognized so far in family C. CFU-E colony growth was defective in seven of the eight persons studied. The formation of BFU-E colonies was normal in family A and increased in family C, whereas of the two members of family B one showed normal and one decreased BFU-E colony growth. Hemin in 30-120 microM concentration increased significantly both BFU-E (p less than 0.01) and CFU-E (p less than 0.005) colony formation in family C. No effect was seen in family A, and in family B the only effect was normalization of the decreased BFU-E colony growth by the highest hemin concentration in one person. This study indicates that differences exist between families with HSA in erythroid colony formation and in response to hemin in vitro, but the low number of investigated members in each family does not permit a conclusive evaluation of the impact of the carrier versus patient status or of sex on the results.

  9. High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection.

    PubMed

    Choudhry, Priya

    2016-01-01

    Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays.

  10. High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

    PubMed Central

    Choudhry, Priya

    2016-01-01

    Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays. PMID:26848849

  11. Giant vesicles "colonies": a model for primitive cell communities.

    PubMed

    Carrara, Paolo; Stano, Pasquale; Luisi, Pier Luigi

    2012-07-09

    Current research on the origin of life typically focuses on the self-organisation of molecular components in individual cell-like compartments, thereby bringing about the emergence of self-sustaining minimal cells. This is justified by the fact that single cells are the minimal forms of life. No attempts have been made to investigate the cooperative mechanisms that could derive from the assembly of individual compartments. Here we present a novel experimental approach based on vesicles "colonies" as a model of primitive cell communities. Experiments show that several advantages could have favoured primitive cell colonies when compared with isolated primitive cells. In fact there are two novel unexpected features typical of vesicle colonies, namely solute capture and vesicle fusion, which can be seen as the basic physicochemical mechanisms at the origin of life.

  12. Modest stimulatory effect of recombinant human GM-CSF on colony growth from peripheral blood human megakaryocyte progenitor cells.

    PubMed

    Mazur, E M; Cohen, J L; Wong, G G; Clark, S C

    1987-12-01

    Recombinant human granulocyte-macrophage colony-stimulating factor (rGM-CSF) has been previously demonstrated to stimulate colony formation from human myeloid, erythroid, and multipotential stem cells. In this investigation, we evaluated the effects of rGM-CSF on colony growth by human megakaryocyte progenitors (CFU-Meg). rGM-CSF was tested at concentrations of 0.1-100 U/ml in plasma clot cultures of adherent-depleted normal peripheral blood mononuclear cells. Control cultures were concurrently prepared containing either no stimulator or megakaryocyte colony-stimulating factor (Meg-CSF) partially purified from aplastic canine serum. rGM-CSF increased megakaryocyte colony numbers from a baseline of 4.3 +/- 1.4 (+/- SEM) in the unstimulated cultures to a maximum of 21.0 +/- 5.3 colonies at an rGM-CSF concentration of 1.0 U/ml. Corresponding megakaryocytic colony size increased from 4.4 to 8.3 cells/colony. Further increasing the rGM-CSF concentration resulted in decreasing megakaryocyte colony growth, reaching 6.8 +/- 2.9 colonies at 100 U/ml. The maximum number of megakaryocyte colonies stimulated by rGM-CSF was only 23.3% of that achieved in the control cultures containing optimal concentrations of serum-derived Meg-CSF protein (2.0 mg/ml). Megakaryocyte colonies stimulated by rGM-CSF consisted of predominantly low ploidy cells approximately equally distributed in 2N, 4N, and 8N ploidy classes. There was no increase in ploidy with any rGM-CSF concentration. These data indicate that rGM-CSF has modest activity in stimulating human megakaryocyte colony growth that is substantially less than that present in serum-derived Meg-CSF. rGM-CSF appears to primarily affect the early mitotic phase of megakaryocyte colony development with little influence on megakaryocyte endoreduplication.

  13. Random mitotic activities across human embryonic stem cell colonies.

    SciTech Connect

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L.

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  14. Sequestration and Distribution Characteristics of Cd(II) by Microcystis aeruginosa and Its Role in Colony Formation

    PubMed Central

    Bi, Xiangdong; Yan, Ran; Li, Fenxiang; Dai, Wei; Jiao, Kewei; Liu, Qi

    2016-01-01

    To investigate the sequestration and distribution characteristics of Cd(II) by Microcystis aeruginosa and its role in Microcystis colony formation, M. aeruginosa was exposed to six different Cd(II) concentrations for 10 days. Cd(II) exposure caused hormesis in the growth of M. aeruginosa. Low concentrations of Cd(II) significantly induced formation of small Microcystis colonies (P < 0.05) and increased the intracellular polysaccharide (IPS) and bound extracellular polysaccharide (bEPS) contents of M. aeruginosa significantly (P < 0.05). There was a linear relationship between the amount of Cd(II) sequestrated by algal cells and the amount added to cultures in the rapid adsorption process that occurred during the first 5 min of exposure. After 10 d, M. aeruginosa sequestrated nearly 80% of 0.2 mg L−1 added Cd(II), while >93% of Cd(II) was sequestrated in the groups with lower added concentrations of Cd(II). More than 80% of the sequestrated Cd(II) was bioadsorbed by bEPS. The Pearson correlation coefficients of exterior and interior factors related to colony formation of M. aeruginosa revealed that Cd(II) could stimulate the production of IPS and bEPS via increasing Cd(II) bioaccumulation and bioadsorption. Increased levels of cross-linking between Cd(II) and bEPS stimulated algal cell aggregation, which eventually promoted the formation of Microcystis colonies. PMID:27777956

  15. Sequestration and Distribution Characteristics of Cd(II) by Microcystis aeruginosa and Its Role in Colony Formation.

    PubMed

    Bi, Xiangdong; Yan, Ran; Li, Fenxiang; Dai, Wei; Jiao, Kewei; Zhou, Qixing; Liu, Qi

    2016-01-01

    To investigate the sequestration and distribution characteristics of Cd(II) by Microcystis aeruginosa and its role in Microcystis colony formation, M. aeruginosa was exposed to six different Cd(II) concentrations for 10 days. Cd(II) exposure caused hormesis in the growth of M. aeruginosa. Low concentrations of Cd(II) significantly induced formation of small Microcystis colonies (P < 0.05) and increased the intracellular polysaccharide (IPS) and bound extracellular polysaccharide (bEPS) contents of M. aeruginosa significantly (P < 0.05). There was a linear relationship between the amount of Cd(II) sequestrated by algal cells and the amount added to cultures in the rapid adsorption process that occurred during the first 5 min of exposure. After 10 d, M. aeruginosa sequestrated nearly 80% of 0.2 mg L(-1) added Cd(II), while >93% of Cd(II) was sequestrated in the groups with lower added concentrations of Cd(II). More than 80% of the sequestrated Cd(II) was bioadsorbed by bEPS. The Pearson correlation coefficients of exterior and interior factors related to colony formation of M. aeruginosa revealed that Cd(II) could stimulate the production of IPS and bEPS via increasing Cd(II) bioaccumulation and bioadsorption. Increased levels of cross-linking between Cd(II) and bEPS stimulated algal cell aggregation, which eventually promoted the formation of Microcystis colonies.

  16. Pattern Formation of Bacterial Colonies by Escherichia coli

    NASA Astrophysics Data System (ADS)

    Tokita, Rie; Katoh, Takaki; Maeda, Yusuke; Wakita, Jun-ichi; Sano, Masaki; Matsuyama, Tohey; Matsushita, Mitsugu

    2009-07-01

    We have studied the morphological diversity and change in bacterial colonies, using the bacterial species Escherichia coli, as a function of both agar concentration Ca and nutrient concentration Cn. We observed various colony patterns, classified them into four types by pattern characteristics and established a morphological diagram by dividing it into four regions. They are regions A [diffusion-limited aggregation (DLA)-like], B (Eden-like), C (concentric-ring), and D (fluid-spreading). In particular, we have observed a concentric-ring colony growth for E. coli. We focused on the periodic growth in region C and obtained the following results: (i) A colony grows cyclically with the growing front repeating an advance (migration phase) and a momentary rest (consolidation phase) alternately. (ii) The growth width L and the bulge width W in one cycle decrease asymptotically to certain values, when Ca is increased. (iii) L does not depend on Cn, while W is an increasing function of Cn. Plausible mechanisms are proposed to explain the experimental results, by comparing them with those obtained for other bacterial species such as Proteus mirabilis and Bacillus subtilis.

  17. The effect of dimethyl sulfoxide on the induction of DNA strand breaks in plasmid DNA and colony formation of PC Cl3 mammalian cells by alpha-, beta-, and Auger electron emitters (223)Ra, (188)Re, and (99m)Tc.

    PubMed

    Runge, Roswitha; Oehme, Liane; Kotzerke, Jörg; Freudenberg, Robert

    2016-12-01

    DNA damage occurs as a consequence of both direct and indirect effects of ionizing radiation. The severity of DNA damage depends on the physical characteristics of the radiation quality, e.g., the linear energy transfer (LET). There are still contrary findings regarding direct or indirect interactions of high-LET emitters with DNA. Our aim is to determine DNA damage and the effect on cellular survival induced by (223)Ra compared to (188)Re and (99m)Tc modulated by the radical scavenger dimethyl sulfoxide (DMSO). Radioactive solutions of (223)Ra, (188)Re, or (99m)Tc were added to either plasmid DNA or to PC Cl3 cells in the absence or presence of DMSO. Following irradiation, single strand breaks (SSB) and double strand breaks (DSB) in plasmid DNA were analyzed by gel electrophoresis. To determine the radiosensitivity of the rat thyroid cell line (PC Cl3), survival curves were performed using the colony formation assay. Exposure to 120 Gy of (223)Ra, (188)Re, or (99m)Tc leads to maximal yields of SSB (80 %) in plasmid DNA. Irradiation with 540 Gy (223)Ra and 500 Gy (188)Re or (99m)Tc induced 40, 28, and 64 % linear plasmid conformations, respectively. DMSO prevented the SSB and DSB in a similar way for all radionuclides. However, with the α-emitter (223)Ra, a low level of DSB could not be prevented by DMSO. Irradiation of PC Cl3 cells with (223)Ra, (188)Re, and (99m)Tc pre-incubated with DMSO revealed enhanced survival fractions (SF) in comparison to treatment without DMSO. Protection factors (PF) were calculated using the fitted survival curves. These factors are 1.23 ± 0.04, 1.20 ± 0.19, and 1.34 ± 0.05 for (223)Ra, (188)Re, and (99m)Tc, respectively. For (223)Ra, as well as for (188)Re and (99m)Tc, dose-dependent radiation effects were found applicable for plasmid DNA and PC Cl3 cells. The radioprotection by DMSO was in the same range for high- and low-LET emitter. Overall, the results indicate the contribution of mainly indirect radiation

  18. Reversible Commitment to Differentiation by Human Multipotent Stromal Cells (MSCs) in Single-Cell Derived Colonies

    PubMed Central

    Ylöstalo, Joni; Bazhanov, Nikolay; Prockop, Darwin J

    2008-01-01

    Objective Human multipotent stromal cells (MSCs) readily form single-cell derived colonies when plated at clonal densities. However, the colonies are heterogeneous since the cells from a colony form new colonies that vary in size and differentiation potential when re-plated at clonal densities. The experiments here tested the hypothesis that the cells in the inner regions of colonies are partially differentiated but the differentiation is reversible. Materials and Methods Cells were separately isolated from the dense inner regions (IN) and less dense outer regions (OUT) of single-cell derived colonies. The cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation. Results The IN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when re-plated at clonal densities and assayed for rates of propagation and clonogenicity. Also, the colonies formed by IN cells were similar to colonies formed by OUT cells in that they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30-75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded. Conclusions The results demonstrated that the cells in the inner region of single-derived colonies are partially differentiated but the differentiation can be reversed by re-plating the cells at clonal densities. PMID:18619725

  19. Longevity of U cells of differentiated yeast colonies grown on respiratory medium depends on active glycolysis.

    PubMed

    Čáp, Michal; Váchová, Libuše; Palková, Zdena

    2015-01-01

    Colonies of Saccharomyces cerevisiae laboratory strains pass through specific developmental phases when growing on solid respiratory medium. During entry into the so-called alkali phase, in which ammonia signaling is initiated, 2 prominent cell types are formed within the colonies: U cells in upper colony regions, which have a longevity phenotype and activate the expression of a large number of metabolic genes, and L cells in lower regions, which die more quickly and exhibit a starvation phenotype. Here, we performed a detailed analysis of the activities of enzymes of central carbon metabolism in lysates of both cell types and determined several fermentation end products, showing that previously reported expression differences are reflected in the different enzymatic capabilities of each cell type. Hence, U cells, despite being grown on respiratory medium, behave as fermenting cells, whereas L cells rely on respiratory metabolism and possess active gluconeogenesis. Using a spectrum of different inhibitors, we showed that glycolysis is essential for the formation, and particularly, the survival of U cells. We also showed that β-1,3-glucans that are released from the cell walls of L cells are the most likely source of carbohydrates for U cells.

  20. In vitro effects of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity.

    PubMed

    Jantová, S; Theiszová, M; Letasiová, S; Birosová, L; Palou, T M

    2008-04-30

    The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard-tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH(-) groups and F(-) ions. The aim of this experimental investigation was to evaluate cytotoxic, genotoxic and mutagenic effects of FHA and FA eluates on Chinese hamster V79 cells and to compare them with the effects of hydroxyapatite (HA) eluate. Cytotoxicity of the biomaterials tested was evaluated by use of the cell colony-formation assay and by direct counting of the cells in each colony. Genotoxicity was assessed by single-cell gel electrophoresis (comet assay) and mutagenicity was evaluated by the Hprt gene-mutation assay and in bacterial mutagenicity tests using Salmonella typhimurium TA100. The results show that the highest test concentrations of the biomaterials (100% and 75% eluates) induced very weak inhibition of colony growth (about 10%). On the other hand, the reduction of cell number per colony induced by these concentrations was in the range from 43% to 31%. The comet assay showed that biomaterials induced DNA breaks, which increased with increasing test concentrations in the order HAcell division in V79 cell colonies.

  1. Edges of human embryonic stem cell colonies display distinct mechanical properties and differentiation potential

    PubMed Central

    Rosowski, Kathryn A.; Mertz, Aaron F.; Norcross, Samuel; Dufresne, Eric R.; Horsley, Valerie

    2015-01-01

    In order to understand the mechanisms that guide cell fate decisions during early human development, we closely examined the differentiation process in adherent colonies of human embryonic stem cells (hESCs). Live imaging of the differentiation process reveals that cells on the outer edge of the undifferentiated colony begin to differentiate first and remain on the perimeter of the colony to eventually form a band of differentiation. Strikingly, this band is of constant width in all colonies, independent of their size. Cells at the edge of undifferentiated colonies show distinct actin organization, greater myosin activity and stronger traction forces compared to cells in the interior of the colony. Increasing the number of cells at the edge of colonies by plating small colonies can increase differentiation efficiency. Our results suggest that human developmental decisions are influenced by cellular environments and can be dictated by colony geometry of hESCs. PMID:26391588

  2. Role of gas vesicles and intra-colony spaces during the process of algal bloom formation.

    PubMed

    Zhang, Yongsheng; Zheng, Binghui; Jiang, Xia; Zheng, Hao

    2013-06-01

    Aggregation morphology, vertical distribution, and algal density were analyzed during the algal cell floating process in three environments. The role of gas vesicles and intra-colony spaces was distinguished by algal blooms treated with ultrasonic waves and high pressure. Results demonstrated that the two buoyancy providers jointly provide buoyancy for floating algal cells. The results were also confirmed by force analysis. In the simulation experiment, the buoyancy acting on algal cells was greater than its gravity at sample ports 2 and 3 of a columnar-cultivated cell vessel, and intra-colony spaces were not detected. In Taihu Lake, gas vesicle buoyancy was notably less than total algal cell gravity. Buoyancy provided by intra-colony spaces exceeded total algal cell gravity at the water surface, but not at other water depths. In the Daning River, total buoyancies provided by the two buoyancy providers were less than total algal cell gravity at different water depths.

  3. Emergent structures and dynamics of cell colonies by contact inhibition of locomotion.

    PubMed

    Smeets, Bart; Alert, Ricard; Pešek, Jiří; Pagonabarraga, Ignacio; Ramon, Herman; Vincent, Romaric

    2016-12-20

    Cells in tissues can organize into a broad spectrum of structures according to their function. Drastic changes of organization, such as epithelial-mesenchymal transitions or the formation of spheroidal aggregates, are often associated either to tissue morphogenesis or to cancer progression. Here, we study the organization of cell colonies by means of simulations of self-propelled particles with generic cell-like interactions. The interplay between cell softness, cell-cell adhesion, and contact inhibition of locomotion (CIL) yields structures and collective dynamics observed in several existing tissue phenotypes. These include regular distributions of cells, dynamic cell clusters, gel-like networks, collectively migrating monolayers, and 3D aggregates. We give analytical predictions for transitions between noncohesive, cohesive, and 3D cell arrangements. We explicitly show how CIL yields an effective repulsion that promotes cell dispersal, thereby hindering the formation of cohesive tissues. Yet, in continuous monolayers, CIL leads to collective cell motion, ensures tensile intercellular stresses, and opposes cell extrusion. Thus, our work highlights the prominent role of CIL in determining the emergent structures and dynamics of cell colonies.

  4. Slowing the Onset of Hypoxia Increases Colony Forming Efficiency of Connective Tissue Progenitor Cells In Vitro.

    PubMed

    Heylman, Christopher M; Caralla, Tonya N; Boehm, Cynthia A; Patterson, Thomas E; Muschler, George F

    2013-09-26

    Survival and colony formation by transplanted tissue derived connective tissue progenitor cells (CTPs) are thought to be important factors in the success of clinical tissue engineering strategies for bone regeneration. Transplantation of cells into defects larger than a few millimeters expose cells to a profoundly hypoxic environment. This study tested the hypothesis that delaying the onset of hypoxia will improve the survival and performance of CTPs in vitro. To mimic declines seen in an avascular in vivo bone defect, colony forming efficiency by marrow derived nucleated cells was assessed under osteogenic conditions. Variation in the rate of oxygen decline from an oxygen tension of 21% to 0.1% oxygen was explored using an incubator with programmable active control of gas concentrations. The effect of doping cultures with defined concentrations of RBCs was also used to evaluate the potential for RBCs to serve as a natural buffer in the setting of declining oxygen levels. A delay in onset of hypoxia over 96 hours resulted in a 3-fold increase in the relative colony forming efficiency (rCFE) of CTPs as compared to an immediate onset of hypoxia. The presence of RBCs in vitro inhibited the rCFE of CTPs. Given the negative effects of RBCs, methods of RBC removal were evaluated and compared for their effectiveness of RBC removal and retention of colony forming efficiency. These data suggest that conditions of hypoxia compromise colony forming efficiency in marrow derived CTPs. However, slowing the rate of decline of oxygen preserved colony forming efficiency at levels achieved in a stable normoxic (3% O2) environment. These data also suggest that RBCs are detrimental to the rCFE of CTPs and that buffy coat is an effective and preferred method for removing RBCs from marrow aspirates while preserving CTPs. These findings may inform clinical strategies for CTP transplantation.

  5. Emergent structures and dynamics of cell colonies by contact inhibition of locomotion

    PubMed Central

    Smeets, Bart; Pešek, Jiří; Pagonabarraga, Ignacio; Ramon, Herman; Vincent, Romaric

    2016-01-01

    Cells in tissues can organize into a broad spectrum of structures according to their function. Drastic changes of organization, such as epithelial–mesenchymal transitions or the formation of spheroidal aggregates, are often associated either to tissue morphogenesis or to cancer progression. Here, we study the organization of cell colonies by means of simulations of self-propelled particles with generic cell-like interactions. The interplay between cell softness, cell–cell adhesion, and contact inhibition of locomotion (CIL) yields structures and collective dynamics observed in several existing tissue phenotypes. These include regular distributions of cells, dynamic cell clusters, gel-like networks, collectively migrating monolayers, and 3D aggregates. We give analytical predictions for transitions between noncohesive, cohesive, and 3D cell arrangements. We explicitly show how CIL yields an effective repulsion that promotes cell dispersal, thereby hindering the formation of cohesive tissues. Yet, in continuous monolayers, CIL leads to collective cell motion, ensures tensile intercellular stresses, and opposes cell extrusion. Thus, our work highlights the prominent role of CIL in determining the emergent structures and dynamics of cell colonies. PMID:27930287

  6. Reaction-diffusion model for pattern formation in E. coli swarming colonies with slime

    NASA Astrophysics Data System (ADS)

    Zorzano, M.-P.; Hochberg, D.; Cuevas, M.-T.; Gómez-Gómez, J.-M.

    2005-03-01

    A new experimental colonial pattern and pattern transition observed in E. coli MG1655 swarming cells grown on semisolid agar are described. We present a reaction-diffusion model that, taking into account the slime generated by these cells and its influence on the bacterial differentiation and motion, reproduces the pattern and successfully predicts the observed changes when the colonial collective motility is limited. In spite of having small nonhyperflagellated swarming cells, under these experimental conditions E. coli MG1655 can very rapidly colonize a surface, with a low branching rate, thanks to a strong fluid production and a locally incremented density of motile, lubricating cells.

  7. Cell Differentiation and Spatial Organization in Yeast Colonies: Role of Cell-Wall Integrity Pathway.

    PubMed

    Piccirillo, Sarah; Morales, Rita; White, Melissa G; Smith, Keston; Kapros, Tamas; Honigberg, Saul M

    2015-12-01

    Many microbial communities contain organized patterns of cell types, yet relatively little is known about the mechanism or function of this organization. In colonies of the budding yeast Saccharomyces cerevisiae, sporulation occurs in a highly organized pattern, with a top layer of sporulating cells sharply separated from an underlying layer of nonsporulating cells. A mutant screen identified the Mpk1 and Bck1 kinases of the cell-wall integrity (CWI) pathway as specifically required for sporulation in colonies. The CWI pathway was induced as colonies matured, and a target of this pathway, the Rlm1 transcription factor, was activated specifically in the nonsporulating cell layer, here termed feeder cells. Rlm1 stimulates permeabilization of feeder cells and promotes sporulation in an overlying cell layer through a cell-nonautonomous mechanism. The relative fraction of the colony apportioned to feeder cells depends on nutrient environment, potentially buffering sexual reproduction against suboptimal environments.

  8. A local PDE model of aggregation formation in bacterial colonies

    NASA Astrophysics Data System (ADS)

    Chavy-Waddy, Paul-Christopher; Kolokolnikov, Theodore

    2016-10-01

    We study pattern formation in a model of cyanobacteria motion recently proposed by Galante, Wisen, Bhaya and Levy. By taking a continuum limit of their model, we derive a novel fourth-order nonlinear parabolic PDE equation that governs the behaviour of the model. This PDE is {{u}t}=-{{u}xx}-{{u}xxxx}+α {{≤ft(\\frac{{{u}x}{{u}xx}}{u}\\right)}x} . We then derive the instability thresholds for the onset of pattern formation. We also compute analytically the spatial profiles of the steady state aggregation density. These profiles are shown to be of the form \\text{sec}{{\\text{h}}p} where the exponent p is related to the parameters of the model. Full numerical simulations give a favorable comparison between the continuum and the underlying discrete system, and show that the aggregation profiles are stable above the critical threshold.

  9. Burkholderia cenocepacia ShvR-regulated genes that influence colony morphology, biofilm formation, and virulence.

    PubMed

    Subramoni, Sujatha; Nguyen, David T; Sokol, Pamela A

    2011-08-01

    Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn5 mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates, l-arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia.

  10. Multiscale modeling of bacterial colonies: how pili mediate the dynamics of single cells and cellular aggregates

    NASA Astrophysics Data System (ADS)

    Pönisch, Wolfram; Weber, Christoph A.; Juckeland, Guido; Biais, Nicolas; Zaburdaev, Vasily

    2017-01-01

    Neisseria gonorrhoeae is the causative agent of one of the most common sexually transmitted diseases, gonorrhea. Over the past two decades there has been an alarming increase of reported gonorrhea cases where the bacteria were resistant to the most commonly used antibiotics thus prompting for alternative antimicrobial treatment strategies. The crucial step in this and many other bacterial infections is the formation of microcolonies, agglomerates consisting of up to several thousands of cells. The attachment and motility of cells on solid substrates as well as the cell-cell interactions are primarily mediated by type IV pili, long polymeric filaments protruding from the surface of cells. While the crucial role of pili in the assembly of microcolonies has been well recognized, the exact mechanisms of how they govern the formation and dynamics of microcolonies are still poorly understood. Here, we present a computational model of individual cells with explicit pili dynamics, force generation and pili-pili interactions. We employ the model to study a wide range of biological processes, such as the motility of individual cells on a surface, the heterogeneous cell motility within the large cell aggregates, and the merging dynamics and the self-assembly of microcolonies. The results of numerical simulations highlight the central role of pili generated forces in the formation of bacterial colonies and are in agreement with the available experimental observations. The model can quantify the behavior of multicellular bacterial colonies on biologically relevant temporal and spatial scales and can be easily adjusted to include the geometry and pili characteristics of various bacterial species. Ultimately, the combination of the microbiological experimental approach with the in silico model of bacterial colonies might provide new qualitative and quantitative insights on the development of bacterial infections and thus pave the way to new antimicrobial treatments.

  11. Isolation and Characterization of Circulating Lymphatic Endothelial Colony Forming Cells

    PubMed Central

    DiMaio, Terri A.; Wentz, Breanna L.; Lagunoff, Michael

    2016-01-01

    Rationale The identification of circulating endothelial progenitor cells has led to speculation regarding their origin as well as their contribution to neovascular development. Two distinct types of endothelium make up the blood and lymphatic vessel system. However, it has yet to be determined whether there are distinct lymphatic-specific circulating endothelial progenitor cells. Objective This study aims to isolate and characterize the cellular properties and global gene expression of lymphatic-specific endothelial progenitor cells. Methods and Results We isolated circulating endothelial colony forming cells (ECFCs) from whole peripheral blood. These cells are endothelial in nature, as defined by their expression of endothelial markers and their ability to undergo capillary morphogenesis in three-dimensional culture. A subset of isolated colonies express markers of lymphatic endothelium, including VEGFR-3 and Prox-1, with low levels of VEGFR-1, a blood endothelial marker, while the bulk of the isolated cells express high VEGFR-1 levels with low VEGFR-3 and Prox-1 expression. The different isolates have differential responses to VEGF-C, a lymphatic endothelial specific cytokine, strongly suggesting that there are lymphatic specific and blood specific ECFCs. Global analysis of gene expression revealed key differences in the regulation of pathways involved in cellular differentiation between blood and lymphatic-specific ECFCs. Conclusion These data indicate that there are two distinguishable circulating ECFC types, blood and lymphatic, which are likely to have discrete functions during neovascularization. PMID:26597759

  12. Enhancement of ’In Vitro’ Granulocyte-Macrophage Colony Formation by Normal Human Serum or Plasma

    DTIC Science & Technology

    1976-08-01

    the CSA dose - response curve . . .. 10 LIST OF TABLES Table 1. Enhancement of Human and Murine In Vitro Bone Marrow Colony Formation by Normal Hluman...presence of NUS. *-, Dose - response curve for murine bone marrow colony formation using PMUE as a source of CSA. --- Dose - response curve for human...bone marrow colony for- mation using human peripheral blood leukocytes as a source of CSA. o---o Partial dose - response curve for human bone mar- row

  13. Defective transient endogenous spleen colony formation in S1/S1d mice.

    PubMed

    Wiktor-Jedrzejczak, W; Ahmed, A; Sharkis, S J; McKee, A; Sell, K W

    1979-04-01

    WCB6F1 mice of the genotype S1/S1d did not form transient 5-day endogenous spleen colonies following midlethal irradiation, either spontaneously or in response to postirradiation bleeding. Their hematologically normal (+/+) littermates produced colonies equivalent in number and morphologic type to a normal strain (D2B6F1), as evaluated by both macroscopic and microscopic criteria. Bone marrow cells from S1/S1d mice, when transplanted into lethally irradiated +/+ mice, were able to generate equivalent numbers of transient endogenous spleen colonies (TE-CFUs), as compared to that obtained when syngeneic +/+ marrow cells were injected into lethally irradiated +/+ recipients. A defective growth of an early class of hematopoietic progenitor cells, resulting in the clinical course of the S1/S1d anemia is suggested and confirms previous reports on the microenvironmental nature of this abnormality.

  14. Agent Based Modelling Helps in Understanding the Rules by Which Fibroblasts Support Keratinocyte Colony Formation

    PubMed Central

    Sun, Tao; McMinn, Phil; Holcombe, Mike; Smallwood, Rod; MacNeil, Sheila

    2008-01-01

    Background Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine

  15. Hepcidin inhibits in vitro erythroid colony formation at reduced erythropoietin concentrations

    PubMed Central

    Dallalio, Gail; Law, Erin; Means, Robert T.

    2006-01-01

    The anemia of chronic disease (ACD) results from 3 major processes: slightly shortened red cell survival, impaired reticuloendothelial system iron mobilization, and impaired erythropoiesis. Hepcidin is an acute-phase protein with specific iron regulatory properties, which, along with the anemia seen with increased hepcidin expression, have led many to consider it the major mediator of ACD. However, if hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired erythropoiesis observed in this syndrome. Erythroid colony formation in vitro was inhibited by hepcidin at erythropoietin (Epo) concentrations less than or equal to 0.5 U/mL but not at Epo 1.0 U/mL. At Epo concentrations of 0.3 U/mL, HCD57 erythroleukemia cells exposed to hepcidin exhibit decreased expression of the antiapoptotic protein pBad compared with controls. These studies suggest that hepcidin may contribute to anemia in ACD not only through effects on iron metabolism, but also through inhibition of erythroid progenitor proliferation and survival. PMID:16332970

  16. The isolation and culture of endothelial colony-forming cells from human and rat lungs.

    PubMed

    Alphonse, Rajesh S; Vadivel, Arul; Zhong, Shumei; Zong, Shumei; McConaghy, Suzanne; Ohls, Robin; Yoder, Mervin C; Thébaud, Bernard

    2015-11-01

    Blood vessels are crucial for the normal development, lifelong repair and homeostasis of tissues. Recently, vascular progenitor cell-driven 'postnatal vasculogenesis' has been suggested as an important mechanism that contributes to new blood vessel formation and organ repair. Among several described progenitor cell types that contribute to blood vessel formation, endothelial colony-forming cells (ECFCs) have received widespread attention as lineage-specific 'true' vascular progenitors. Here we describe a protocol for the isolation of pulmonary microvascular ECFCs from human and rat lung tissue. Our technique takes advantage of an earlier protocol for the isolation of circulating ECFCs from the mononuclear cellular fraction of peripheral blood. We adapted the earlier protocol to isolate resident ECFCs from the distal lung tissue. After enzymatic dispersion of rat or human lung samples into a cellular suspension, CD31-expressing cells are positively selected using magnetic-activated cell sorting and plated in endothelial-specific growth conditions. The colonies arising after 1-2 weeks in culture are carefully separated and expanded to yield pure ECFC cultures after a further 2-3 weeks. The resulting cells demonstrate the defining characteristics of ECFCs such as (i) 'cobblestone' morphology of cultured cell monolayers; (ii) acetylated low-density lipoprotein uptake and Ulex europaeus lectin binding; (iii) tube-like network formation in Matrigel; (iv) expression of endothelial cell-specific surface markers and the absence of hematopoietic or myeloid surface antigens; (v) self-renewal potential displayed by the most proliferative cells; and (vi) contribution to de novo vessel formation in an in vivo mouse implant model. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 4 weeks. Isolation and culture of lung vascular ECFCs will allow assessment of the functional state of these cells in experimental and human

  17. Pure and mixed erythroid colony formation in vitro stimulated by spleen conditioned medium with no detectable erythropoietin.

    PubMed Central

    Johnson, G R; Metcalf, D

    1977-01-01

    Cells from CBA fetal mouse liver formed pure or mixed erythroid colonies in semisolid agarculture after stimulation by medium conditioned by pokeweed mitogen-stimulated mouse spleen cells. In general shape, the erythroid colonies resembled typical 7-day single or multiple (burst) colonies. However one-third to one-half contained, in addition to erythroid cells, macrophages and neutrophils and, less commonly, megakaryocytes or eosinophils. Culture of micro manipulated single colony-forming cells showed these erythroid colonies to be clones. Colony-forming cells declined in frequency with advancing fetal age, but low numbers were detectable in adult bone marrow. Assays of spleen conditioned medium in polycythemic mice failed to detect erythropoietin; the cloning system may detect a fetal type of erythropoietin-independent, erythropoietic cell since few were detected in adult marrow. Images PMID:269439

  18. High nutrient concentration and temperature alleviated formation of large colonies of Microcystis: Evidence from field investigations and laboratory experiments.

    PubMed

    Zhu, Wei; Zhou, Xiaohua; Chen, Huaimin; Gao, Li; Xiao, Man; Li, Ming

    2016-09-15

    Correlations between Microcystis colony size and environmental factors were investigated in Meiliang Bay and Gonghu Bay of Lake Taihu (China) from 2011 to 2013. Compared with Gonghu Bay, both nutrient concentrations and Microcystis colony sizes were greater in Meiliang Bay. The median colony size (D50: 50% of the total mass of particles smaller than this size) increased from April to August and then decreased until November. In both bays, the average D50 of Microcystis colonies were <100 μm in spring, but colonies within moderate-size (100-500 μm) dominated in summer. The differences in colony size in Meiliang Bay and Gonghu Bay were probably due to horizontal drift driven by the prevailing south wind in summer. Redundancy analysis (RDA) of field data indicated that colony size was negatively related to nutrient concentrations but positively related to air temperature, suggesting that low nutrient concentrations and high air temperature promoted formation of large colonies. To validate the field survey, Microcystis colonies collected from Lake Taihu were cultured at different temperatures (15, 20, 25 and 30 °C) under high and low nutrient concentrations for 9 days. The size of Microcystis colonies significantly decreased when temperature was above 20 °C but had no significant change at 15 °C. The differences in temperature effects on colony formation shown from field and laboratory suggested that the larger colonies in summer were probably due to the longer growth period rather than the higher air temperature and light intensity. In addition, colony size decreased more significantly at high nutrient levels. Therefore, it could be concluded that high nutrient concentration and temperature may alleviate formation of large colonies of Microcystis.

  19. The role of gravity in the nutrition and formation of Bacillus colonies

    NASA Astrophysics Data System (ADS)

    Puzyr, A.; Tirranen, L.; Krylova, T.

    The soil-like substrate is used to cultivate higher plants in man-made closed ecosystems. It allows increasing the closeness of the systems and decreasing the plant solid residues and human wastes. Unusual funnel-shaped bacterial colonies of Bacillus species have been observed during analysis of microflora of plant nutritional solution. The colonies have the following characteristics: a) the diameter of "funnel socket" (the biomass contacting with nutritional agar) is 10.0-15.0 mm; b) the thickness of "funnel socket" is 0.5-2.5 mm; c) the diameter of the middle part of the "funnel spout" (the biomass contacting with the gas phase) is 1,0-1,5 mm; d) the length of the "funnel spout" is 10.0-15.0 mm. In the socket and the middle part of the "funnel spout" there is a gas cavity which is most probably formed by bacterial gas metabolites. It has been shown that: i) the surface of these funnel-shaped colonies of Bacillus species is hydrophobic, as is the surface of other Bacillus species ( . brevis, B. cellulomonos, B. flavus, B.B formosus, B. subtilis); ii) the forms of colonies can be changed by varying the position of the growing biomass in relation to the gravitation forces. The experiment proved that the form of the "funnel sockets" and the length of the "funnel spouts" of the colonies are determined by hydrophobic air-contacting surface layer, which does not leak and stretches under the weight of accumulated water. A hypothesis has been suggested that the gravity force plays the role of a "pump" supplying and holding water within the colony. Thus, the water that comes under the gravity force contains dissolved nutrients and bacterial cells in the hydrophobic layer. These cells that are situated far away from the nutrient agar have no nutrient deficiency. The water accumulated by the colonies might be free water of agar media or it can be produced by metabolic disruption of medium fat. Hence, when growing a colony in agar media the water-soluble nutrient substances

  20. Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells

    PubMed Central

    Lianou, Alexandra

    2013-01-01

    Conventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells of Salmonella enterica serotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μmax) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (N0). For bacterial populations with N0 of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations. PMID:23354712

  1. Development of serum-free quality and quantity control culture of colony-forming endothelial progenitor cell for vasculogenesis.

    PubMed

    Masuda, Haruchika; Iwasaki, Hiroto; Kawamoto, Atsuhiko; Akimaru, Hiroshi; Ishikawa, Masakazu; Ii, Masaaki; Shizuno, Tomoko; Sato, Atsuko; Ito, Rie; Horii, Miki; Ishida, Hideyuki; Kato, Shunichi; Asahara, Takayuki

    2012-02-01

    Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum-free quality and quantity control culture system for colony-forming EPCs to enhance their regenerative potential. A culture with serum-free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin-6, and Flt-3 ligand was determined as optimal quality and quantity culture (QQc) in terms of the most vasculogenic colony-forming EPC expansion, evaluated by the newly established EPC colony formation assay. The QQc of umbilical cord blood-CD133(+) cells for 7 days produced a 52.9-fold increase in total cell number and 3.28-fold frequency in definitive EPC colony development, resulting in a 203.9-fold increase in estimated total definitive EPC colony number in vitro. Pre- or post-QQc cells were intramyocardially transplanted into nude rats with myocardial infarction (MI). Echocardiographic and micromanometer-tipped conductance catheter examinations 28 days post-MI revealed significant preservation of left ventricular (LV) function in rats receiving pre- or post-QQc cells compared with those receiving phosphate-buffered saline. Assessments of global LV contractility indicated a dose-dependent effect of pre- or post-QQc cells and the superior potency of post-QQc cells over pre-QQc cells. Furthermore, immunohistochemistry showed more abundant formation of both human and rat endothelial cells and cardiomyocytes in the infarcted myocardium following transplantation of post-QQc cells compared with pre-QQc cells. Our optimal serum-free quality and quantity culture may enhance the therapeutic potential of EPCs in both quantitative and qualitative aspects for cardiovascular regeneration.

  2. Development of Serum-Free Quality and Quantity Control Culture of Colony-Forming Endothelial Progenitor Cell for Vasculogenesis

    PubMed Central

    Masuda, Haruchika; Iwasaki, Hiroto; Kawamoto, Atsuhiko; Akimaru, Hiroshi; Ishikawa, Masakazu; Ii, Masaaki; Shizuno, Tomoko; Sato, Atsuko; Ito, Rie; Horii, Miki; Ishida, Hideyuki; Kato, Shunichi

    2012-01-01

    Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum-free quality and quantity control culture system for colony-forming EPCs to enhance their regenerative potential. A culture with serum-free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin-6, and Flt-3 ligand was determined as optimal quality and quantity culture (QQc) in terms of the most vasculogenic colony-forming EPC expansion, evaluated by the newly established EPC colony formation assay. The QQc of umbilical cord blood-CD133+ cells for 7 days produced a 52.9-fold increase in total cell number and 3.28-fold frequency in definitive EPC colony development, resulting in a 203.9-fold increase in estimated total definitive EPC colony number in vitro. Pre- or post-QQc cells were intramyocardially transplanted into nude rats with myocardial infarction (MI). Echocardiographic and micromanometer-tipped conductance catheter examinations 28 days post-MI revealed significant preservation of left ventricular (LV) function in rats receiving pre- or post-QQc cells compared with those receiving phosphate-buffered saline. Assessments of global LV contractility indicated a dose-dependent effect of pre- or post-QQc cells and the superior potency of post-QQc cells over pre-QQc cells. Furthermore, immunohistochemistry showed more abundant formation of both human and rat endothelial cells and cardiomyocytes in the infarcted myocardium following transplantation of post-QQc cells compared with pre-QQc cells. Our optimal serum-free quality and quantity culture may enhance the therapeutic potential of EPCs in both quantitative and qualitative aspects for cardiovascular regeneration. PMID:23197763

  3. [Significance of the colonial components for the medusa formation and differentiation inPodocoryne carnea M. Sars].

    PubMed

    Brändle, Elisabeth

    1971-09-01

    1. All axial regions of the gonozoid and the medusa buds ofPodocaryne carnea M. Sars incorporate(3)H-thymidine. Medusa buds grow by mitosis and by migration of cells from the colony into the buds. 2. Stolonization inhibits the formation and differentiation of medusa buds: non stolonizing chimerae formed by a gonozoid and an autozoid produce more buds than stolonizing ones. 3. When isolated gonozoids and likewise isolated budding regions are not connected with an nutritive autozoid, the formation and differentiation of medusa buds are restricted. Young medusa buds (stage 3, Frey, 1968) transplanted onto autozoids may differentiate into medusae, while isolated buds of the same stage are transformed into stolons. 4. If the hypostome of the gonozoid is separated from the subhypostomal budding region by ligature and does not regenerate, the young buds already present are resorbed and no new ones are formed. 5. Heads of gonozoids transplanted onto cauli of adult autozoids may induce the formation of medusa buds in the subtentacular axial region. These buds differentiate into normal medusae. 6. Isolated adult autozoids, treated with extract taken from hypostomes of gonozoids, form medusa buds which complete normal differentiation. 7. Treatment of autozoid colonies with extract from gonozoids brings about a standstill of colony growth and resorption of autozoids. After transfer to normal sea-water a compensatory increase in growth and head formation takes place. 8. The results are discussed. It is suggested that an "activator" substance is produced in the hypostome of the gonozoid which induces and maintains the budding region in the subtentacular zone. Furthermore, budding would be dependent on the nutritional state of the gonozoid, food being supplied by the autozoid, and on the extent of inhibition by intensive stolonization.

  4. Are self-ligating brackets related to less formation of Streptococcus mutans colonies? A systematic review.

    PubMed

    do Nascimento, Leonard Euler Andrade Gomes; de Souza, Margareth Maria Gomes; Azevedo, Angela Rita Pontes; Maia, Lucianne Cople

    2014-01-01

    To verify, by means of a systematic review, whether the design of brackets (conventional or self-ligating) influences adhesion and formation of Streptococcus mutans colonies. four databases (Cochrane Central Register of Controlled Trials, Ovid ALL EMB Reviews, PubMed and BIREME) were selected to search for relevant articles covering the period from January 1965 to December 2012. in first consensus by reading the title and abstract. The full text was obtained from publications that met the inclusion criteria. Two reviewers independently extracted data using the following keywords: conventional, self-ligating, biofilm, Streptococcus mutans, and systematic review; and independently evaluated the quality of the studies. In case of divergence, the technique of consensus was adopted. The search strategy resulted in 1,401 articles. The classification of scientific relevance revealed the high quality of the 6 eligible articles of which outcomes were not unanimous in reporting not only the influence of the design of the brackets (conventional or self-ligating) over adhesion and formation of colonies of Streptococcus mutans, but also that other factors such as the quality of the bracket type, the level of individual oral hygiene, bonding and age may have greater influence. Statistical analysis was not feasible because of the heterogeneous methodological design. Within the limitations of this study, it was concluded that there is no evidence for a possible influence of the design of the brackets (conventional or self-ligating) over colony formation and adhesion of Streptococcus mutans.

  5. A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability.

    PubMed

    Welch, Aaron Z; Koshland, Douglas E

    2013-12-01

    Here we describe the first high-throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high-throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10(8) dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high-throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities < 1%. Thus, we show that tadpoling provides an easy, inexpensive, space-saving method, amenable to high-throughput screens, for accurately measuring yeast cell viability.

  6. Umbilical Cord Blood Circulating Progenitor Cells and Endothelial Colony-Forming Cells Are Decreased in Preeclampsia.

    PubMed

    Gumina, Diane L; Black, Claudine P; Balasubramaniam, Vivek; Winn, Virginia D; Baker, Christopher D

    2016-01-01

    Preeclampsia (PE) is a pregnancy-specific disease characterized by the new onset of hypertension and proteinuria. Mothers with PE are known to develop endothelial dysfunction, but its effect on infants has been understudied, as newborns are often asymptomatic. Recent studies indicate that infants born from preeclamptic pregnancies develop endothelial dysfunction including higher blood pressure during childhood and an increased risk of stroke later in life. We hypothesize that PE reduces the number and function of fetal angiogenic progenitor cells and may contribute to this increased risk. We quantified 2 distinct types of angiogenic progenitors, pro-angiogenic circulating progenitor cells (CPCs) and endothelial colony-forming cells (ECFCs), from the umbilical cord blood of preeclamptic pregnancies and normotensive controls. Pro-angiogenic and nonangiogenic CPCs were enumerated via flow cytometry and ECFCs by cell culture. Additionally, we studied the growth, migration, and tube formation of ECFCs from PE and gestational age-matched normotensive control pregnancies. We found that PE resulted in decreased cord blood pro-angiogenic CPCs and ECFCs. Nonangiogenic CPCs were also decreased. Preeclamptic ECFCs demonstrated decreased growth and migration but formed tube-like structures in vitro similar to controls. Our results suggest that the preeclamptic environment alters the number and function of angiogenic progenitor cells and may increase the risk of later vascular disease.

  7. Modulation of colony stimulating factor release and apoptosis in human colon cancer cells by anticancer drugs

    PubMed Central

    Calatayud, S; Warner, T D; Mitchell, J A

    2002-01-01

    Modulation of the immune response against tumour cells is emerging as a valuable approach for cancer treatment. Some experimental studies have shown that secretion of colony stimulating factors by cancer cells reduces their tumorigenicity and increases their immunogenicity probably by promoting the cytolitic and antigen presenting activities of leukocytes. We have observed that human colon cancer cells (HT-29) are able to secrete granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor when stimulated with cytokines (IL-1β and TNF-α). In this study we assessed, for the first time, the effects of several anticancer drugs on colony stimulating factor release or apoptosis in HT-29 cells. Cytokine-induced release of granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor was significantly increased by cisplatin and 6-mercaptopurine. Taxol only increased macrophage-colony stimulating factor release while reduced that of granulocyte-colony stimulating factor. No changes in colony stimulating factor secretion were observed after treatment with methotrexate. Only cisplatin and taxol induced apoptosis in these cells. Secretion of colony stimulating factors by colon cancer cells may contribute to the immune host response against them. Anticancer drugs such as cisplatin and 6-mercaptopurine increase colony stimulating factor secretion by cytokine stimulated cancer cells probably through mechanisms different to those leading to cell apoptosis, an effect that may contribute to their anti-neoplasic action. British Journal of Cancer (2002) 86, 1316–1321. DOI: 10.1038/sj/bjc/6600240 www.bjcancer.com © 2002 Cancer Research UK PMID:11953891

  8. Isolation and characterization of a plasmid DNA from periodontopathogenic bacterium, Eikenella corrodens 1073, which affects pilus formation and colony morphology.

    PubMed

    Azakami, Hiroyuki; Akimichi, Hiromi; Usui, Masakatsu; Yumoto, Hiromichi; Ebisu, Shigeyuki; Kato, Akio

    2005-05-23

    Eikenella corrodens (Ec) is one of a group of periodontopathogenic bacteria. A plasmid DNA (8.7 kb) isolated from Ec 1073 was designated pMU1. Agarose gel electrophoresis and Southern analysis suggested that pMU1-like plasmids were carried in 2 Ec strains, including 1073, with higher hemagglutination (HA) activity than other strains. We determined the nucleotide sequence of this plasmid and identified 7 ORFs. A homology search revealed that 4 ORFs of pMU1 were homologous to ORFs in pJTPS1, found in a spontaneous avirulent mutant of the phytopathogenic bacterium, Ralstonia solanacearum. pJTPS1 is a putative hypovirulent plasmid, which is thought to control the virulence of R. solanacearum. We also found the ORF to be homologous to the recombinase specific to the type IV pilin gene. We introduced a part of pMU1 into the Ec 23834 strain, which has a pilus structure on its cell surface and forms corroding colonies on solid medium. No pilus structure was observed on the surface of transformants, most of which formed non-corroding colonies. When such transformants (or Ec 1073) were cured of pMU1 with acridine orange, they remained non-foliated and non-corroding. The results suggest that pMU1 might irreversibly affect pilus formation and colony morphology, and might be involved in the pathogenicity and virulence of Ec.

  9. Generation of Colonies of Induced Trophoblast Cells During Standard Reprogramming of Porcine Fibroblasts to Induced Pluripotent Stem Cells1

    PubMed Central

    Ezashi, Toshihiko; Matsuyama, Haruyo; Telugu, Bhanu Prakash V.L.; Roberts, R. Michael

    2011-01-01

    During reprogramming of porcine mesenchymal cells with a four-factor (POU5F1/SOX2/KLF4/MYC) mixture of vectors, a fraction of the colonies had an atypical phenotype and arose earlier than the recognizable porcine induced pluripotent stem (iPS) cell colonies. Within days after each passage, patches of cells with an epithelial phenotype formed raised domes, particularly under 20% O2 conditions. Relative to gene expression of the iPS cells, there was up-regulation of genes for transcription factors associated with trophoblast (TR) lineage emergence, e.g., GATA2, PPARG, MSX2, DLX3, HAND1, GCM1, CDX2, ID2, ELF5, TCFAP2C, and TEAD4 and for genes required for synthesis of products more typical of differentiated TR, such as steroids (HSD17B1, CYP11A1, and STAR), pregnancy-associated glycoproteins (PAG6), and select cytokines (IFND, IFNG, and IL1B). Although POU5F1 was down-regulated relative to that in iPS cells, it was not silenced in the induced TR (iTR) cells over continued passage. Like iPS cells, iTR cells did not senesce on extended passage and displayed high telomerase activity. Upon xenografting into immunodeficient mice, iTR cells formed nonhemorrhagic teratomas composed largely of layers of epithelium expressing TR markers. When cultured under conditions that promoted embryoid body formation, iTR cells formed floating spheres consisting of a single epithelial sheet whose cells were tethered laterally by desmosome-like structures. In conclusion, reprogramming of porcine fibroblasts to iPS cells generates, as a by-product, colonies composed of self-renewing populations of TR cells, possibly containing TR stem cells. PMID:21734265

  10. Generation of colonies of induced trophoblast cells during standard reprogramming of porcine fibroblasts to induced pluripotent stem cells.

    PubMed

    Ezashi, Toshihiko; Matsuyama, Haruyo; Telugu, Bhanu Prakash V L; Roberts, R Michael

    2011-10-01

    During reprogramming of porcine mesenchymal cells with a four-factor (POU5F1/SOX2/KLF4/MYC) mixture of vectors, a fraction of the colonies had an atypical phenotype and arose earlier than the recognizable porcine induced pluripotent stem (iPS) cell colonies. Within days after each passage, patches of cells with an epithelial phenotype formed raised domes, particularly under 20% O(2) conditions. Relative to gene expression of the iPS cells, there was up-regulation of genes for transcription factors associated with trophoblast (TR) lineage emergence, e.g., GATA2, PPARG, MSX2, DLX3, HAND1, GCM1, CDX2, ID2, ELF5, TCFAP2C, and TEAD4 and for genes required for synthesis of products more typical of differentiated TR, such as steroids (HSD17B1, CYP11A1, and STAR), pregnancy-associated glycoproteins (PAG6), and select cytokines (IFND, IFNG, and IL1B). Although POU5F1 was down-regulated relative to that in iPS cells, it was not silenced in the induced TR (iTR) cells over continued passage. Like iPS cells, iTR cells did not senesce on extended passage and displayed high telomerase activity. Upon xenografting into immunodeficient mice, iTR cells formed nonhemorrhagic teratomas composed largely of layers of epithelium expressing TR markers. When cultured under conditions that promoted embryoid body formation, iTR cells formed floating spheres consisting of a single epithelial sheet whose cells were tethered laterally by desmosome-like structures. In conclusion, reprogramming of porcine fibroblasts to iPS cells generates, as a by-product, colonies composed of self-renewing populations of TR cells, possibly containing TR stem cells.

  11. Vibrio cholerae O1 Strain TSI-4 Produces the Exopolysaccharide Materials That Determine Colony Morphology, Stress Resistance, and Biofilm Formation

    PubMed Central

    Wai, Sun Nyunt; Mizunoe, Yoshimitsu; Takade, Akemi; Kawabata, Shun-Ichiro; Yoshida, Shin-Ichi

    1998-01-01

    Vibrio cholerae O1 strain TSI-4 (El Tor, Ogawa) can shift to a rugose colony morphology from its normal translucent colony morphology in response to nutrient starvation. We have investigated differences between the rugose and translucent forms of V. cholerae O1 strain TSI-4. Electron microscopic examination of the rugose form of TSI-4 (TSI-4/R) revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the ferritin-stained material was absent around the translucent form of TSI-4 (TSI-4/T). The exopolysaccharide produced by V. cholerae TSI-4/R was found to have a composition of N-acetyl-d-glucosamine, d-mannose, 6-deoxy-d-galactose, and d-galactose (7.4:10.2:2.4:3.0). The expression of an amorphous exopolysaccharide promotes biofilm development under static culture conditions. Biofilm formation by the rugose strain was determined by scanning electron microscopy, and most of the surface of the film was colonized by actively dividing rod cells. The corresponding rugose and translucent strains were compared for stress resistance. By having exopolysaccharide materials, the rugose strains acquired resistance to osmotic and oxidative stress. Our data indicated that an exopolysaccharide material on the surface of the rugose strain promoted biofilm formation and resistance to the effects of two stressing agents. PMID:9758780

  12. Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation.

    PubMed

    Wai, S N; Mizunoe, Y; Takade, A; Kawabata, S I; Yoshida, S I

    1998-10-01

    Vibrio cholerae O1 strain TSI-4 (El Tor, Ogawa) can shift to a rugose colony morphology from its normal translucent colony morphology in response to nutrient starvation. We have investigated differences between the rugose and translucent forms of V. cholerae O1 strain TSI-4. Electron microscopic examination of the rugose form of TSI-4 (TSI-4/R) revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the ferritin-stained material was absent around the translucent form of TSI-4 (TSI-4/T). The exopolysaccharide produced by V. cholerae TSI-4/R was found to have a composition of N-acetyl-D-glucosamine, D-mannose, 6-deoxy-D-galactose, and D-galactose (7.4:10.2:2.4:3.0). The expression of an amorphous exopolysaccharide promotes biofilm development under static culture conditions. Biofilm formation by the rugose strain was determined by scanning electron microscopy, and most of the surface of the film was colonized by actively dividing rod cells. The corresponding rugose and translucent strains were compared for stress resistance. By having exopolysaccharide materials, the rugose strains acquired resistance to osmotic and oxidative stress. Our data indicated that an exopolysaccharide material on the surface of the rugose strain promoted biofilm formation and resistance to the effects of two stressing agents.

  13. Pattern formation in a growing bacterial colony facilitated by extra-cellular polymeric substances

    NASA Astrophysics Data System (ADS)

    Ghosh, Pushpita; Mondal, Jagannath; Ben-Jacob, Eshel; Levine, Herbert

    2015-03-01

    Self-organization in bacterial colony is quite pervasive and diverse phenomena. Bacteria are known to self-organize into multicellular communities, commonly known as biofilms, in which microbial cells live in close association with a solid surface and are embedded in a self-produced extracellular polymeric substances(EPS). In such dense systems mechanical interactions among the structural components can be expected to significantly contribute to the morphological properties. By a simple particle-based simulation model of nonmotile rod-shaped bacterial cells and EPS secreted in a growing colony, we investigate how the combined mechanical effects can give rise naturally spatial heterogeneity observed in a biofilm. In our individual-based simulation model all the components interact mechanically via repulsive forces by pushing each other away as bacterial cells grow and divide consuming diffusing nutrient and produce EPS. We show that mechanical interactions control the collective behavior of the system, particularly, we show that the presence of non-adsorbing EPS leads spontaneous aggregation of bacterial cells by depletion attraction and generates phase separated patterns in a nonequilibrium growing colony.

  14. Characterization of endothelial colony-forming cells from peripheral blood samples of adult horses.

    PubMed

    Salter, Margaret M; Seeto, Wen J; DeWitt, Blake B; Hashimi, Sarah A; Schwartz, Dean D; Lipke, Elizabeth A; Wooldridge, Anne A

    2015-02-01

    To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses. Jugular venous blood samples from 24 adult horses. Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates. Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry. ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.

  15. Involvement of allelopathy in the formation of monospecific colonies of ferns.

    PubMed

    Kato-Noguchi, Hisashi

    2015-05-01

    Some fern species often dominate plant communities by forming large monospecific colonies. However, the potential mechanism for this domination of the ferns remains obscure. Many plants secrete a wide range of compounds into the rhizosphere and change the chemical and physical properties of the rhizosphere soil. Through the secretion of compounds, such as allelopathic substances, plants inhibit the germination and growth of neighboring plants to compete more effectively for the resources. Ferns contain a variety of secondary metabolites and some of those compounds are released from the ferns into the rhizosphere soil, either as exudates from living ferns or by decomposition of fern residues in sufficient quantities to affect the germination and growth of neighboring plants as allelopathic substances. Therefore, allelopathic chemical interaction of the ferns with neighboring plants may play an important role in the formation of the monospecific colonies of the ferns.

  16. Automated cell colony counting and analysis using the circular Hough image transform algorithm (CHiTA)

    NASA Astrophysics Data System (ADS)

    Bewes, J. M.; Suchowerska, N.; McKenzie, D. R.

    2008-11-01

    We present an automated cell colony counting method that is flexible, robust and capable of providing more in-depth clonogenic analysis than existing manual and automated approaches. The full form of the Hough transform without approximation has been implemented, for the first time. Improvements in computing speed have facilitated this approach. Colony identification was achieved by pre-processing the raw images of the colonies in situ in the flask, including images of the flask edges, by erosion, dilation and Gaussian smoothing processes. Colony edges were then identified by intensity gradient field discrimination. Our technique eliminates the need for specialized hardware for image capture and enables the use of a standard desktop scanner for distortion-free image acquisition. Additional parameters evaluated included regional colony counts, average colony area, nearest neighbour distances and radial distribution. This spatial and qualitative information extends the utility of the clonogenic assay, allowing analysis of spatially-variant cytotoxic effects. To test the automated system, two flask types and three cell lines with different morphology, cell size and plating density were examined. A novel Monte Carlo method of simulating cell colony images, as well as manual counting, were used to quantify algorithm accuracy. The method was able to identify colonies with unusual morphology, to successfully resolve merged colonies and to correctly count colonies adjacent to flask edges.

  17. Characterization of stroma-dependent blast colony-forming cells in human marrow

    SciTech Connect

    Gordon, M.Y.; Dowding, C.R.; Riley, G.P.; Greaves, M.F.

    1987-01-01

    Human bone marrow contains a population of haemopoietic progenitor cells that can be distinguished by their ability to adhere to preformed stromal layers (cultured in the presence of methylprednisolone (MP/sup +/) and form blast cell colonies. The stromal layers function in the colony assay after they have been heavily irradiated but not after they have been passaged. The binding of the progenitor cells to the stromal cells is complete after 2 hours of coincubation, and stromal layers of 9.6 cm/sup 2/ can provide adhesion sites for at least 2000 blast colony-forming cells. The blast colony-forming cells were shown by micromanipulation to self-renew as well as the give rise to multipotential and lineage-committed colony-forming progenitor cells.

  18. Colony-forming progenitor cells in the postnatal mouse liver and pancreas give rise to morphologically distinct insulin-expressing colonies in 3D cultures.

    PubMed

    Jin, Liang; Feng, Tao; Chai, Jing; Ghazalli, Nadiah; Gao, Dan; Zerda, Ricardo; Li, Zhuo; Hsu, Jasper; Mahdavi, Alborz; Tirrell, David A; Riggs, Arthur D; Ku, Hsun Teresa

    2014-01-01

    In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed "Dark" colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency (~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133⁺CD49f(low)CD107b(low) phenotype, while pancreatic CFU-Dark are CD133⁻. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth.

  19. Scaling of Traction Forces with the Size of Cohesive Cell Colonies

    PubMed Central

    Mertz, Aaron F.; Banerjee, Shiladitya; Che, Yonglu; German, Guy K.; Xu, Ye; Hyland, Callen; Marchetti, M. Cristina; Horsley, Valerie; Dufresne, Eric R.

    2014-01-01

    To understand how the mechanical properties of tissues emerge from interactions of multiple cells, we measure traction stresses of cohesive colonies of 1–27 cells adherent to soft substrates. We find that traction stresses are generally localized at the periphery of the colony and the total traction force scales with the colony radius. For large colony sizes, the scaling appears to approach linear, suggesting the emergence of an apparent surface tension of the order of 10−3 N/m. A simple model of the cell colony as a contractile elastic medium coupled to the substrate captures the spatial distribution of traction forces and the scaling of traction forces with the colony size. PMID:23003091

  20. Phase field simulations of autocatalytic formation of alpha lamellar colonies in Ti-6Al-4V

    DOE PAGES

    Radhakrishnan, Bala; Gorti, Sarma; Babu, Suresh Sudharsanam

    2016-09-13

    Here, we present phase field simulations incorporating energy contributions due to thermodynamics, and anisotropic interfacial and strain energies, to demonstrate the nucleation and growth of multiple variants of alpha from beta in Ti-6Al-4V under isothermal conditions. The simulations focused on the effect of thermodynamic driving force and nucleation rate on the morphology of the transformed alpha assuming that the partitioning of V between beta and alpha is negligible for short isothermal holds. The results indicate that a high nucleation rate favors the formation of the basket-weave structure. However, at a lower nucleation rate the simulations show the intragranular nucleation ofmore » a colony structure by an autocatalytic nucleation mechanism adjacent to a pre-existing alpha variant. New side-plates of the same variant appear to nucleate progressively and grow to form the colony. The isothermal simulation results are used to offer a possible explanation for the transition from a largely basket weave structure to a colony structure inside narrow layer bands occurring during continuous heating and cooling conditions encountered during laser additive manufacturing of Ti-6Al-4V.« less

  1. Phase field simulations of autocatalytic formation of alpha lamellar colonies in Ti-6Al-4V

    SciTech Connect

    Radhakrishnan, Bala; Gorti, Sarma; Babu, Suresh Sudharsanam

    2016-09-13

    Here, we present phase field simulations incorporating energy contributions due to thermodynamics, and anisotropic interfacial and strain energies, to demonstrate the nucleation and growth of multiple variants of alpha from beta in Ti-6Al-4V under isothermal conditions. The simulations focused on the effect of thermodynamic driving force and nucleation rate on the morphology of the transformed alpha assuming that the partitioning of V between beta and alpha is negligible for short isothermal holds. The results indicate that a high nucleation rate favors the formation of the basket-weave structure. However, at a lower nucleation rate the simulations show the intragranular nucleation of a colony structure by an autocatalytic nucleation mechanism adjacent to a pre-existing alpha variant. New side-plates of the same variant appear to nucleate progressively and grow to form the colony. The isothermal simulation results are used to offer a possible explanation for the transition from a largely basket weave structure to a colony structure inside narrow layer bands occurring during continuous heating and cooling conditions encountered during laser additive manufacturing of Ti-6Al-4V.

  2. Phase field simulations of autocatalytic formation of alpha lamellar colonies in Ti-6Al-4V

    SciTech Connect

    Radhakrishnan, Bala; Gorti, Sarma; Babu, Suresh Sudharsanam

    2016-09-13

    Here, we present phase field simulations incorporating energy contributions due to thermodynamics, and anisotropic interfacial and strain energies, to demonstrate the nucleation and growth of multiple variants of alpha from beta in Ti-6Al-4V under isothermal conditions. The simulations focused on the effect of thermodynamic driving force and nucleation rate on the morphology of the transformed alpha assuming that the partitioning of V between beta and alpha is negligible for short isothermal holds. The results indicate that a high nucleation rate favors the formation of the basket-weave structure. However, at a lower nucleation rate the simulations show the intragranular nucleation of a colony structure by an autocatalytic nucleation mechanism adjacent to a pre-existing alpha variant. New side-plates of the same variant appear to nucleate progressively and grow to form the colony. The isothermal simulation results are used to offer a possible explanation for the transition from a largely basket weave structure to a colony structure inside narrow layer bands occurring during continuous heating and cooling conditions encountered during laser additive manufacturing of Ti-6Al-4V.

  3. Are self-ligating brackets related to less formation of Streptococcus mutans colonies? A systematic review

    PubMed Central

    do Nascimento, Leonard Euler Andrade Gomes; de Souza, Margareth Maria Gomes; Azevedo, Angela Rita Pontes; Maia, Lucianne Cople

    2014-01-01

    Objective To verify, by means of a systematic review, whether the design of brackets (conventional or self-ligating) influences adhesion and formation of Streptococcus mutans colonies. Methods Search strategy: four databases (Cochrane Central Register of Controlled Trials, Ovid ALL EMB Reviews, PubMed and BIREME) were selected to search relevant articles covering the period from January 1965 to December 2012. Selection Criteria: in first consensus by reading the title and abstract. The full text was obtained from publications that met the inclusion criteria. Data collection and analysis: Two reviewers independently extracted data using the keywords: conventional, self-ligating, biofilm, Streptococcus mutans, and systematic review; and independently evaluated the quality of the studies. In case of divergence, the technique of consensus was adopted. Results The search strategy resulted in 1,401 articles. The classification of scientific relevance revealed the high quality of the 6 eligible articles of which outcomes were not unanimous in reporting not only the influence of the design of the brackets (conventional or self-ligating) over adhesion and formation of colonies of Streptococcus mutans, but also that other factors such as the quality of the bracket type, the level of individual oral hygiene, bonding and age may have greater influence. Statistical analysis was not feasible because of the heterogeneous methodological design. Conclusions Within the limitations of this study, it was concluded that there is no evidence for a possible influence of the design of the brackets (conventional or self-ligating) over colony formation and adhesion of Streptococcus mutans. PMID:24713561

  4. Manufacture of Clinical-Grade Human Clonal Mesenchymal Stem Cell Products from Single Colony Forming Unit-Derived Colonies Based on the Subfractionation Culturing Method.

    PubMed

    Yi, TacGhee; Kim, Si-na; Lee, Hyun-Joo; Kim, Junghee; Cho, Yun-Kyoung; Shin, Dong-Hee; Tak, Sun-Ji; Moon, Sun-Hwa; Kang, Ji-Eun; Ji, In-Mi; Lim, Huyn-Ja; Lee, Dong-Soon; Jeon, Myung-Shin; Song, Sun U

    2015-12-01

    Stem cell products derived from mesenchymal stem cells (MSCs) have been widely used in clinical trials, and a few products have been already commercialized. However, the therapeutic effects of clinical-grade MSCs are still controversial owing to mixed results from recent clinical trials. A potential solution to overcome this hurdle may be to use clonal stem cells as the starting cell material to increase the homogeneity of the final stem cell products. We have previously developed an alternative isolation and culture protocol for establishing a population of clonal MSCs (cMSCs) from single colony forming unit (CFU)-derived colonies. In this study, we established a good manufacturing practice (GMP)-compatible procedure for the clinical-grade production of human bone marrow-derived cMSCs based on the subfractionation culturing method. We optimized the culture procedures to expand and obtain a clonal population of final MSC products from single CFU-derived colonies in a GMP facility. The characterization results of the final cMSC products met our preset criteria. Animal toxicity tests were performed in a good laboratory practice facility, and showed no toxicity or tumor formation in vivo. These tests include single injection toxicity, multiple injection toxicity, biodistribution analysis, and tumorigenicity tests in vivo. No chromosomal abnormalities were detected by in situ karyotyping using oligo-fluorescence in situ hydridization (oligo-FISH), providing evidence of genetic stability of the clinical-grade cMSC products. The manufacture and quality control results indicated that our GMP methodology could produce sufficient clonal population of MSC products from a small amount of bone marrow aspirate to treat a number of patients.

  5. Pure erythropoietic colony and burst formations in serum-free culture and their enhancement by insulin-like growth factor I.

    PubMed

    Akahane, K; Tojo, A; Urabe, A; Takaku, F

    1987-08-01

    Recombinant human insulin-like growth factor I (IGF-I) increased human and murine erythropoietic colony formation in serum-free culture. In order to investigate the effects of purified factors such as IGF-I on hemopoietic progenitor cells, we have established a serum-free culture system which supports the clonal growth of CFU-E- and BFU-E-derived colonies. Exogenously supplied ingredients were bovine serum albumin (BSA), transferrin, lipid suspensions, 2-mercaptoethanol, and recombinant human erythropoietin (epo). Among these, BSA and cholesterol were found to be essential ingredients. The optimum concentration of BSA sufficient to grow BFU-E was 3%. Erythroid colony and burst formation of human and murine marrow cells was enhanced twofold (p less than 0.05) by a physiological concentration of recombinant human IGF-I. Potentiation was observed in a dose-dependent manner between 10(-9) and 10(-7) M. A few murine CFU-E colonies were formed in the absence of epo. These results suggest that IGF-I has a supportive effect on the proliferation and differentiation of erythroid precursor cells stimulated by epo and that its action is synergistic with that of epo.

  6. Colony formation by sublethally heat-injured Zygosaccharomyces rouxii as affected by solutes in the recovery medium and procedure for sterilizing medium.

    PubMed Central

    Golden, D A; Beuchat, L R

    1990-01-01

    Recovery and colony formation by healthy and sublethally heat-injured cells of Zygosaccharomyces rouxii as influenced by the procedure for sterilizing recovery media (YM agar [YMA], wort agar, cornmeal agar, and oatmeal agar) were investigated. Media were supplemented with various concentrations of glucose, sucrose, glycerol, or sorbitol and sterilized by autoclaving (110 degrees C, 15 min) and by repeated treatment with steam (100 degrees C). An increase in sensitivity was observed when heat-injured cells were plated on glucose-supplemented YMA at an aw of 0.880 compared with aws of 0.933 and 0.998. Colonies which developed from unheated and heated cells on YMA at aws of 0.998 and 0.933 generally exceeded 0.5 mm in diameter within 3.5 to 4 days of incubation at 25 degrees C, whereas colonies formed on YMA at an aw of 0.880 typically did not exceed 0.5 mm in diameter until after 5.5 to 6.5 days of incubation. The number of colonies exceeding 0.5 mm in diameter which were formed by heat-injured cells on YMA at an aw of 0.880 was 2 to 3 logs less than the total number of colonies detected, i.e., on YMA at an aw of 0.933 and using no limits of exclusion based on colony diameter. A substantial portion of cells which survived heat treatment were sublethally injured as evidenced by increased sensitivity to a suboptimum aw (0.880). In no instance was recovery of Z. rouxii significantly affected by medium sterilization procedure when glucose or sorbitol was used as the aw-suppressing solute.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2403251

  7. Electrospun Scaffolds: Enhanced Lineage-Specific Differentiation Efficiency of Human Induced Pluripotent Stem Cells by Engineering Colony Dimensionality Using Electrospun Scaffolds (Adv. Healthcare Mater. 12/2016).

    PubMed

    Maldonado, Maricela; Ico, Gerardo; Low, Karen; Luu, Rebeccah J; Nam, Jin

    2016-06-01

    Electrospun scaffolds provide soft nanofibrous networks pliable by human induced pluripotent stem cells. J. Nam and co-workers show on page 1408 that such compliant scaffolding leads to the formation of stem cell colonies with a distinctive three-dimensional morphology. The morphological modulation resulted in the lineage-specific differentiation, suggesting a potential means to enhance translational applications of the stem cells.

  8. Granulocyte colony-stimulating factor-based stem cell mobilization in patients with sickle cell disease.

    PubMed

    Rosenbaum, Cara; Peace, David; Rich, Elizabeth; Van Besien, Koen

    2008-06-01

    Granulocyte colony-stimulating factor (G-CSF) has been reported to exacerbate vaso-occlusive crises in sickle cell disease. It has been recommended to avoid its use for stem cell mobilization in this population, yet autologous transplant is the standard of care and at times a life-saving treatment for patients with various hematologic malignancies such as relapsed aggressive lymphoma or multiple myeloma. We report 5 cases of patients with sickle cell disease and related hemoglobinopathies who underwent granulocyte-colony stimulating factor (G-CSF)-mobilization of peripheral blood stem cells (PBSC). Three of them developed manageable vaso-occlusive pain symptoms requiring parenteral narcotics alone. The 2 others had no complications. These cases demonstrate that stem cell mobilization using G-CSF, although complicated and not without risk, is feasible in patients with sickle cell syndromes.

  9. Characterization of ectopic colonies that form in widespread areas of the nervous system with neural stem cell transplants into the site of a severe spinal cord injury.

    PubMed

    Steward, Oswald; Sharp, Kelli G; Yee, Kelly Matsudaira; Hatch, Maya N; Bonner, Joseph F

    2014-10-15

    We reported previously the formation of ectopic colonies in widespread areas of the nervous system after transplantation of fetal neural stem cells (NSCs) into spinal cord transection sites. Here, we characterize the incidence, distribution, and cellular composition of the colonies. NSCs harvested from E14 spinal cords from rats that express GFP were treated with a growth factor cocktail and grafted into the site of a complete spinal cord transection. Two months after transplant, spinal cord and brain tissue were analyzed histologically. Ectopic colonies were found at long distances from the transplant in the central canal of the spinal cord, the surface of the brainstem and spinal cord, and in the fourth ventricle. Colonies were present in 50% of the rats, and most rats had multiple colonies. Axons extended from the colonies into the host CNS. Colonies were strongly positive for nestin, a marker for neural precursors, and contained NeuN-positive cells with processes resembling dendrites, GFAP-positive astrocytes, APC/CC1-positive oligodendrocytes, and Ki-67-positive cells, indicating ongoing proliferation. Stereological analyses revealed an estimated 21,818 cells in a colony in the fourth ventricle, of which 1005 (5%) were Ki-67 positive. Immunostaining for synaptic markers (synaptophysin and VGluT-1) revealed large numbers of synaptophysin-positive puncta within the colonies but fewer VGluT-1 puncta. Continuing expansion of NSC-derived cell masses in confined spaces in the spinal cord and brain could produce symptoms attributable to compression of nearby tissue. It remains to be determined whether other cell types with self-renewing potential can also form colonies.

  10. Quantification of nucleated cells, CD34-positive cells and CFU-GM colonies in single bone marrow samples and bone marrow harvests derived from healthy children.

    PubMed

    Schündeln, Michael M; Walde, Gabriele; Basu, Oliver; Havers, Werner; Kremens, Bernhard

    2014-05-01

    Little is known regarding bone marrow (BM) cellularity, CD34+ fraction, and CFU-GM colony formation in relation to age and whether healthy children require a reference range distinct from healthy adults. We therefore analyzed a series of single BM aspirates from 45 healthy children who were evaluated as potential BM donors. Thirty-three of these children subsequently donated BM. We quantified the nucleated cell count, fraction of CD34+ cells, and number of CFU-GM colonies in single aspirates and BM harvests. Single aspirates displayed a mean nucleated cell count of 31.3 × 10(6) cells/mL, a mean fraction of 1.17% CD34+ cells, and a mean colony forming potential of 66.6 CFU-GM/10(5) cells. Harvests yielded the same number of nucleated cells but increased numbers of CD34+ cells and CFU-GM compared with single aspirates. The mean nucleated cell count in BM harvests was 31.1 × 10(6) /mL with a mean fraction of 1.95% CD34+ cells and a mean of 112.4 CFU-GM colonies/10(5) cells. The concentration of nucleated cells was elevated compared with reported adult counts, while CD34+ percentage and CFU-GM counts were similar. In this series of healthy children, the fraction of CD34+ cells, CFU-GM colonies, and nucleated cells decreased with age. We did not identify gender specific differences. To our knowledge, this represents the first comprehensive study of CD34+ cell fraction, CFU-GM counts, and nucleated cell numbers in the BM of healthy children. The findings provide valuable information for practical use for BM transplantation and contribute to the understanding of hematopoiesis from birth to adulthood.

  11. Cell-Surface Phenol Soluble Modulins Regulate Staphylococcus aureus Colony Spreading

    PubMed Central

    Kizaki, Hayato; Omae, Yosuke; Tabuchi, Fumiaki; Saito, Yuki; Sekimizu, Kazuhisa

    2016-01-01

    Staphylococcus aureus produces phenol-soluble modulins (PSMs), which are amphipathic small peptides with lytic activity against mammalian cells. We previously reported that PSMα1–4 stimulate S. aureus colony spreading, the phenomenon of S. aureus colony expansion on the surface of soft agar plates, whereas δ-toxin (Hld, PSMγ) inhibits colony-spreading activity. In this study, we revealed the underlying mechanism of the opposing effects of PSMα1–4 and δ-toxin in S. aureus colony spreading. PSMα1–4 and δ-toxin are abundant on the S. aureus cell surface, and account for 18% and 8.5% of the total amount of PSMα1–4 and δ-toxin, respectively, in S. aureus overnight cultures. Knockout of PSMα1–4 did not affect the amount of cell surface δ-toxin. In contrast, knockout of δ-toxin increased the amount of cell surface PSMα1–4, and decreased the amount of culture supernatant PSMα1–4. The δ-toxin inhibited PSMα3 and PSMα2 binding to the S. aureus cell surface in vitro. A double knockout strain of PSMα1–4 and δ-toxin exhibited decreased colony spreading compared with the parent strain. Expression of cell surface PSMα1–4, but not culture supernatant PSMα1–4, restored the colony-spreading activity of the PSMα1-4/δ-toxin double knockout strain. Expression of δ-toxin on the cell surface or in the culture supernatant did not restore the colony-spreading activity of the PSMα1-4/δ-toxin double knockout strain. These findings suggest that cell surface PSMα1–4 promote S. aureus colony spreading, whereas δ-toxin suppresses colony-spreading activity by inhibiting PSMα1–4 binding to the S. aureus cell surface. PMID:27723838

  12. Allelopathy is involved in the formation of pure colonies of the fern Gleichenia japonica.

    PubMed

    Kato-Noguchi, Hisashi; Saito, Yoshihumi; Ohno, Osamu; Suenaga, Kiyotake

    2013-04-15

    The fern Gleichenia japonica is one of the most widely distributed fern and occurs throughout East to South Asia. The species often dominates plant communities by forming large monospecific colonies. However, the potential mechanism for this domination has not yet been described. The objective of this study was to test the hypothesis that allelochemicals are involved in the formation of G. japonica colonies. An aqueous methanol extract of G. japonica inhibited the growth of seedlings of garden cress (Lepidium sativum), lettuce (Lactuca sativa), ryegrass (Lolium multiflorum) and timothy (Phleum pratense). Increasing extract concentration increased the inhibition. These results suggest that G. japonica contain allelopathic substances. The extract was then purified by several chromatographies with monitoring the inhibitory activity and two growth inhibitory substances causing the allelopathic effect were isolated. The chemical structures of the two substances were determined by spectral data to be a novel compound 3-O-β-allopyranosyl-13-O-β-fucopyranosyl-3β-hydroxymanool (1) and 18-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-13-epitorreferol (2). These compounds inhibited the shoot and root growth of garden cress, lettuce, alfalfa (Medicago sativa), timothy, ryegrass and barnyardgrass (Echinochloa crus-galli) at concentrations greater than 0.1-1.0mM. The concentrations required for 50% growth inhibition of root and shoot growth of these test plants ranged from 0.72 to 3.49mM and 0.79 to 3.51mM for compounds 1 and 2, respectively. Concentration of compounds 1 and 2 in soil under the pure colony of G. japonica was 4.9 and 5.7mM, respectively, indicating concentrations over those required for 50% growth inhibition are potentially available under monocultural stands of these ferns. Therefore, these compounds may contribute to the allelopathic effects caused by presence of G. japonica and may thus contribute to the establishment of monocultural stands by this

  13. The effects of electrospun substrate-mediated cell colony morphology on the self-renewal of human induced pluripotent stem cells.

    PubMed

    Maldonado, Maricela; Wong, Lauren Y; Echeverria, Cristina; Ico, Gerardo; Low, Karen; Fujimoto, Taylor; Johnson, Jed K; Nam, Jin

    2015-05-01

    The development of xeno-free, chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard, various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal, i.e., proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly, when surface chemistry of the substrates was uniformly controlled by collagen conjugation, the stiffness of substrate was inversely related to the sphericity, a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture, implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall, we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Hairdressing and nursing: presentation of self and professional formation in colonial Australia.

    PubMed

    Nelson, S

    2001-04-01

    When Lucy Osburn led her team of Nightingale sisters to the Sydney Infirmary in 1868 she knew that a challenge awaited her. Her goal was to transform the colony's only public hospital into a respectable, ordered environment where, according to the Sanitarian view of the universe espoused by Miss Nightingale, the patient would find the resources to heal himself (sic). The prime difficulty was not the filth and disorder of the institution, it was the calibre of the nurses. This paper offers a case study into the issues of presentation of self, institutional shaping and the professional formation of nurses in the late nineteenth and early twentieth century. The aesthetics of bodily grooming and the class and ethnic issues embedded in the professionalisation of nursing will be discussed. The story of nursing provides an exemplar of the disaggregation of a domain of female expertise. The translation of this expertise to a mass occupation embodied significant difficulties. Not the least of these difficulties was the problem nursing leaders encountered when attempting to instil certain personal attributes and vocational values in non pious common women. It will be argued here that it was the inculcation of a specific set of attributes that created the nurse. It is this persona of the nurse, and the challenge that it presented for colonial nurses in the late nineteenth and early twentieth centuries, that this paper explores.

  15. [Effects of Branchionus calyciflorus culture media filtrate on Microcystis aeruginosa, Scenedesmus obliquus and Chlorella vulgaris colony formation and growth].

    PubMed

    Yang, Zhou; Kong, Fanxiang; Shi, Xiaoli; Yang, Jiaxin

    2005-06-01

    To examine the possible information transfer by chemicals between zooplankton and algae, this paper studied the effects of Brachionus calyciflorus culture media filtrate on the colony formation and growth of Microcystis aeruginosa, Scenedesmus obliquus and Chlorella vulgaris. The results showed that the test filtrate could significantly promote the colony formation and population growth of S. obliquus, while no significant effect was observed on M. aeruginosa and C. vulgaris. The induced colony formation of S. obliquus increased its resistance to grazing, and thus, reduced the risk of its being grazed, which could be viewed as a kind of inducing defense. The accelerated growth of C. vulgaris and the toxin production of M. aeruginosa could also be interpreted as a defense mechanism against grazing. It maybe concluded that M. aeruginosa, S. obliquus and C. vulgaris could adopt different ecological strategies to resist the potential grazing by rotifer B. calyciflorus, and thus, to keep their population on a certain scale.

  16. Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells.

    PubMed

    Decaris, Martin L; Lee, Chang I; Yoder, Mervin C; Tarantal, Alice F; Leach, J Kent

    2009-01-01

    Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues.

  17. CKbeta8-1 alters expression of cyclin E in colony forming units-granulocyte macrophage (CFU-GM) lineage from human cord blood CD34+ cells.

    PubMed

    Noh, Eui Kyu; Ra, Jae Sun; Lee, Seong Ae; Kwon, Byoung S; Han, In Seob

    2005-12-31

    A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34+ cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34+ cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34+ cells were compared with those in untreated CD34+ cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34+ cells. Here we describe an in vitro system in which CB CD34+ cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.

  18. Bionomics and formation of "Bonsai" colonies with long term rearing of Coptotermes formosanus (Isoptera: Rhinotermitidae)

    USDA-ARS?s Scientific Manuscript database

    This laboratory study reports the ability of Formosan subterranean termite, Coptotermes formosanus Shiraki, colonies to survive for at least 9-yr while restricted to a sweater box. Colonies survived by limiting queen size and worker numbers, allowing these bonsai colonies to thrive. Queen physogastr...

  19. Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control

    PubMed Central

    Kato, Ryuji; Matsumoto, Megumi; Sasaki, Hiroto; Joto, Risako; Okada, Mai; Ikeda, Yurika; Kanie, Kei; Suga, Mika; Kinehara, Masaki; Yanagihara, Kana; Liu, Yujung; Uchio-Yamada, Kozue; Fukuda, Takayuki; Kii, Hiroaki; Uozumi, Takayuki; Honda, Hiroyuki; Kiyota, Yasujiro; Furue, Miho K

    2016-01-01

    Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of ‘hPSC colony morphology’, permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity. PMID:27667091

  20. Impact of chlorine on the cell integrity and toxin release and degradation of colonial Microcystis.

    PubMed

    Fan, Jiajia; Rao, La; Chiu, Yi-Ting; Lin, Tsair-Fuh

    2016-10-01

    The occurrence of toxic cyanobacteria in drinking water sources is problematic for water authorities as they can impair drinking water quality. Chlorine as a commonly used oxidant in water treatment plants has shown the potential to lyse cyanobacterial cells, resulting in the release of secondary metabolites which are hard to be removed during conventional water treatment processes. The majority of cyanobacterial species such as Microcystis, often occur in colonial forms under natural conditions. However, previous studies have mainly focused on the influence of chlorination on individual cyanobacterial cells due to technique limitations. A syringe dispersion method combined with a fluorescence technique (SYTOX Green stain with flow cytometry), was successfully developed for the evaluation of cell integrity of colonial Microcystis. Chlorination of Microcystis-laden water was conducted at different chlorine dosages for different colonial sizes (<37, 37-270 and 270-550 μm). The results indicated that colonial Microcystis cells were more resistant to chlorine oxidation than individual cells, which may be attributed to protection from the cell-bound mucilage. There was a lag phase before cell rupture occurred and a Delayed Chick Watson Model describes the experimental data very well for the kinetics of cyanobacterial cell rupture. The growing colonial size caused increases in the lag phases but decreases in the cell lysis rates. Chlorination also induced the release of microcystins (MCs) from colonial Microcystis cells. In particular, increased levels of dissolved MCs were observed in Cheng Kung Lake (CKL) water. In summary, the reaction of chlorine with colonial cyanobacteria is more complicated than with individual cells. The efficiency of chlorine oxidation could be reduced by the cell-bound mucilage and natural water matrix. These observations may provide insights for water authorities to assess the risk to drinking water quality posed by chlorination under

  1. Early Developmental Program Shapes Colony Morphology in Bacteria

    PubMed Central

    Mamou, Gideon; Malli Mohan, Ganesh Babu; Rouvinski, Alex; Rosenberg, Alex; Ben-Yehuda, Sigal

    2016-01-01

    Summary When grown on a solid surface, bacteria form highly organized colonies, yet little is known about the earliest stages of colony establishment. Following Bacillus subtilis colony development from a single progenitor cell, a sequence of highly ordered spatiotemporal events was revealed. Colony was initiated by the formation of leading-cell chains, deriving from the colony center and extending in multiple directions, typically in a “Y-shaped” structure. By eradicating particular cells during these early stages, we could influence the shape of the resulting colony and demonstrate that Y-arm extension defines colony size. A mutant in ymdB encoding a phosphodiesterase displayed unordered developmental patterns, indicating a role in guiding these initial events. Finally, we provide evidence that intercellular nanotubes contribute to proper colony formation. In summary, we reveal a “construction plan” for building a colony and provide the initial molecular basis for this process. PMID:26904951

  2. Early Developmental Program Shapes Colony Morphology in Bacteria.

    PubMed

    Mamou, Gideon; Malli Mohan, Ganesh Babu; Rouvinski, Alex; Rosenberg, Alex; Ben-Yehuda, Sigal

    2016-03-01

    When grown on a solid surface, bacteria form highly organized colonies, yet little is known about the earliest stages of colony establishment. Following Bacillus subtilis colony development from a single progenitor cell, a sequence of highly ordered spatiotemporal events was revealed. Colony was initiated by the formation of leading-cell chains, deriving from the colony center and extending in multiple directions, typically in a "Y-shaped" structure. By eradicating particular cells during these early stages, we could influence the shape of the resulting colony and demonstrate that Y-arm extension defines colony size. A mutant in ymdB encoding a phosphodiesterase displayed unordered developmental patterns, indicating a role in guiding these initial events. Finally, we provide evidence that intercellular nanotubes contribute to proper colony formation. In summary, we reveal a "construction plan" for building a colony and provide the initial molecular basis for this process.

  3. Glycosaminoglycan mimetic improves enrichment and cell functions of human endothelial progenitor cell colonies.

    PubMed

    Chevalier, Fabien; Lavergne, Mélanie; Negroni, Elisa; Ferratge, Ségolène; Carpentier, Gilles; Gilbert-Sirieix, Marie; Siñeriz, Fernando; Uzan, Georges; Albanese, Patricia

    2014-05-01

    Human circulating endothelial progenitor cells isolated from peripheral blood generate in culture cells with features of endothelial cells named late-outgrowth endothelial colony-forming cells (ECFC). In adult blood, ECFC display a constant quantitative and qualitative decline during life span. Even after expansion, it is difficult to reach the cell dose required for cell therapy of vascular diseases, thus limiting the clinical use of these cells. Glycosaminoglycans (GAG) are components from the extracellular matrix (ECM) that are able to interact and potentiate heparin binding growth factor (HBGF) activities. According to these relevant biological properties of GAG, we designed a GAG mimetic having the capacity to increase the yield of ECFC production from blood and to improve functionality of their endothelial outgrowth. We demonstrate that the addition of [OTR(4131)] mimetic during the isolation process of ECFC from Cord Blood induces a 3 fold increase in the number of colonies. Moreover, addition of [OTR(4131)] to cell culture media improves adhesion, proliferation, migration and self-renewal of ECFC. We provide evidence showing that GAG mimetics may have great interest for cell therapy applied to vascular regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of ECFC.

  4. Hepatic differentiation of human embryonic stem cells as microscaled multilayered colonies leading to enhanced homogeneity and maturation.

    PubMed

    Yao, Rui; Wang, Jingyu; Li, Xiaokang; Jung Jung, Da; Qi, Hao; Kee, Keh Kooi; Du, Yanan

    2014-11-12

    Directed differentiation of human embryonic stem cells (hESCs) towards hepatocyte-like cells on planar tissue culture plates has been extensively investigated with great promise to provide alternative cell sources for drug metabolism/toxicity testing. Recently, hepatic differentiation of hESCs in 3D configuration with better mimicry of embryonic liver development represents incremental efforts to improve the differentiation efficiency and cellular maturation. However, most of the present 3D differentiation configurations involved interruptive operations during the multi-staged differentiation process, which might impose unwanted influence on cellular differentiation. Most of the current researches resulted in generation of hepatocytes with high expression of AFP, which is minimally expressed in primary hepatocytes. Here, off-the-shelf micro-stencil arrays are developed to generate adherent multilayered colonies composed of hESCs-derived cells. Uninterrupted cellular differentiation and proliferation is achieved to recapitulate the continuous and multi-stage liver development. Compared with conventional 2D format, the micro-scaled multilayered colonies with uniform and defined sizes constrained within the microwells are composed of more homogenous and mature hepatocyte-like cells with significantly lowered AFP expression and elevated hepatic functions. The multilayered colonies as novel 3D configuration for hepatic differentiation of hESCs represent a significant step toward efficient generation of functional hepatocytes for regenerative medicine and drug discovery.

  5. Role of gravity in the formation of bacterial colonies with a hydrophobic surface layer

    NASA Astrophysics Data System (ADS)

    Puzyr, A. P.; Tirranen, L. K.; Krylova, T. Y.; Borodina, E. V.

    A simple technique for determining hydrophobic-hydrophilic properties of bacterial colonies surface, which involves putting a drop of liquid with known properties (e.g. water, oil) on their surface, has been described. This technique allows quick estimate of wettability of bacterial colony surface, i.e. its hydrophobic-hydrophilic properties. The behaviour of water drops on colonies of bacteria Bacillus five strains (of different types) has been studied. It was revealed that 1) orientation in the Earth gravity field during bacterial growth can define the form of colonies with hydrophobic surface; 2) the form and size of the colony are dependent on the extention ability, most probably, of the hydrophobic layer; 3) the Earth gravity field (gravity) serves as a 'pump' providing and keeping water within the colony. We suppose that at growing colonies on agar media the inflow of water-soluble nutrient materials takes place both due to diffusion processes and directed water current produced by the gravity. The revealed effect probably should be taken into consideration while constructing the models of colonies growing on dense nutrient media. The easily determined hydrophobic properties of colonies surface can become a systematic feature after collecting more extensive data on the surface hydrophobic-hydrophilic properties of microorganism colonies of other types and species.

  6. Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on glial scar formation after spinal cord injury in rats.

    PubMed

    Chung, Joonho; Kim, Moon Hang; Yoon, Yong Je; Kim, Kil Hwan; Park, So Ra; Choi, Byung Hyune

    2014-12-01

    This study investigated the effects of granulocyte colony-stimulating factor (G-CSF) on glial scar formation after spinal cord injury (SCI) in rats and compared the therapeutic effects between G-CSF and granulocytemacrophage colony-stimulating factor (GM-CSF) to evaluate G-CSF as a potential substitute for GM-CSF in clinical application. Rats were randomly assigned to 1 of 4 groups: a sham-operated group (Group 1), an SCI group without treatment (Group 2), an SCI group treated with G-CSF (Group 3), and an SCI group treated with GM-CSF (Group 4). G-CSF and GM-CSF were administered via intraperitoneal injection immediately after SCI. The effects of G-CSF and GM-CSF on functional recovery, glial scar formation, and axonal regeneration were evaluated and compared. The rats in Groups 3 and 4 showed better functional recovery and more decreased cavity sizes than those in Group 2 (p < 0.05). Both G-CSF and GM-CSF suppressed intensive expression of glial fibrillary acidic protein around the cavity at 4 weeks and reduced the expression of chondroitin sulfate proteoglycans (p < 0.05). Also, early administration of G-CSF and GM-CSF protected axon fibers from destructive injury and facilitated axonal regeneration. There were no significant differences in comparisons of functional recovery, glial scar formation, and axonal regeneration between G-CSF and GM-CSF. G-CSF suppressed glial scar formation after SCI in rats, possibly by restricting the expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycans, which might facilitate functional recovery from SCI. GM-CSF and G-CSF had similar effects on glial scar formation and functional recovery after SCI, suggesting that G-CSF can potentially be substituted for GM-CSF in the treatment of SCI.

  7. Tracking of replicative senescence in mesenchymal stem cells by colony-forming unit frequency.

    PubMed

    Schellenberg, Anne; Hemeda, Hatim; Wagner, Wolfgang

    2013-01-01

    Long-term culture of mesenchymal stem cells (MSC) has major impact on cellular characteristics and differentiation potential. Numerous clinical trials raise high hopes in regenerative medicine and this necessitates reliable quality control of the cellular products-also with regard to replicative senescence. The maximum number of population doublings before entering the senescent state depends on the cell type, tissue of origin, culture medium as well as cell culture methods. Therefore, it would be valuable to predict the remaining proliferative potential in the course of culture expansion. Here, we describe a refined fibroblastic colony forming unit (CFU-f) assay which can be performed at any passage during culture expansion with simple cell culture techniques. This method is based on limiting dilutions in the 96-well format to determine the proportion of highly proliferative and clonogenic cells. The number of CFU-f declines rapidly during culture expansion. Especially at higher passages the CFU-f frequency correlates very well with the remaining cumulative population doublings. This approach can be used as quality measure to estimate the remaining proliferative potential of MSC in culture.

  8. The sulfated polysaccharide fucoidan rescues senescence of endothelial colony-forming cells for ischemic repair.

    PubMed

    Lee, Jun Hee; Lee, Sang Hun; Choi, Sung Hyun; Asahara, Takayuki; Kwon, Sang-Mo

    2015-06-01

    The efficacy of cell therapy using endothelial colony-forming cells (ECFCs) in the treatment of ischemia is limited by the replicative senescence of isolated ECFCs in vitro. Such senescence must therefore be overcome in order for such cell therapies to be clinically applicable. This study aimed to investigate the potential of sulfated polysaccharide fucoidan to rescue ECFCs from cellular senescence and to improve in vivo vascular repair by ECFCs. Fucoidan-preconditioning of senescent ECFCs was shown by flow cytometry to restore the expression of functional ECFC surface markers (CD34, c-Kit, VEGFR2, and CXCR4) and stimulate the in vitro tube formation capacity of ECFCs. Fucoidan also promoted the expression of cell cycle-associated proteins (cyclin E, Cdk2, cyclin D1, and Cdk4) in senescent ECFCs, significantly reversed cellular senescence, and increased the proliferation of ECFCs via the FAK, Akt, and ERK signaling pathways. Fucoidan was found to enhance the survival, proliferation, incorporation, and endothelial differentiation of senescent ECFCs transplanted in ischemic tissues in a murine hind limb ischemia model. Moreover, ECFC-induced functional recovery and limb salvage were markedly improved by fucoidan pretreatment of ECFCs. To our knowledge, the findings of our study are the first to demonstrate that fucoidan enhances the neovasculogenic potential of ECFCs by rescuing them from replicative cellular senescence. Pretreatment of ECFCs with fucoidan may thus provide a novel strategy for the application of senescent stem cells to therapeutic neovascularization.

  9. Morphology and dynamic scaling analysis of cell colonies with linear growth fronts

    NASA Astrophysics Data System (ADS)

    Huergo, M. A. C.; Pasquale, M. A.; Bolzán, A. E.; Arvia, A. J.; González, P. H.

    2010-09-01

    The growth of linear cell colony fronts is investigated from the morphology of cell monolayer colonies, the cell size and shape distribution, the front displacement velocity, and the dynamic scaling analysis of front roughness fluctuations. At the early growth stages, colony patterns consist of rather ordered compact domains of small cells, whereas at advanced stages, an uneven distribution of cells sets in, and some large cells and cells exhibiting large filopodia are produced. Colony front profiles exhibit overhangs and behave as fractals with the dimension DF=1.25±0.05 . The colony fronts shift at 0.22±0.02μmmin-1 average constant linear velocity and their roughness (w) increases with time (t) . Dynamic scaling analysis of experimental and overhang-corrected growth profile data shows that w versus system width l log-log plots collapse to a single curve when l exceeds a certain threshold value lo , a width corresponding to the average diameter of few cells. Then, the influence of overhangs on the roughness dynamics becomes negligible, and a growth exponent β=0.33±0.02 is derived. From the structure factor analysis of overhang-corrected profiles, a global roughness exponent αs=0.50±0.05 is obtained. For l>200μm , this set of exponents fulfills the Family-Vicsek relationship. It is consistent with the predictions of the continuous Kardar-Parisi-Zhang model.

  10. Effects of lead(II) on the extracellular polysaccharide (EPS) production and colony formation of cultured Microcystis aeruginosa.

    PubMed

    Bi, Xiang-dong; Zhang, Shu-lin; Dai, Wei; Xing, Ke-zhing; Yang, Fan

    2013-01-01

    To investigate the effects of lead(II) on the production of extracellular polysaccharides (EPS), including bound extracellular polysaccharides (bEPS) and soluble extracellular polysaccharides (sEPS), and the colony formation of Microcystis aeruginosa, cultures of M. aeruginosa were exposed to four concentrations (5.0, 10.0, 20.0 and 40.0 mg/L) of lead(II) for 10 d under controlled laboratory conditions. The results showed that 5.0 and 10.0 mg/L lead(II) stimulated M. aeruginosa growth throughout the experiment while 20.0 and 40.0 mg/L lead(II) inhibited M. aeruginosa growth in the first 2 d exposure and then stimulated it. As compared to the control group, significant increases in the bEPS and sEPS production were observed in 20.0 and 40.0 mg/L lead(II) treatments (P < 0.05). Large colony formations were not observed throughout the experiment. However, four tested concentrations of lead(II) could significantly promote the formation of small and middle colonies after 10 d exposure (P < 0.05), and 40.0 mg/L lead(II) had the best stimulatory effect. Lead(II) could stimulate bEPS production, which conversely promoted colony formation, suggesting that heavy metals might be contributing to the bloom-forming of M. aeruginosa in natural conditions.

  11. The Legacy of Literacy Practices in Colonial Taiwan. Japanese-Taiwanese-Chinese: Language Interaction and Identity Formation

    ERIC Educational Resources Information Center

    Heylen, Ann

    2005-01-01

    This paper offers a historical and sociolinguistic interrogation of Taiwanese to demonstrate the significance of language continuum in relation to identity formation. To this end, Taiwanese is discussed as a particular variety of language. Literacy practices in the Japanese colonial period (1895-1945) are contrasted with the precolonial and…

  12. Influence of Gelatin-Thrombin Matrix Tissue Sealant on Bacterial Colony Formation and Risk of Pelvic Infection

    PubMed Central

    Jarrett, Michael J.; Vázquez-Torres, Andres; Frank, Daniel N.; McCollister, Bruce D.; Henthorn, Patrick K.; Ir, Diana; Sheeder, Jeanelle; Guy, Michael S.; Anwar, Hiba Q.; Behbakht, Kian

    2016-01-01

    Objective. Gelatin-thrombin matrix (GTM) tissue sealant use was previously identified as an independent predictor of pelvic infection following hysterectomies. We aim to elucidate contributing factors by assessing influence of GTM on bacterial colony formation and characterizing bacteria present at the vaginal cuff. Methods. Escherichia coli was incubated in phosphate-buffered saline (PBS) and pelvic washings with and without GTM to assess influence on colony formation. Pelvic washings of the vaginal cuff were collected from hysterectomies occurring from June through October 2015. In vitro techniques, 16S rRNA gene qPCR, and 16S amplicon sequencing were performed with washings to characterize bacteria at the vaginal cuff. Results. Mean bacterial colony formation in PBS was greater for E. coli incubated in the presence of GTM (1.48 × 107 CFU/mL) versus without (9.95 × 105 CFU/mL) following 20-hour incubation (p = 0.001). Out of 61 pelvic washings samples, 3 were culture positive (≥5000 CFU/mL) with Enterococcus faecalis. Conclusion. In vitro experiments support a facilitating role of GTM on colony formation of E. coli in PBS. However, given the negative results of surgical site washings following adequate disinfection, the role of GTM in promoting posthysterectomy pelvic infections may be limited. Analysis of pelvic washings revealed presence of E. faecalis, but results were inconclusive. Further studies are recommended. PMID:27199534

  13. Evolution and development of budding by stem cells: ascidian coloniality as a case study.

    PubMed

    Brown, Federico D; Swalla, Billie J

    2012-09-15

    The evolution of budding in metazoans is not well understood on a mechanistic level, but is an important developmental process. We examine the evolution of coloniality in ascidians, contrasting the life histories of solitary and colonial forms with a focus on the cellular and developmental basis of the evolution of budding. Tunicates are an excellent group to study colonial transitions, as all solitary larvae develop with determinant and invariant cleavage patterns, but colonial species show robust developmental flexibility during larval development. We propose that acquiring new stem cell lineages in the larvae may be a preadaptation necessary for the evolution of budding. Brooding in colonial ascidians allows increased egg size, which in turn allows greater flexibility in the specification of cells and cell numbers in late embryonic and pre-metamorphic larval stages. We review hypotheses for changes in stem cell lineages in colonial species, describe what the current data suggest about the evolution of budding, and discuss where we believe further studies will be most fruitful.

  14. DECREASED LEVEL OF CORD BLOOD CIRCULATING ENDOTHELIAL COLONY-FORMING CELLS IN PREECLAMPSIA

    PubMed Central

    Muñoz-Hernandez, Rocio; Miranda, Maria L.; Stiefel, Pablo; Lin, Ruei-Zeng; Praena-Fernández, Juan M.; Dominguez-Simeon, Maria J.; Villar, Jose; Moreno-Luna, Rafael; Melero-Martin, Juan M.

    2014-01-01

    Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=15) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation and migration towards VEGF-A and FGF-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies (P = .04), a reduction that was independent of other obstetric factors. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events. PMID:24752434

  15. Decreased level of cord blood circulating endothelial colony-forming cells in preeclampsia.

    PubMed

    Muñoz-Hernandez, Rocio; Miranda, Maria L; Stiefel, Pablo; Lin, Ruei-Zeng; Praena-Fernández, Juan M; Dominguez-Simeon, Maria J; Villar, Jose; Moreno-Luna, Rafael; Melero-Martin, Juan M

    2014-07-01

    Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=15) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation, and migration toward vascular endothelial growth factor-A and fibroblast growth factor-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies (P=0.04), a reduction that was independent of other obstetric factors. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events.

  16. Periodic growth of bacterial colonies

    NASA Astrophysics Data System (ADS)

    Yamazaki, Yoshihiro; Ikeda, Takemasa; Shimada, Hirotoshi; Hiramatsu, Fumiko; Kobayashi, Naoki; Wakita, Jun-ichi; Itoh, Hiroto; Kurosu, Sayuri; Nakatsuchi, Michio; Matsuyama, Tohey; Matsushita, Mitsugu

    2005-06-01

    The formation of concentric ring colonies by bacterial species Bacillus subtilis and Proteus mirabilis has been investigated experimentally, focusing our attention on the dependence of local cell density upon the bacterial motility. It has been confirmed that these concentric ring colonies reflect the periodic change of the bacterial motility between motile cell state and immotile cell state. We conclude that this periodic change is macroscopically determined neither by biological factors (i.e., biological clock) nor by chemical factors (chemotaxis as inhibitor). And our experimental results strongly suggest that the essential factor for the change of the bacterial motility during concentric ring formation is the local cell density.

  17. Persistence of a Staphylococcus aureus small colony variants (S. aureus SCV) within bovine mammary epithelial cells.

    PubMed

    Atalla, Heba; Gyles, Carlton; Mallard, Bonnie

    2010-07-14

    Persistent bovine Staphylococcus aureus mastitis is attributable to the versatility of this pathogen within the mammary gland environment and to the formation of small colony variants (SCVs) that can survive within host cells. Previous studies had shown that S. aureus SCV Heba3231, isolated from a cow with chronic mastitis, had invaded and persisted in primary bovine aortic endothelial cells but caused minimal deleterious effects. The objective of this study was to investigate the interaction of SCV Heba3231 with bovine mammary epithelial cells (MAC-T cells) compared to its parent strain 3231 and to prototype strain Newbould 305. Monolayer cells were infected with each strain at various multiplicity of infections (MOIs) for 1 and 3.5h, followed by 20 min incubation with lysostaphin. Recovery of the SCV was significantly higher (P<0.05) after 3.8h with MOI of 100 compared to recovery of strains 3231 and Newbould 305. Upon further incubation, viable SCV were detected up to 96 h while 3231 were not isolated at 24h or later. Transmission electron microscopy demonstrated SCV uptake by MAC-T cells following a series of events similar to those for strain 3231. At 24h, multiple SCV were seen within enclosed vacuoles, while the 3231 parent strain was released extracellularly and the monolayer cells were damaged. The ability of SCV Heba3231 to survive inside vacuoles could be related to up-regulation of protective mechanisms. These findings highlight the potential role of bovine mammary epithelial cells and S. aureus SCV in persistent bovine mastitis.

  18. Highly metastatic 13762NF rat mammary adenocarcinoma cell clones stimulate bone marrow by secretion of granulocyte-macrophage colony-stimulating factor/interleukin-3 activity.

    PubMed

    McGary, C T; Miele, M E; Welch, D R

    1995-12-01

    Circulating neutrophil (polymorphonuclear leukocyte levels rise 50-fold in 13762NF tumor-bearing rats in proportion to the tumor's metastatic potential. Purified tumor-elicited neutrophils enhance metastasis of syngeneic tumor cells when co-injected intravenously; however, circulating and phorbol ester-activated polymorphonuclear neutrophils do not. The purpose of this study was to elucidate the source of tumor-elicited neutrophils in metastatic tumor-bearing rats. We examined the bone marrow in rats bearing tumors of poorly, moderately, and highly metastatic cell clones. Marrow from rats with highly metastatic tumors had increased cellularity (100%), myeloid to erythroid ratio (10:1), and megakaryocytes compared with control rats (cellularity, approximately 80%; myeloid to erythroid ratio, 5:1), with marrows from rats with moderately metastatic tumors having intermediate values. This suggested production of a colony-stimulating factor by the metastatic cells. To confirm this, bone marrow colony formation from control and tumor-bearing rats was compared. Colony number increased in proportion to the metastatic potential of the tumor. Conditioned medium from metastatic cells supported growth of the granulocyte-macrophage colony-stimulating factor/interleukin-3-dependent 32Dcl3 cell line, but media from nonmetastatic or moderately metastatic cells did not. Antibodies to murine granulocyte-macrophage colony-stimulating factor neutralized 32Dcl3 growth in tumor cell conditioned medium. These results suggest production of a granulocyte-macrophage colony-stimulating factor or interleukin-3-like activity by highly metastatic 13762NF clones and implicate a possible role for colony-stimulating factors in regulating the metastatic potential of mammary adenocarcinoma cell clones.

  19. Role of Vasa, Piwi, and Myc-expressing coelomic cells in gonad regeneration of the colonial tunicate, Botryllus primigenus.

    PubMed

    Kawamura, Kaz; Sunanaga, Takeshi

    2011-01-01

    In the colonial tunicate, Botryllus primigenus Oka, gonads consist of indifferent germline precursor cells, the primordial testis and ovary, and mature gonads, of which the immature gonad components can be reconstructed de novo in vascular buds that arise from the common vascular system, although the mechanism is uncertain. In this study, we investigated how and what kinds of cells regenerated the gonad components. We found that few Vasa-positive cells in the hemocoel entered the growing vascular bud, where their number increased, and finally developed exclusively into female germ cells. Simultaneously, small cell aggregates consisting of Vasa(-) and Vasa(±) cells appeared de novo in the lateral body cavity of developing vascular buds. Double fluorescent in situ hybridization showed that these cell aggregates were both Piwi- and Myc-positive. They could form germline precursor cells and a primordial testis and ovary that strongly expressed Vasa. Myc knockdown by RNA interference conspicuously lowered Piwi expression and resulted in the loss of germline precursor cells without affecting Vasa(+) oocyte formation. Myc may contribute to gonad tissue formation via Piwi maintenance. When human recombinant BMP 4 was injected in the test vessel, coelomic Piwi(+) cells were induced to express Vasa in the blood. We conclude, therefore, that in vascular buds of B. primigenus, female germ cells can develop from homing Vasa(+) cells in the blood, and that other gonad components can arise from coelomic Vasa(-)/Piwi(+)/Myc(+) cells.

  20. The hematopoietic chemokine CXCL12 promotes integration of human endothelial colony forming cell-derived cells into immature vessel networks.

    PubMed

    Newey, Sarah E; Tsaknakis, Grigorios; Khoo, Cheen P; Athanassopoulos, Thanassi; Camicia, Rosalba; Zhang, Youyi; Grabowska, Rita; Harris, Adrian L; Roubelakis, Maria G; Watt, Suzanne M

    2014-11-15

    Proangiogenic factors, vascular endothelial growth factor (VEGF), and fibroblast growth factor-2 (FGF-2) prime endothelial cells to respond to "hematopoietic" chemokines and cytokines by inducing/upregulating expression of the respective chemokine/cytokine receptors. Coculture of human endothelial colony forming cell (ECFC)-derived cells with human stromal cells in the presence of VEGF and FGF-2 for 14 days resulted in upregulation of the "hematopoietic" chemokine CXCL12 and its CXCR4 receptor by day 3 of coculture. Chronic exposure to the CXCR4 antagonist AMD3100 in this vasculo/angiogenesis assay significantly reduced vascular tubule formation, an observation recapitulated by delayed AMD3100 addition. While AMD3100 did not affect ECFC-derived cell proliferation, it did demonstrate a dual action. First, over the later stages of the 14-day cocultures, AMD3100 delayed tubule organization into maturing vessel networks, resulting in enhanced endothelial cell retraction and loss of complexity as defined by live cell imaging. Second, at earlier stages of cocultures, we observed that AMD3100 significantly inhibited the integration of exogenous ECFC-derived cells into established, but immature, vascular networks. Comparative proteome profiler array analyses of ECFC-derived cells treated with AMD3100 identified changes in expression of potential candidate molecules involved in adhesion and/or migration. Blocking antibodies to CD31, but not CD146 or CD166, reduced the ECFC-derived cell integration into these extant vascular networks. Thus, CXCL12 plays a key role not only in endothelial cell sensing and guidance, but also in promoting the integration of ECFC-derived cells into developing vascular networks.

  1. Colony Dimorphism in Bradyrhizobium Strains

    PubMed Central

    Sylvester-Bradley, Rosemary; Thornton, Philip; Jones, Peter

    1988-01-01

    Ten isolates of Bradyrhizobium spp. which form two colony types were studied; the isolates originated from a range of legume species. The two colony types differed in the amount of gum formed or size or both, depending on the strain. Whole 7-day-old colonies of each type were subcultured to determine the proportion of cells which had changed to the other type. An iterative computerized procedure was used to determine the rate of switching per generation between the two types and to predict proportions reached at equilibrium for each strain. The predicted proportions of the wetter (more gummy) or larger colony type at equilibrium differed significantly between strains, ranging from 0.9999 (strain CIAT 2383) to 0.0216 (strain CIAT 2469), because some strains switched faster from dry to wet (or small to large) and others switched faster from wet to dry (or large to small). Predicted equilibrium was reached after about 140 generations in strain USDA 76. In all but one strain (CIAT 3030) the growth rate of the wetter colony type was greater than or similar to that of the drier type. The mean difference in generation time between the two colony types was 0.37 h. Doubling times calculated for either colony type after 7 days of growth on the agar surface ranged from 6.0 to 7.3 h. The formation of two persistent colony types by one strain (clonal or colony dimorphism) may be a common phenomenon among Bradyrhizobium strains. Images PMID:16347599

  2. Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer.

    PubMed

    PUCK, T T; MARCUS, P I; CIECIURA, S J

    1956-02-01

    Two methods for simple and rapid plating of single HeLa cells, human, carcinomatous cells, are described. These result in growth and formation of colonies from each single cell. One of these procedures uses irradiated, non-multiplying "feeder" cells to condition the medium. The second requires more gentle handling of the cells, but otherwise is virtually the same as that used in plating bacteria on semisolid, nutrient media. By extension of these methods, it is possible to isolate single mutant colonies and grow pure clonal stocks of animal cells. These genetically uniform strains are much more homogeneous in their behavior than the parental HeLa cell population. Growth curves obtained from developing colonies are highly reproducible. The most active mutant stocks so far isolated display a generation time of 18 to 20 hours. In pooled human serum HeLa cells assume a highly stretched, ameboid form, with marked motility; whereas growth of the same cells in a variety of non-human sera results in tightly packed, columnar, epithelial-like morphology. The two cell types possess volumes, nuclear cross-sections, plating efficiencies, and generation times which are identical within experimental error, but display widely different cross-sectional areas, suggesting that the basic change occurs in the cell surface. It is conceivable that this change may be related to that which enables the cells of a compact tumor to become invasive. Animal cells subjected to the standard trypsinization procedures which involve mechanical trauma and repeated washings in incomplete media leak large amounts of P and suffer impaired ability to reproduce as isolated cells. Application of the methods described in this paper as a tool for quantitative study of normal mammalian cell growth, physiology, genetics, and biochemistry, and the response of cells to drugs, viruses, high energy radiation, and other agents have been indicated.

  3. Gestational diabetes induces alterations in the function of neonatal endothelial colony forming cells

    PubMed Central

    Blue, Emily K.; DiGiuseppe, Robert; Derr-Yellin, Ethel; Acosta, Juan Carlos; Pay, S. Louise; Hanenberg, Helmut; Schellinger, Megan M.; Quinney, Sara K.; Mund, Julie A.; Case, Jamie; Haneline, Laura S.

    2014-01-01

    Background Children born to mothers with gestational diabetes mellitus (GDM) experience increased risk of developing hypertension, type 2 diabetes mellitus, and obesity. Disrupted function of endothelial colony forming cells (ECFCs) may contribute to this enhanced risk. The goal of this study was to determine if cord blood ECFCs from GDM pregnancies exhibit altered functionality. Methods ECFCs isolated from the cord blood of control and GDM pregnancies were assessed for proliferation, senescence, and Matrigel network formation. The requirement for p38MAPK in hyperglycemia-induced senescence was determined using inhibitor and overexpression studies. Results GDM ECFCs were more proliferative than control ECFCs. However, GDM ECFCs exhibited decreased network forming ability in Matrigel. Aging of ECFCs by serial passaging led to increased senescence and reduced proliferation of GDM ECFCs. ECFCs from GDM pregnancies were resistant to hyperglycemia-induced senescence compared to controls. In response to hyperglycemia, control ECFCs activated p38MAPK, which was required for hyperglycemia-induced senescence. In contrast, GDM ECFCs had no change in p38MAPK activation under equivalent conditions. Conclusion Intrauterine exposure of ECFCs to GDM induces unique phenotypic alterations. The resistance of GDM ECFCs to hyperglycemia-induced senescence and decreased p38MAPK suggest that these progenitor cells have undergone changes to induce tolerance to a hyperglycemic environment. PMID:24232636

  4. The Antiproliferative and Colony-suppressive Activities of STAT3 Inhibitors in Human Cancer Cells Is Compromised Under Hypoxic Conditions.

    PubMed

    Tian, Jilai; Xiao, Hui; Wu, Ruohan; Cao, Yang; Li, Chenglong; Xu, Ronald; Pierson, Christopher R; Finlay, Jonathan L; Yang, Fang; Gu, Ning; Lin, Jiayuh

    2017-02-01

    Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been indicated as a novel cancer drug target, since it plays an important role in diverse oncogenic processes including survival, cell proliferation and migration. Emerging STAT3 inhibitors have demonstrated efficacy in cancer cells and animal tumor models. It is well known that most solid tumors are characterized by hypoxia, but it is not clear if hypoxic conditions affect activity of STAT3 inhibitors. To examine this, two STAT3 inhibitors were tested to investigate their inhibitory efficacy in cancer cells grown under hypoxic conditions compared with those without hypoxia. Cell proliferation, colony formation and western blot assays were performed to examine the differences in the cell viability, proliferation and proteins in the STAT3 pathway. Under hypoxic conditions, the half-maximal inhibitory concentration values for both STAT3 inhibitors were increased compared to normoxic conditions in human pancreatic cancer, medulloblastoma and sarcoma cell lines. In addition, the ability of both STAT3 inhibitors to inhibit colony formation in pancreatic cancer, medulloblastoma and sarcoma cell lines was reduced under hypoxic conditions when compared to cells under normoxic conditions. Furthermore, there was an increase in phosphorylated STAT3 levels in cancer cells under hypoxic conditions, suggesting this may be one of the mechanisms of resistance. In summary, the results presented here provide a novel finding of STAT3 inhibitor activity under hypoxic conditions and indicate that under such low oxygen conditions, the anticancer efficacy of STAT3 inhibitors was indeed hampered. These results highlight the need to develop new therapeutic strategies to overcome the resistance of cancer cells to STAT3 inhibitors under hypoxic conditions. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  5. Cytosine arabinoside reduces the numbers of granulocyte macrophage colony forming cells (GM-CFC) and high proliferative potential colony forming cells (HPP-CFC) in vivo in mice.

    PubMed

    Teleka, Stanley; Chijuwa, Alexander; Senga, Edward; Chisi, John E

    2011-12-01

    Cytosine arabinoside (Ara-C) is an S-phase specific cytotoxic drug used in the treatment of malignancies. It is converted to Cytosine Arabinoside triphosphate (Ara-CTP) in the cell. Cytosine Arabinoside triphosphate, reversibly displaces deoxy cytidine triphosphate from DNA polymerase for incorporation into DNA. This process leads to cell death. To investigate the in vivo effects of Ara-C on the Granulocyte Macrophage Colony Forming Cells (GM-CFC) and High Proliferative Potential Colony Forming Cells (HPP-CFC) respectively in mice. Ara-C (150mg/kg) was administered intraperitoneally (i.p) once to mice and bone marrow cells sampled on days 1, 3 and 6. Ara-C reduced the numbers of both GM-CFC and HPP-CFC in the bone marrow. HPP-CFCs were initially more sensitive to Ara-C treatment than GM-CFCs. In the six days after treatment the effect on GM-CFC persisted, while there was a partial recovery in the number of HPP-CFCs. It is possible that Ara-C disturbs the stem cells niche by damaging the stromal cells of the bone marrow microenvironment. This would result in derangement of HPP-CFC proliferation.

  6. A Mechanistic Collective Cell Model for Epithelial Colony Growth and Contact Inhibition

    PubMed Central

    Aland, Sebastian; Hatzikirou, Haralambos; Lowengrub, John; Voigt, Axel

    2015-01-01

    We present a mechanistic hybrid continuum-discrete model to simulate the dynamics of epithelial cell colonies. Collective cell dynamics are modeled using continuum equations that capture plastic, viscoelastic, and elastic deformations in the clusters while providing single-cell resolution. The continuum equations can be viewed as a coarse-grained version of previously developed discrete models that treat epithelial clusters as a two-dimensional network of vertices or stochastic interacting particles and follow the framework of dynamic density functional theory appropriately modified to account for cell size and shape variability. The discrete component of the model implements cell division and thus influences cell size and shape that couple to the continuum component. The model is validated against recent in vitro studies of epithelial cell colonies using Madin-Darby canine kidney cells. In good agreement with experiments, we find that mechanical interactions and constraints on the local expansion of cell size cause inhibition of cell motion and reductive cell division. This leads to successively smaller cells and a transition from exponential to quadratic growth of the colony that is associated with a constant-thickness rim of growing cells at the cluster edge, as well as the emergence of short-range ordering and solid-like behavior. A detailed analysis of the model reveals a scale invariance of the growth and provides insight into the generation of stresses and their influence on the dynamics of the colonies. Compared to previous models, our approach has several advantages: it is independent of dimension, it can be parameterized using classical elastic properties (Poisson’s ratio and Young’s modulus), and it can easily be extended to incorporate multiple cell types and general substrate geometries. PMID:26445436

  7. A Mechanistic Collective Cell Model for Epithelial Colony Growth and Contact Inhibition.

    PubMed

    Aland, Sebastian; Hatzikirou, Haralambos; Lowengrub, John; Voigt, Axel

    2015-10-06

    We present a mechanistic hybrid continuum-discrete model to simulate the dynamics of epithelial cell colonies. Collective cell dynamics are modeled using continuum equations that capture plastic, viscoelastic, and elastic deformations in the clusters while providing single-cell resolution. The continuum equations can be viewed as a coarse-grained version of previously developed discrete models that treat epithelial clusters as a two-dimensional network of vertices or stochastic interacting particles and follow the framework of dynamic density functional theory appropriately modified to account for cell size and shape variability. The discrete component of the model implements cell division and thus influences cell size and shape that couple to the continuum component. The model is validated against recent in vitro studies of epithelial cell colonies using Madin-Darby canine kidney cells. In good agreement with experiments, we find that mechanical interactions and constraints on the local expansion of cell size cause inhibition of cell motion and reductive cell division. This leads to successively smaller cells and a transition from exponential to quadratic growth of the colony that is associated with a constant-thickness rim of growing cells at the cluster edge, as well as the emergence of short-range ordering and solid-like behavior. A detailed analysis of the model reveals a scale invariance of the growth and provides insight into the generation of stresses and their influence on the dynamics of the colonies. Compared to previous models, our approach has several advantages: it is independent of dimension, it can be parameterized using classical elastic properties (Poisson's ratio and Young's modulus), and it can easily be extended to incorporate multiple cell types and general substrate geometries.

  8. Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells.

    PubMed

    Smadja, David M; Levy, Marilyne; Huang, Lan; Rossi, Elisa; Blandinières, Adeline; Israel-Biet, Dominique; Gaussem, Pascale; Bischoff, Joyce

    2015-10-01

    Pulmonary vasodilators and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary hypertension (PH). Endothelial dysfunction is a key feature of PH, and we previously reported that treprostinil therapy increases number and proliferative potential of endothelial colony forming cells (ECFC) isolated from PH patients' blood. In the present study, the objective was to determine how treprostinil contributes to the proangiogenic functions of ECFC. We examined the effect of treprostinil on ECFC obtained from cord blood in terms of colony numbers, proliferative and clonogenic properties in vitro, as well as in vivo vasculogenic properties. Surprisingly, treprostinil inhibited viability of cultured ECFC but did not modify their clonogenic properties or the endothelial differentiation potential from cord blood stem cells. Treprostinil treatment significantly increased the vessel-forming ability of ECFC combined with mesenchymal stem cells (MSC) in Matrigel implanted in nude mice. In vitro, ECFC proliferation was stimulated by conditioned media from treprostinil-pretreated MSC, and this effect was inhibited either by the use of VEGF-A blocking antibodies or siRNA VEGF-A in MSC. Silencing VEGF-A gene in MSC also blocked the pro-angiogenic effect of treprostinil in vivo. In conclusion, increased VEGF-A produced by MSC can account for the increased vessel formation observed during treprostinil treatment. The clinical relevance of these data was confirmed by the high level of VEGF-A detected in plasma from patients with paediatric PH who had been treated with treprostinil. Moreover, our results suggest that VEGF-A level in patients could be a surrogate biomarker of treprostinil efficacy.

  9. Identification and enrichment of colony-forming cells from the adult murine pituitary

    SciTech Connect

    Lepore, D.A.; Roeszler, K.; Wagner, J.; Ross, S.A.; Bauer, K.; Thomas, P.Q. , E-Mail: paul.thomas@mcri.edu.au

    2005-08-01

    Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, {beta}-Ala-Lys-N{epsilon}-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA{sup +} cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA{sup +} population contains rare cells that are GH{sup +} or PRL{sup +}. GH{sup +} cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH{sup +} cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments.

  10. Isolation of endothelial colony-forming cells from blood samples collected from the jugular and cephalic veins of healthy adult horses.

    PubMed

    Sharpe, Ashley N; Seeto, Wen J; Winter, Randolph L; Zhong, Qiao; Lipke, Elizabeth A; Wooldridge, Anne A

    2016-10-01

    OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor-2, and von Willebrand factor) and hematopoietic (CD14) cell markers by use of indirect immunofluorescence assay (IFA) and flow cytometry. RESULTS Colonies with cobblestone morphology were isolated from 15 of 17 horses. Blood collected from the cephalic vein yielded colonies significantly more often (14/17 horses) than did blood collected from the jugular vein (8/17 horses). Of 14 cephalic blood samples with colonies, 13 were obtained with DGC and 8 with WBA. Of 8 jugular blood samples with colonies, 8 were obtained with DGC and 4 with WBA. Colony frequency (colonies per milliliter of blood) was significantly higher for cephalic blood samples and samples isolated with DGC. Cells formed vascular tubules, had uptake of DiI-Ac-LDL, and expressed endothelial markers by use of IFA and flow cytometry, which confirmed their identity as ECFCs. CONCLUSIONS AND CLINICAL RELEVANCE Maximum yield of ECFCs was obtained for blood samples collected from both the jugular and cephalic veins and use of DGC to isolate cells. Consistent yield of ECFCs from peripheral blood of horses will enable studies to evaluate diagnostic and therapeutic uses.

  11. LitR Is a Repressor of syp Genes and Has a Temperature-Sensitive Regulatory Effect on Biofilm Formation and Colony Morphology in Vibrio (Aliivibrio) salmonicida

    PubMed Central

    Bjelland, Ane Mohn; Ronessen, Maria; Robertsen, Espen; Willassen, Nils Peder

    2014-01-01

    Vibrio (Aliivibrio) salmonicida is the etiological agent of cold water vibriosis, a disease in farmed Atlantic salmon (Salmo salar) that is kept under control due to an effective vaccine. A seawater temperature below 12°C is normally required for disease development. Quorum sensing (QS) is a cell density-regulated communication system that bacteria use to coordinate activities involved in colonization and pathogenesis, and we have previously shown that inactivation of the QS master regulator LitR attenuates the V. salmonicida strain LFI1238 in a fish model. We show here that strain LFI1238 and a panel of naturally occurring V. salmonicida strains are poor biofilm producers. Inactivation of litR in the LFI1238 strain enhances medium- and temperature-dependent adhesion, rugose colony morphology, and biofilm formation. Chemical treatment and electron microscopy of the biofilm identified an extracellular matrix consisting mainly of a fibrous network, proteins, and polysaccharides. Further, by microarray analysis of planktonic and biofilm cells, we identified a number of genes regulated by LitR and, among these, were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. The syp genes were regulated by LitR in both planktonic and biofilm lifestyle analyses. Disruption of syp genes in the V. salmonicida ΔlitR mutant alleviated adhesion, rugose colony morphology, and biofilm formation. Hence, LitR is a repressor of syp transcription that is necessary for expression of the phenotypes examined. The regulatory effect of LitR on colony morphology and biofilm formation is temperature sensitive and weak or absent at temperatures above the bacterium's upper threshold for pathogenicity. PMID:24973072

  12. Requirement for Cell Dispersion Prior to Selection of Induced Azaguanine-Resistant Colonies of Chinese Hamster Cells

    PubMed Central

    Myhr, B. C.; DiPaolo, J. A.

    1975-01-01

    With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79–4 cells were exposed to 500 µM MMS, 7 µM AFAA, or 10 µM MNNG and allowed to multiply before being reseeded at 4 x 104 cells/60 mm dish and selected with 10 µg/ml AZG. Maximum frequencies of 4 x 10-5, 4 x 10-4, and 2.4 x 10-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.—AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0–20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies. PMID:1093934

  13. Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

    SciTech Connect

    Joo, Hyung Joon; Seo, Ha-Rim; Jeong, Hyo Eun; Choi, Seung-Cheol; Park, Jae Hyung; Yu, Cheol Woong; Hong, Soon Jun; Chung, Seok; Lim, Do-Sun

    2014-07-11

    Highlights: • Two distinct vascular progenitor cells are induced from adult peripheral blood. • ECFCs induce vascular structures in vitro and in vivo. • SMPCs augment the in vitro and in vivo angiogenic potential of ECFCs. • Both cell types have synergistic therapeutic potential in ischemic hindlimb model. - Abstract: Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood.

  14. Granulocyte colony-stimulating factor versus granulocyte-macrophage colony-stimulating factor for collection of peripheral blood progenitor cells from healthy donors.

    PubMed

    Fischmeister, G; Gadner, H

    2000-05-01

    The harvesting of peripheral blood progenitor cells (PBPCs) after granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor stimulation instead of bone marrow in healthy donors has become increasingly popular. Donors, given the choice between bone marrow and PBPC donation, often prefer cytapheresis because of the easier access, no necessity for general anesthesia, and no multiple bone marrow punctures. In addition, accelerated engraftment and immunomodulation by granulocyte colony-stimulating factor-mobilized PBPCs are advantageous for the recipient. However, because of donor inconvenience and poor mobilization, there is a need to develop improved procedures. Aspects such as durability of hematopoietic engraftment, characterization of the earliest stem cell, and composition of PBPCs are not yet well defined, and international donor registration and follow-up must be considered when evaluating long-term safety profiles in healthy donors. This review concentrates on the most significant developments on mobilization of PBPCs published during the past year.

  15. Cord Blood Endothelial Colony-Forming Cells from Newborns with Congenital Diaphragmatic Hernia

    PubMed Central

    Baker, Christopher D.; Black, Claudine P.; Ryan, Sharon L.; Balasubramaniam, Vivek; Abman, Steven H.

    2013-01-01

    Endothelial colony-forming cells (ECFC) are decreased in the cord blood of preterm infants with moderate-to-severe bronchopulmonary dysplasia (BPD). We quantified ECFC from infants with congenital diaphragmatic hernia (CDH), a neonatal disorder with severe lung hypoplasia. Unlike newborns who develop BPD, those with CDH had increased and highly-proliferative cord blood ECFC. PMID:23684109

  16. Enhanced Lineage-Specific Differentiation Efficiency of Human Induced Pluripotent Stem Cells by Engineering Colony Dimensionality Using Electrospun Scaffolds.

    PubMed

    Maldonado, Maricela; Ico, Gerardo; Low, Karen; Luu, Rebeccah J; Nam, Jin

    2016-06-01

    Electrospun scaffolds with varied stiffness promote distinct colony morphology of human induced pluripotent stem cells, which affects their subsequent differentiation. On soft scaffolds, induced pluripotent stem cells develop 3D colonies due to the pliability of the electrospun fibrous networks, leading to greater differentiation tendency to ectodermal lineage. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Hyaluronic Acid Hydrogels Support Cord-Like Structures from Endothelial Colony-Forming Cells

    PubMed Central

    Yee, Derek; Hanjaya-Putra, Donny; Bose, Vivek; Luong, Eli

    2011-01-01

    The generation of functional vascular networks has the potential to improve treatment for vascular diseases and to facilitate successful organ transplantation. Endothelial colony-forming cells (ECFCs) have robust proliferative potential and can form vascular networks in vivo. ECFCs are recruited from a bone marrow niche to the site of vascularization, where cues from the extracellular matrix instigate vascular morphogenesis. Although this process has been elucidated using natural matrix, little is known about vascular morphogenesis by ECFCs in synthetic matrix, a xeno-free scaffold that can provide a more controllable and clinically relevant alternative for regenerative medicine. We sought to study hyaluronic acid (HA) hydrogels as three-dimensional scaffolds for capillary-like structure formation from ECFCs, and to determine the crucial parameters needed to design such synthetic scaffolds. We found that ECFCs express HA-specific receptors and that vascular endothelial growth factor stimulates hyaluronidase expression in ECFCs. Using a well-defined and controllable three-dimensional HA culture system, we were able to decouple the effect of matrix viscoelasticity from changes in adhesion peptide density. We determined that decreasing matrix viscoelasticity, which corresponds to a loose ultrastructure, significantly increases ECFC vascular tube length and area, and that the effect of local delivery of vascular endothelial growth factor within the hydrogel depends on the makeup of the synthetic environment. Collectively, these results set forth initial design criteria that need to be considered in developing vascularized tissue constructs. PMID:21247340

  18. In vitro culture of mesenchymal lineage cells established from the colonial tunicate Botryllus primigenus.

    PubMed

    Kawamura, Kazuo; Takeoka, Sae; Takahashi, Show; Sunanaga, Takeshi

    2006-03-01

    Body trunks were isolated from juvenile zooids of the Japanese colonial tunicate Botryllus primigenus and cultured in vitro to establish tissue-specific cell lines. Epidermal cells from some explants spread and formed a flat sheet consisting of vacuolated cells. They then dissociated into single cells, and their growth stopped within two weeks. Continuously proliferating cells were established from four explants. After the 20th implantation, nuclear and mitochondrial DNAs were extracted from these cells. The nucleotide sequences of proliferating cell nuclear antigen (PCNA) and mitochondrial large ribosomal RNA (mtlrRNA) completely matched the PCNA and mtlrRNA taken from living colonies of B. primigenus; this shows that the four independently proliferating cells were indeed of the Botryllus origin. One cell line (Bp0306E10) comprised round-shaped cells with a diameter of 8-10 microm. These cells have been cultured in vitro with a doubling time of approximately 24 hours since June, 2003. The BrdU labeling index was approximately 2%. Monoclonal antibodies raised against the cultured cells recognized a 28 kDa polypeptide and stained free mesenchymal cells in vivo. G418-resistant subclonal cells could be established by introducing a tunicate retrotransposon loaded with the neomycin resistance gene into the cells by electroporation. This study is the first to succeed in producing a sustainable cell culture of Botryllus.

  19. Multipotent epithelial cells in the process of regeneration and asexual reproduction in colonial tunicates.

    PubMed

    Kawamura, Kazuo; Sugino, Yasuo; Sunanaga, Takeshi; Fujiwara, Shigeki

    2008-01-01

    The cellular and molecular features of multipotent epithelial cells during regeneration and asexual reproduction in colonial tunicates are described in the present study. The epicardium has been regarded as the endodermal tissue-forming epithelium in the order Enterogona, because only body fragments having the epicardium exhibit the regenerative potential. Epicardial cells in Polycitor proliferus have two peculiar features; they always accompany coelomic undifferentiated cells, and they contain various kinds of organelles in the cytoplasm. During strobilation a large amount of organelles are discarded in the lumen, and then, each tissue-forming cell takes an undifferentiated configuration. Septum cells in the stolon are also multipotent in Enterogona. Free cells with a similar configuration to the septum inhabit the hemocoel. They may provide a pool for epithelial septum cells. At the distal tip of the stolon, septum cells are columnar in shape and apparently undifferentiated. They are the precursor of the stolonial bud. In Pleurogona, the atrial epithelium of endodermal origin is multipotent. In Polyandrocarpa misakiensis, it consists of pigmented squamous cells. The cells have ultrastructurally fine granules in the cytoplasm. During budding, coelomic cells with similar morphology become associated with the atrial epithelium. Then, cells of organ placodes undergo dedifferentiation, enter a cell division cycle, and commence morphogenesis. Retinoic acid-related molecules are involved in this dedifferentiation process of multipotent cells. We conclude that in colonial tunicates two systems support the flexibility of tissue remodeling during regeneration and asexual reproduction; dedifferentiation of epithelial cells and epithelial transformation of coelomic free cells.

  20. Detection of normal B-cell precursors that give rise to colonies producing both kappa and lambda light immunoglobulin chains.

    PubMed Central

    Sauter, H; Paige, C J

    1987-01-01

    The pre-B-cell cloning assay is an in vitro differentiation system in which B-lymphocyte precursors expand and generate colonies containing immunoglobulin-secreting cells. Analysis of surface characteristics, growth requirements, and kinetics suggested that these cells represent early stages of the B-cell differentiation pathway. Here we describe a modification of the assay, which allowed us to determine the differentiative potential of these clonable pre-B cells. Using a nitrocellulose protein-transfer technique, we studied immunoglobulin light chain expression in colonies derived from fetal mouse liver B-cell precursors; in particular, we explored whether the B-cell precursors are already committed to the expression of a particular light chain gene at the initiation of culture. Our results show that fetal liver-derived B-cell progenitors generate colonies in vitro that secrete kappa and lambda light chains at a ratio similar to that found in colonies derived from adult splenic B cells. Further, we document the existence of colonies that are derived from single cells and that simultaneously secrete both types of light chains. This indicates that the progenitors of (kappa + lambda)-producing colonies are light chain-uncommitted at the initiation of culture. These cells are able to rearrange their light chain genes in vitro and differentiate along the B-cell pathway to form colonies secreting both kappa and lambda chains. PMID:3110779

  1. GPR87 mediates lysophosphatidic acid-induced colony dispersal in A431 cells.

    PubMed

    Ochiai, Shoichi; Furuta, Daisuke; Sugita, Kazuya; Taniura, Hideo; Fujita, Norihisa

    2013-09-05

    We have previously reported that an orphan G protein-coupled receptor GPR87 was activated by lysophosphatidic acid (LPA) and that it induced an increase in the intracellular Ca(2+) levels in the CHO cells genetically engineered to express GPR87-Gα16 fusion protein. Because the Ca(2+) response was blocked by the LPA receptor antagonist Ki16425, GPR87 was suggested to be a putative LPA receptor. However, further studies are required to confirm whether GPR87 is an LPA receptor. A previous study showed that colonies of A431 cells treated with LPA showed rapid and synchronized dissociation. Because A431 cells have been shown to express GPR87, we used these cells to examine whether GPR87 acted as an LPA receptor. When A431 cells were treated with gpr87-specific siRNA, the expression of GPR87 was decreased and LPA-induced colony dispersal was significantly reduced. Treatment of the cells with lpa1 siRNA had an additive effect in decrease in the colony dispersal. Studies on the LPA-mediated signaling pathway in A431 cells indicated that transactivation of the epidermal growth factor receptor (EGFR) by LPA led to cell scattering. PD153035, an inhibitor of tyrosine-kinase of EGFR, and BB94, an inhibitor of metalloprotease which produces a ligand for EGFR, significantly prevented the LPA-induced scattering of A431 cells pretreated with lpa1 or gpr87-siRNA. These results strongly suggested that GPR87 acts as an LPA receptor and induces colony dispersal via the transactivation of EGFR in A431 cells. © 2013 Elsevier B.V. All rights reserved.

  2. Endothelial cell colony forming units derived from malignant breast diseases are resistant to tumor necrosis factor-α-induced apoptosis

    PubMed Central

    Chou, Chen-Pin; Jiang, Shih Sheng; Pan, Huay-Ben; Yen, Yi-Chen; Tseng, Hui-Hwa; Hung, Yu-Ting; Wang, Ssu-Han; Chen, Yu-Lin; Chen, Ya-Wen

    2016-01-01

    Mobilisation of endothelial progenitor cells (EPCs) from the bone marrow is a crucial step in the formation of de novo blood vessels, and levels of peripheral blood EPCs have been shown to be elevated in certain malignant states. Using flow cytometry and a Hill-based colony forming unit (CFU) assay, the present study indicated that higher levels of CD34 and vascular endothelial growth factor receptor 2 (VEGFR2) double-positive EPCs, as well as increased formation of endothelial cell colony-forming units (EC-CFUs) are associated with benign and malignant breast diseases, providing possible indicators for breast disease detection. Gene expression profiles revealed a genetic difference between CD34+ VEGFR2+ EPCs and EC-CFUs. Decreased expression of tumour necrosis factor receptor 2 (TNFR2) signalling-related genes and inhibition of tumour necrosis factor (TNF)-induced signalling were demonstrated in EC-CFUs derived from patients with malignant breast disease in comparison with those from healthy controls. Interestingly, our data provided the first evidence that EC-CFUs derived from patients with malignant breast disease were resistant to TNF-α-induced apoptosis, indicating a plausible target for future therapeutic interventions. PMID:27881867

  3. Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

    PubMed

    Bhadriraju, Kiran; Halter, Michael; Amelot, Julien; Bajcsy, Peter; Chalfoun, Joe; Vandecreme, Antoine; Mallon, Barbara S; Park, Kye-Yoon; Sista, Subhash; Elliott, John T; Plant, Anne L

    2016-07-01

    Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events.

  4. Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens

    PubMed Central

    Kjærgaard, Kristian; Schembri, Mark A.; Hasman, Henrik; Klemm, Per

    2000-01-01

    Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation of E. coli cells in static cultures. Additionally, surface display of Ag43 is associated with a distinct frizzy colony morphology in E. coli. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important Pseudomonas fluorescens strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between E. coli and P. fluorescens. Furthermore, Ag43 expression enhances biofilm formation in P. fluorescens to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other gram-negative bacteria. Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants. PMID:10940019

  5. Antigen 43 from Escherichia coli induces inter- and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens.

    PubMed

    Kjaergaard, K; Schembri, M A; Hasman, H; Klemm, P

    2000-09-01

    Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation of E. coli cells in static cultures. Additionally, surface display of Ag43 is associated with a distinct frizzy colony morphology in E. coli. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important Pseudomonas fluorescens strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between E. coli and P. fluorescens. Furthermore, Ag43 expression enhances biofilm formation in P. fluorescens to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other gram-negative bacteria. Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants.

  6. Maternal Body-Mass Index and Cord Blood Circulating Endothelial Colony-Forming Cells

    PubMed Central

    Lin, Ruei-Zeng; Miranda, Maria L.; Vallejo-Vaz, Antonio J.; Stiefel, Pablo; Praena-Fernández, Juan M.; Bernal-Bermejo, Jose; Jimenez-Jimenez, Luis M.; Villar, Jose; Melero-Martin, Juan M.

    2013-01-01

    Objective Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that are particularly abundant in umbilical cord blood. We sought to determine whether ECFC abundance in cord blood is associated with maternal body-mass index (BMI) in non-pathological pregnancies. Study design We measured the level of ECFCs in the cord blood of neonates (n=27) born from non-obese healthy mothers with non-pathological pregnancies and examined whether ECFC abundance correlated with maternal BMI. We also examined the effect of maternal BMI on ECFC phenotype and function using angiogenic and vasculogenic assays. Results We observed variation in ECFC abundance among subjects and found a positive correlation between pre-pregnancy maternal BMI and ECFC content (r=0.51, P=0.007), which was independent of other obstetric factors. Despite this variation, ECFC phenotype and functionality were deemed normal and highly similar between subjects with maternal BMI <25 kg/m2 and BMI between 25–30 kg/m2, including the ability to form vascular networks in vivo. Conclusions This study underlines the need to consider maternal BMI as a potential confounding factor for cord blood levels of ECFCs in future comparative studies between healthy and pathological pregnancies. Endothelial colony-forming cells (ECFCs) are a subset of progenitor cells that circulate in peripheral blood and can give rise to endothelial cells (1,2), contributing to the formation of new vasculature and the maintenance of vascular integrity (3–5). The mechanisms that regulate the abundance of these cells in vivo remain poorly understood. ECFCs are rare in adult peripheral blood (1,2,10). In contrast, there is an elevated number of these cells in fetal blood during the third trimester of pregnancy (11–13). Emerging evidence indicates that deleterious conditions during fetal life can impair ECFC content and function. For instance, offspring of diabetic mothers have been shown to have

  7. Assessing the potential of colony morphology for dissecting the CFU-F population from human bone marrow stromal cells.

    PubMed

    Gothard, D; Dawson, J I; Oreffo, R O C

    2013-05-01

    Mesenchymal stem cells (MSCs) provide an ideal cell source for bone tissue engineering strategies. However, bone marrow stromal cell (BMSC) populations that contain MSCs are highly heterogeneous expressing a wide variety of proliferative and differentiation potentials. Current MSC isolation methods employing magnetic-activated and fluorescent-activated cell sorting can be expensive and time consuming and, in the absence of specific MSC markers, fail to generate homogeneous populations. We have investigated the potential of various colony morphology descriptors to provide correlations with cell growth potential. Density-independent colony forming unit-fibroblastic (CFU-F) capacity is a MSC prerequisite and resultant colonies display an array of shapes and sizes that might be representative of cell function. Parent colonies were initially categorised according to their diameter and cell density and grouped before passage for the subsequent assessment of progeny colonies. Whereas significant morphological differences between distinct parent populations indicated a correlation with immunophenotype, enhanced CFU-F capacity was not observed when individual colonies were isolated according to these morphological parameters. Colony circularity, an alternative morphological measure, displayed a strong correlation with subsequent cell growth potential. The current study indicates the potential of morphological descriptors for predicting cell growth rate and suggests new directions for research into dissection of human BMSC CFU-F populations.

  8. Colony Expansion of Socially Motile Myxococcus xanthus Cells Is Driven by Growth, Motility, and Exopolysaccharide Production

    PubMed Central

    Patra, Pintu; Kissoon, Kimberley; Cornejo, Isabel; Kaplan, Heidi B.; Igoshin, Oleg A.

    2016-01-01

    Myxococcus xanthus, a model organism for studies of multicellular behavior in bacteria, moves exclusively on solid surfaces using two distinct but coordinated motility mechanisms. One of these, social (S) motility is powered by the extension and retraction of type IV pili and requires the presence of exopolysaccharides (EPS) produced by neighboring cells. As a result, S motility requires close cell-to-cell proximity and isolated cells do not translocate. Previous studies measuring S motility by observing the colony expansion of cells deposited on agar have shown that the expansion rate increases with initial cell density, but the biophysical mechanisms involved remain largely unknown. To understand the dynamics of S motility-driven colony expansion, we developed a reaction-diffusion model describing the effects of cell density, EPS deposition and nutrient exposure on the expansion rate. Our results show that at steady state the population expands as a traveling wave with a speed determined by the interplay of cell motility and growth, a well-known characteristic of Fisher’s equation. The model explains the density-dependence of the colony expansion by demonstrating the presence of a lag phase–a transient period of very slow expansion with a duration dependent on the initial cell density. We propose that at a low initial density, more time is required for the cells to accumulate enough EPS to activate S-motility resulting in a longer lag period. Furthermore, our model makes the novel prediction that following the lag phase the population expands at a constant rate independent of the cell density. These predictions were confirmed by S motility experiments capturing long-term expansion dynamics. PMID:27362260

  9. FTIR imaging of the 3D extracellular matrix used to grow colonies of breast cancer cell lines.

    PubMed

    Smolina, Margarita; Goormaghtigh, Erik

    2016-01-21

    Infrared imaging was applied to investigate a reconstituted basement membrane, known as Matrigel, in three-dimensional cell cultures. Matrigel, in the vicinity of the colonies, was examined for four breast cancer cell lines presenting different 3D colony morphologies. MCF-7 and T-47D present mass colonies, SKBR-3 grape-like colonies and MDA-MB-231 stellate colonies associated with a more invasive phenotype. The edge of the cell colonies was found to be significantly depleted in Matrigel. Except in a limited number of cases, Matrigel appeared to be thinner at the edges of the colonies but not completely destroyed or torn off as it would be for a purely mechanical effect. When a PCA was run on the spectra of one or several colonies, the score images on PC#3 and PC#4 presented structures in the Matrigel areas which appeared as fringes, lines, dots or regular patterns. This effect represents a very small fraction of the total variance but is reproducible for all the 4 cell lines. PC#4 presents systematically a maximum near 1624 cm(-1) and a minimum around 1700 cm(-1). When spectra are normalized, the effect is less marked but does not disappear. The nature of the variations that exist in the Matrigel layer is therefore not solely related to thickness but also to the chemical composition. At this stage, the weakness of the effect prevents a thorough investigation.

  10. In Vivo Growing of New Cell Colonies in a Portion of Bone Marrow: Potential Use for Indirect Cell Therapy

    PubMed Central

    Manzanedo, Ana; Rodriguez, Fidel; Obeso, Jose A.; Rodriguez, Manuel

    2010-01-01

    The ability of bone marrow cells (BMCs) to migrate to different organs can be used for indirect cell therapy, a procedure based on the engraftment of therapeutic cells in a different place from where they will finally move to and perform their action and which could be particularly useful for chronic illness where a persistent and long-lasting therapeutic action is required. Thus, establishing a stable colony of engineered BMCs is a requisite for the chronic provision of damaged tissues with engineered cells. Reported here is a procedure for creating such a cell colony in a portion of the bone marrow (BM). The study was performed in C57BL/6j mice and consisted of developing a focal niche in a portion of the bone marrow with focal irradiation so that it could be selectively colonized by BM cells (C57BL/6-FG-VC-GFP mice) injected in the blood stream. Both the arrival of cells coming from the nonirradiated BM (week 1 after irradiation) and the proliferation of cells in the irradiated BM (week 2) prevented the homing of injected cells in the BM niche. However, when BMCs were injected in a time window about 48 h after irradiation they migrated to the BM niche where they established a cell colony able to: 1) survive for a long period of time [the percentage of injected cells increased in the BM from day 30 postinjection (15%) to day 110 postinjection 28%)]; 2) express cell differentiation markers (90% of them were lineage committed 4 weeks after engraftment); and 3) colonize to the blood stream (with 5% and 9% of all blood cells being computed 1 and 3 months after engraftment, respectively). The intravenous injection of BMCs in combination with a previous transitory focal myeloablation is a safe and easy method for creating the long-lasting colony of modified BMCs needed for treating chronic and progressive illness with indirect cell therapy. PMID:26966633

  11. Micronucleus formation induced by dielectric barrier discharge plasma exposure in brain cancer cells

    NASA Astrophysics Data System (ADS)

    Kaushik, Nagendra K.; Uhm, Hansup; Ha Choi, Eun

    2012-02-01

    Induction of micronucleus formation (cytogenetic damage) in brain cancer cells upon exposure of dielectric barrier discharge plasma has been investigated. We have investigated the influence of exposure and incubation times on T98G brain cancer cells by using growth kinetic, clonogenic, and micronucleus formation assay. We found that micronucleus formation rate directly depends on the plasma exposure time. It is also shown that colony formation capacity of cells has been inhibited by the treatment of plasma at all doses. Cell death and micronucleus formation are shown to be significantly elevated by 120 and 240 s exposure of dielectric barrier discharge plasma.

  12. A mathematical description of a growing cell colony based on the mechanical bidomain model

    NASA Astrophysics Data System (ADS)

    Auddya, Debabrata; Roth, Bradley J.

    2017-03-01

    The mechanical bidomain model is used to describe a colony of cells growing on a substrate. Analytical expressions are derived for the intracellular and extracellular displacements. Mechanotransduction events are driven by the difference between the displacements in the two spaces, corresponding to the force acting on integrins. The equation for the displacement consists of two terms: one proportional to the radius that is the same in the intracellular and extracellular spaces (the monodomain term) and one that is proportional to a modified Bessel function that is responsible for mechanotransduction (the bidomain term). The model predicts that mechanotransduction occurs within a few length constants of the colony’s edge, and an expression for the length constant contains the intracellular and extracellular shear moduli and the spring constant of the integrins coupling the two spaces. The model predictions are qualitatively consistent with experiments on human embryonic stem cell colonies, in which differentiation is localized near the edge.

  13. Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture.

    PubMed

    Azizi, Hossein; Skutella, Thomas; Shahverdi, Abdolhossein

    2017-01-01

    The properties of self-renewal and division in spermatogonial stem cells (SSCs) support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and selfrenewal of germline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system. In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco's modified Eagle's medium (DMEM) medium (CTRL group), 10% fetal bovine serum (FBS)+DMEM (10% group), and growth factor (G group) that contained 2% FBS, glial cell-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry against the germ cell markers α6, β1, c-Kit, Thy-1, c-Ret, Plzf, and Oct4. The statistical significance between mean values in different groups was determined by one-way analysis of variance (ANOVA). We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression (P<0.05) of germ cell markers in the G and 10% groups versus the testis cells (T). Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermato- gonial stem-like colonies were partially positive. A non

  14. Some observations on the three-dimensional growth of L5178Y cell colonies in soft agar culture.

    NASA Technical Reports Server (NTRS)

    Dalen, H.; Burki, H. J.

    1971-01-01

    The three-dimensional organization of spherical colonies formed by L5178Y cells grown in soft agar cultures was investigated by light and scanning electron microscopy. Visible colonies were formed after 7 days of incubation and increased in size for more than 2 weeks. At this time the colonies contained a central core of necrotic cells surrounded by an outer shell of normal-looking cells in loose contact with each other. Cross sectional radioautographs revealed that tritiated precursors were incorporated only into those cells in the ?viable cell' shell and not in the necrotic center of the colony. It is pointed out that increased knowledge of the factors leading to this type of three-dimensional organization is of particular interest, since it is similar to the conditions found in certain types of solid tumors (Thomlinson and Gray, 1955).

  15. Reduced mitotic activity at the periphery of human embryonic stem cell colonies cultured in vitro with mitotically-inactivated murine embryonic fibroblast feeder cells.

    PubMed

    Heng, Boon Chin; Cao, Tong; Liu, Hua; Rufaihah, Abdul Jalil

    2005-01-01

    This study attempted to investigate whether different levels of mitotic activity exist within different physical regions of a human embryonic stem (hES) cell colony. Incorporation of 5-bromo-2-deoxyuridine (BrdU) within newly-synthesized DNA, followed by immunocytochemical staining was used as a means of detecting mitotically-active cells within hES colonies. The results showed rather surprisingly that the highest levels of mitotic activity are primarily concentrated within the central regions of hES colonies, whereas the peripheral regions exhibited reduced levels of cellular proliferation. Two hypothetical mechanisms are therefore proposed for hES colony growth and expansion. Firstly, it is envisaged that the less mitotically-active hES cells at the periphery of the colony are continually migrating outwards, thereby providing space for newly-divided daughter cells within the more mitotically-active central region of the hES colony. Secondly, it is proposed that the newly-divided hES cells within the central region of the colony somehow migrate to the outer periphery. This could possibly explain why the periphery of hES colonies are less mitotically-active, since there would obviously be an extended time-lag before newly-divided daughter cells are ready again for the next cell division. Further investigations need to be carried out to characterize the atypical mechanisms by which hES colonies grow and expand in size.

  16. Recovery of hematopoietic colony-forming cells in irradiated mice pretreated with interleukin 1 (IL-1)

    SciTech Connect

    Schwartz, G.N.; Neta, R.; Vigneulle, R.M.; Patchen, M.L.; MacVittie, T.J.

    1988-01-01

    Data in this report determined the effect of a single injection of recombinant interleukin 1 a (rIL-1) prior to irradiation of B6D2F1 mice on the recovery of colony-forming cells (CFC) at early and late times after sublethal and lethal doses of radiation, Injection of rIL-1 promoted an earlier recovery of mature cells in the blood and CFC in the bone marrow and spleen. For example, 8 days after 6.5-Gy irradiation, the number of CFU-E (colony-forming units erythroid), BFU-E (burst-forming units-erythroid), and GMCFC (granulocyte-macrophage colony-forming cells) per femur was approximately 1.5-fold higher in rIL-injected mice than in saline-injected mice. Also, 5, 9, and 12 days after irradiation, the number of both day 8 and day 12 CFU-S (colony-forming units-spleen) was almost twofold greater in bone marrow from rIL1-injected mice. The earlier recovery of CFU-S in rIL-1 injected mice was not associated with an increase in the number of CFU-S that survived immediately after irradiation. Also, 7 months after irradiation, the number of CFU-S per femur of both saline-and rIL-1 injected mice was still < 50% of normal values. Data in this report demonstrate that a single injection of rIL-1 prior to irradiation accelerates early hematopoietic recovery in irradiation mice, but does not prevent expression of radiation-induced front-end damage damage to hematopoietic tissues.

  17. Interplay between Intrinsic Noise and the Stochasticity of the Cell Cycle in Bacterial Colonies

    PubMed Central

    Canela-Xandri, Oriol; Sagués, Francesc; Buceta, Javier

    2010-01-01

    Abstract Herein we report on the effects that different stochastic contributions induce in bacterial colonies in terms of protein concentration and production. In particular, we consider for what we believe to be the first time cell-to-cell diversity due to the unavoidable randomness of the cell-cycle duration and its interplay with other noise sources. To that end, we model a recent experimental setup that implements a protein dilution protocol by means of division events to characterize the gene regulatory function at the single cell level. This approach allows us to investigate the effect of different stochastic terms upon the total randomness experimentally reported for the gene regulatory function. In addition, we show that the interplay between intrinsic fluctuations and the stochasticity of the cell-cycle duration leads to different constructive roles. On the one hand, we show that there is an optimal value of protein concentration (alternatively an optimal value of the cell cycle phase) such that the noise in protein concentration attains a minimum. On the other hand, we reveal that there is an optimal value of the stochasticity of the cell cycle duration such that the coherence of the protein production with respect to the colony average production is maximized. The latter can be considered as a novel example of the recently reported phenomenon of diversity induced resonance. PMID:20513389

  18. Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images

    PubMed Central

    Haponen, Markus; Rasku, Jyrki

    2016-01-01

    The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient's cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using a k-NN classifier showing improved accuracy compared to earlier studies. PMID:27493680

  19. Plasma erythropoietin assay by a fetal mouse liver cell culture method with special reference to effective elimination of erythroid colony inhibitor(s) in plasma.

    PubMed

    Sakata, S; Enoki, Y; Nakatani, A; Kohzuki, H; Ohga, Y; Shimizu, S

    1987-03-01

    Methods for the elimination of an inhibitor(s) of erythroid colony formation from plasma were examined in an attempt to measure genuine plasma erythropoietin (Epo) activities with an erythroid colony-forming assay using fetal mouse liver cells. Acid-boiling-chloroform (ABC) treatment was concluded to be the best method because the plasma thus treated stimulated colony formation most and contained the least protein. The dose-response curve for the plasma was parallel to that for the standard Epo preparation. The "erythroid colony-stimulating activity" in the plasma was completely additive to that in the standard Epo, and appeared to be a relatively heat-stable and acid protein with an isoelectric point lower than 5.0. These results suggest that the activity in the plasma is identical to that in the standard Epo. Stability of the plasma Epo activity was dependent on storage temperature and enhanced by adding 1% bovine serum albumin (BSA). Average Epo titers for normal adult, full-term cord, and murine plasmas, all ABC-treated and with 1% BSA added, were 192.4, 184.5, and 150.6 mU/ml, respectively. These values were much higher than those measured by the in vivo standard polycythemic mouse assay.

  20. Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells

    PubMed Central

    Al-Maleki, Anis Rageh; Mariappan, Vanitha; Vellasamy, Kumutha Malar; Tay, Sun Tee; Vadivelu, Jamuna

    2015-01-01

    Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV]) to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA) and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno) is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk), ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis. PMID:25996927

  1. A Novel Combinatorial Therapy With Pulp Stem Cells and Granulocyte Colony-Stimulating Factor for Total Pulp Regeneration

    PubMed Central

    Iohara, Koichiro; Murakami, Masashi; Takeuchi, Norio; Osako, Yohei; Ito, Masataka; Ishizaka, Ryo; Utunomiya, Shinji; Nakamura, Hiroshi; Matsushita, Kenji

    2013-01-01

    Treatment of deep caries with pulpitis is a major challenge in dentistry. Stem cell therapy represents a potential strategy to regenerate the dentin-pulp complex, enabling conservation and restoration of teeth. The objective of this study was to assess the efficacy and safety of pulp stem cell transplantation as a prelude for the impending clinical trials. Clinical-grade pulp stem cells were isolated and expanded according to good manufacturing practice conditions. The absence of contamination, abnormalities/aberrations in karyotype, and tumor formation after transplantation in an immunodeficient mouse ensured excellent quality control. After autologous transplantation of pulp stem cells with granulocyte-colony stimulating factor (G-CSF) in a dog pulpectomized tooth, regenerated pulp tissue including vasculature and innervation completely filled in the root canal, and regenerated dentin was formed in the coronal part and prevented microleakage up to day 180. Transplantation of pulp stem cells with G-CSF yielded a significantly larger amount of regenerated dentin-pulp complex compared with transplantation of G-CSF or stem cells alone. Also noteworthy was the reduction in the number of inflammatory cells and apoptotic cells and the significant increase in neurite outgrowth compared with results without G-CSF. The transplanted stem cells expressed angiogenic/neurotrophic factors. It is significant that G-CSF together with conditioned medium of pulp stem cells stimulated cell migration and neurite outgrowth, prevented cell death, and promoted immunosuppression in vitro. Furthermore, there was no evidence of toxicity or adverse events. In conclusion, the combinatorial trophic effects of pulp stem cells and G-CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications. PMID:23761108

  2. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions

    PubMed Central

    Beers, Jeanette; Gulbranson, Daniel R.; George, Nicole; Siniscalchi, Lauren I.; Jones, Jeffrey; Thomson, James A.; Chen, Guokai

    2013-01-01

    This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (iPSCs). In this protocol, passaging one six-well or 10 cm plate of cells takes about 6–7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation, or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human pluripotent stem cells in E8 medium. PMID:23099485

  3. A mutagen-testing assay based on heterogeneity in diameter and integrated optical density of mammalian cell colonies.

    PubMed Central

    Dairkee, S H; Glaser, D A

    1984-01-01

    We investigated the effects of the well-known mutagenic agents ethyl methanesulfonate (EtMes), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and ICR-191 on colonies of the Chinese hamster ovary line CHO cultured on a semisolid substrate. These agents induced heterogeneity in diameter and integrated optical density of colonies as determined by computer-assisted photography and subsequent analysis of the images of the colonies. When CHO colonies were exposed to agents such as urethane that are not known to be mutagenic in mammalian systems or to activation-requiring mutagens such as cyclophosphamide, there was no noticeable effect on the distribution of colony diameter and volume. Similarly, nonmutagenic agents such as dimethyl sulfoxide (Me2SO) also did not induce heterogeneity in colony diameter and integrated optical density. Our observations recommend the use of agar-grown mammalian cell colonies for predictive testing of chemical mutagens and carcinogens in a simple, in vitro mammalian cell assay. This assay system, unlike other mammalian cell culture assays, allows detection and measurement of the simultaneous effects of chemical mutagens on several genetic and non-genetic targets and, thus, may emulate more closely the potential hazards of these agents in vivo. PMID:6585791

  4. Enhancement of Umbilical Cord Blood Cell Hematopoiesis by Maitake Beta-Glucan Is Mediated by Granulocyte Colony-Stimulating Factor Production▿

    PubMed Central

    Lin, Hong; Cheung, Sandy W. Y.; Nesin, Mirjana; Cassileth, Barrie R.; Cunningham-Rundles, Susanna

    2007-01-01

    Maitake beta-glucan (MBG) is an extract from the fruit body of the Grifola frondosa mushroom that is being widely used to treat cancer in Asia. We have previously reported that MBG enhances mouse bone marrow cell (BMC) hematopoiesis in vitro and protects BMC from doxorubicin (DOX) toxicity. In the current study, we investigated the ability of MBG to enhance hematopoiesis and to reduce the toxic effects of DOX on fresh human umbilical cord blood (CB) cells. MBG treatment significantly enhanced the colony formation unit (CFU) response of granulocytes-macrophages (CFU-GM response) over the whole dose range of 12.5 to 100 μg/ml (P < 0.05). The addition of MBG to DOX-treated CB cells significantly protected granulocyte-macrophage colony formation from the toxicity of DOX, which otherwise produced strong hematopoietic repression. MBG also partially replaced recombinant human granulocyte colony-stimulating factor (rhG-CSF), as shown by a significant augmentation of the CFU-GM response in the absence of rhG-CSF. We found that MBG induces granulocyte colony-stimulating factor (G-CSF) production in CB CD33+ monocytes, as detected by intracellular cytokine flow cytometric assessment. In contrast, we found that adult peripheral blood monocytes did not produce a significant G-CSF response to MBG, whereas both adult and CB monocytes produced G-CSF in response to lipopolysaccharide. These studies provide the first evidence that MBG induces hematopoietic stem cell proliferation and differentiation of CFU-GM in umbilical CB cells and acts directly to induce G-CSF. PMID:17093103

  5. [Knockdown of Larp4b in Lin(-) cells does not affect the colony forming ability of mouse hematopoietic cells].

    PubMed

    Wang, Xiao-Juan; Pang, Ya-Kun; Cheng, Hui; Dong, Fang; Liang, Hao-Yue; Zhang, Ying-Chi; Wang, Xiao-Min; Xu, Jing; Cheng, Tao; Yuan, Wei-Ping

    2013-06-01

    Larp4b is a member of the LARP family, which can interact with RNA and generally stimulate the translation of mRNA. Abnormal expression of Larp4b can be found in leukemia patients in our previous study. This study was purposed to detect the relative expression of Larp4b mRNA in different subpopulations of mouse hematopoietic cells, to construct lentivirus vector containing shLarp4b targeting mouse gene Larp4b and to explore its effects on mouse Lin(-) cells infected with shLarp4b by lentivirus. SF-LV-shLarP4b-EGFP and control vectors were constructed and two-plasmid lentivirus packing system was used to transfect 293T cells. After 48 h and 72 h, lentivirus SF-LV-shLarp4b-EGFP was harvested and was used to infect Lin(-) cells. After 48 h, EGFP(+) cells was sorted by flow cytometry (FCM). Meanwhile, semi-quantitative real time-PCR, AnnexinV-PE/7-AAD staining, PI staining and colony forming cell assay (CFC) were performed to determine the expression of Larp4b and its effect on the proliferation of hematopoietic progenitor cells. The results showed that Larp4b was highly expressed in myeloid cells. SF-LV-shLarp4b-EGFP was successfully constructed according to the restriction endonuclease digestion assay. RT-PCR confirmed that Larp4b was efficiently knockdown in mouse Lin(-) cells. The low expression of Larp4b did not affect the colony forming number, the apoptosis and cell cycle of Lin(-) cells. It is concluded that knockdown of Larp4b in mouse Lin(-) cells do not contribute to the colony forming ability and the growth of Lin(-) cells in vitro. This useful knockdown system will be used to study in vivo Larp4b in future.

  6. Error-Correcting Output Codes in Classification of Human Induced Pluripotent Stem Cell Colony Images.

    PubMed

    Joutsijoki, Henry; Haponen, Markus; Rasku, Jyrki; Aalto-Setälä, Katriina; Juhola, Martti

    2016-01-01

    The purpose of this paper is to examine how well the human induced pluripotent stem cell (hiPSC) colony images can be classified using error-correcting output codes (ECOC). Our image dataset includes hiPSC colony images from three classes (bad, semigood, and good) which makes our classification task a multiclass problem. ECOC is a general framework to model multiclass classification problems. We focus on four different coding designs of ECOC and apply to each one of them k-Nearest Neighbor (k-NN) searching, naïve Bayes, classification tree, and discriminant analysis variants classifiers. We use Scaled Invariant Feature Transformation (SIFT) based features in classification. The best accuracy (62.4%) is obtained with ternary complete ECOC coding design and k-NN classifier (standardized Euclidean distance measure and inverse weighting). The best result is comparable with our earlier research. The quality identification of hiPSC colony images is an essential problem to be solved before hiPSCs can be used in practice in large-scale. ECOC methods examined are promising techniques for solving this challenging problem.

  7. Error-Correcting Output Codes in Classification of Human Induced Pluripotent Stem Cell Colony Images

    PubMed Central

    Haponen, Markus; Rasku, Jyrki

    2016-01-01

    The purpose of this paper is to examine how well the human induced pluripotent stem cell (hiPSC) colony images can be classified using error-correcting output codes (ECOC). Our image dataset includes hiPSC colony images from three classes (bad, semigood, and good) which makes our classification task a multiclass problem. ECOC is a general framework to model multiclass classification problems. We focus on four different coding designs of ECOC and apply to each one of them k-Nearest Neighbor (k-NN) searching, naïve Bayes, classification tree, and discriminant analysis variants classifiers. We use Scaled Invariant Feature Transformation (SIFT) based features in classification. The best accuracy (62.4%) is obtained with ternary complete ECOC coding design and k-NN classifier (standardized Euclidean distance measure and inverse weighting). The best result is comparable with our earlier research. The quality identification of hiPSC colony images is an essential problem to be solved before hiPSCs can be used in practice in large-scale. ECOC methods examined are promising techniques for solving this challenging problem. PMID:27847810

  8. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

    PubMed Central

    Lee, Kyung Hoon; Lee, Won Young; Kim, Jin Hoi; Park, Chan Kyu; Do, Jeong Tae; Kim, Jae Hwan; Choi, Young Suk; Kim, Nam Hyung; Song, Hyuk

    2016-01-01

    Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months) limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells. PMID:26880974

  9. Relevance of density, size and DNA content of tumour cells to the lung colony assay.

    PubMed Central

    Grdina, D. J.; Hittelman, W. N.; White, R. A.; Meistrich, M. L.

    1977-01-01

    Mouse fibrosarcoma tumours were dissociated and divided into subpopulations of viable cells by centrifugation in linear density gradients of Renografin. Two of these subpopulations, designated Band 2 and Band 4, differed in their clonogenic ability in lung colony assay. The less dense Band 2 cells were significantly more clonogenic than the Band 4 cells (2.9 percent vs 1.4 percent respectively). Each band was further separated on the basis of cell size by centrifugal elutriation. Each size class of cells comprising Band 2 showed higher clonogenic ability than the corresponding size class in Band 4. Thus cell size differences were not responsible for the clonogenic differences between these bands. To determine whether cell-cycle distribution of the tumour cells was responsible for differences in cloning efficiency, flow microfluorometric and premature chromosome condensation methods were utilized. The unseparated and Band 4 populations showed a higher percentage of cells in S and G2 than did the Band 2 populations, but many of the S and G2 tumour cells showed extensive chromosome damage. From this study we conclude that the increased clonogenic ability of the lighter tumour cells is not due to differences in cell size or cell-cycle parameters. Images Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:563726

  10. A system for automatically scanning tissue culture dishes to detect fluorescently labeled cell colonies

    NASA Astrophysics Data System (ADS)

    Goldstein, Seth R.; Guan-Xiong, Zhou; Siegel, Ruth E.; Brownstein, Michael J.

    1989-07-01

    A microprocessor-controlled system has been developed to automatically scan tissue culture dishes in order to detect and locate fluorescently labeled cell colonies for subsequent cloning. An X-Y recorder mechanism moves an 80-mm-diam dish in a raster scan through a stationary laser beam adjacent to a photomultiplier tube equipped with an emission filter. Front-end electronics processes the PMT signal to screen out small-scale fluorescent artifacts of selectable size (e.g., <0.2 mm). Appropriate signals are input to the computer which then interrupts the raster scan to perform a limited fine scan of the dish to accurately localize the fluorescent spot position. An additional artifact test using a different filter is then performed. The underside of the dish is scribed at the location of spots that pass this test. Using fluorescein as a label, fluorescence as low as 25% above intrinsic cell background fluorescence can be detected. The fluorescence signal level threshhold can be set (e.g., 70% above intrinsic cell background) to within an accuracy of approximately 15% of intrinsic cell background fluorescence, and the system will reliably find colonies with a signal exceeding that level. With the above typical values, the number of false positives is typically less than five per dish and the time to completely scan an 80-mm-diam tissue culture dish is 2-4 min.

  11. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.

    PubMed

    Beers, Jeanette; Gulbranson, Daniel R; George, Nicole; Siniscalchi, Lauren I; Jones, Jeffrey; Thomson, James A; Chen, Guokai

    2012-11-01

    This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol, passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium.

  12. Increase in Bacterial Colony Formation from a Permafrost Ice Wedge Dosed with a Tomitella biformata Recombinant Resuscitation-Promoting Factor Protein.

    PubMed

    Puspita, Indun Dewi; Kitagawa, Wataru; Kamagata, Yoichi; Tanaka, Michiko; Nakatsu, Cindy H

    2015-01-01

    Resuscitation-promoting factor (Rpf) is a protein that has been found in a number of different Actinobacteria species and has been shown to promote the growth of active cells and resuscitate dormant (non-dividing) cells. We previously reported the biological activity of an Rpf protein in Tomitella biformata AHU 1821(T), an Actinobacteria isolated from a permafrost ice wedge. This protein is excreted outside the cell; however, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. Therefore, the aim of the present study was to determine whether Rpf from T. biformata impacted the cultivation of other bacteria from the permafrost ice wedge from which it was originally isolated. All experiments used recombinant Rpf proteins produced using a Rhodococcus erythropolis expression system. Dilutions of melted surface sterilized ice wedge samples mixed with different doses of the purified recombinant Rpf (rRpf) protein indicated that the highest concentration tested, 1250 pM, had a significantly (p <0.05) higher number of CFUs on agar plates after 8 d, approximately 14-fold higher than that on control plates without rRpf. 16S rRNA gene sequences revealed that all the colonies on plates were mainly related to Brevibacterium antiquum strain VKM Ac-2118 (AY243344), with 98-99% sequence identity. This species is also a member of the phylum Actinobacteria and was originally isolated from Siberian permafrost sediments. The results of the present study demonstrated that rRpf not only promoted the growth of T. biformata from which it was isolated, but also enhanced colony formation by another Actinobacteria in an environmental sample.

  13. Increase in Bacterial Colony Formation from a Permafrost Ice Wedge Dosed with a Tomitella biformata Recombinant Resuscitation-Promoting Factor Protein

    PubMed Central

    Puspita, Indun Dewi; Kitagawa, Wataru; Kamagata, Yoichi; Tanaka, Michiko; Nakatsu, Cindy H.

    2015-01-01

    Resuscitation-promoting factor (Rpf) is a protein that has been found in a number of different Actinobacteria species and has been shown to promote the growth of active cells and resuscitate dormant (non-dividing) cells. We previously reported the biological activity of an Rpf protein in Tomitella biformata AHU 1821T, an Actinobacteria isolated from a permafrost ice wedge. This protein is excreted outside the cell; however, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. Therefore, the aim of the present study was to determine whether Rpf from T. biformata impacted the cultivation of other bacteria from the permafrost ice wedge from which it was originally isolated. All experiments used recombinant Rpf proteins produced using a Rhodococcus erythropolis expression system. Dilutions of melted surface sterilized ice wedge samples mixed with different doses of the purified recombinant Rpf (rRpf) protein indicated that the highest concentration tested, 1250 pM, had a significantly (p <0.05) higher number of CFUs on agar plates after 8 d, approximately 14-fold higher than that on control plates without rRpf. 16S rRNA gene sequences revealed that all the colonies on plates were mainly related to Brevibacterium antiquum strain VKM Ac-2118 (AY243344), with 98–99% sequence identity. This species is also a member of the phylum Actinobacteria and was originally isolated from Siberian permafrost sediments. The results of the present study demonstrated that rRpf not only promoted the growth of T. biformata from which it was isolated, but also enhanced colony formation by another Actinobacteria in an environmental sample. PMID:25843055

  14. Acute leukemia after radiotherapy in a patient with Turcot's syndrome. Impaired colony formation in skin fibroblast cultures after irradiation

    SciTech Connect

    Li, F.P.; Little, J.B.; Bech-Hansen, N.T.; Paterson, M.C.; Arlett, C.; Garnick, M.B.; Mayer, R.J.

    1983-02-01

    Colonic polyposis and carcinoma developed in a woman with Turcot's syndrome at the age of 31 years; astrocytoma developed when she was 37. Her brother and sister had died of astrocytoma at the ages of 18 and 33 years, respectively. Progressive neutropenia developed in the patient three months after radiotherapy for her brain tumor and acute myelomonocytic leukemia 19 months after treatment. Three laboratories independently evaluated cultures of her skin fibroblasts for in vitro sensitivity to cell killing (loss of colony-forming ability) by x-rays. Survival assays consistently revealed slight but significant radiosensitivity in an early-passage (six to 10 doublings) fibroblast subculture. A later subculture (21 to 29 doublings) showed no abnormality, a possible effect of selective in vitro loss of radiosensitive cells.

  15. An integrated systems biology approach to understanding the rules of keratinocyte colony formation.

    PubMed

    Sun, Tao; McMinn, Phil; Coakley, Simon; Holcombe, Mike; Smallwood, Rod; Macneil, Sheila

    2007-12-22

    Closely coupled in vitro and in virtuo models have been used to explore the self-organization of normal human keratinocytes (NHK). Although it can be observed experimentally, we lack the tools to explore many biological rules that govern NHK self-organization. An agent-based computational model was developed, based on rules derived from literature, which predicts the dynamic multicellular morphogenesis of NHK and of a keratinocyte cell line (HaCat cells) under varying extracellular Ca++ concentrations. The model enables in virtuo exploration of the relative importance of biological rules and was used to test hypotheses in virtuo which were subsequently examined in vitro. Results indicated that cell-cell and cell-substrate adhesions were critically important to NHK self-organization. In contrast, cell cycle length and the number of divisions that transit-amplifying cells could undergo proved non-critical to the final organization. Two further hypotheses, to explain the growth behaviour of HaCat cells, were explored in virtuo-an inability to differentiate and a differing sensitivity to extracellular calcium. In vitro experimentation provided some support for both hypotheses. For NHKs, the prediction was made that the position of stem cells would influence the pattern of cell migration post-wounding. This was then confirmed experimentally using a scratch wound model.

  16. Differentiation-Independent Fluctuation of Pluripotency-Related Transcription Factors and Other Epigenetic Markers in Embryonic Stem Cell Colonies

    PubMed Central

    Šustáčková, Gabriela; Legartová, Soňa; Kozubek, Stanislav; Stixová, Lenka; Pacherník, Jiří

    2012-01-01

    Embryonic stem cells (ESCs) maintain their pluripotency through high expression of pluripotency-related genes. Here, we show that differing levels of Oct4, Nanog, and c-myc proteins among the individual cells of mouse ESC (mESC) colonies and fluctuations in these levels do not disturb mESC pluripotency. Cells with strong expression of Oct4 had low levels of Nanog and c-myc proteins and vice versa. In addition, cells with high levels of Nanog tended to occupy interior regions of mESC colonies. In contrast, peripherally positioned cells within colonies had dense H3K27-trimethylation, especially at the nuclear periphery. We also observed distinct levels of endogenous and exogenous Oct4 in particular cell cycle phases. The highest levels of Oct4 occurred in G2 phase, which correlated with the pKi-67 nuclear pattern. Moreover, the Oct4 protein resided on mitotic chromosomes. We suggest that there must be an endogenous mechanism that prevents the induction of spontaneous differentiation, despite fluctuations in protein levels within an mESC colony. Based on the results presented here, it is likely that cells within a colony support each other in the maintenance of pluripotency. PMID:21609209

  17. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells.

    PubMed

    Choudhary, Geetika S; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A; Ren, Dacheng

    2015-11-30

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics.

  18. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells

    PubMed Central

    Choudhary, Geetika S.; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A.; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  19. Growth and morphology of colonies of Chinese hamster ovary cells growing on agar is affected by insulin

    PubMed Central

    Aidells, Bruce D.; Konrad, Michael W.; Glaser, Donald A.

    1979-01-01

    As a model for the effect of hormones and growth factors on three-dimensional growth of mammalian cells, we have analyzed the effect of insulin on the three-dimensional growth and morphology of Chinese hamster ovary (CHO) colonies grown on the surface of agar. Sequential photographs in dark-field illumination of growing colonies have been analyzed with computer-assisted techniques. In this analysis the entire shape of each colony in a sizeable population (up to 105 colonies per experiment) can be measured and distributions of parameters derived from these measurements can be studied. In fetal calf serum (FCS), insulin has a dose-related stimulatory effect on cell growth that is most pronounced when growth has slowed down. In 10% FCS, insulin has a similar but diminished effect. When CHO cells are grown conventionally on plastic substrata or in suspension, insulin has little effect on cell growth at 4% serum concentration. Computer analysis of changes in the distribution of colony morphology proved to be a sensitive, dose-dependent, and reproducible assay of a hormonal effect. As little as 5 ng of insulin per ml added to 10% FCS causes a shift in the distribution of colony morphologies. In 4% FCS, 50 ng of insulin per ml is required to produce a detectable change in the colony morphology distribution. Computer analysis of cells grown three-dimensionally on agar provides a powerful approach to studying the effects of hormones and provides observations not available when cells are grown on plastic substrata. Images PMID:287027

  20. Area-based cell colony surviving fraction evaluation: A novel fully automatic approach using general-purpose acquisition hardware.

    PubMed

    Militello, Carmelo; Rundo, Leonardo; Conti, Vincenzo; Minafra, Luigi; Cammarata, Francesco Paolo; Mauri, Giancarlo; Gilardi, Maria Carla; Porcino, Nunziatina

    2017-09-05

    The current methodology for the Surviving Fraction (SF) measurement in clonogenic assay, which is a technique to study the anti-proliferative effect of treatments on cell cultures, involves manual counting of cell colony forming units. This procedure is operator-dependent and error-prone. Moreover, the identification of the exact colony number is often not feasible due to the high growth rate leading to the adjacent colony merging. As a matter of fact, conventional assessment does not deal with the colony size, which is generally correlated with the delivered radiation dose or the administered cytotoxic agent. Considering that the Area Covered by Colony (ACC) is proportional to the colony number and size as well as to the growth rate, we propose a novel fully automatic approach exploiting Circle Hough Transform, to automatically detect the wells in the plate, and local adaptive thresholding, which calculates the percentage of ACC for the SF quantification. This measurement relies just on this covering percentage and does not consider the colony number, preventing inconsistencies due to intra- and inter-operator variability. To evaluate the accuracy of the proposed approach, we compared the SFs obtained by our automatic ACC-based method against the conventional counting procedure. The achieved results (r = 0.9791 and r = 0.9682 on MCF7 and MCF10A cells, respectively) showed values highly correlated with the measurements using the traditional approach based on colony number alone. The proposed computer-assisted methodology could be integrated in laboratory practice as an expert system for the SF evaluation in clonogenic assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system are qualified dendritic cells.

    PubMed

    Li, Gong-Bo; Lu, Guang-Xiu

    2010-01-01

    A widely-used method for generating dendritic cell (DC) is to culture bone marrow cells in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium for 6-10 days. Usually, non-adherent cells are used as qualified dendritic cells while the adherent ones are discarded as "non-dendritic cells" or macrophages. In this study, we show that the adherent cells are nearly identical to the non-adherent cells in both dendritic cell surface markers expression and main dendritic cell-related functions, hence to prove that these "junk cells" are actually qualified dendritic cells.

  2. Prolonged Growth of a Clinical Staphylococcus aureus Strain Selects for a Stable Small-Colony-Variant Cell Type

    PubMed Central

    Bui, Long M. G.; Hoffmann, Peter; Turnidge, John D.; Zilm, Peter S.

    2014-01-01

    An undetermined feature of Staphylococcus aureus pathogenesis is its persistence and then relapse of disease. This has been explained by its switch to alternative lifestyles, mainly as biofilm or small-colony variants (SCVs). Studying the native characteristics of SCVs has been problematic due to their reversion to the parental lifestyle. We have observed that for a number of S. aureus strains as they switch to an SCV lifestyle, there is the formation of an extracellular matrix. We focused our analysis on one strain, WCH-SK2. For bacterial survival in the host, the combination of low nutrients and the prolonged time frame forms a stress that selects for a specific cell type from the population. In this context, we used steady-state growth conditions with low nutrients and a controlled low growth rate for a prolonged time and with methylglyoxal. These conditions induced S. aureus WCH-SK2 into a stable SCV cell type; the cells did not revert after subculturing. Analysis revealed these cells possessed a metabolic and surface profile that was different from those of previously described SCVs or biofilm cells. The extracellular matrix was protein and extracellular DNA but not polysaccharide. The SCV cells induced expression of certain surface proteins (such as Ebh) and synthesis of lantibiotics while downregulating factors that stimulate the immune response (leucocidin, capsule, and carotenoid). Our data reveal cell heterogeneity within an S. aureus population and under conditions that resemble long-term survival in the host have identified a previously unnoticed S. aureus cell type with a distinctive metabolic and molecular profile. PMID:25385795

  3. Effect of leukaemic sera & cell-extracts on splenic colony counts (CFU-S).

    PubMed

    Gupta, S; Rusia, U; Agarwal, S; Sood, S K

    1991-08-01

    Sera and leukaemic cell extracts from patients of acute leukaemia were evaluated for their effect on the repopulating ability of the pluripotent stem cells and erythroid differentiation by an in vivo splenic colony count (CFU-S) technique. Normal donor marrow cells of mice were treated with sera and cell extracts from patients of acute leukaemic and healthy controls and injected in the recipient mice. The CFU-S performed on the seventh day to assess repopulating ability of the stem cell showed consistently lower CFU-S counts in the test groups, with leukaemic sera (P less than 0.01) as well as leukaemic cell-extracts (P less than 0.001). The erythroid differentiation assessed by 59Fe uptake by the spleens also showed significantly reduced counts in the two test groups (P less than 0.01 and less than 0.001 respectively). The results indicate that both leukaemic sera and cell-extracts exert a significant suppressive effect on the repopulating ability of the stem cells and on their erythroid differentiation.

  4. Impaired colony-forming capacity of circulating endothelial progenitor cells in patients with emphysema.

    PubMed

    Kim, Eun-Kyung; Lee, Ji-Hyun; Jeong, Hye-Cheol; Oh, Doyeon; Hwang, Seong-Gyu; Cho, Yong-Wook; Lee, Seon-Ju; Oh, Yeon-Mok; Lee, Sang-Do

    2012-01-01

    Chronic obstructive pulmonary disease (COPD) is classified into emphysema and chronic bronchitis, which are thought to result from different pathophysiological pathways. Smoking-induced lung parenchymal destruction and inadequate repair are involved in the pathogenesis of emphysema. In addition, decreased expression of vascular endothelial growth factor and increased endothelial cell apoptosis in the lung may participate in emphysema pathogenesis. As stem cells, circulating endothelial progenitor cells (EPCs) may play a key role in the maintenance of vascular integrity by replacing and repairing the damaged endothelial cells in the tissues. To determine whether the lack of appropriate repair by circulating EPCs in cases of smoking-induced endothelial cell injury participates in emphysema pathogenesis, we determined the association between the colony-forming or migratory capacity of circulating EPCs and the presence of emphysema in 51 patients with COPD. The patients were divided into emphysema (n = 23) and non-emphysema groups (n = 28) based on high-resolution computed tomography. Twenty-two smokers with normal lung function and 14 normal non-smokers served as controls. Circulating EPCs isolated from patients with emphysema showed significantly lower colony-forming units (CFUs) than those from patients with non-emphysema group, smokers with normal lung function, and normal non-smokers. EPCs from patients with emphysema showed significantly lower migratory capacity than those from normal non-smoking controls (p < 0.05). On multivariate analysis, the EPC-CFU was independently associated with emphysema (OR 0.944, 95% CI = 0.903-0.987, p = 0.011). Thus, impaired functions of circulating EPCs may contribute to the development of emphysema.

  5. A new computational approach to simulate pattern formation in Paenibacillus dendritiformis bacterial colonies

    NASA Astrophysics Data System (ADS)

    Tucker, Laura Jane

    Under the harsh conditions of limited nutrient and hard growth surface, Paenibacillus dendritiformis in agar plates form two classes of patterns (morphotypes). The first class, called the dendritic morphotype, has radially directed branches. The second class, called the chiral morphotype, exhibits uniform handedness. The dendritic morphotype has been modeled successfully using a continuum model on a regular lattice; however, a suitable computational approach was not known to solve a continuum chiral model. This work details a new computational approach to solving the chiral continuum model of pattern formation in P. dendritiformis. The approach utilizes a random computational lattice and new methods for calculating certain derivative terms found in the model.

  6. Macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis.

    PubMed Central

    Clinton, S. K.; Underwood, R.; Hayes, L.; Sherman, M. L.; Kufe, D. W.; Libby, P.

    1992-01-01

    The infiltration of monocytes into the vascular wall and their transformation into lipid-laden foam cells characterizes early atherogenesis. Macrophages are also present in more advanced human atherosclerotic plaques and can produce many mediators that may contribute to lesion formation and progression. Macrophage colony-stimulating factor (MCSF) enhances the proliferation and differentiation of monocyte progenitors and is required for the survival and activation of mature monocytes and macrophages. The authors therefore examined the expression of the MCSF gene in cultured human vascular endothelial (EC) and smooth muscle cells (SMC) as well as in atheromatous lesions from rabbits and humans. Growth arrested EC and SMC contain a low level of MCSF mRNA. Bacterial lipopolysaccharide (LPS), recombinant human interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) induced MCSF mRNA accumulation in a concentration-dependent manner in both EC and SMC. These stimuli induced large increases in MCSF mRNA with peak induction between 4-8 hours after treatment. LPS, IL-1 alpha, and TNF alpha stimulated EC and SMC also showed increased fluorescent antibody staining for MCSF protein and released immunoreactive MCSF in a time-dependent manner. In contrast, phorbol 12-myristate 13-acetate (PMA) was a less potent inducer of MCSF gene expression and iron-oxidized low-density lipoproteins (ox-LDL) did not increase consistently MCSF mRNA or the synthesis and secretion of immunoreactive protein. Northern analysis of mRNA isolated from the atheromatous aorta of rabbits fed a 1% cholesterol diet for 10 weeks showed elevated MCSF mRNA compared with controls. Immunostaining of atheromatous arterial lesions of rabbits demonstrated MCSF protein in association with intimal SMC as well as macrophages. Furthermore, polymerase chain reaction (PCR) analysis of MCSF mRNA in human atheromata showed higher levels than found in nonatherosclerotic arteries and veins. Since the

  7. Soft agar colony formation assay for in vitro testing of sensitivity to chemotherapy of gynecologic malignancies.

    PubMed

    Williams, T J; Lieber, M M; Podratz, K C; Malkasian, G D

    1983-04-15

    In vitro growth of tumor cells may provide a way of testing the effectiveness of chemotherapeutic agents in the treatment of cancer. In 1980 and 1981, operations for gynecologic malignancy were performed on 610 Mayo Clinic patients, and malignant tissue and fluids were obtained from 204 cancers that involved the vulva, uterus, fallopian tubes, and ovaries. These yielded 76 clonogenic stem-cell preparations; and various chemotherapeutic agents were tested against these 76 tumors on soft agar. Considered in this study were the overall process of culturing the samples of tumors and, especially, the data from the preparations that showed sufficient growth of tumor cells for testing. Our guiding concerns were the usefulness of this method to gynecologists and the possible benefits to patients.

  8. Identification of vitamin B1 metabolism as a tumor-specific radiosensitizing pathway using a high-throughput colony formation screen

    PubMed Central

    Buffa, Francesca M.; Yu, Sheng; Ebner, Daniel V.; Howarth, Alison; Folkes, Lisa K.; Budwal, Balam; Chu, Kwun-Ye; Durrant, Lisa; Muschel, Ruth J.; McKenna, W. Gillies; Higgins, Geoff S.

    2015-01-01

    Colony formation is the gold standard assay for determining reproductive cell death after radiation treatment, since effects on proliferation often do not reflect survival. We have developed a high-throughput radiosensitivity screening method based on clonogenicity and screened a siRNA library against kinases. Thiamine pyrophosphokinase-1 (TPK1), a key component of Vitamin B1/thiamine metabolism, was identified as a target for radiosensitization. TPK1 knockdown caused significant radiosensitization in cancer but not normal tissue cell lines. Other means of blocking this pathway, knockdown of thiamine transporter-1 (THTR1) or treatment with the thiamine analogue pyrithiamine hydrobromide (PyrH) caused significant tumor specific radiosensitization. There was persistent DNA damage in cells irradiated after TPK1 and THTR1 knockdown or PyrH treatment. Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitization target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumor specific radiosensitization. PMID:25788274

  9. Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0.

    PubMed

    Maire, Marie-Aline; Rast, Claudine; Vasseur, Paule

    2012-04-11

    This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 7.0. It is intended as a visual aid for the identification and the scoring of cell colonies in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.

  10. Cooperation between human fibrocytes and endothelial colony-forming cells increases angiogenesis via the CXCR4 pathway.

    PubMed

    Smadja, David M; Dorfmüller, Peter; Guerin, Coralie L; Bieche, Ivan; Badoual, Cécile; Boscolo, Elisa; Kambouchner, Marianne; Cazes, Aurélie; Mercier, Olaf; Humbert, Marc; Gaussem, Pascale; Bischoff, Joyce; Israël-Biet, Dominique

    2014-11-01

    Fibrotic diseases of the lung are associated with a vascular remodelling process. Fibrocytes (Fy) are a distinct population of blood-borne cells that co-express haematopoietic cell antigens and fibroblast markers, and have been shown to contribute to organ fibrosis. The purpose of this study was to determine whether fibrocytes cooperate with endothelial colony-forming cells (ECFC) to induce angiogenesis. We isolated fibrocytes from blood of patient with idiopathic pulmonary fibrosis (IPF) and characterised them by flow cytometry, quantitative reverse transcriptase PCR (RTQ-PCR), and confocal microscopy. We then investigated the angiogenic interaction between fibrocytes and cord-blood-derived ECFC, both in vitro and in an in vivo Matrigel implant model. Compared to fibroblast culture medium, fibrocyte culture medium increased ECFC proliferation and differentiation via the SDF-1/CXCR4 pathway. IPF-Fy co-implanted with human ECFC in Matrigel plugs in immunodeficient mice formed functional microvascular beds, whereas fibroblasts did not. Evaluation of implants after two weeks revealed an extensive network of erythrocyte-containing blood vessels. CXCR4 blockade significantly inhibited this blood vessel formation. The clinical relevance of these data was confirmed by strong CXCR4 expression in vessels close to fibrotic areas in biopsy specimens from patients with IPF, by comparison with control lungs. In conclusion, circulating fibrocytes might contribute to the intense remodelling of the pulmonary vasculature in patients with idiopathic pulmonary fibrosis.

  11. Granulocyte colony-stimulating factor receptor expression on human transitional cell carcinoma of the bladder.

    PubMed Central

    Tachibana, M.; Miyakawa, A.; Uchida, A.; Murai, M.; Eguchi, K.; Nakamura, K.; Kubo, A.; Hata, J. I.

    1997-01-01

    Receptors for granulocyte colony-stimulating factor (G-CSFRs) have been confirmed on the cell surfaces of several non-haematopoietic cell types, including bladder cancer cells. This observation has naturally led to the hypothesis that the expression of G-CSFR on these cells may enhance their growth by G-CSF. In this study, the expression of G-CSFR was determined in both established human bladder cancer cell lines and primary bladder cancers. We studied five different human bladder cancer cell lines (KU-1, KU-7, T-24, NBT-2 and KK) and 26 newly diagnosed bladder tumours. G-CSFR mRNA expressions on cultured cell lines were determined using the reverse transcriptase polymerase chain reaction (RT-PCR) method. Furthermore, the G-CSFR binding experiments on the cultured cell lines were conducted using the Na(125)I-labelled G-CSF ligand-binding assay method. Moreover, the G-CSFR mRNA expressions on primary bladder tumour specimens were assessed using the in situ RT-PCR method. Three out of the five cultured cell lines (KU-1, NBT-2 and KK) exhibited G-CSFR mRNA signals when the RT-PCR method was used. The G-CSFR binding experiments showed an equilibrium dissociation constant (K[d]) of 490 pM for KU-1, 340 pM for NBT-2 and 103 pM for KK cells. With in situ RT-PCR, the tumour cells of 6 out of 26 primary bladder tumour specimens (23.1%) presented positive G-CSFR mRNA signals. Thus, in this study, G-CSFR expression was frequently observed on bladder cancer cells. Therefore, the clinical use of G-CSF for patients with bladder cancer should be selected with great care. Images Figure 1 Figure 3 Figure 4 PMID:9166942

  12. MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1.

    PubMed

    Salvucci, Ombretta; Jiang, Kan; Gasperini, Paola; Maric, Dragan; Zhu, Jinfang; Sakakibara, Shuhei; Espigol-Frigole, Georgina; Wang, Shushang; Tosato, Giovanna

    2012-06-01

    Mobilization of hematopoietic stem/progenitor cells from the bone marrow to the peripheral blood by granulocyte colony-stimulating factor is the primary means to acquire stem cell grafts for hematopoietic cell transplantation. Since hematopoietic stem/progenitor cells represent a minority of all blood cells mobilized by granulocyte colony-stimulating factor, the underlying mechanisms need to be understood in order to develop selective drugs. We analyzed phenotypic, biochemical and genetic changes in bone marrow cell populations from granulocyte colony-stimulating factor-mobilized and control mice, and linked such changes to effective mobilization of hematopoietic stem/progenitor cells. We show that granulocyte colony-stimulating factor indirectly reduces expression of surface vascular cell adhesion molecule 1 on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles in the bone marrow extracellular compartment. We found that hematopoietic stem/progenitor cells, stromal cells and endothelial cells readily incorporate these miR126-loaded microvescicles, and that miR126 represses vascular cell adhesion molecule 1 expression on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells. In line with this, miR126-null mice displayed a reduced mobilization response to granulocyte colony-stimulating factor. Our results implicate miR126 in the regulation of hematopoietic stem/progenitor cell trafficking between the bone marrow and peripheral sites, clarify the role of vascular cell adhesion molecule 1 in granulocyte colony-stimulating factor-mediated mobilization, and have important implications for improved approaches to selective mobilization of hematopoietic stem/progenitor cells.

  13. A cell-based model for quorum sensing in heterogeneous bacterial colonies.

    PubMed

    Melke, Pontus; Sahlin, Patrik; Levchenko, Andre; Jönsson, Henrik

    2010-06-17

    Although bacteria are unicellular organisms, they have the ability to act in concert by synthesizing and detecting small diffusing autoinducer molecules. The phenomenon, known as quorum sensing, has mainly been proposed to serve as a means for cell-density measurement. Here, we use a cell-based model of growing bacterial microcolonies to investigate a quorum-sensing mechanism at a single cell level. We show that the model indeed predicts a density-dependent behavior, highly dependent on local cell-clustering and the geometry of the space where the colony is evolving. We analyze the molecular network with two positive feedback loops to find the multistability regions and show how the quorum-sensing mechanism depends on different model parameters. Specifically, we show that the switching capability of the network leads to more constraints on parameters in a natural environment where the bacteria themselves produce autoinducer than compared to situations where autoinducer is introduced externally. The cell-based model also allows us to investigate mixed populations, where non-producing cheater cells are shown to have a fitness advantage, but still cannot completely outcompete producer cells. Simulations, therefore, are able to predict the relative fitness of cheater cells from experiments and can also display and account for the paradoxical phenomenon seen in experiments; even though the cheater cells have a fitness advantage in each of the investigated groups, the overall effect is an increase in the fraction of producer cells. The cell-based type of model presented here together with high-resolution experiments will play an integral role in a more explicit and precise comparison of models and experiments, addressing quorum sensing at a cellular resolution.

  14. YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa.

    PubMed

    Malone, Jacob G; Jaeger, Tina; Spangler, Christian; Ritz, Daniel; Spang, Anne; Arrieumerlou, Cécile; Kaever, Volkhard; Landmann, Regine; Jenal, Urs

    2010-03-12

    During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important

  15. Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

    PubMed

    Besse, A; Trimoreau, F; Faucher, J L; Praloran, V; Denizot, Y

    1999-07-08

    Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.

  16. Autophagosome formation in mammalian cells.

    PubMed

    Burman, Chloe; Ktistakis, Nicholas T

    2010-12-01

    Autophagy is a fundamental intracellular trafficking pathway conserved from yeast to mammals. It is generally thought to play a pro-survival role, and it can be up regulated in response to both external and intracellular factors, including amino acid starvation, growth factor withdrawal, low cellular energy levels, endoplasmic reticulum (ER) stress, hypoxia, oxidative stress, pathogen infection, and organelle damage. During autophagy initiation a portion of the cytosol is surrounded by a flat membrane sheet known as the isolation membrane or phagophore. The isolation membrane then elongates and seals itself to form an autophagosome. The autophagosome fuses with normal endocytic traffic to mature into a late autophagosome, before fusing with lysosomes. The molecular machinery that enables formation of an autophagosome in response to the various autophagy stimuli is almost completely identified in yeast and-thanks to the observed conservation-is also being rapidly elucidated in higher eukaryotes including mammals. What are less clear and currently under intense investigation are the mechanism by which these various autophagy components co-ordinate in order to generate autophagosomes. In this review, we will discuss briefly the fundamental importance of autophagy in various pathophysiological states and we will then review in detail the various players in early autophagy. Our main thesis will be that a conserved group of heteromeric protein complexes and a relatively simple signalling lipid are responsible for the formation of autophagosomes in mammalian cells.

  17. Enhanced activation of B cells in a granulocyte colony-stimulating factor-mobilized peripheral blood stem cell graft.

    PubMed

    Tayebi, H; Lapierre, V; Saas, P; Lienard, A; Sutton, L; Milpied, N; Attal, M; Cahn, J Y; Kuentz, M; Blaise, D; Hervé, P; Tiberghien, P; Robinet, E

    2001-09-01

    In a randomized study that compared human leucocyte antigen-identical allogeneic granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cell (PBSC) versus bone marrow (BM) transplantation, the expression of activation markers, CD23, CD25 and CD45RO by B cells, was compared in blood before and after G-CSF mobilization and in PBSC versus BM grafts. The fractions of CD23+ and CD25+ B cells were higher in PBSC than in BM grafts. Moreover, we observed a G-CSF-induced increase in B-cell fractions in blood as well as in PBSC grafts when compared with BM grafts. Such an enhanced B-cell activation could contribute to the accelerated kinetics of immuno-haematological reconstitution, the occurrence of acute haemolysis in the ABO minor incompatibility setting, as well as the increased incidence of chronic graft-versus-host disease observed after PBSC transplantation.

  18. High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization

    PubMed Central

    Nijmeh, Hala; Balasubramaniam, Vivek; Burns, Nana; Ahmad, Aftab; Stenmark, Kurt R.

    2014-01-01

    Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations. However, their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony-forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from the PA of hypoxic (VVEC-Hx) compared with control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization. PMID:24508729

  19. High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization.

    PubMed

    Nijmeh, Hala; Balasubramaniam, Vivek; Burns, Nana; Ahmad, Aftab; Stenmark, Kurt R; Gerasimovskaya, Evgenia V

    2014-04-01

    Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations. However, their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony-forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from the PA of hypoxic (VVEC-Hx) compared with control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization.

  20. Human papillomavirus 16 E5 induces bi-nucleated cell formation by cell-cell fusion

    SciTech Connect

    Hu Lulin; Plafker, Kendra; Vorozhko, Valeriya; Zuna, Rosemary E.; Hanigan, Marie H.; Gorbsky, Gary J.; Plafker, Scott M.; Angeletti, Peter C.; Ceresa, Brian P.

    2009-02-05

    Human papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes - E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis.

  1. Formation of STAT5-containing DNA binding complexes in response to colony-stimulating factor-1 and platelet-derived growth factor.

    PubMed

    Novak, U; Mui, A; Miyajima, A; Paradiso, L

    1996-08-02

    Colony-stimulating factor (CSF-1) activates several members belonging to the STAT (signal transducers and activators of transcription) family of transcription factors. We investigated the DNA binding complexes activated by CSF-1 in several cell lines and compared them with complexes activated by platelet-derived growth factor and interleukin 3. Our results indicate that the SIF-A complex activated by CSF-1 and platelet-derived growth factor may contain STAT3/STAT5 heterodimers binding to the high affinity SIF binding site, m67. In addition, both growth factors activate one or several STAT5-containing protein complexes binding to the prolactin-inducible element, PIE. The formation of these complexes was cell type and growth factor specific. Interleukin 3 activated only PIE binding complexes containing STAT5A and STAT5B and did not activate m67 binding complexes. It appears, therefore, that STAT5 cannot bind to m67 as a homodimer, but it can bind if it is dimerized with STAT3, whereas it can bind to the PIE element without being either complexed with STAT3 or any other known STAT protein, possibly as a homodimer or as STAT5A/STAT5B heterodimer. However, in addition, STAT5 may heterodimerize with other proteins and form novel PIE binding complexes.

  2. A lineage of diploid platelet-forming cells precedes polyploid megakaryocyte formation in the mouse embryo.

    PubMed

    Potts, Kathryn S; Sargeant, Tobias J; Markham, John F; Shi, Wei; Biben, Christine; Josefsson, Emma C; Whitehead, Lachlan W; Rogers, Kelly L; Liakhovitskaia, Anna; Smyth, Gordon K; Kile, Benjamin T; Medvinsky, Alexander; Alexander, Warren S; Hilton, Douglas J; Taoudi, Samir

    2014-10-23

    In this study, we test the assumption that the hematopoietic progenitor/colony-forming cells of the embryonic yolk sac (YS), which are endowed with megakaryocytic potential, differentiate into the first platelet-forming cells in vivo. We demonstrate that from embryonic day (E) 8.5 all megakaryocyte (MK) colony-forming cells belong to the conventional hematopoietic progenitor cell (HPC) compartment. Although these cells are indeed capable of generating polyploid MKs, they are not the source of the first platelet-forming cells. We show that proplatelet formation first occurs in a unique and previously unrecognized lineage of diploid platelet-forming cells, which develop within the YS in parallel to HPCs but can be specified in the E8.5 Runx1-null embryo despite the absence of the progenitor cell lineage.

  3. Endothelial colony-forming cells ameliorate endothelial dysfunction via secreted factors following ischemia-reperfusion injury.

    PubMed

    Collett, Jason A; Mehrotra, Purvi; Crone, Allison; Shelley, W Christopher; Yoder, Mervin C; Basile, David P

    2017-05-01

    Damage to endothelial cells contributes to acute kidney injury (AKI) by leading to impaired perfusion. Endothelial colony-forming cells (ECFC) are endothelial precursor cells with high proliferative capacity, pro-angiogenic activity, and in vivo vessel forming potential. We hypothesized that ECFC may ameliorate the degree of AKI and/or promote repair of the renal vasculature following ischemia-reperfusion (I/R). Rat pulmonary microvascular endothelial cells (PMVEC) with high proliferative potential were compared with pulmonary artery endothelial cells (PAEC) with low proliferative potential in rats subjected to renal I/R. PMVEC administration reduced renal injury and hastened recovery as indicated by serum creatinine and tubular injury scores, while PAEC did not. Vehicle-treated control animals showed consistent reductions in renal medullary blood flow (MBF) within 2 h of reperfusion, while PMVEC protected against loss in MBF as measured by laser Doppler. Interestingly, PMVEC mediated protection occurred in the absence of homing to the kidney. Conditioned medium (CM) from human cultured cord blood ECFC also conveyed beneficial effects against I/R injury and loss of MBF. Moreover, ECFC-CM significantly reduced the expression of ICAM-1 and decreased the number of differentiated lymphocytes typically recruited into the kidney following renal ischemia. Taken together, these data suggest that ECFC secrete factors that preserve renal function post ischemia, in part, by preserving microvascular function. Copyright © 2017 the American Physiological Society.

  4. Granulocyte colony-stimulating factor increases the platelet volume in peripheral stem cell apheresis donors.

    PubMed

    Ihara, Akihiro; Matsui, Keiko; Minami, Ryouta; Uchida, Shuzou; Ueda, Shuji; Nishiura, Tetsuo

    2008-01-01

    We investigated the short-term influence of granulocyte colony-stimulating factor (G-CSF) administration on platelet counts and platelet indices in 12 donors (8 males and 4 females; median age 34 years, range 16-49) for peripheral stem cell transplantation using an automated blood cell analyzer. On day 3 (D3) compared with D0, 11 donors with normal laboratory and physical findings showed increases in platelet indices (chi(2) = 12.0, p = 0.0025). Furthermore, mean platelet volume (MPV) was significantly increased (p = 0.04). Also, platelet count decreased, and platelet distribution width and platelet-large cell ratio were increased, but these were not significant. On the contrary, 1 donor with abnormal laboratory findings who had large platelets (MPV 11.4 fl) before G-CSF administration showed decreases in platelet indices (MPV 10.3 fl) on D3, although platelet count (18.2 x 10(4)/microl) decreased after G-CSF administration. G-CSF administration induces an inflammatory process with endothelial cell activation. This is probably the reason why platelet volume increases after G-CSF use. This is the first report showing that G-CSF administration immediately induces increases in large platelets in peripheral stem cell transplant donors before harvest.

  5. CD44 expression in endothelial colony-forming cells regulates neurovascular trophic effect

    PubMed Central

    Sakimoto, Susumu; Marchetti, Valentina; Aguilar, Edith; Lee, Kelsey; Usui, Yoshihiko; Bucher, Felicitas; Trombley, Jennifer K.; Fallon, Regis; Wagey, Ravenska; Peters, Carrie; Scheppke, Elizabeth L.; Westenskow, Peter D.

    2017-01-01

    Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations, since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina, it may be possible to use one cell type to rescue another cell type in the face of severe stress, such as hypoxia or genetically encoded cell-specific degenerations. Here, we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore, we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44, the hyaluronic acid receptor, and IGFBPs (insulin-like growth factor–binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases. PMID:28138561

  6. Synergy of interleukin 1 and granulocyte colony-stimulating factor: in vivo stimulation of stem-cell recovery and hematopoietic regeneration following 5-fluorouracil treatment of mice

    SciTech Connect

    Moore, M.A.S.; Warren, D.J.

    1987-10-01

    The human bladder carcinoma cell line 5637 produces hematopoietic growth factors (granulocyte and granulocyte/macrophage colony-stimulating factors (G-CSF and GM-CSF)) and hemopoietin 1, which synergizes with CSFs to stimulate colony formation by primitive hematopoietic stem cells in 5-fluorouracil-treated mouse bone marrow. Molecular and functional properties of hemopoietin 1 identified it as identical to interleukin 1..cap alpha.. (IL-1..cap alpha..). When bone marrow cells from 5-fluorouracil-treated mice were cultured in suspension for 7 days with recombinant human IL-1..cap alpha.. and/or G-CSF, it was found that the two factors synergized to enhance recovery of myelopoietic cells and colony-forming cells of both high and low proliferative potential. G-CSF alone did not sustain these populations, but the combination had greater-than-additive stimulating capacity. In vivo, 5-fluorouracil (150 mg/kg) produced profound myelosuppression and delayed neutrophil regeneration for up to 2 weeks in C3H/HeJ mice. Daily administration of recombinant human G-CSF or human IL-1..cap alpha.. accelerated recovery of stem cells, progenitor cells, and blood neutrophils by up to 4 days in 5-fluorouracil-treated C3H/HeJ and B6D2F/sub 1/ mice. The combination of IL-1..cap alpha.. and G-CSF acted synergistically, reducing neutropenia and accelerating recovery of normal neutrophil numbers by up to 7 days. These results indicate the possible therapeutic potential of combination therapy with IL-1 and hematopoietic growth factors such as G-CSF in the treatment of chemotherapy- or radiation-induced myelosuppression.

  7. The use of granulocyte-colony stimulating factor induced mobilization for isolation of dental pulp stem cells with high regenerative potential.

    PubMed

    Murakami, Masashi; Horibe, Hiroshi; Iohara, Koichiro; Hayashi, Yuki; Osako, Yohei; Takei, Yoshifumi; Nakata, Kazuhiko; Motoyama, Noboru; Kurita, Kenichi; Nakashima, Misako

    2013-12-01

    Human dental pulp stem cells (DPSCs) contain subsets of progenitor/stem cells with high angiogenic, neurogenic and regenerative potential useful for cell therapy. It is essential to develop a safe and efficacious method to isolate the clinical-grade DPSCs subsets from a small amount of pulp tissue without using conventional flow cytometry. Thus, a method for isolation of DPSCs subsets based on their migratory response to optimized concentration of 100 ng/ml of granulocyte-colony stimulating factor (G-CSF) was determined in this study. The DPSCs mobilized by G-CSF (MDPSCs) were enriched for CD105, C-X-C chemokine receptor type 4 (CXCR-4) and G-CSF receptor (G-CSFR) positive cells, demonstrating stem cell properties including high proliferation rate and stability. The absence of abnormalities/aberrations in karyotype and lack of tumor formation after transplantation in an immunodeficient mouse were demonstrated. The conditioned medium of MDPSCs exhibited anti-apoptotic activity, enhanced migration and immunomodulatory properties. Furthermore, transplantation of MDPSCs accelerated vasculogenesis in an ischemic hindlimb model and augmented regenerated pulp tissue in an ectopic tooth root model compared to that of colony-derived DPSCs, indicating higher regenerative potential of MDPSCs. In conclusion, this isolation method for DPSCs subsets is safe and efficacious, having utility for potential clinical applications to autologous cell transplantation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Enhanced Viability of Endothelial Colony Forming Cells in Fibrin Microbeads for Sensor Vascularization.

    PubMed

    Gandhi, Jarel K; Zivkovic, Lada; Fisher, John P; Yoder, Mervin C; Brey, Eric M

    2015-09-18

    Enhanced vascularization at sensor interfaces can improve long-term function. Fibrin, a natural polymer, has shown promise as a biomaterial for sensor coating due to its ability to sustain endothelial cell growth and promote local vascularization. However, the culture of cells, particularly endothelial cells (EC), within 3D scaffolds for more than a few days is challenging due to rapid loss of EC viability. In this manuscript, a robust method for developing fibrin microbead scaffolds for long-term culture of encapsulated ECs is described. Fibrin microbeads are formed using sodium alginate as a structural template. The size, swelling and structural properties of the microbeads were varied with needle gauge and composition and concentration of the pre-gel solution. Endothelial colony-forming cells (ECFCs) were suspended in the fibrin beads and cultured within a perfusion bioreactor system. The perfusion bioreactor enhanced ECFCs viability and genome stability in fibrin beads relative to static culture. Perfusion bioreactors enable 3D culture of ECs within fibrin beads for potential application as a sensor coating.

  9. Growth of a radiation-transformed clone of C3H 1OT1/2 cells into melanin-producing colonies

    SciTech Connect

    Szekely, J.G.; Raaphorst, G.P.; Lobreau, A.U.; Azzam, E.I.; Vadasz, J.A.

    1985-01-01

    When R25, a radiation-transformed clone of C3H 1OT1/2 cells, was plated in soft agarose, a fraction of the colonies became pigmented. The morphologies of the white and dark colonies and their cells were compared by optical, transmission and scanning electron microscopy. The transformed R25 cells had apparently differentiated into melanin-producing cells in the soft agarose, with the white colonies containing actively growing cells having only a few melanosomes, and the dark colonies being made up of stationary-phase cells filled with electronopaque melanosomes. Exposure of the R25 cells to 4.0 Gy of X-rays decreased the percentage of dark colonies, while exposure to 1% DMSO had no effect.

  10. Enhancement of erythroid colony growth by triiodothyronine in cell cultures from bone marrow of normal and anemic rats with chronic renal failure.

    PubMed

    Malgor, L A; Valsecia, M E; Verges, E G; de Markowsky, E E

    1995-01-01

    In order to make a contribution in clarifying the role of thyroid hormones on modulation of erythropoiesis and to gain a further insight on the effects of these hormones in the anemia of chronic renal failure (CRF), we studied the action of triiodo-1-thyronine (LT3) and DT3, a dextrorotary non-calorigenic isomer of T3 on late (CFU-E) and early (BFU-E) committed erythroid precursor cells from bone marrow of normal and anemic uremic rats. Cultures were prepared using the methylcellulose technique containing a standard dose (182 mU/ml) of erythropoietin (Ep), LT3 and DT3 in doses of 0.5 and 1.5 micrograms/ml. Thyroid hormones were added to cultures in the absence of Ep. Our results demonstrated that LT3 and DT3 produced a direct and significant stimulation of CFU-E formation and a moderate increase of BFU-E. A dose-correlation was apparent in cultures containing thyroid hormones. DT3 was somewhat less active than LT3. As expected, Ep also produced a significant increase in erythroid colony formation, mainly CFU-E. It is notheworthy that the effects of LT3, DT3 and Ep on erythroid colony growth were significantly higher in marrow cultures from anemic rats with CRF, indicating an increased proliferative cell kinetics of committed erythroid cells in response to these drugs.

  11. CD34 expression modulates tube-forming capacity and barrier properties of peripheral blood-derived endothelial colony-forming cells (ECFCs).

    PubMed

    Tasev, Dimitar; Konijnenberg, Lara S F; Amado-Azevedo, Joana; van Wijhe, Michiel H; Koolwijk, Pieter; van Hinsbergh, Victor W M

    2016-07-01

    Endothelial colony-forming cells (ECFC) are grown from circulating CD34(+) progenitors present in adult peripheral blood, but during in vitro expansion part of the cells lose CD34. To evaluate whether the regulation of CD34 characterizes the angiogenic phenotypical features of PB-ECFCs, we investigated the properties of CD34(+) and CD34(-) ECFCs with respect to their ability to form capillary-like tubes in 3D fibrin matrices, tip-cell gene expression, and barrier integrity. Selection of CD34(+) and CD34(-) ECFCs from subcultured ECFCs was accomplished by magnetic sorting (FACS: CD34(+): 95 % pos; CD34(-): 99 % neg). Both fractions proliferated at same rate, while CD34(+) ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD3(4-) cells. However, during cell culture CD34(-) cells re-expressed CD34. Cell-seeding density, cell-cell contact formation, and serum supplements modulated CD34 expression. CD34 expression in ECFCs was strongly suppressed by newborn calf serum. Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression. Silencing of CD34 with siRNA resulted in strengthening of cell-cell contacts and increased barrier function of ECFC monolayers as measured by ECIS. Furthermore, CD34 siRNA reduced tube formation by ECFC, but did not affect tip-cell gene expression. These findings demonstrate that CD34(+) and CD34(-) cells are different phenotypes of similar cells and that CD34 (1) can be regulated in ECFC; (2) is positively involved in capillary-like sprout formation; (3) is associated but not causally related to tip-cell gene expression; and (4) can affect endothelial barrier function.

  12. Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor

    SciTech Connect

    Messner, H.A.; Yamasaki, K.; Jamal, N.; Minden, M.M.; Yang, Y.C.; Wong, G.G.; Clark, S.C.

    1987-10-01

    Supernatants of COS-1 cells transfected with gibbon cDNA encoding interleukin 3 (IL-3) with homology to sequences for human IL-3 were tested for ability to promote growth of various human hemopoietic progenitors. The effect of these supernatants as a source of recombinant IL-3 was compared to that of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) as well as to that of medium conditioned by phytohemagglutinin-stimulated leukocytes. The frequency of multilineage colonies, erythroid bursts, and megakaryocyte colonies in cultures containing the COS-1 cell supernatant was equivalent to the frequency observed in the controls and significantly higher than found in cultures plated with recombinant GM-CSF. G-CSF did not support the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. In contrast, growth of granulocyte-macrophage colonies was best supported with GM-CSF, while recombinant IL-3 yielded colonies at lower or at best equivalent frequency. The simultaneous addition of higher concentrations of GM-CSF to cultures containing IL-3 in optimal amounts did not enhance the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. However, the frequency of such colonies and bursts increased with GM-CSF when cultures were plated with suboptimal concentrations of IL-3. Growth of colonies within the granulocyte-macrophage lineage is optimally supported by GM-CSF and does not increase with further addition of IL-3.

  13. The use of granulocyte-colony-stimulating factor in volunteer unrelated hemopoietic stem cell donors.

    PubMed

    Pamphilon, Derwood; Nacheva, Elisabeth; Navarrete, Cristina; Madrigal, Alejandro; Goldman, John

    2008-07-01

    Granulocyte-colony-stimulating factor (G-CSF) is used for the mobilization of hemopoietic stem cells in healthy donors. It has a number of common side effects such as bone pain, which resolve rapidly after administration is discontinued. Recent publications have raised concern that it might act as a trigger for the development of hematologic malignancy in susceptible individuals, possibly by causing genomic instability, but to date there is no evidence that healthy volunteer donors who receive G-CSF are at any increased risk. Ongoing studies aim to confirm whether or not G-CSF can cause chromosomal abnormalities in healthy donors. In the UK, the British Bone Marrow Registry and Anthony Nolan Trust give G-CSF to donors who have agreed to donate peripheral blood stem cells. It is recommended by the UK Registries at present that all stem cell donors are given updated information explaining the current uncertainties with regard to the use of G-CSF before they give informed consent to its administration. This information is based on a statement agreed by the World Marrow Donor Association for use by individual donor registries. Further, it is our current practice that all donors who have received G-CSF, as well as marrow donors who do not, should be under regular review for at least 10 years to allow the occurrence of any long-term adverse events to be documented.

  14. Proteomic analysis of oxidative modification in endothelial colony-forming cells treated by hydrogen peroxide.

    PubMed

    Wei, Jun; Liu, Ying; Chang, Ming; Sun, Chong-Ling; Li, Da-Wei; Liu, Zhi-Qiang; Hu, Lin-Sen

    2012-06-01

    Endothelial progenitor cells (EPCs) which circulate in the peripheral blood and reside in blood vessels are proven to promote the repair of damaged endothelium and improve the function of endothelial cells after vascular injury. Recently, EPCs have been extensively studied as risk biomarkers and a potential therapeutic tool for cardiovascular disease. It is known that oxidative stress is one of the most important pathogenetic factors impairing endothelial function. During the repair process after endothelial injury, EPCs are exposed to oxidative stress. In this study, we treated endothelial colony-forming cells (ECFCs) with hydrogen peroxide (H₂O₂) as an oxidative stress model and observed the changes in cytology and morphology of ECFCs. In addition, we investigated the alterations in oxidative levels of proteins associated with H₂O₂-induced morphological and cytological changes in ECFCs by proteomic analysis of oxidative modification. The results showed that H₂O₂ treatment led to a decreased proliferation, increased apoptosis and impaired tube-forming ability of ECFCs in a dose-dependent manner. Five proteins with upregulated oxidative levels were identified successfully. The upregulated oxidative levels of these five proteins may be responsible for the dysfunction of ECFCs under oxidative stress. Our results may provide some novel insights into the molecular mechanisms of oxidative stress action on ECFCs.

  15. Granulocyte colony-stimulating factor (G-CSF) depresses angiogenesis in vivo and in vitro: implications for sourcing cells for vascular regeneration therapy.

    PubMed

    Tura, O; Crawford, J; Barclay, G R; Samuel, K; Hadoke, P W F; Roddie, H; Davies, J; Turner, M L

    2010-07-01

    The most common source of hematopoietic progenitor cells (HPCs) for hematopoietic reconstitution comprises granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs). It has been proposed that endothelial progenitor cells (EPCs) share precursors with HPCs, and that EPC release may accompany HPC mobilization to the circulation following G-CSF administration. To investigate EPC activity following HPC mobilization, and the direct effects of exogenous G-CSF administration on human umbilical vein endothelial cells (HUVECs) and endothelial outgrowth cells (EOCs), using in vitro and in vivo correlates of angiogenesis. Heparinized venous blood samples were collected from healthy volunteers and from cord blood at parturition. G-CSF-mobilized samples were collected before administration, at apheresis harvest, and at follow-up. PBSCs were phenotyped by flow cytometry, and cultured in standard colony-forming unit (CFU)-EPC and EOC assays. The effect of exogenous G-CSF was investigated by addition of it to HUVECs and EOCs in standard tubule formation and aortic ring assays, and in an in vivo sponge implantation model. Our data show that G-CSF mobilization of PBSCs produces a profound, reversible depression of circulating CFU-EPCs. Furthermore, G-CSF administration did not mobilize CD34+CD133- cells, which include precursors of EOCs. No EOCs were cultured from any mobilized PBSCs studied. Exogenous G-CSF inhibited CFU-EPC generation, HUVEC and EOC tubule formation, microvessel outgrowth, and implanted sponge vascularization in mice. G-CSF administration depresses both endothelial cell angiogenesis and monocyte proangiogenic activity, and we suggest that any angiogenic benefit observed following implantation of cells mobilized by G-CSF may come only from a paracrine effect from HPCs.

  16. Relationship of the Major Constituents of the Neurospora crassa Cell Wall to Wild-Type and Colonial Morphology

    PubMed Central

    Mahadevan, P. R.; Tatum, E. L.

    1965-01-01

    Mahadevan, P. R. (The Rockefeller Institute, New York, N.Y.), and E. L. Tatum. Relationship of the major constituents of the Neurospora crassa cell wall to wild-type and colonial morphology. J. Bacteriol. 90:1073–1081. 1965.—The relationship of cell wall to morphology in Neurospora crassa was studied by correlating the levels of structural polymers of the cell wall with wild-type and colonial morphology. The cell wall of N. crassa contains at least four major complexes: a peptide-polysaccharide complex; two glucose polymers, one of which was found to be a laminarinlike β-1,3-glucan; and, lastly, chitin. The levels of one or more of these structural polymers are consistently altered in single-gene mutants with colonial growth, and in sorbose-induced colonial growth. The proportions of these polymers, particularly of the peptide-polysaccharide complex and the β-1,3-glucan, appear to be important to morphology. Images PMID:5847797

  17. [The expression of antigens, detectable with ICO-series monoclonal antibodies, on the surfaces of cells forming granulocyte and macrophage colonies in semiliquid agar].

    PubMed

    Savel'eva, E V; Kharlamova, L A; Baryshnikov, A Iu; Agafonov, V A; Tupitsyn, N N

    1989-01-01

    The expression of antigens on granulocyte-macrophagal colony-forming cells of patients with nonhematological diseases was studied. Treatment of bone marrow cells with murine monoclonal antibodies ICO-1 and ICO-11 led to statistically significant inhibition of the number of growing colonies. Monoclonal antibodies ICO-02, ICO-10, ICO-GM-1 and ICO-G-2 had no such effect.

  18. Endoplasmic Reticulum Ca(2+) Handling and Apoptotic Resistance in Tumor-Derived Endothelial Colony Forming Cells.

    PubMed

    Poletto, Valentina; Dragoni, Silvia; Lim, Dmitry; Biggiogera, Marco; Aronica, Adele; Cinelli, Mariapia; De Luca, Antonio; Rosti, Vittorio; Porta, Camillo; Guerra, Germano; Moccia, Francesco

    2016-10-01

    Truly endothelial progenitor cells (EPCs) can be mobilized from bone marrow to support the vascular network of growing tumors, thereby sustaining the metastatic switch. Endothelial colony forming cells (ECFCs) are the only EPC subtype belonging to the endothelial phenotype and capable of incorporating within neovessels. The intracellular Ca(2+) machinery plays a key role in ECFC activation and is remodeled in renal cellular carcinoma-derived ECFCs (RCC-ECFCs). Particularly, RCC-ECFCs seems to undergo a drop in endoplasmic reticulum (ER) Ca(2+) concentration ([Ca(2+) ]ER ). This feature is remarkable when considering that inositol-1,4,5-trisphosphate (InsP3 )-dependent ER-to-mitochondria Ca(2+) transfer regulates the intrinsic apoptosis pathway. Herein, we sought to assess whether: (1) the [Ca(2+) ]ER and the InsP3 -induced ER-mitochondria Ca(2+) shuttle are reduced in RCC-ECFCs; and (2) the dysregulation of ER Ca(2+) handling leads to apoptosis resistance in tumor-derived cells. RCC-ECFCs displayed a reduction both in [Ca(2+) ]ER and in the InsP3 -dependent mitochondrial Ca(2+) uptake, while they expressed normal levels of Bcl-2 and Bak. The decrease in [Ca(2+) ]ER was associated to a remarkable ER expansion in RCC-ECFCs, which is a hallmark of ER stress, and did not depend on the remodeling of the Ca(2+) -transporting and the ER Ca(2+) -storing systems. As expected, RCC-ECFCs were less sensitive to rapamycin- and thapsigargin-induced apoptosis; however, buffering intracellular Ca(2+) levels with BAPTA dampened apoptosis in both cell types. Finally, store-operated Ca(2+) entry was seemingly uncoupled from the apoptotic machinery in RCC-ECFCs. Thus, the chronic underfilling of the ER Ca(2+) pool could confer a survival advantage to RCC-ECFCs and underpin RCC resistance to pharmacological treatment. J. Cell. Biochem. 117: 2260-2271, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Characterization of the Maize Stalk Rot Pathogens Fusarium subglutinans and F. temperatum and the Effect of Fungicides on Their Mycelial Growth and Colony Formation

    PubMed Central

    Shin, Jong-Hwan; Han, Joon-Hee; Lee, Ju Kyong; Kim, Kyoung Su

    2014-01-01

    Maize is a socioeconomically important crop in many countries. Recently, a high incidence of stalk rot disease has been reported in several maize fields in Gangwon province. In this report, we show that maize stalk rot is associated with the fungal pathogens Fusarium subglutinans and F. temperatum. Since no fungicides are available to control these pathogens on maize plants, we selected six fungicides (tebuconazole, difenoconazole, fluquinconazole, azoxystrobin, prochloraz and kresoxim-methyl) and examined their effectiveness against the two pathogens. The in vitro antifungal effects of the six fungicides on mycelial growth and colony formation were investigated. Based on the inhibition of mycelial growth, the most toxic fungicide was tebuconazole with 50% effective concentrations (EC50) of <0.1 μg/ml and EC90 values of 0.9 μg/ml for both pathogens, while the least toxic fungicide was azoxystrobin with EC50 values of 0.7 and 0.5 μg/ml for F. subglutinans and F. temperatum, respectively, and EC90 values of >3,000 μg/ml for both pathogens. Based on the inhibition of colony formation by the two pathogens, kresoxim-methyl was the most toxic fungicide with complete inhibition of colony formation at concentrations of 0.1 and 0.01 μg/ml for F. subglutinans and F. temperatum, respectively, whereas azoxystrobin was the least toxic fungicide with complete inhibition of colony formation at concentrations >3,000 μg/ml for both pathogens. PMID:25506304

  20. Maternal body-mass index and cord blood circulating endothelial colony-forming cells.

    PubMed

    Moreno-Luna, Rafael; Muñoz-Hernandez, Rocio; Lin, Ruei-Zeng; Miranda, Maria L; Vallejo-Vaz, Antonio J; Stiefel, Pablo; Praena-Fernández, Juan M; Bernal-Bermejo, Jose; Jimenez-Jimenez, Luis M; Villar, Jose; Melero-Martin, Juan M

    2014-03-01

    Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that are particularly abundant in umbilical cord blood. We sought to determine whether ECFC abundance in cord blood is associated with maternal body-mass index (BMI) in nonpathologic pregnancies. We measured the level of ECFCs in the cord blood of neonates (n = 27) born from non-obese healthy mothers with nonpathologic pregnancies and examined whether ECFC abundance correlated with maternal BMI. We also examined the effect of maternal BMI on ECFC phenotype and function using angiogenic and vasculogenic assays. We observed variation in ECFC abundance among subjects and found a positive correlation between prepregnancy maternal BMI and ECFC content (r = 0.51, P = .007), which was independent of other obstetric factors. Despite this variation, ECFC phenotype and functionality were deemed normal and highly similar between subjects with maternal BMI <25 kg/m(2) and BMI between 25-30 kg/m(2), including the ability to form vascular networks in vivo. This study underlines the need to consider maternal BMI as a potential confounding factor for cord blood levels of ECFCs in future comparative studies between healthy and pathologic pregnancies. Copyright © 2014 Mosby, Inc. All rights reserved.

  1. Role of macrophage colony-stimulating factor (M-CSF) in human granulosa cells.

    PubMed

    Xu, Song; Zhang, Zhifen; Xia, Li-Xia; Huang, Jian

    2016-12-01

    Macrophage colony-stimulating factor (M-CSF) has been proved to have a positive role in the follicular development. We investigated its effect on human granulosa cells and found that M-CSF could stimulate the production of E2. The production of FSH receptors was enhanced by M-CSF in vitro in a dose-dependent manner with or without the addition of tamoxifen (p <0.05). Correspondingly, FSH was also able to coordinate the expression of M-CSF and its receptor (p <0.05). That maybe important to maintain the level of Nppc and the meiotic arrest of the oocyte. The protein p-JAK2 and p-STAT3 in JAK/STAT-signaling pathway elevated after the influence of M-CSF (p < 0.05). These results suggest that M-CSF has a role in regulating the response of granulosa cells to gonadotropins. Its function is associated with JAK/STAT-signaling pathway.

  2. Long-active granulocyte colony-stimulating factor for peripheral blood hematopoietic progenitor cell mobilization.

    PubMed

    Martino, Massimo; Laszlo, Daniele; Lanza, Francesco

    2014-06-01

    Peg-filgrastim (PEG-FIL), a polyethylene glycol-conjugated form of granulocyte colony-stimulating factor (G-CSF), has been introduced in clinical practice and is effective in shortening the time of neutropenia after cytotoxic chemotherapy. G-CSF has emerged as the preferred cytokine for hematopoietic progenitor cells' (HPC) mobilization. Nevertheless, data on the ability of PEG-FIL in this field have been published. We review publications in the field with the goal of providing an overview of this approach. PEG-FIL may be able to mobilize CD34(+) cells in a more timely fashion than G-CSF, with the advantages of only a single-dose administration, an earlier start and a reduction in the number of apheresis procedures. The main controversies concern the dosage of the drug and the optimal dose. In the context of chemo-mobilization, a single dose of 6 mg PEG-FIL seems effective in terms of HPC's mobilization and there is no increase in this effect if the dose is doubled to 12 mg. Steady-state mobilization requires higher doses of PEG-FIL and this approach is not cost-effective when compared with G-CSF. The experiences with PEG-FIL in the healthy donor setting are very limited.

  3. Effect of adrenalectomy on recipients of allogeneic lymphocytes on inactivation of endogenous colony-forming cells in mice

    SciTech Connect

    Semenkov, V.F.

    1985-06-01

    This paper presents a study of the killer functions of lymph node cells directed against endogenous colony-forming cells in adrenalectomized recipients in a genetic system with one-way incompatibility: parental line - F/sub 1/ hybrid. Mice were irradiated with Co 60 gamma rays on the EGO-2 apparatus with dose rate from 200 to 250 R/min. The results were subjected to statistical analysis by Student's test. It can be tentatively suggested that the killer action of T lymphocytes on endogenous colonies was intensified in adrenal-ectomized recipients with endogenous hypocorticism, as a result of cooperation with the cortisol-sensitive subpopulation of T helper cells, of a change in the properties of the antigen-recognizing receptors, or an increase in the sensitivity of target cells to the killer action of T lymphocytes.

  4. Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle.

    PubMed

    Kang, Kyu-Tae; Lin, Ruei-Zeng; Kuppermann, David; Melero-Martin, Juan M; Bischoff, Joyce

    2017-04-10

    Here we investigated whether endothelial colony forming cells (ECFC) and mesenchymal progenitor cells (MPC) form vascular networks and restore blood flow in ischemic skeletal muscle, and whether host myeloid cells play a role. ECFC + MPC, ECFC alone, MPC alone, or vehicle alone were injected into the hind limb ischemic muscle one day after ligation of femoral artery and vein. At day 5, hind limbs injected with ECFC + MPC showed greater blood flow recovery compared with ECFC, MPC, or vehicle. Tail vein injection of human endothelial specific Ulex europaeus agglutinin-I demonstrated an increased number of perfused human vessels in ECFC + MPC compared with ECFC. In vivo bioluminescence imaging showed ECFC persisted for 14 days in ECFC + MPC-injected hind limbs. Flow cytometric analysis of ischemic muscles at day 2 revealed increased myeloid lineage cells in ECFC + MPC-injected muscles compared to vehicle-injected muscles. Neutrophils declined by day 7, while the number of myeloid cells, macrophages, and monocytes did not. Systemic myeloid cell depletion with anti-Gr-1 antibody blocked the improved blood flow observed with ECFC + MPC and reduced ECFC and MPC retention. Our data suggest that ECFC + MPC delivery could be used to reestablish blood flow in ischemic tissues, and this may be enhanced by coordinated recruitment of host myeloid cells.

  5. Granulocyte, granulocyte–macrophage, and macrophage colony-stimulating factors can stimulate the invasive capacity of human lung cancer cells

    PubMed Central

    Pei, X-H; Nakanishi, Y; Takayama, K; Bai, F; Hara, N

    1999-01-01

    We and other researchers have previously found that colony-stimulating factors (CSFs), which generally include granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), promote invasion by lung cancer cells. In the present study, we studied the effects of these CSFs on gelatinase production, urokinase plasminogen activator (uPA) production and their activity in human lung cancer cells. Gelatin zymographs of conditioned media derived from human lung adenocarcinoma cell lines revealed two major bands of gelatinase activity at 68 and 92 kDa, which were characterized as matrix metalloproteinase (MMP)-2 and MMP-9 respectively. Treatment with CSFs increased the 68- and 92-kDa activity and converted some of a 92-kDa proenzyme to an 82-kDa enzyme that was consistent with an active form of the MMP-9. Plasminogen activator zymographs of the conditioned media from the cancer cells showed that CSF treatment resulted in an increase in a 48–55 kDa plasminogen-dependent gelatinolytic activity that was characterized as human uPA. The conditioned medium from the cancer cells treated with CSFs stimulated the conversion of plasminogen to plasmin, providing a direct demonstration of the ability of enhanced uPA to increase plasmin-dependent proteolysis. The enhanced invasive behaviour of the cancer cells stimulated by CSFs was well correlated with the increase in MMPs and uPA activities. These data suggest that the enhanced production of extracellular matrix-degrading proteinases by the cancer cells in response to CSF treatment may represent a biochemical mechanism which promotes the invasive behaviour of the cancer cells. © 1999 Cancer Research Campaign PMID:10408691

  6. Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes

    PubMed Central

    2013-01-01

    Background The objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development. Methods Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at −50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose–response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid (SOF, n = 212); 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) tissue culture medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days. Results Immunoreactivity for both α and β GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control (SOF alone, P = 0.2). Conclusions GM-CSF enhanced cumulus

  7. Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes.

    PubMed

    Peralta, Oscar A; Bucher, Danai; Fernandez, Ana; Berland, Marco; Strobel, Pablo; Ramirez, Alfredo; Ratto, Marcelo H; Concha, Ilona

    2013-06-24

    The objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development. Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at -50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose-response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid (SOF, n = 212); 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) tissue culture medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days. Immunoreactivity for both α and β GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control (SOF alone, P = 0.2). GM-CSF enhanced cumulus cell expansion of in vitro matured bovine

  8. Human endothelial colony-forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold-based tissue engineering.

    PubMed

    Denecke, Bernd; Horsch, Liska D; Radtke, Stefan; Fischer, Johannes C; Horn, Peter A; Giebel, Bernd

    2015-11-01

    One of the major challenges in tissue engineering is to supply larger three-dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)-derived endothelial colony-forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue-culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo-expanded ECFCs are a novel, very potent cell source for scaffold-based tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Selection of Small-Colony Variants of Salmonella enterica Serovar Typhimurium in Nonphagocytic Eucaryotic Cells

    PubMed Central

    Cano, David A.; Pucciarelli, M. Graciela; Martínez-Moya, Marina; Casadesús, Josep; García-del Portillo, Francisco

    2003-01-01

    Salmonella enterica strains are enteropathogenic bacteria that survive and proliferate within vacuolar compartments of epithelial and phagocytic cells. Recently, it has been reported that fibroblast cells are capable of restricting S. enterica serovar Typhimurium intracellular growth. Here, we show that prolonged residence of bacteria in the intracellular environment of fibroblasts results in the appearance of genetically stable small-colony variants (SCV). A total of 103 SCV isolates, obtained from four independent infections, were subjected to phenotypic analysis. The following phenotypes were observed: (i) δ-aminolevulinic acid auxotrophy; (ii) requirement for acetate or succinate for growth in glucose minimal medium; (iii) auxotrophy for aromatic amino acids; and (iv) reduced growth rate under aerobic conditions not linked to nutrient auxotrophy. The exact mutations responsible for the SCV phenotype in three representative isolates were mapped in the lpd, hemL, and aroD genes, which code for dihydrolipoamide dehydrogenase, glutamate-1-semyaldehyde aminotransferase, and 3-dehydroquinate dehydratase, respectively. The lpd, hemL, and aroD mutants had intracellular persistence rates in fibroblasts that were 3 to 4 logs higher than that of the parental strain and decreased susceptibility to aminoglycoside antibiotics. All three of these SCV isolates were attenuated in the BALB/c murine typhoid model. Complementation with lpd+, hem+, and aroD+ genes restored the levels of intracellular persistence and antibiotic susceptibility to levels of the wild-type strain. However, virulence was not exhibited by any of the complemented strains. Altogether, our data demonstrate that similar to what it has been reported for SCV isolates of other pathogens, S. enterica SCV display enhanced intracellular persistence in eucaryotic cells and are impaired in the ability to cause overt disease. In addition, they also suggest that S. enterica SCV may be favored in vivo. PMID:12819049

  10. Granulocyte-Macrophage Colony-Stimulating Factor Priming plus Papillomavirus E6 DNA Vaccination: Effects on Papilloma Formation and Regression in the Cottontail Rabbit Papillomavirus-Rabbit Model

    PubMed Central

    Leachman, Sancy A.; Tigelaar, Robert E.; Shlyankevich, Mark; Slade, Martin D.; Irwin, Michele; Chang, Ed; Wu, T. C.; Xiao, Wei; Pazhani, Sundaram; Zelterman, Daniel; Brandsma, Janet L.

    2000-01-01

    A cottontail rabbit papillomavirus (CRPV) E6 DNA vaccine that induces significant protection against CRPV challenge was used in a superior vaccination regimen in which the cutaneous sites of vaccination were primed with an expression vector encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that induces differentiation and local recruitment of professional antigen-presenting cells. This treatment induced a massive influx of major histocompatibility complex class II-positive cells. In a vaccination-challenge experiment, rabbit groups were treated by E6 DNA vaccination, GM-CSF DNA inoculation, or a combination of both treatments. After two immunizations, rabbits were challenged with CRPV at low, moderate, and high stringencies and monitored for papilloma formation. As expected, all clinical outcomes were monotonically related to the stringency of the viral challenge. The results demonstrate that GM-CSF priming greatly augmented the effects of CRPV E6 vaccination. First, challenge sites in control rabbits (at the moderate challenge stringency) had a 0% probability of remaining disease free, versus a 50% probability in E6-vaccinated rabbits, and whereas GM-CSF alone had no effect, the interaction between GM-CSF priming and E6 vaccination increased disease-free survival to 67%. Second, the incubation period before papilloma onset was lengthened by E6 DNA vaccination alone or to some extent by GM-CSF DNA inoculation alone, and the combination of treatments induced additive effects. Third, the rate of papilloma growth was reduced by E6 vaccination and, to a lesser extent, by GM-CSF treatment. In addition, the interaction between the E6 and GM-CSF treatments was synergistic and yielded more than a 99% reduction in papilloma volume. Finally, regression occurred among the papillomas that formed in rabbits treated with the E6 vaccine and/or with GM-CSF, with the highest regression frequency occurring in rabbits that received the combination

  11. Biosimilar granulocyte-colony-stimulating factor for healthy donor stem cell mobilization: need we be afraid?

    PubMed

    Bonig, Halvard; Becker, Petra S; Schwebig, Arnd; Turner, Matthew

    2015-02-01

    Biosimilars are approved biologics with comparable quality, safety, and efficacy to a reference product. Unlike generics, which are chemically manufactured copies of small-molecule drugs with relatively simple chemical structures, the biosimilar designation is applied to drugs that are produced by living organisms, implying much more difficult to control manufacturing and purification procedures. To account for these complexities, the European Medicines Agency (EMA), the US Food and Drug Administration, the Australian Therapeutic Goods Administration, and other regulatory authorities have devised and implemented specific, markedly more demanding pathways for the evaluation and approval of biosimilars. To date, several biosimilars have been approved, including versions of somatropin, erythropoietin, and granulocyte-colony-stimulating factor (G-CSF), and several biosimilar monoclonal antibodies are currently in development. The reference G-CSF product (Neupogen, Amgen) has been used for many years for prevention and treatment of neutropenia and also for mobilization of peripheral blood stem cells (PBSCs). However, concerns have been raised about the safety and efficacy of biosimilar G-CSF during PBSC mobilization procedures, especially in healthy donors. This article reviews the available evidence on the use of biosimilar G-CSF in this setting. Aggregate clinical evidence supports the assessment by the EMA of biosimilar and originator G-CSF as highly biologically similar, with respect to desired and undesired effects.

  12. Tomatidine inhibits replication of Staphylococcus aureus small-colony variants in cystic fibrosis airway epithelial cells.

    PubMed

    Mitchell, Gabriel; Gattuso, Mariza; Grondin, Gilles; Marsault, Éric; Bouarab, Kamal; Malouin, François

    2011-05-01

    Small-colony variants (SCVs) often are associated with chronic Staphylococcus aureus infections, such as those encountered by cystic fibrosis (CF) patients. We report here that tomatidine, the aglycon form of the plant secondary metabolite tomatine, has a potent growth inhibitory activity against SCVs (MIC of 0.12 μg/ml), whereas the growth of normal S. aureus strains was not significantly altered by tomatidine (MIC, >16 μg/ml). The specific action of tomatidine was bacteriostatic for SCVs and was clearly associated with their dysfunctional electron transport system, as the presence of the electron transport inhibitor 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) caused normal S. aureus strains to become susceptible to tomatidine. Inversely, the complementation of SCVs' respiratory deficiency conferred resistance to tomatidine. Tomatidine provoked a general reduction of macromolecular biosynthesis but more specifically affected the incorporation of radiolabeled leucine in proteins of HQNO-treated S. aureus at a concentration corresponding to the MIC against SCVs. Furthermore, tomatidine inhibited the intracellular replication of a clinical SCV in polarized CF-like epithelial cells. Our results suggest that tomatidine eventually will find some use in combination therapy with other traditional antibiotics to eliminate persistent forms of S. aureus.

  13. Ionizing Radiation Induces Macrophage Foam Cell Formation and Aggregation Through JNK-Dependent Activation of CD36 Scavenger Receptors

    SciTech Connect

    Katayama, Ikuo; Hotokezaka, Yuka; Matsuyama, Toshifumi; Sumi, Tadateru; Nakamura, Takashi

    2008-03-01

    Purpose: Irradiated arteries of cancer patients can be associated with atherosclerosis-like lesions containing cholesterol-laden macrophages (foam cells). Endothelial cell damage by irradiation does not completely explain the foam cell formation. We investigated the possible underlying mechanisms for ionizing radiation (IR)-induced foam cell formation. Methods and Materials: Human peripheral blood monocytes were activated by macrophage colony-stimulating factor and then treated with varying doses of IR in vitro in the absence of endothelial cells. Scavenger receptor expression and foam cell formation of IR-treated macrophages were investigated in the presence or absence of oxidized low-density lipoprotein. We also assessed the importance of mitogen-activated protein kinase activity in the macrophage colony-stimulating factor-activated human monocytes (macrophages) for the foam cell formation. Results: We found that IR treatment of macrophage colony-stimulating factor-activated human peripheral blood monocytes resulted in the enhanced expression of CD36 scavenger receptors and that cholesterol accumulated in the irradiated macrophages with resultant foam cell formation in the presence of oxidized low-density lipoprotein. Furthermore, when cultured on collagen gels, human macrophages formed large foam cell aggregates in response to IR. Antibodies against CD36 inhibited the IR-induced foam cell formation and aggregation, indicating that the IR-induced foam cell formation and the subsequent aggregation are dependent on functional CD36. In addition, we found that IR of human macrophages resulted in c-Jun N-terminal kinase activation and that c-Jun N-terminal kinase inhibition suppressed IR-induced CD36 expression and the subsequent foam cell formation and aggregation. Conclusion: Taken together, these results suggest that IR-induced foam cell formation is mediated by c-Jun N-terminal kinase-dependent CD36 activation.

  14. The Self-Identity Protein IdsD Is Communicated between Cells in Swarming Proteus mirabilis Colonies.

    PubMed

    Saak, Christina C; Gibbs, Karine A

    2016-12-15

    Proteus mirabilis is a social bacterium that is capable of self (kin) versus nonself recognition. Swarming colonies of this bacterium expand outward on surfaces to centimeter-scale distances due to the collective motility of individual cells. Colonies of genetically distinct populations remain separate, while those of identical populations merge. Ids proteins are essential for this recognition behavior. Two of these proteins, IdsD and IdsE, encode identity information for each strain. These two proteins bind in vitro in an allele-restrictive manner. IdsD-IdsE binding is correlated with the merging of populations, whereas a lack of binding is correlated with the separation of populations. Key questions remained about the in vivo interactions of IdsD and IdsE, specifically, whether IdsD and IdsE bind within single cells or whether IdsD-IdsE interactions occur across neighboring cells and, if so, which of the two proteins is exchanged. Here we demonstrate that IdsD must originate from another cell to communicate identity and that this nonresident IdsD interacts with IdsE resident in the recipient cell. Furthermore, we show that unbound IdsD in recipient cells does not cause cell death and instead appears to contribute to a restriction in the expansion radius of the swarming colony. We conclude that P. mirabilis communicates IdsD between neighboring cells for nonlethal kin recognition, which suggests that the Ids proteins constitute a type of cell-cell communication.

  15. Swarming and complex pattern formation in Paenibacillus vortex studied by imaging and tracking cells.

    PubMed

    Ingham, Colin J; Ben Jacob, Eshel

    2008-02-25

    Swarming motility allows microorganisms to move rapidly over surfaces. The Gram-positive bacterium Paenibacillus vortex exhibits advanced cooperative motility on agar plates resulting in intricate colonial patterns with geometries that are highly sensitive to the environment. The cellular mechanisms that underpin the complex multicellular organization of such a simple organism are not well understood. Swarming by P. vortex was studied by real-time light microscopy, by in situ scanning electron microscopy and by tracking the spread of antibiotic-resistant cells within antibiotic-sensitive colonies. When swarming, P. vortex was found to be peritrichously flagellated. Swarming by the curved cells of P. vortex occurred on an extremely wide range of media and agar concentrations (0.3 to 2.2% w/v). At high agar concentrations (> 1% w/v) rotating colonies formed that could be detached from the main mass of cells by withdrawal of cells into the latter. On lower percentage agars, cells moved in an extended network composed of interconnected "snakes" with short-term collision avoidance and sensitivity to extracts from swarming cells. P. vortex formed single Petri dish-wide "supercolonies" with a colony-wide exchange of motile cells. Swarming cells were coupled by rapidly forming, reversible and non-rigid connections to form a loose raft, apparently connected via flagella. Inhibitors of swarming (p-Nitrophenylglycerol and Congo Red) were identified. Mitomycin C was used to trigger filamentation without inhibiting growth or swarming; this facilitated dissection of the detail of swarming. Mitomycin C treatment resulted in malcoordinated swarming and abortive side branch formation and a strong tendency by a subpopulation of the cells to form minimal rotating aggregates of only a few cells. P. vortex creates complex macroscopic colonies within which there is considerable reflux and movement and interaction of cells. Cell shape, flagellation, the aversion of cell masses to fuse

  16. Order and instabilities in dense bacterial colonies

    NASA Astrophysics Data System (ADS)

    Tsimring, Lev

    2012-02-01

    The structure of cell colonies is governed by the interplay of many physical and biological factors, ranging from properties of surrounding media to cell-cell communication and gene expression in individual cells. The biomechanical interactions arising from the growth and division of individual cells in confined environments are ubiquitous, yet little work has focused on this fundamental aspect of colony formation. By combining experimental observations of growing monolayers of non-motile strain of bacteria Escherichia coli in a shallow microfluidic chemostat with discrete-element simulations and continuous theory, we demonstrate that expansion of a dense colony leads to rapid orientational alignment of rod-like cells. However, in larger colonies, anisotropic compression may lead to buckling instability which breaks perfect nematic order. Furthermore, we found that in shallow cavities feedback between cell growth and mobility in a confined environment leads to a novel cell streaming instability. Joint work with W. Mather, D. Volfson, O. Mondrag'on-Palomino, T. Danino, S. Cookson, and J. Hasty (UCSD) and D. Boyer, S. Orozco-Fuentes (UNAM, Mexico).

  17. Molecular Mechanisms Regulating Cell Fusion and Heterokaryon Formation in Filamentous Fungi.

    PubMed

    Daskalov, Asen; Heller, Jens; Herzog, Stephanie; Fleißner, André; Glass, N Louise

    2017-03-01

    For the majority of fungal species, the somatic body of an individual is a network of interconnected cells sharing a common cytoplasm and organelles. This syncytial organization contributes to an efficient distribution of resources, energy, and biochemical signals. Cell fusion is a fundamental process for fungal development, colony establishment, and habitat exploitation and can occur between hyphal cells of an individual colony or between colonies of genetically distinct individuals. One outcome of cell fusion is the establishment of a stable heterokaryon, culminating in benefits for each individual via shared resources or being of critical importance for the sexual or parasexual cycle of many fungal species. However, a second outcome of cell fusion between genetically distinct strains is formation of unstable heterokaryons and the induction of a programmed cell death reaction in the heterokaryotic cells. This reaction of nonself rejection, which is termed heterokaryon (or vegetative) incompatibility, is widespread in the fungal kingdom and acts as a defense mechanism against genome exploitation and mycoparasitism. Here, we review the currently identified molecular players involved in the process of somatic cell fusion and its regulation in filamentous fungi. Thereafter, we summarize the knowledge of the molecular determinants and mechanism of heterokaryon incompatibility and place this phenomenon in the broader context of biotropic interactions and immunity.

  18. Colonial America.

    ERIC Educational Resources Information Center

    Web Feet K-8, 2001

    2001-01-01

    Presents resources for grades K-8, on the subject of Colonial America. Describes Web sites; CD-ROMs and software; videos; books; audios; magazines; and professional resources. Includes two articles, "Native Americans in the Colonies," and "The Golden Age of Pirates," which also highlight resources. Presents a Web activity focusing on daily life in…

  19. Radiation promotes invasiveness of non-small-cell lung cancer cells through granulocyte-colony-stimulating factor.

    PubMed

    Cui, Y-H; Suh, Y; Lee, H-J; Yoo, K-C; Uddin, N; Jeong, Y-J; Lee, J-S; Hwang, S-G; Nam, S-Y; Kim, M-J; Lee, S-J

    2015-10-16

    Despite ionizing radiation (IR) is being widely used as a standard treatment for lung cancer, many evidences suggest that IR paradoxically promotes cancer malignancy. However, its molecular mechanisms underlying radiation-induced cancer progression remain obscure. Here, we report that exposure to fractionated radiation (2 Gy per day for 3 days) induces the secretion of granulocyte-colony-stimulating factor (G-CSF) that has been commonly used in cancer therapies to ameliorate neutropenia. Intriguingly, radiation-induced G-CSF promoted the migratory and invasive properties by triggering the epithelial-mesenchymal cell transition (EMT) in non-small-cell lung cancer cells (NSCLCs). By irradiation, G-CSF was upregulated transcriptionally by β-catenin/TCF4 complex that binds to the promoter region of G-CSF as a transcription factor. Importantly, irradiation increased the stability of β-catenin through the activation of PI3K/AKT (phosphatidylinositol 3-kinase/AKT), thereby upregulating the expression of G-CSF. Radiation-induced G-CSF is recognized by G-CSFR and transduced its intracellular signaling JAK/STAT3 (Janus kinase/signal transducers and activators of transcription), thereby triggering EMT program in NSCLCs. Taken together, our findings suggest that the application of G-CSF in cancer therapies to ameliorate neutropenia should be reconsidered owing to its effect on cancer progression, and G-CSF could be a novel therapeutic target to mitigate the harmful effect of radiotherapy for the treatment of NSCLC.

  20. Interactions between colon cancer cells and tumor-infiltrated macrophages depending on cancer cell-derived colony stimulating factor 1

    PubMed Central

    Wang, Huayang; Shao, Qianqian; Sun, Jintang; Ma, Chao; Gao, Wenjuan; Wang, Qingjie; Zhao, Lei; Qu, Xun

    2016-01-01

    ABSTRACT Tumor-infiltrated macrophages were potential targets of the immune therapy for patients with colon cancer. Colony stimulating factor 1 (CSF1) is a primary chemoattractant and functional regulator for macrophages, and therefore would be a feasible intervention for the macrophage-targeting therapeutics. However, the expression of CSF1 in colon cancer microenvironment and its roles in cancer development is largely unknown. In the present study, we found that CSF1 was over-expressed exclusively in colon cancer cells and was correlated with macrophages infiltration. The high CSF1 expression and macrophages infiltration were related to the tumor–node-metastasis (TNM) stage of colon cancer, and suggested to be positively associated with survival of colon cancer patients. In the in vitro studies based on an indirect Transwell system, we found that co-culture with macrophage promoted CSF1 production in colon cancer cells. Further investigation on regulatory mechanisms suggested that CSF1 production in colon cancer cells was dependent on PKC pathway, which was activated by IL-8, mainly produced by macrophages. Moreover, colon cancer cell-derived CSF1 drove the recruitment of macrophages and re-educated their secretion profile, including the augment of IL-8 production. The mice tumor xenografts study also found that over-expression of CSF1 in colon cancer cells promoted intratumoral infiltration of macrophages, and partially suppressed tumor growth. In all, our results demonstrated that CSF1 was an important factor in the colon cancer microenvironment, involving in the interactions between colon cancer cells and tumor-infiltrated macrophages. PMID:27141406

  1. Interactions between colon cancer cells and tumor-infiltrated macrophages depending on cancer cell-derived colony stimulating factor 1.

    PubMed

    Wang, Huayang; Shao, Qianqian; Sun, Jintang; Ma, Chao; Gao, Wenjuan; Wang, Qingjie; Zhao, Lei; Qu, Xun

    2016-04-01

    Tumor-infiltrated macrophages were potential targets of the immune therapy for patients with colon cancer. Colony stimulating factor 1 (CSF1) is a primary chemoattractant and functional regulator for macrophages, and therefore would be a feasible intervention for the macrophage-targeting therapeutics. However, the expression of CSF1 in colon cancer microenvironment and its roles in cancer development is largely unknown. In the present study, we found that CSF1 was over-expressed exclusively in colon cancer cells and was correlated with macrophages infiltration. The high CSF1 expression and macrophages infiltration were related to the tumor-node-metastasis (TNM) stage of colon cancer, and suggested to be positively associated with survival of colon cancer patients. In the in vitro studies based on an indirect Transwell system, we found that co-culture with macrophage promoted CSF1 production in colon cancer cells. Further investigation on regulatory mechanisms suggested that CSF1 production in colon cancer cells was dependent on PKC pathway, which was activated by IL-8, mainly produced by macrophages. Moreover, colon cancer cell-derived CSF1 drove the recruitment of macrophages and re-educated their secretion profile, including the augment of IL-8 production. The mice tumor xenografts study also found that over-expression of CSF1 in colon cancer cells promoted intratumoral infiltration of macrophages, and partially suppressed tumor growth. In all, our results demonstrated that CSF1 was an important factor in the colon cancer microenvironment, involving in the interactions between colon cancer cells and tumor-infiltrated macrophages.

  2. Silicon Formation for Solar Cells

    NASA Technical Reports Server (NTRS)

    Sancier, K.

    1985-01-01

    Highly pure silicon obtained for solar cells by proposed technique that sprays liquid-sodium droplets into SiF4 gas. Resulting freely flowing powder of silicon and sodium fluoride will not adhere to reactor walls and easily transferred to melt separator to recover silicon.

  3. Dynamics of Superoxide Production and Decay in Natural Trichodesmium Colonies from the Sargasso Sea: Implications for Cell Signaling

    NASA Astrophysics Data System (ADS)

    Hansel, C. M.; Buchwald, C.; Diaz, J. M.; Dyhrman, S.; Van Mooy, B. A. S.

    2014-12-01

    Reactive oxygen species (ROS) are key players in the biogeochemistry of the ocean, where they serve a critical role in the cycling of carbon and metals. Research in the past decade has introduced phytoplankton and, most recently, heterotrophic bacteria as significant sources of ROS, including superoxide, within both photic and aphotic regions of the ocean. ROS are both beneficial and detrimental to life. For instance, superoxide is a vital inter- and intra-cellular signaling molecule, yet at high concentrations it induces lipid peroxidation and initiates programmed cell death (PCD). In fact, superoxide has been implicated in PCD in the nitrogen-fixing diazotroph Trichodesmium, presumably leading to the demise of blooms within oligotrophic marine systems. Here, we explore the rates of superoxide production and decay by natural Trichodesmium populations obtained from various surface waters in the Sargasso Sea. We investigate also the role of light and colony density and morphology (puff v. raft) on superoxide fluxes. We find that Trichodesmium colonies produce extracellular superoxide at extremely high rates in the dark that are on par with those of the toxic raphidophyte Chattonella. The rates of superoxide production, however, rapidly decline with increasing cell density pointing to a role for superoxide in cell signaling in these organisms. We also find extremely rapid extracellular superoxide degradation by Trichodesmium. Together, this likely reflects a need for these organisms to maintain ROS at levels that will support signaling but below the threshold level that triggers PCD or oxidative damage. We also show differences in the effect of light on superoxide fluxes as a function of Trichodesmium colony morphology, suggesting differences in either colony physiology or associated bacterial symbionts. These findings point to complex physiological, ecological, and physical influences on ROS dynamics in phytoplankton that require further exploration.

  4. Cell proliferation dynamics of somatic and germline tissues during zooidal life span in the colonial tunicate Botryllus primigenus.

    PubMed

    Kawamura, Kazuo; Tachibana, Miki; Sunanaga, Takeshi

    2008-07-01

    Botryllus primigenus is a colonial tunicate in which three successive generations develop synchronously. To identify proliferation centers and possible adult stem cells during asexual reproduction, somatic and germline cells were labeled with 5-bromo-2'-deoxyuridine (BrdU). In the youngest generation, multipotent epithelial cells exhibited an average labeling index (LI) of 30% 24 hr after BrdU injection. In the middle generation, the LI of organ rudiments decreased gradually and reached zero by the beginning of the eldest generation. Exceptionally, cells of specialized tissues such as the pharyngeal inner longitudinal vessel and the posterior end of the endostyle continued DNA synthesis and mitosis even in the eldest generation. Proliferating somatic and germline cells of younger generations expressed a Botryllus myc homolog (BpMyc), but adult tissues did not. This result strongly suggests that in B. primigenus undifferentiated progenitor cells are discernible from possible adult stem cells by the presence or absence of BpMyc.

  5. Direct formate fuel cells: A review

    NASA Astrophysics Data System (ADS)

    An, L.; Chen, R.

    2016-07-01

    Direct formate fuel cells (DFFC), which convert the chemical energy stored in formate directly into electricity, are recently attracting more attention, primarily because of the use of the carbon-neutral fuel and the low-cost electrocatalytic and membrane materials. As an emerging energy technology, the DFFC has made a rapid progress in recent years (currently, the state-of-the-art power density is 591 mW cm-2 at 60 °C). This article provides a review of past research on the development of this type of fuel cell, including the working principle, mechanisms and materials of the electrocatalytic oxidation of formate, singe-cell designs and performance, as well as innovative system designs. In addition, future perspectives with regard to the development of this fuel cell system are also highlighted.

  6. Interleukin 3 perfusion in W/Wv mice allows the development of macroscopic hematopoietic spleen colonies and restores cutaneous mast cell number

    SciTech Connect

    Ody, C.; Kindler, V.; Vassalli, P. )

    1990-07-01

    The genetically anemic W/Wv mice are characterized by the inability of their bone marrow cells to form macroscopic pluripotent hematopoietic colonies in the spleen of irradiated recipients upon transfer (colony-forming units). Furthermore, they almost totally lack mast cells, notably in the skin. In the present study, we have tested the effect of recombinant murine interleukin 3 (rmIL-3) on W/Wv mice hematopoiesis. Transfer of W/Wv bone marrow cells into lethally irradiated recipients perfused with rmIL-3 is followed by the appearance of macroscopic spleen colonies. Moreover, perfusion of rmIL-3 in W/Wv mice: (a) restores almost normal total numbers of hematopoietic precursors (colony-forming cells), but without modification of anemia; and (b) leads to the appearance of a normal number of mastocytes in the skin.

  7. Growth of Bacterial Colonies

    NASA Astrophysics Data System (ADS)

    Warren, Mya; Hwa, Terence

    2013-03-01

    On hard agar gel, there is insufficient surface hydration for bacteria to swim or swarm. Instead, growth occurs in colonies of close-packed cells, which expand purely due to repulsive interactions: individual bacteria push each other out of the way through the force of their growth. In this way, bacterial colonies represent a new type of ``active'' granular matter. In this study, we investigate the physical, biochemical, and genetic elements that determine the static and dynamic aspects of this mode of bacterial growth for E. coli. We characterize the process of colony expansion empirically, and use discrete and continuum models to examine the extent to which our observations can be explained by the growth characteristics of non-communicating cells, coupled together by physical forces, nutrients, and waste products. Our results challenge the commonly accepted modes of bacterial colony growth and provide insight into sources of growth limitation in crowded bacterial communities.

  8. Mast cells mediate malignant pleural effusion formation.

    PubMed

    Giannou, Anastasios D; Marazioti, Antonia; Spella, Magda; Kanellakis, Nikolaos I; Apostolopoulou, Hara; Psallidas, Ioannis; Prijovich, Zeljko M; Vreka, Malamati; Zazara, Dimitra E; Lilis, Ioannis; Papaleonidopoulos, Vassilios; Kairi, Chrysoula A; Patmanidi, Alexandra L; Giopanou, Ioanna; Spiropoulou, Nikolitsa; Harokopos, Vaggelis; Aidinis, Vassilis; Spyratos, Dionisios; Teliousi, Stamatia; Papadaki, Helen; Taraviras, Stavros; Snyder, Linda A; Eickelberg, Oliver; Kardamakis, Dimitrios; Iwakura, Yoichiro; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Kalomenidis, Ioannis; Blackwell, Timothy S; Agalioti, Theodora; Stathopoulos, Georgios T

    2015-06-01

    Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1β, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.

  9. Cooperatively Generated Stresslet Flows Supply Fresh Fluid to Multicellular Choanoflagellate Colonies

    NASA Astrophysics Data System (ADS)

    Roper, Marcus; Dayel, Mark J.; Pepper, Rachel E.; Koehl, M. A. R.

    2013-05-01

    The flagellated protozoan Salpingoeca rosetta is one of the closest relatives of multicellular animals. Unicellular S. rosetta can be induced to form multicellular colonies, but colonies swim more slowly than individual cells so the advantages conferred by colony formation are uncertain. Here we use theoretical models to show that hydrodynamic cooperation between cells can increase the fluid supply to the colony, an important predictor of feeding rate. Our results suggest that hydrodynamic benefits may have been an important selective factor in the evolution of early multicellular animals.

  10. Colony Size of Phaeocystis Antarctica (Prymnesiophyceae) as Influenced by Zooplankton Grazers

    EPA Science Inventory

    The haptophyte Phaeocystis antarctica is a dominant phytoplankton species in the Ross Sea, Antarctica, and exists as solitary cells and mucilaginous colonies that differ by several orders of magnitude in size. Recent studies with P. globosa suggested that colony formation and enl...

  11. Colony Size of Phaeocystis Antarctica (Prymnesiophyceae) as Influenced by Zooplankton Grazers

    EPA Science Inventory

    The haptophyte Phaeocystis antarctica is a dominant phytoplankton species in the Ross Sea, Antarctica, and exists as solitary cells and mucilaginous colonies that differ by several orders of magnitude in size. Recent studies with P. globosa suggested that colony formation and enl...

  12. Dynamic scaling analysis of two-dimensional cell colony fronts in a gel medium: A biological system approaching a quenched Kardar-Parisi-Zhang universality

    NASA Astrophysics Data System (ADS)

    Huergo, M. A. C.; Muzzio, N. E.; Pasquale, M. A.; González, P. H. Pedro; Bolzán, A. E.; Arvia, A. J.

    2014-08-01

    The interfacial two-dimensional spreading dynamics of quasilinear Vero cell colony fronts in methylcellulose (MC)-containing culture medium, under a constant average front displacement velocity regime, was investigated. Under comparable experimental conditions, the average colony front displacement velocity becomes lower than that reported for a standard culture medium. Initially, the presence of MC in the medium hinders both the colony spreading, due to a gradual change in the average size and shape of cells and their distribution in the colony, and the cell motility in the gelled medium. Furthermore, at longer culture times enlarged cells appear at random in the border region of the colony. These cells behave as obstacles (pinning sites) for the displacement of smaller cells towards the colony front. The dynamic scaling analysis of rough fronts yields the set of exponents α =0.63±0.04,β =0.75±0.05, and z =0.84±0.05, which is close to that expected for a quenched Kardar-Parisi-Zhang model.

  13. Dynamic scaling analysis of two-dimensional cell colony fronts in a gel medium: a biological system approaching a quenched Kardar-Parisi-Zhang universality.

    PubMed

    Huergo, M A C; Muzzio, N E; Pasquale, M A; Pedro González, P H; Bolzán, A E; Arvia, A J

    2014-08-01

    The interfacial two-dimensional spreading dynamics of quasilinear Vero cell colony fronts in methylcellulose (MC)-containing culture medium, under a constant average front displacement velocity regime, was investigated. Under comparable experimental conditions, the average colony front displacement velocity becomes lower than that reported for a standard culture medium. Initially, the presence of MC in the medium hinders both the colony spreading, due to a gradual change in the average size and shape of cells and their distribution in the colony, and the cell motility in the gelled medium. Furthermore, at longer culture times enlarged cells appear at random in the border region of the colony. These cells behave as obstacles (pinning sites) for the displacement of smaller cells towards the colony front. The dynamic scaling analysis of rough fronts yields the set of exponents α=0.63±0.04,β=0.75±0.05, and z=0.84±0.05, which is close to that expected for a quenched Kardar-Parisi-Zhang model.

  14. Prospective surface marker-based isolation and expansion of fetal endothelial colony-forming cells from human term placenta.

    PubMed

    Patel, Jatin; Seppanen, Elke; Chong, Mark S K; Yeo, Julie S L; Teo, Erin Y L; Chan, Jerry K Y; Fisk, Nicholas M; Khosrotehrani, Kiarash

    2013-11-01

    The term placenta is a highly vascularized tissue and is usually discarded upon birth. Our objective was to isolate clinically relevant quantities of fetal endothelial colony-forming cells (ECFCs) from human term placenta and to compare them to the well-established donor-matched umbilical cord blood (UCB)-derived ECFCs. A sorting strategy was devised to enrich for CD45-CD34+CD31Lo cells prior to primary plating to obtain pure placental ECFCs (PL-ECFCs) upon culture. UCB-ECFCs were derived using a well-described assay. PL-ECFCs were fetal in origin and expressed the same cell surface markers as UCB-ECFCs. Most importantly, a single term placenta could yield as many ECFCs as 27 UCB donors. PL-ECFCs and UCB-ECFCs had similar in vitro and in vivo vessel forming capacities and restored mouse hind limb ischemia in similar proportions. Gene expression profiles were only minimally divergent between PL-ECFCs and UCB-ECFCs, probably reflecting a vascular source versus a circulating source. Finally, PL-ECFCs and UCB-ECFCs displayed similar hierarchies between high and low proliferative colonies. We report a robust strategy to isolate ECFCs from human term placentas based on their cell surface expression. This yielded much larger quantities of ECFCs than UCB, but the cells were comparable in immunophenotype, gene expression, and in vivo functional ability. We conclude that PL-ECFCs have significant bio-banking and clinical translatability potential.

  15. Mast cells mediate malignant pleural effusion formation

    PubMed Central

    Giannou, Anastasios D.; Marazioti, Antonia; Spella, Magda; Kanellakis, Nikolaos I.; Apostolopoulou, Hara; Psallidas, Ioannis; Prijovich, Zeljko M.; Vreka, Malamati; Zazara, Dimitra E.; Lilis, Ioannis; Papaleonidopoulos, Vassilios; Kairi, Chrysoula A.; Patmanidi, Alexandra L.; Giopanou, Ioanna; Spiropoulou, Nikolitsa; Harokopos, Vaggelis; Aidinis, Vassilis; Spyratos, Dionisios; Teliousi, Stamatia; Papadaki, Helen; Taraviras, Stavros; Snyder, Linda A.; Eickelberg, Oliver; Kardamakis, Dimitrios; Iwakura, Yoichiro; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Kalomenidis, Ioannis; Blackwell, Timothy S.; Agalioti, Theodora; Stathopoulos, Georgios T.

    2015-01-01

    Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1β, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell–induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable. PMID:25915587

  16. Differential spheroid formation by oral cancer cells.

    PubMed

    Lee, Carlin; Lee, Casey; Atakilit, Amha; Siu, Amanda; Ramos, Daniel M

    2014-12-01

    Squamous cell carcinomas (SCC) make up 96% of all oral cancers. Most laboratory SCC studies grow cells as a monolayer, which does not accurately represent the disease in vivo. We used a more relevant multicellular spheroid (MCS) model to study this disease. The SCC9β6KDFyn cell line, which expresses full-length β6 and a kinase dead Fyn formed the largest MCS. Cell adhesive properties are dynamic and N-cadherin was increased in the largest MCS. c-Raf mediates the survival of tumor cells and was consistently expressed both in monolayers and in the MCS by SCC9β6D1 cells which lack the β6 cytoplasmic tail and, do not activate Fyn. SCC9β6KDFyn cells also express high levels of c-Raf when grown as spheroids in which Fyn suppression stimulates MCS formation. Tumor microenvironment and growth patterns modulate cell behavior and suppression of Fyn kinase may promote MCS growth.

  17. Cell cycle tracking for irradiated and unirradiated bystander cells in a single colony with exposure to a soft X-ray microbeam.

    PubMed

    Kaminaga, Kiichi; Noguchi, Miho; Narita, Ayumi; Hattori, Yuya; Usami, Noriko; Yokoya, Akinari

    2016-11-01

    To establish a new experimental technique to explore the photoelectric and subsequent Auger effects on the cell cycles of soft X-ray microbeam-irradiated cells and unirradiated bystander cells in a single colony. Several cells located in the center of a microcolony of HeLa-Fucci cells consisting of 20-80 cells were irradiated with soft X-ray (5.35 keV) microbeam using synchrotron radiation as a light source. All cells in the colony were tracked for 72 h by time-lapse microscopy imaging. Cell cycle progression, division, and death of each cell in the movies obtained were analyzed by pedigree assay. The number of cell divisions in the microcolony was also determined. The fates of these cells were clarified by tracking both irradiated and unirradiated bystander cells. Irradiated cells showed significant cell cycle retardation, explosive cell death, or cell fusion after a few divisions. These serious effects were also observed in 15 and 26% of the bystander cells for 10 and 20 Gy irradiation, respectively, and frequently appeared in at least two daughter or granddaughter cells from a single-parent cell. We successfully tracked the fates of microbeam-irradiated cells and unirradiated bystander cells with live cell recordings, which have revealed the dynamics of soft X-ray irradiated and unirradiated bystander cells for the first time. Notably, cell deaths or cell cycle arrests frequently arose in closely related cells. These details would not have been revealed by a conventional immunostaining imaging method. Our approach promises to reveal the dynamic cellular effects of soft X-ray microbeam irradiation and subsequent Auger processes from various endpoints in future studies.

  18. Associative memory cells: Formation, function and perspective

    PubMed Central

    Wang, Jin-Hui; Cui, Shan

    2017-01-01

    Associative learning and memory are common activities in life, and their cellular infrastructures constitute the basis of cognitive processes. Although neuronal plasticity emerges after memory formation, basic units and their working principles for the storage and retrieval of associated signals remain to be revealed. Current reports indicate that associative memory cells, through their mutual synapse innervations among the co-activated sensory cortices, are recruited to fulfill the integration, storage and retrieval of multiple associated signals, and serve associative thinking and logical reasoning. In this review, we aim to summarize associative memory cells in their formation, features and functional impacts. PMID:28408978

  19. Combination of stem cell factor and granulocyte colony-stimulating factor mobilizes the highest number of primitive haemopoietic progenitors as shown by pre-colony-forming unit (pre-CFU) assay.

    PubMed

    Horsfall, M J; Hui, C H; To, L B; Begley, C G; Basser, R L; Simmons, P J

    2000-06-01

    Fifty-two patients with poor prognosis carcinoma of the breast underwent peripheral blood stem cell (PBSC) mobilization using five different regimens. The yields of primitive haemopoietic progenitors were quantified by a recently described pre-colony-forming unit (pre-CFU) assay using limiting dilution analysis (LDA). Results of days 14 and 35 pre-CFU were also correlated with conventional CD34+ cell enumeration, CFU-GM (granulocyte-macrophage) and long-term culture-initiating cell (LTCIC) assays. The yield of pre-CFUs with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) was significantly higher than with G-CSF alone, cyclophosphamide (Cyclo) and granulocyte-monocyte colony-stimulating factor (GM-CSF), interleukin (IL)-3 and GM-CSF, or Cyclo alone. No significant correlation between neutrophil engraftment and pre-CFU could be demonstrated. Furthermore, CFU-GM was shown to bear a stronger correlation with pre-CFU and LTCIC than CD34+ cell measurement; thus, CFU-GM remains a useful biological tool for haemopoietic stem cell assay. We conclude that the combination of G-CSF and SCF mobilizes the highest number of pre-CFUs as measured by functional pre-CFU assay, which provides an alternative measurement of primitive haemopoietic progenitors to the LTCIC assay.

  20. Diatom Cell Size, Coloniality and Motility: Trade-Offs between Temperature, Salinity and Nutrient Supply with Climate Change

    PubMed Central

    Svensson, Filip; Norberg, Jon; Snoeijs, Pauline

    2014-01-01

    Reduction in body size has been proposed as a universal response of organisms, both to warming and to decreased salinity. However, it is still controversial if size reduction is caused by temperature or salinity on their own, or if other factors interfere as well. We used natural benthic diatom communities to explore how “body size” (cells and colonies) and motility change along temperature (2–26°C) and salinity (0.5–7.8) gradients in the brackish Baltic Sea. Fourth-corner analysis confirmed that small cell and colony sizes were associated with high temperature in summer. Average community cell volume decreased linearly with 2.2% per °C. However, cells were larger with artificial warming when nutrient concentrations were high in the cold season. Average community cell volume increased by 5.2% per °C of artificial warming from 0 to 8.5°C and simultaneously there was a selection for motility, which probably helped to optimize growth rates by trade-offs between nutrient supply and irradiation. Along the Baltic Sea salinity gradient cell size decreased with decreasing salinity, apparently mediated by nutrient stoichiometry. Altogether, our results suggest that climate change in this century may polarize seasonality by creating two new niches, with elevated temperature at high nutrient concentrations in the cold season (increasing cell size) and elevated temperature at low nutrient concentrations in the warm season (decreasing cell size). Higher temperature in summer and lower salinity by increased land-runoff are expected to decrease the average cell size of primary producers, which is likely to affect the transfer of energy to higher trophic levels. PMID:25279720

  1. Diatom cell size, coloniality and motility: trade-offs between temperature, salinity and nutrient supply with climate change.

    PubMed

    Svensson, Filip; Norberg, Jon; Snoeijs, Pauline

    2014-01-01

    Reduction in body size has been proposed as a universal response of organisms, both to warming and to decreased salinity. However, it is still controversial if size reduction is caused by temperature or salinity on their own, or if other factors interfere as well. We used natural benthic diatom communities to explore how "body size" (cells and colonies) and motility change along temperature (2-26°C) and salinity (0.5-7.8) gradients in the brackish Baltic Sea. Fourth-corner analysis confirmed that small cell and colony sizes were associated with high temperature in summer. Average community cell volume decreased linearly with 2.2% per °C. However, cells were larger with artificial warming when nutrient concentrations were high in the cold season. Average community cell volume increased by 5.2% per °C of artificial warming from 0 to 8.5°C and simultaneously there was a selection for motility, which probably helped to optimize growth rates by trade-offs between nutrient supply and irradiation. Along the Baltic Sea salinity gradient cell size decreased with decreasing salinity, apparently mediated by nutrient stoichiometry. Altogether, our results suggest that climate change in this century may polarize seasonality by creating two new niches, with elevated temperature at high nutrient concentrations in the cold season (increasing cell size) and elevated temperature at low nutrient concentrations in the warm season (decreasing cell size). Higher temperature in summer and lower salinity by increased land-runoff are expected to decrease the average cell size of primary producers, which is likely to affect the transfer of energy to higher trophic levels.

  2. Successful mobilization of peripheral blood stem cells in children with cancer using plerixafor (Mozobil) and granulocyte-colony stimulating factor.

    PubMed

    Avramova, Boryana E; Yordanova, Maya N; Konstantinov, Dobrin N; Bobev, Dragan G

    2011-01-01

    This paper describes the successful mobilization of peripheral blood stem cells for autologous transplantation in three children with malignant diseases by using plerixafor (Mozobil; Genzyme Corporation, Cambridge, MA) and granulocyte-colony stimulating factor (G-CSF) after failed previous mobilizations. A median sixfold increase in the number of circulating CD34+ cells after plerixafor treatment as compared with the baseline level was observed. An optimal CD34+ cell count for transplantation with one or two leukapheresis sessions was achieved. Mobilization using plerixafor was found to be safe with no adverse events. Therefore, the combination of G-CSF and plerixafor in children results in effective increases in peripheral CD34+ cell counts and reduces the risk of mobilization failure.

  3. Successful mobilization of peripheral blood stem cells in children with cancer using plerixafor (Mozobil™) and granulocyte-colony stimulating factor

    PubMed Central

    Avramova, Boryana E; Yordanova, Maya N; Konstantinov, Dobrin N; Bobev, Dragan G

    2011-01-01

    This paper describes the successful mobilization of peripheral blood stem cells for autologous transplantation in three children with malignant diseases by using plerixafor (Mozobil™; Genzyme Corporation, Cambridge, MA) and granulocyte-colony stimulating factor (G-CSF) after failed previous mobilizations. A median sixfold increase in the number of circulating CD34+ cells after plerixafor treatment as compared with the baseline level was observed. An optimal CD34+ cell count for transplantation with one or two leukapheresis sessions was achieved. Mobilization using plerixafor was found to be safe with no adverse events. Therefore, the combination of G-CSF and plerixafor in children results in effective increases in peripheral CD34+ cell counts and reduces the risk of mobilization failure. PMID:21966213

  4. Presence of Calcium Lowers the Expansion of Bacillus subtilis Colony Biofilms

    PubMed Central

    Mhatre, Eisha; Sundaram, Anandaroopan; Hölscher, Theresa; Mühlstädt, Mike; Bossert, Jörg; Kovács, Ákos T.

    2017-01-01

    Robust colony formation by Bacillus subtilis is recognized as one of the sessile, multicellular lifestyles of this bacterium. Numerous pathways and genes are responsible for the architecturally complex colony structure development. Cells in the biofilm colony secrete extracellular polysaccharides (EPS) and protein components (TasA and the hydrophobin BslA) that hold them together and provide a protective hydrophobic shield. Cells also secrete surfactin with antimicrobial as well as surface tension reducing properties that aid cells to colonize the solid surface. Depending on the environmental conditions, these secreted components of the colony biofilm can also promote the flagellum-independent surface spreading of B. subtilis, called sliding. In this study, we emphasize the influence of Ca2+ in the medium on colony expansion of B. subtilis. Interestingly, the availability of Ca2+ has no major impact on the induction of complex colony morphology. However, in the absence of this divalent ion, peripheral cells of the colony expand radially at later stages of development, causing colony size to increase. We demonstrate that the secreted extracellular compounds, EPS, BslA, and surfactin facilitate colony expansion after biofilm maturation. We propose that Ca2+ hinders biofilm colony expansion by modifying the amphiphilic properties of surfactin. PMID:28212310

  5. Recognition of Epstein-Barr virus (EBV)-infected cells by T cell colonies from a human chimera: restriction by allogeneic determinants.

    PubMed Central

    Plotnicky, H; Touraine, J L

    1993-01-01

    The anti-EBV T cell response was studied in a severe combined immunodeficiency patient (PS) who received two transplants of fetal liver cells. His peripheral blood mononuclear cells (PBMC) were incubated with EBV and cultured during 15 days. Eleven colonies were derived from the T lymphocytes causing the regression of the infected cell foci: nine were constituted with CD3+ CD4+ CD8- lymphocytes and two with CD3+ CD4- CD8+ cells. HLA typing of six colonies showed that two of them derived from the first transplant and four from the second one. The colonies killed the cells of the lymphoblastoid line (LCL) derived from the recipient (PS-LCL), but failed to kill the LCL matched with the transplants. With only one exception, they all lysed also the LCL derived from the mother or from the father, but they were ineffective on the EBV-negative lymphoblasts. Two colonies recognized determinants which did not appear to be HLA antigens, although they were shared by PS and by one of his parents, two (CD4- CD8+) reacted against the LCL which shared HLA-A3 or -A33 with PS-LCL, and four (CD4+ CD8-) lysed the LCL sharing HLA-A3, -A33 or -DR5 with PS-LCL, among which only one was demonstrated to interact directly with host HLA-class I determinants. These data indicate that T lymphocytes differentiating in contact with histo-incompatible determinants may express the capability to recognize viral antigens and to lyse virus-infected cells in the context of allogeneic MHC or non-MHC molecules. PMID:7504600

  6. OpenCFU, a new free and open-source software to count cell colonies and other circular objects.

    PubMed

    Geissmann, Quentin

    2013-01-01

    Counting circular objects such as cell colonies is an important source of information for biologists. Although this task is often time-consuming and subjective, it is still predominantly performed manually. The aim of the present work is to provide a new tool to enumerate circular objects from digital pictures and video streams. Here, I demonstrate that the created program, OpenCFU, is very robust, accurate and fast. In addition, it provides control over the processing parameters and is implemented in an intuitive and modern interface. OpenCFU is a cross-platform and open-source software freely available at http://opencfu.sourceforge.net.

  7. Contribution of Vascular Cells to Neointimal Formation

    PubMed Central

    Yuan, Falei; Wang, Dong; Xu, Kang; Wang, Jixian; Zhang, Zhijun; Yang, Li; Yang, Guo-Yuan; Li, Song

    2017-01-01

    The de-differentiation and proliferation of smooth muscle cells (SMCs) are widely accepted as the major contributor to vascular remodeling. However, recent studies indicate that vascular stem cells (VSCs) also play an important role, but their relative contribution remains to be elucidated. In this study, we used genetic lineage tracing approach to further investigate the contribution of SMCs and VSCs to neointimal thickening in response to endothelium denudation injury or artery ligation. In vitro and in vivo analysis of MYH11-cre/Rosa-loxP-RFP mouse artery showed that SMCs proliferated at a much slower rate than non-SMCs. Upon denudation or ligation injury, two distinct types of neointima were identified: Type-I neointimal cells mainly involved SMCs, while Type II mainly involved non-SMCs. Using Sox10-cre/Rosa-loxP-LacZ mice, we found that Sox10+ cells were one of the cell sources in neointima. In addition, lineage tracing using Tie2-cre/Rosa-LoxP-RFP showed that endothelial cells also contributed to the neointimal formation, but rarely transdifferentiated into mesenchymal lineages. These results provide a novel insight into the contribution of vascular cells to neointima formation, and have significant impact on the development of more effective therapies that target specific vascular cell types. PMID:28060852

  8. Macrophage Colony Stimulating Factor Derived from CD4+ T Cells Contributes to Control of a Blood-Borne Infection

    PubMed Central

    de Melo, Gabrielly L.; Anidi, Chioma; Hamburger, Rebecca; Pham, Jennifer

    2016-01-01

    Dynamic regulation of leukocyte population size and activation state is crucial for an effective immune response. In malaria, Plasmodium parasites elicit robust host expansion of macrophages and monocytes, but the underlying mechanisms remain unclear. Here we show that myeloid expansion during P. chabaudi infection is dependent upon both CD4+ T cells and the cytokine Macrophage Colony Stimulating Factor (MCSF). Single-cell RNA-Seq analysis on antigen-experienced T cells revealed robust expression of Csf1, the gene encoding MCSF, in a sub-population of CD4+ T cells with distinct transcriptional and surface phenotypes. Selective deletion of Csf1 in CD4+ cells during P. chabaudi infection diminished proliferation and activation of certain myeloid subsets, most notably lymph node-resident CD169+ macrophages, and resulted in increased parasite burden and impaired recovery of infected mice. Depletion of CD169+ macrophages during infection also led to increased parasitemia and significant host mortality, confirming a previously unappreciated role for these cells in control of P. chabaudi. This work establishes the CD4+ T cell as a physiologically relevant source of MCSF in vivo; probes the complexity of the CD4+ T cell response during type 1 infection; and delineates a novel mechanism by which T helper cells regulate myeloid cells to limit growth of a blood-borne intracellular pathogen. PMID:27923070

  9. Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

    SciTech Connect

    Islam, Shamima; Hassan, Ferdaus; Tumurkhuu, Gantsetseg; Dagvadorj, Jargalsaikhan; Koide, Naoki; Naiki, Yoshikazu; Mori, Isamu; Yoshida, Tomoaki; Yokochi, Takashi . E-mail: yokochi@aichi-med-u.ac.jp

    2007-08-24

    Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-{alpha} antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-{kappa}B ligand (RANKL). TNF-{alpha} might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-{kappa}B and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.

  10. Site-specific regulation of tissue dendritic cell function by granulocyte–macrophage colony-stimulating-factor

    PubMed Central

    Lees, Joanne; Boam, David; Proungvitaya, Tanakorn; Wood, Peter J

    2004-01-01

    Tissue dendritic cells (DC) are usually associated with phagocytic function but poor T-cell immunostimulatory capacity. Following activation, dendritic cells are stimulated to leave tissue sites and migrate to lymphoid tissue, acquiring immunostimulatory capacity during the process. We provide evidence that the immunostimulatory capacity of tissue DC, but not spleen cells, can be affected in situ by granulocyte–macrophage colony-stimulating-factor (GM-CSF). Initially it was found that islet cells from non-obese diabetic and BALB/c mice, which produce GM-CSF, showed significantly higher immunostimulatory capacity than islets from C3H and C57BL/6 mice, which do not produce GM-CSF. Second, pretreatment of nonobese diabetic mice with anti-GM-CSF antibody significantly reduced the immunostimulatory capacity of islet cells, but not spleen cells, although it had no effect on the numbers of cells expressing DC-associated antigens. Therefore the immunostimulatory function of islet DC is partially dependent on GM-CSF. By contrast, spleen DC immunostimulatory function does not show the same dependence on GM-CSF. This may affect the ability of dendritic cells to stimulate autoimmune responses or tolerance. PMID:15554926

  11. Kinetics of human hemopoietic cells after in vivo administration of granulocyte-macrophage colony-stimulating factor.

    PubMed Central

    Aglietta, M; Piacibello, W; Sanavio, F; Stacchini, A; Aprá, F; Schena, M; Mossetti, C; Carnino, F; Caligaris-Cappio, F; Gavosto, F

    1989-01-01

    The kinetic changes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) on hemopoietic cells were assessed in physiological conditions by administering GM-CSF (8 micrograms/kg per d) for 3 d to nine patients with solid tumors and normal bone marrow (BM), before chemotherapy. GM-CSF increased the number of circulating granulocytes and monocytes; platelets, erythrocytes, lymphocyte number, and subsets were unmodified. GM-CSF increased the percentage of BM S phase BFU-E (from 32 +/- 7 to 79 +/- 16%), day 14 colony-forming unit granulocyte-macrophage (CFU-GM) (from 43 +/- 20 to 82 +/- 11%) and day 7 CFU-GM (from 41 +/- 14 to 56 +/- 20%). The percentage of BM myeloblasts, promyelocytes, and myelocytes in S phase increased from 26 +/- 14 to 41 +/- 6%, and that of erythroblasts increased from 25 +/- 12 to 30 +/- 12%. This suggests that GM-CSF activates both erythroid and granulomonopoietic progenitors but that, among the morphologically recognizable BM precursors, only the granulomonopoietic lineage is a direct target of the molecule. GM-CSF increased the birth rate of cycling cells from 1.3 to 3.4 cells %/h and decreased the duration of the S phase from 14.3 to 9.1 h and the cell cycle time from 86 to 26 h. After treatment discontinuation, the number of circulating granulocytes and monocytes rapidly fell. The proportion of S phase BM cells dropped to values lower than pretreatment levels, suggesting a period of relative refractoriness to cell cycle-active antineoplastic agents. PMID:2643633

  12. Transport vesicle formation in plant cells.

    PubMed

    Hwang, Inhwan; Robinson, David G

    2009-12-01

    In protein trafficking, transport vesicles bud from donor compartments and carry cargo proteins to target compartments with which they fuse. Thus, vesicle formation is an essential step in protein trafficking. As for mammals, plant cells contain the three major types of vesicles: COPI, COPII, and CCV and the major molecular players in vesicle-mediated protein transport are also present. However, plant cells generally contain more isoforms of the coat proteins, ARF GTPases and their regulatory proteins, as well as SNAREs. In addition, plants have established some unique subfamilies, which may reflect plant cell-specific conditions such as the absence of an ER-Golgi intermediate compartment and the combined activities of the TGN and early endosome. Thus, even though we are still at an early stage in understanding the physiological function of these proteins, it is already clear that vesicle-mediated protein transport in plant cells displays both similarities as well as differences in animal cells.

  13. Radiosensitivity of kidney colony-forming cells: a short-term assay in situ in the mouse

    SciTech Connect

    Ewen, C.; Hendry, J.H.

    1989-04-01

    A short-term colony assay for renal tubule epithelium has been developed. Uranyl nitrate (UN) is a heavy metal nephrotoxin that induces acute tubule necrosis followed by a large compensatory increase in the rate of cell proliferation in the nephron. UN was used to precipitate latent damage following renal irradiation. Using a subcapsular colony count at 14 days after unilateral irradiation, a single-dose cell survival curve was obtained with a D0 of 4.2 +/- 0.3 Gy. High-dose irradiation of an exteriorized kidney resulted in a survival curve which was biphasic, with a plateau in survival between 18 and 40 Gy. Subtraction of this plateau level from all the survival data gave D0 values of 2.5 +/- 0.2 Gy (data analyzed between 7.5 and 16 Gy) or 2.0 +/- 0.2 Gy (over range 12-16 Gy). The D0 value obtained at 20 months after bilateral (or unilateral) kidney irradiation, without the use of UN, was 2.9 +/- 1.1 Gy (over range 10-14 Gy).

  14. A hypothesis of target cell formation in sickle cell disease.

    PubMed

    Wong, P

    2016-08-01

    A fraction of erythrocytes appear as target cells in stained blood smears in sickle cell disease, due to a inheritance of the hemoglobin variant Hb S, polymerizing upon deoxygenation. These cells appear in a three dimension as thin cups. A process of their formation in this disease is proposed based on a band 3-based mechanism of the erythrocyte shape control, able to explain the erythrocyte echinocytosis by glucose depletion. It indicates that their formation is due to a stomatocytogenic slow outward transport of the dibasic form of endogenous Pi with an H(+) by band 3, promoted by the decrease of the Donnan ratio, which decreases cell pH and volume, attributed by a decrease of cell KCl concentration by the higher efflux of K(+)Cl(-) cotransport and Ca(2+) activation of the Gardos channel. Its implications are briefly discussed with respect to target cells per se, target cell formation in other hemoglobinopathies, acquired and inherited disorders of the lipid metabolism and dehydrated hereditary stomatocytosis as well as a stomatocyte presence in a double heterozygote of Hb S and Hb C and of an involvement of the process of target cell formation in acanthocytosis in acquired and inherited disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Continuum Theory of Dislocations: Cell Structure Formation

    NASA Astrophysics Data System (ADS)

    Limkumnerd, Surachate; Sethna, James P.

    2005-03-01

    Line-like topological defects inside metals are called dislocations. These dislocations in late stages of hardening form patterns called cell structures. We are developing a mesoscale theory for the formation of cell structures that systematically derives the order parameter fields and evolution laws from the conserved topological Burgers vector density or the Nye dislocation tensor. (In classical plasticity theories, describing scales large compared to these cells, one normally bypasses the complicated motions of the dislocations by supplying yield surface and plastic hardening function in order to determine the evolution of state variables.) Using Landau approach and a closure approximation, an evolution equation for the dislocation density tensor is obtained by employing simple symmetry arguments and the constraint that the elastic energy must decrease with time at fixed stress. The evolution laws lead to singularity formation at finite times, which we expect will be related to the formation of cell walls. Implementation of finite difference simulations using the upwind scheme and the results in one and higher dimensions will be discussed.

  16. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

    PubMed

    Fortunato, Tiago M; Vara, Dina S; Wheeler-Jones, Caroline P; Pula, Giordano

    2014-01-01

    Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25-100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.

  17. Commensal Microbiota and CD8+ T Cells Shape the Formation of Invariant NKT Cells

    PubMed Central

    Wei, Bo; Wingender, Gerhard; Fujiwara, Daisuke; Chen, Diana YuHui; McPherson, Michael; Brewer, Sarah; Borneman, James; Kronenberg, Mitchell; Braun, Jonathan

    2012-01-01

    Commensal bacteria play an important role in formation of the immune system, but the mechanisms involved are incompletely understood. In this study, we analyze CD1d-restricted invariant NKT (iNKT) cells in germfree mice and in two colonies of C57BL/6 mice termed conventional flora and restricted flora (RF), stably bearing commensal microbial communities of diverse but distinct composition. In germfree mice, iNKT cells were moderately reduced, suggesting that commensal microbiota were partially required for the antigenic drive in maintaining systemic iNKT cells. Surprisingly, even greater depletion of iNKT cell population occurred in RF mice. This was in part attributable to reduced RF levels of intestinal microbial taxa (Sphingomonas spp.) known to express antigenic glycosphingolipid products. However, memory and activated CD8+ T cells were also expanded in RF mice, prompting us to test whether CD8+ T cell activity might be further depleting iNKT cells. Indeed, iNKT cell numbers were restored in RF mice bearing the CD8α−/− genotype or in adult wild-type RF mice acutely depleted with anti-CD8 Ab. Moreover, iNKT cells were restored in RF mice bearing the Prf1−/− phenotype, a key component of cytolytic function. These findings indicate that commensal microbiota, through positive (antigenic drive) and negative (cytolytic depletion by CD8+ T cells) mechanisms, profoundly shape the iNKT cell compartment. Because individuals greatly vary in the composition of their microbial communities, enteric microbiota may play an important epigenetic role in the striking differences in iNKT cell abundance in humans and therefore in their potential contribution to host immune status. PMID:20048124

  18. Solar cell contact formation using laser ablation

    DOEpatents

    Harley, Gabriel; Smith, David D.; Cousins, Peter John

    2015-07-21

    The formation of solar cell contacts using a laser is described. A method of fabricating a back-contact solar cell includes forming a poly-crystalline material layer above a single-crystalline substrate. The method also includes forming a dielectric material stack above the poly-crystalline material layer. The method also includes forming, by laser ablation, a plurality of contacts holes in the dielectric material stack, each of the contact holes exposing a portion of the poly-crystalline material layer; and forming conductive contacts in the plurality of contact holes.

  19. Solar cell contact formation using laser ablation

    DOEpatents

    Harley, Gabriel; Smith, David D.; Cousins, Peter John

    2014-07-22

    The formation of solar cell contacts using a laser is described. A method of fabricating a back-contact solar cell includes forming a poly-crystalline material layer above a single-crystalline substrate. The method also includes forming a dielectric material stack above the poly-crystalline material layer. The method also includes forming, by laser ablation, a plurality of contacts holes in the dielectric material stack, each of the contact holes exposing a portion of the poly-crystalline materiat layer; and forming conductive contacts in the plurality of contact holes.

  20. Solar cell contact formation using laser ablation

    DOEpatents

    Harley, Gabriel; Smith, David; Cousins, Peter

    2012-12-04

    The formation of solar cell contacts using a laser is described. A method of fabricating a back-contact solar cell includes forming a poly-crystalline material layer above a single-crystalline substrate. The method also includes forming a dielectric material stack above the poly-crystalline material layer. The method also includes forming, by laser ablation, a plurality of contacts holes in the dielectric material stack, each of the contact holes exposing a portion of the poly-crystalline material layer; and forming conductive contacts in the plurality of contact holes.

  1. Prognostic utility of spontaneous erythroid colony formation and JAK2 mutational analysis for thrombotic events in essential thrombocythaemia.

    PubMed

    Weston, H; Cowell, V; Grimmett, K; Saal, R; Jones, M; Mills, T; Gill, D; Marlton, P; Bird, R; Mollee, P

    2011-05-01

    Thrombotic events in essential thrombocythaemia (ET) are difficult to predict with current risk stratification based on age and prior history of thrombosis. We aimed to assess the predictive value of the JAK2 V617F mutation (JAK2) and spontaneous erythroid colony (SEC) growth for the development of thrombotic events post diagnosis in patients with ET. Consecutive patients with ET were retrospectively identified, and clinical and laboratory correlates were evaluated. Thrombotic events were categorized according to their occurrence at or prior to diagnosis (prior thrombosis), and any time post diagnosis of ET (subsequent thrombosis). JAK2 analysis was performed by allele-specific PCR on whole blood or bone marrow. A total of 62 patients was identified, median age 63 years; 67% (41/61) JAK2-positive and 47% (25/53) SEC-positive. Median follow-up was 33 months (range, 1 to 137). JAK2-positive patients showed a trend to increased prior thrombosis (27% vs 5%, P= 0.08), and a significant increase in the development of subsequent thrombosis (5-year event rate 31% vs 6%, P= 0.04), which persisted when stratified for a history of prior thrombosis (P= 0.04). Survival was not affected by JAK2 status. The SEC assay predicted an increased rate of baseline thrombosis (16% vs 0%, P= 0.04), but was not found to be predictive of any subsequent thrombotic events. Patients with ET who are JAK2-positive by whole blood allele-specific PCR appear to be at increased risk of thrombotic complications, which is independent of a prior history of thrombosis. © 2011 The Authors. Internal Medicine Journal © 2011 Royal Australasian College of Physicians.

  2. Chemical microenvironments and single-cell carbon and nitrogen uptake in field-collected colonies of Trichodesmium under different pCO2.

    PubMed

    Eichner, Meri J; Klawonn, Isabell; Wilson, Samuel T; Littmann, Sten; Whitehouse, Martin J; Church, Matthew J; Kuypers, Marcel Mm; Karl, David M; Ploug, Helle

    2017-06-01

    Gradients of oxygen (O2) and pH, as well as small-scale fluxes of carbon (C), nitrogen (N) and O2 were investigated under different partial pressures of carbon dioxide (pCO2) in field-collected colonies of the marine dinitrogen (N2)-fixing cyanobacterium Trichodesmium. Microsensor measurements indicated that cells within colonies experienced large fluctuations in O2, pH and CO2 concentrations over a day-night cycle. O2 concentrations varied with light intensity and time of day, yet colonies exposed to light were supersaturated with O2 (up to ~200%) throughout the light period and anoxia was not detected. Alternating between light and dark conditions caused a variation in pH levels by on average 0.5 units (equivalent to 15 nmol l(-1) proton concentration). Single-cell analyses of C and N assimilation using secondary ion mass spectrometry (SIMS; large geometry SIMS and nanoscale SIMS) revealed high variability in metabolic activity of single cells and trichomes of Trichodesmium, and indicated transfer of C and N to colony-associated non-photosynthetic bacteria. Neither O2 fluxes nor C fixation by Trichodesmium were significantly influenced by short-term incubations under different pCO2 levels, whereas N2 fixation increased with increasing pCO2. The large range of metabolic rates observed at the single-cell level may reflect a response by colony-forming microbial populations to highly variable microenvironments.

  3. Follow-up of healthy donors receiving granulocyte colony-stimulating factor for peripheral blood progenitor cell mobilization and collection. Results of the Spanish Donor Registry.

    PubMed

    de la Rubia, Javier; de Arriba, Felipe; Arbona, Cristina; Pascual, María J; Zamora, Concha; Insunza, Andrés; Martínez, Dorleta; Paniagua, Carmen; Díaz, Miguel A; Sanz, Miguel A

    2008-05-01

    Information about the long-term follow-up and safety of granulocyte colony-stimulating factor administration to healthy donors is limited. The aims of this study were to analyze the side effects of granulocyte colony-stimulating factor administration in donors included in a Spanish Registry of hematopoietic stem cell donors and to determine the long-term outcome of these donors. The Spanish National Donor Registry was developed to record the short- and long-term results of granulocyte colony-stimulating factor administration to mobilize peripheral blood progenitor cells in normal donors. To date, 1436 donors (771 males, 665 females) with a median age of 37 years (range, 1 to 74 years) have been registered. Granulocyte colony-stimulating factor was the only cytokine administered. A baseline investigation was performed in every donor before granulocyte colony-stimulating factor administration and follow-up investigations (controls) were planned at 4 weeks and annually thereafter for up to 5 years after the mobilization. At least one of the scheduled controls was performed in 736 donors, while 320 donors have been followed for 2 years or more. The peripheral white blood cell count decreased significantly from 6.8 x 10(9)/L at baseline to 5.9 x 10(9)/L at 4 weeks after leukapheresis (p<0.0001) and remained at values lower than those observed premobilization until 2 years after mobilization. In contrast, hemoglobin concentration and platelet count returned to normal values within 1 year after mobilization. Bone pain (90%) and headache (33%) were the most frequently reported granulocyte colony-stimulating factor-related side effects. Five patients (0.68%) were diagnosed as having solid tumors (lung cancer in two patients and thyroid carcinoma, choroid melanoma, and colon carcinoma in one patient each) between 10 and 64 months after administration of granulocyte colony-stimulating factor. No hematologic malignancies have been reported. The clinical side effects of

  4. Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms.

    PubMed

    van Gestel, Jordi; Weissing, Franz J; Kuipers, Oscar P; Kovács, Akos T

    2014-10-01

    In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express 'cooperative traits', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.

  5. Phagocytosis and cytokine response to rough and smooth colony variants of Mycobacterium abscessus by human peripheral blood mononuclear cells.

    PubMed

    Jönsson, Bodil; Ridell, Malin; Wold, Agnes E

    2013-01-01

    Mycobacterium abscessus is a non-tuberculous mycobacteria able to cause opportunistic infections in selected patient groups. During the last decades it has emerged as a cause of chronic pulmonary infection in patients with cystic fibrosis (CF). M. abscessus strains exhibit either smooth or rough colony morphology. Strains exhibiting the rough phenotype more often cause pulmonary infections in CF patients than did the smooth ones. Here, we examined phagocytosis and production of cytokines by human peripheral blood mononuclear cells, in response to M. abscessus strains with smooth and rough colony phenotype. The rough isolates all formed multicellular cords, similar to what is observed in Mycobacterium tuberculosis. Monocytes were generally unable to internalize these rough cord isolates, in contrast with the smooth ones. Furthermore, the rough M. abscessus strains induced a distinct cytokine profile differing from that induced by the smooth ones. Rough isolates induced significantly less IL-10 and tumour necrosis factor compared to smooth strains, but more IL-1β. Both varieties induced equal amounts of IFN-γ, IL-17, IL-23, IL-6, IL-8 and equally little IL-12. The ability to withstand phagocytosis might be a virulence factor contributing to the capacity of rough M. abscessus strains to give persistent pulmonary infections. © 2012 The Authors APMIS © 2012 APMIS.

  6. Catheter colonization and abscess formation due to Staphylococcus epidermidis with normal and small-colony-variant phenotype is mouse strain dependent.

    PubMed

    Sander, Gunnar; Börner, Tina; Kriegeskorte, André; von Eiff, Christof; Becker, Karsten; Mahabir, Esther

    2012-01-01

    Coagulase-negative staphylococci (CoNS) form a thick, multilayered biofilm on foreign bodies and are a major cause of nosocomial implant-associated infections. Although foreign body infection models are well-established, limited in vivo data are available for CoNS with small-colony-variant (SCV) phenotype described as causative agents in implant-associated infections. Therefore, we investigated the impact of the Staphylococcus epidermidis phenotype on colonization of implanted PVC catheters and abscess formation in three different mouse strains. Following introduction of a catheter subcutaneously in each flank of 8- to 12-week-old inbred C57BL/6JCrl (B6J), outbred Crl:CD1(ICR) (CD-1), and inbred BALB/cAnNCrl (BALB/c) male mice, doses of S. epidermidis O-47 wild type, its hemB mutant with stable SCV phenotype, or its complemented mutant at concentrations of 10(6) to 10(9) colony forming units (CFUs) were gently spread onto each catheter. On day 7, mice were sacrificed and the size of the abscesses as well as bacterial colonization was determined. A total of 11,500 CFUs of the complemented mutant adhered to the catheter in BALB/c followed by 9,960 CFUs and 9,900 CFUs from S. epidermidis wild type in BALB/c and CD-1, respectively. SCV colonization was highest in CD-1 with 9,500 CFUs, whereas SCVs were not detected in B6J. The minimum dose that led to colonization or abscess formation in all mouse strains was 10(7) or 10(8) CFUs of the normal phenotype, respectively. A minimum dose of 10(8) or 10(9) CFU of the hemB mutant with stable SCV phenotype led to colonization only or abscess formation, respectively. The largest abscesses were detected in BALB/c inoculated with wild type bacteria or SCV (64 mm(2) vs. 28 mm(2)). Our results indicate that colonization and abscess formation by different phenotypes of S. epidermidis in a foreign body infection model is most effective in inbred BALB/c followed by outbred CD-1 and inbred B6J mice.

  7. Identification and characterization of receptors for granulocyte colony-stimulating factor on human placenta and trophoblastic cells

    SciTech Connect

    Uzumaki, Hiroya; Okabe, Tetsuro; Sasaki, Norio; Hagiwara, Koichi; Takaku, Fumimaro; Tobita, Masahito; Yasukawa, Kaoru ); Ito, Seiga ); Umezawa, Yoshimi )

    1989-12-01

    Since radioiodination of human granulocyte colony-stimulating factor (G-CSF) is difficult, the authors synthesized a mutein of human G-CSF that retains full biological activity and receptor-binding capacity for at least 2 weeks after radioiodination. Receptors for human G-CSF were characterized in the plasma membrane fraction from the human term placenta (human placental membranes) and trophoblastic cells by using the {sup 125}I-labeled mutein of human G-CSF (KW-2228). The specific binding of {sup 125}I-labeled KW-2228 to placental membranes was pH-dependent, with maximal specific binding at pH 7.8; it increased linearly with protein to 3.7 mg of protein per ml and was both time- and temperature-dependent, with maximal binding at 4{degree}C after a 24-hr incubation. When the authors examined the ability of hematopoietic growth factors to inhibit {sup 125}I-labeled KW-2228 binding, they found that KW-2228 and intact human G-CSF ihibited {sup 125}I-labeled KW-2228 binding, whereas erythropoietin or granulocyte-macrophage colony-stimulating factor did not. Scatchard analysis revealed a single receptor type. The human G-CSF receptors on human placental membranes were shown to consist of two molecular species that could be specifically cross-linked to {sup 125}I-labeled KW-2228. Human trophoblastic cells, T3M-3, also possessed a single receptor for G-CSF. They have identified the receptor for human G-CSF on human placental membranes and trophoblastic cells.

  8. Do two different stem cell grafts: G-CSF stimulated and unstimulated bone marrow differ according to hematopoietic colony forming capacity?

    PubMed

    Özgüner, Meltem; Azık, Mehmet Fatih; Tavil, Betül; Bozkaya, Ikbal; Köksal, Yasin; Canal, Elif; Uçkan, Duygu; Tunç, Bahattin

    2014-06-01

    The study was designed to compare colony forming capacity of granulocyte-colony stimulating factor (G-CSF) stimulated bone marrow (G-BM) with standard unstimulated bone marrow (U-BM) of healthy donors of pediatric patients. CFU-Assay results of 26 healthy donors of pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) were analyzed retrospectively. 13 of donors received 10 μg/kg per day of G-CSF as a single injection for 3 consecutive days and other 13 of donors had unstimulated BM. Colony forming capacity of hematopoietic stem cells evaluated with Colony Forming Unit-Assay (CFU-Assay) with in semi-solid agar culture medium after 14-18 days of culture period. CFU-Assay results of G-BM and U-BM (expressed as means) were; Burst Forming Unit-Erythroid (BFU-E): 15.20 × 10(4)/kg and 8.38 × 10(4)/kg, Colony Forming Unit-Granulocyte Macrophage (CFU-GM): 10.35 × 10(4)/kg and 5.67 × 10(4)/kg, Colony Forming Unit-Erythroid (CFU-E): 0.59 × 10(4)/kg and 0.33 × 10(4)/kg, CFU-Granulocyte Erythroid Macrophage Megakaryocyte (CFU-GEMM): 0.52 × 10(4)/kg and 0.53 × 10(4)/kg respectively. BFU-E and CFU-GM capacity of G-BM was increased and statistically significantly different than standard U-BM (p ⩽ 0.01). In conclusion, increased colony forming capacity of hematopoietic stem cells of G-BM when compared with standard unstimulated BM could be a major advantage for transplantation.

  9. Intravenous Administration of Endothelial Colony-Forming Cells Overexpressing Integrin β1 Augments Angiogenesis in Ischemic Legs

    PubMed Central

    Goto, Kazuko; Takahashi, Tomoyuki; Okada, Hideshi; Kanamori, Hiromitsu; Kawamura, Itta; Watanabe, Takatomo; Morishita, Kentaro; Tsujimoto, Akiko; Miyazaki, Nagisa; Ushikoshi, Hiroaki; Kawasaki, Masanori; Mikami, Atsushi; Kosai, Ken-ichiro; Minatoguchi, Shinya

    2016-01-01

    When injected directly into ischemic tissue in patients with peripheral artery disease, the reparative capacity of endothelial progenitor cells (EPCs) appears to be limited by their poor survival. We, therefore, attempted to improve the survival of transplanted EPCs through intravenous injection and gene modification. We anticipated that overexpression of integrin β1 will enable injected EPCs to home to ischemic tissue, which abundantly express extracellular matrix proteins, the ligands for integrins. In addition, integrin β1 has an independent angiogenesis-stimulating function. Human endothelial colony-forming cells (ECFCs; late-outgrowth EPCs) were transduced using a lentiviral vector encoding integrin β1 (ITGB1) or enhanced green fluorescent protein (GFP). We then locally or systemically injected phosphate-buffered saline or the genetically modified ECFCs (GFP-ECFCs or ITGB1-ECFCs; 1 × 105 cells each) into NOD/Shi-scid, IL-2Rγnull mice whose right femoral arteries had been occluded 24 hours earlier. Upregulation of extracellular matrix proteins, including fibronectin, was apparent in the ischemic legs. Four weeks later, blood perfusion of the ischemic limb was significantly augmented only in the ITGB1-ECFC group. Scanning electron microscopy of vascular casts revealed increases in the perfused blood vessels in the ischemic legs of mice in the ITGB1-ECFC group and significant increases in the density of both capillaries and arterioles. Transplanted ECFC-derived vessels accounted for 28% ± 4.2% of the vessels in the ITGB1-ECFC group, with no cell fusion. Intravenous administration of ECFCs engineered to home to ischemic tissue appears to efficiently mediate therapeutic angiogenesis in a mouse model of peripheral artery disease. Significance The intravenous administration of endothelial colony-forming cells (ECFCs) genetically modified to overexpress integrin β1 effectively stimulated angiogenesis in ischemic mouse hindlimbs. Transplanted ECFCs were observed

  10. The co-injection of somatic cells with embryonic stem cells affects teratoma formation and the properties of teratoma-derived stem cell-like cells.

    PubMed

    Gong, Seung Pyo; Kim, Boyun; Kwon, Hyo Sook; Yang, Woo Sub; Jeong, Jae-Wook; Ahn, Jiyeon; Lim, Jeong Mook

    2014-01-01

    The aim of this study was to assess the biological reactions triggered by stem cell transplantation related to phenotypic alteration, host-to-cell response, chromosomal stability, transcriptional alteration, and stem cell-like cell re-expansion. B6CBAF1 mouse embryonic stem cells (ESCs) were injected subcutaneously into homologous or heterologous (B6D2F1) recipients, and heterologous injections were performed with or without co-injection of B6D2F1 fetal fibroblasts. All homologous injections resulted in teratoma formation, whereas a sharp decrease in formation was detected after heterologous injection (100 vs. 14%; p<0.05). The co-injection of somatic cells in heterologous injections enhanced teratoma formation significantly (14 vs. 75%; p<0.05). Next, ESC-like cell colonies with the same genotype as parental ESCs were formed by culturing teratoma-dissociated cells. Compared with parental ESCs, teratoma-derived ESC-like cells exhibited significantly increased aneuploidy, regardless of homologous or heterologous injections. Repopulation of the parental ESCs was the main factor that induced chromosomal instability, whereas the co-injection of somatic cells did not restore chromosomal normality. Different genes were expressed in the parental ESCs and teratoma-derived ESC-like cells; the difference was larger with parental vs. heterologous than parental vs. homologous co-injections. The co-injection of somatic cells decreased this difference further. In conclusion, the host-to-cell interactions triggered by ESC transplantation could be modulated by co-injection with somatic cells. A mouse model using homologous or heterologous transplantation of stem cells could help monitor cell adaptability and gene expression after injection.

  11. Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

    SciTech Connect

    Taru Sharma, G.; Dubey, Pawan K.; Verma, Om Prakash; Pratheesh, M.D.; Nath, Amar; Sai Kumar, G.

    2012-08-03

    Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it

  12. Viable Mycoplasma mycoides ssp. mycoides small colony-mediated depression of the bovine cell responsiveness to the mitogen concanavalin A.

    PubMed

    Dedieu, L; Balcer-Rodrigues, V

    2006-10-01

    Mycoplasma mycoides ssp. mycoides biotype Small Colony (MmmSC) is the causative agent of contagious bovine pleuropneumonia (CBPP), which is still a major tropical cattle disease. Development of an efficient vaccine requires an understanding of the immunopathology of CBPP as MmmSC presents a strong ability to escape the host immune response. The objective of this study was to determine whether the presence of MmmSC can modulate the immune response induced by the mitogen Concanavalin A (ConA) on bovine immune cells [peripheral blood mononuclear cells (PBMC) and lymph node (LN) cells]. Comparative analysis of the immunomodulating properties of viable versus heat-killed MmmSC on ConA-stimulated immune cells revealed that while heat-killed MmmSC had no effect, viable MmmSC strongly depressed, in a concentration-dependent manner, the ConA mitogenic activity (blastogenesis and interferon-gamma production). Both B-cell and T-cell activation were affected with the highest impact on the CD4 T cells. The phenotypic analysis showed that the ConA-induced proliferation of CD25(+) cells was strongly reduced when co-exposed to viable MmmSC, confirming that events associated with ConA-induced cell activation were suppressed by the pathogen. This study thus demonstrated that viable MmmSC is able to inhibit the polyclonal mitogenic activity of the ConA on bovine PBMC and LN cells. This finding strongly suggests that the persistence of viable MmmSC may also thus inhibit the bovine immune response directed towards inactivated MmmSC, whether dead or in the form of antigens, also present during infection. This study confirmed that MmmSC has evolved an efficient mechanism to prevent its elimination from the host.

  13. Downregulation of ROS-FIG inhibits cell proliferation, colony‑formation, cell cycle progression, migration and invasion, while inducing apoptosis in intrahepatic cholangiocarcinoma cells.

    PubMed

    Deng, Gang; Hu, Chenghuan; Zhu, Lei; Huang, Feizhou; Huang, Wei; Xu, Hongbo; Nie, Wanpin

    2014-09-01

    Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer with poor responsiveness to existing drug therapies. Therefore, novel treatment strategies against ICC are required to improve survival. The aim of this study was to demonstrate the role of fused-in-glioblastoma-c-ros-oncogene1 (FIG-ROS) fusion gene in ICC. ROS was positively expressed in ICC tissues and HUCCT1 cells. Plasmids expressing ROS- and FIG-specific shRNAs were constructed and transfected into HUCCT1 cells. The results showed that single transfection of ROS- or FIG-specific shRNA inhibited HUCCT1 cell proliferation, colony formation, cell cycle progression, migration and invasion, while inducing apoptosis. Moreover, the co-inhibition of ROS- and FIG-specific shRNA exhibited stronger effects on HUCCT1 cell proliferation, apoptosis, colony formation, cell cycle progression, migration and invasion, when compared to single inhibition of ROS and FIG. Furthermore, findings of this study suggested that the AKT signaling pathway was involved in the ROS-FIG-mediated biological processes of HUCCT1 cells. In summary, the results suggest that FIG-ROS plays an oncogenic role in ICC. Additionally, ROS1-6290 and FIG-363 segments may become effective therapeutic targets for ICC harboring ROS-FIG fusion protein.

  14. T Cell-Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration.

    PubMed

    Dohlman, Thomas H; Ding, Julia; Dana, Reza; Chauhan, Sunil K

    2017-02-01

    Growing evidence suggests that granulocyte-macrophage colony-stimulating factor (GM-CSF) contributes to T helper 17 (Th17) cell-associated immunoinflammatory diseases. The purpose of this study was to evaluate the effect of T cell-derived GM-CSF on CD11b+ myeloid cell function in dry eye disease (DED). In a murine model of DED, quantitative real-time PCR and ELISA were used to measure GM-CSF expression at the ocular surface, and flow cytometry was used to enumerate GM-CSF producing Th17 cells. A granulocyte-macrophage colony-stimulating factor neutralizing antibody was used topically in vivo and in an in vitro culture system to evaluate the role of GM-CSF in recruiting and maturing CD11b+ cells. Clinical disease severity was evaluated after topical administration of GM-CSF neutralizing antibody. In dry eye disease, GM-CSF is significantly upregulated at the ocular surface and the frequency of GM-CSF producing Th17 cells is significantly increased in the draining lymph nodes. In vitro neutralization of GM-CSF from CD4+ T cells derived from DED mice suppresses major histocompatibility complex II expression by CD11b+ cells and CD11b+ cell migration. Topical neutralization of GM-CSF in a murine model of DED suppresses CD11b+ maturation and migration, as well as Th17 cell induction, yielding a reduction in clinical signs of disease. T helper 17 cell-derived GM-CSF contributes to DED pathogenesis by promoting CD11b+ cell activation and migration to the ocular surface.

  15. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice

    PubMed Central

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C. A.; Woll, Petter S.; Jacobsen, Sten Eirik W.

    2016-01-01

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19+ B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R+CD19+ ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R+ myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R+ myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported. PMID:27207794

  16. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice.

    PubMed

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C A; Woll, Petter S; Jacobsen, Sten Eirik W; Sitnicka, Ewa

    2016-07-14

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19(+) B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R(+)CD19(+) ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R(+) myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R(+) myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported. © 2016 by The American Society of Hematology.

  17. Biologic Activity of Autologous, Granulocyte-Macrophage Colony Stimulating Factor Secreting Alveolar Soft Parts Sarcoma and Clear Cell Sarcoma Vaccines

    PubMed Central

    Goldberg, John; Fisher, David E.; Demetri, George D.; Neuberg, Donna; Allsop, Stephen A.; Fonseca, Catia; Nakazaki, Yukoh; Nemer, David; Raut, Chandrajit P.; George, Suzanne; Morgan, Jeffrey A.; Wagner, Andrew J.; Freeman, Gordon J.; Ritz, Jerome; Lezcano, Cecilia; Mihm, Martin; Canning, Christine; Hodi, F. Stephen; Dranoff, Glenn

    2015-01-01

    Purpose Alveolar soft parts sarcoma (ASPS) and clear cell sarcoma (CCS) are rare mesenchymal malignancies driven by chromosomal translocations that activate members of the microphthalmia transcription factor (MITF) family. However, in contrast to malignant melanoma, little is known about their immunogenicity. To learn more about the host response to ASPS and CCS, we conducted a phase I clinical trial of vaccination with irradiated, autologous sarcoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor (GM-CSF). Experimental Design Metastatic tumors from ASPS and CCS patients were resected, processed to single cell suspensions, transduced with a replication defective adenoviral vector encoding GM-CSF, and irradiated. Immunizations were administered subcutaneously and intradermally weekly times three and then every other week. Results Vaccines were successfully manufactured for 11 of the 12 enrolled patients. Eleven subjects received from 3 to 13 immunizations. Toxicities were restricted to grade 1–2 skin reactions at inoculation sites. Vaccination elicited local dendritic cell infiltrates and stimulated T cell mediated delayed type-hypersensitivity reactions to irradiated, autologous tumor cells. Antibody responses to tissue-type plasminogen activator (tTPA) and angiopoietins-1/2 were detected. Tumor biopsies showed programmed death-1 (PD-1) positive CD8+ T cells in association with PD ligand-1 (PD-L1) expressing sarcoma cells. No tumor regressions were observed. Conclusions Vaccination with irradiated, GM-CSF secreting autologous sarcoma cell vaccines is feasible, safe, and biologically active. Concurrent targeting of angiogenic cytokines and antagonism of the PD-1 negative regulatory pathway might intensify immune-mediated tumor destruction. PMID:25805798

  18. Tumor Progression of Skin Carcinoma Cells in Vivo Promoted by Clonal Selection, Mutagenesis, and Autocrine Growth Regulation by Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor

    PubMed Central

    Mueller, Margareta M.; Peter, Wolfgang; Mappes, Marion; Huelsen, Andrea; Steinbauer, Heinrich; Boukamp, Petra; Vaccariello, Michael; Garlick, Jonathan; Fusenig, Norbert E.

    2001-01-01

    Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo-progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations. PMID:11583982

  19. Weekly CODE chemotherapy with recombinant human granulocyte colony-stimulating factor for relapsed or refractory small cell lung cancer.

    PubMed

    Sato, K; Tsuchiya, S; Minato, K; Sunaga, N; Ishihara, S I; Makimoto, T; Naruse, I; Hoshino, H; Watanabe, S; Saitoh, R; Mori, M

    2000-01-01

    We used cisplatin, vincristine, doxorubicin, and etoposide (CODE) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) weekly for salvage chemotherapy in relapsed or refractory small cell lung cancer (SCLC). We reviewed the medical charts of patients between January 1993 and December 1996 at the National Nishi-Gunma Hospital. Twenty patients were treated with salvage chemotherapy. The overall response rate was 55.0%. The median survival time of extensive disease patients from the start of CODE therapy was 23 weeks and the 1-year survival rate was 21.0%. Toxicities were severe, especially in myelosuppression. CODE could be selected as a salvage therapy for chemotherapy- relapsed SCLC cases.

  20. Changes in epidermal radiosensitivity with time associated with increased colony numbers.

    PubMed

    van den Aardweg, G J; Morris, G M; Bywaters, A; Bakker, E J; Mooi, W J

    2001-05-01

    Epidermal clonogenic cell survival and colony formation following irradiation were investigated and related to radiosensitivity. A rapid in vivo/in vitro assay was developed for the quantification of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. Bromodeoxyuridine (BrdU)-labelled cells in full thickness epidermal sheets were visualized using standard immunohistochemistry. In unirradiated skin, approximately 900 BrdU-positive cells mm(-2) were counted. In a time sequence experiment, BrdU-positive cell numbers increased from an average of 900 cells mm(-2) to approximately 1400 cells mm(-2) after BrdU-labelling for 2-24 h. In irradiated skin, colonies containing >/=16 BrdU-positive cells were seen for the first time at days 14/15 after irradiation. The number of these colonies per cm(2) as a function of skin surface dose yielded a cell survival curve with a D(0)-value (+/-SE) of 3.9+/-0.6 Gy. This relatively high D(0)-value is possibly due to a rapid fall off in depth dose distribution for the iridium-192 source and consequently a substantial contribution of hair follicular epithelium to colony formation. At 14/15 days after irradiation, the ED(50) level of 33.6 Gy for the in vivo response of moist desquamation corresponded with 2.7 colonies cm(-2). Surprisingly, the number of colonies increased with time after irradiation with an estimated doubling time of approximately 4 days, while the D(0)-value remained virtually unchanged. This increase in colony numbers could be due to migration of clonogenic cells, to the recruitment of dormant clonogenic cell survivors by elevated levels of cytokines, or to both. Although frequent biopsying caused increased cytokine levels, which had a systemic effect on unirradiated skin, it had no influence on colony formation in irradiated skin. Smaller colonies, containing 4-8 cells or 9-15 cells, were abundant, particularly after higher doses, which resulted in higher D(0)-values. The majority of

  1. PAK6 targets to cell–cell adhesions through its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape

    PubMed Central

    Morse, Elizabeth M.; Sun, Xiaowen; Olberding, Jordan R.; Ha, Byung Hak; Boggon, Titus J.; Calderwood, David A.

    2016-01-01

    ABSTRACT The six serine/threonine kinases in the p21-activated kinase (PAK) family are important regulators of cell adhesion, motility and survival. PAK6, which is overexpressed in prostate cancer, was recently reported to localize to cell–cell adhesions and to drive epithelial cell colony escape. Here we report that PAK6 targeting to cell–cell adhesions occurs through its N-terminus, requiring both its Cdc42/Rac interactive binding (CRIB) domain and an adjacent polybasic region for maximal targeting efficiency. We find PAK6 localization to cell–cell adhesions is Cdc42-dependent, as Cdc42 knockdown inhibits PAK6 targeting to cell–cell adhesions. We further find the ability of PAK6 to drive epithelial cell colony escape requires kinase activity and is disrupted by mutations that perturb PAK6 cell–cell adhesion targeting. Finally, we demonstrate that all type II PAKs (PAK4, PAK5 and PAK6) target to cell–cell adhesions, albeit to differing extents, but PAK1 (a type I PAK) does not. Notably, the ability of a PAK isoform to drive epithelial colony escape correlates with its targeting to cell–cell adhesions. We conclude that PAKs have a broader role in the regulation of cell–cell adhesions than previously appreciated. PMID:26598554

  2. Pattern formation in cell membrane adhesion

    NASA Astrophysics Data System (ADS)

    Discher, Dennis; Hategan, A.; Sengupta, K.; Sackmann, E.

    2004-03-01

    Strong adhesion of highly active cells often nucleates focal adhesions or related structures that are, over time, reinforced by cytoskeleton (actin, etc.). Red cells lack such complex adhesion systems, but they are shown here to also exhibit complex spatial patterns within an adhesive contact zone. While strong adhesion and spreading of the red cell to a dense poly-L-lysine surface appears complete in < 1 s by reflective interference microscopy, over longer times of 10-15 min or more distinct patterns in fluorescently labeled membrane components emerge. The fluorescent lipid Fl-PE (fluorescein phosphoethanolamine), in particular, is seen to diffuse and reorganize (eg. worm-like domains of <500 nm) within the contact zone, independent of whether the cell is intact or ruptured. Lipid patterns are accompanied by visible perturbations in band 3 distribution and weaker perturbations in membrane skeleton actin. Although fluorescent poly-L-lysine is shown to be uniform under cells, pressing down on the membrane quenches the lipid patterns and reveals the topographical basis for pattern formation. Regions of strong contact are thus separated by regions where the membrane is more distant from the surface.

  3. Inhibition of the colony-stimulating-factor-1 receptor affects the resistance of lung cancer cells to cisplatin

    PubMed Central

    Pass, Harvey I.; Lavilla, Carmencita; Canino, Claudia; Goparaju, Chandra; Preiss, Jordan; Noreen, Samrah; Blandino, Giovanni; Cioce, Mario

    2016-01-01

    In the present work we show that multiple lung cancer cell lines contain cisplatin resistant cell subpopulations expressing the Colony-Stimulating-Factor-Receptor-1 (CSF-1R) and surviving chemotherapy-induced stress. By exploiting siRNA-mediated knock down in vitro and the use of an investigational CSF-1R TKI (JNJ-40346527) in vitro and in vivo, we show that expression and function of the receptor are required for the clonogenicity and chemoresistance of the cell lines. Thus, inhibition of the kinase activity of the receptor reduced the levels of EMT-associated genes, stem cell markers and chemoresistance genes. Additionally, the number of high aldehyde dehydrogenase (ALDH) expressing cells was reduced, consequent to the lack of cisplatin-induced increase of ALDH isoforms. This affected the collective chemoresistance of the treated cultures. Treatment of tumor bearing mice with JNJ-40346527, at pharmacologically relevant doses, produced strong chemo-sensitizing effects in vivo. These anticancer effects correlated with a reduced number of CSF-1Rpos cells, in tumors excised from the treated mice. Depletion of the CD45pos cells within the treated tumors did not, apparently, play a major role in mediating the therapeutic response to the TKI. Thus, lung cancer cells express a functional CSF-1 and CSF-1R duo which mediates pro-tumorigenic effects in vivo and in vitro and can be targeted in a therapeutically relevant way. These observations complement the already known role for the CSF-1R at mediating the pro-tumorigenic properties of tumor-infiltrating immune components. PMID:27486763

  4. T cell expression of granulocyte-macrophage colony-stimulating factor in juvenile arthritis is contingent upon Th17 plasticity.

    PubMed

    Piper, Christopher; Pesenacker, Anne M; Bending, David; Thirugnanabalan, Balathas; Varsani, Hemlata; Wedderburn, Lucy R; Nistala, Kiran

    2014-07-01

    Granulocyte-macrophage colony stimulating factor (GM-CSF) is a potent inflammatory mediator that is responsible for recruitment and activation of innate immune cells. Recent data from murine studies have identified Th17 cells as a key source of GM-CSF and suggest that T cell-derived GM-CSF is instrumental in the induction of autoimmune disease. The present study was undertaken to analyze the expression of T cell-derived GM-CSF in the joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the differentiation of Th17 cells and how this relates to GM-CSF+ T helper cells. Synovial fluid (SF) and peripheral blood (PB) samples from 24 patients with JIA were analyzed, by flow cytometry and reverse transcription-polymerase chain reaction, for expression of GM-CSF and the Th17 marker CD161. A cytokine capture assay was used to purify Th17 cells and test the plasticity of cytokine production in response to interleukin-12 (IL-12) and IL-23. The frequency of GM-CSF-producing T helper cells was significantly enriched in SF mononuclear cells compared to PB mononuclear cells from the patients with JIA (24.1% of CD4+ T cells versus 2.9%) and closely correlated with the erythrocyte sedimentation rate (r(2) = 0.91, P < 0.001). Synovial GM-CSF+ T cells were predominantly CD161+ and coexpressed interferon-γ (IFNγ), but not IL-17. Culture of Th17 cells in the presence of IL-12 led to rapid up-regulation of GM-CSF and IFNγ, recapitulating the phenotype of GM-CSF-expressing cells within the joint. Our results identify a novel outcome of Th17 plasticity in humans that may account for the enrichment of GM-CSF-expressing T cells in the joints of patients with JIA. The association of GM-CSF expression with systemic inflammation highlights the potential role of Th17-related cytokines in the pathology of JIA. © 2014 The Authors. Arthritis & Rheumatology is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

  5. Production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture.

    PubMed

    Liu, Yu-Kuo; Huang, Li-Fen; Ho, Shin-Lon; Liao, Chun-Yu; Liu, Hsin-Yi; Lai, Ying-Hui; Yu, Su-May; Lu, Chung-An

    2012-05-01

    To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. Copyright © 2011 Wiley Periodicals, Inc.

  6. Pinning-depinning transition in a stochastic growth model for the evolution of cell colony fronts in a disordered medium

    NASA Astrophysics Data System (ADS)

    Moglia, Belén; Albano, Ezequiel V.; Guisoni, Nara

    2016-11-01

    We study a stochastic lattice model for cell colony growth, which takes into account proliferation, diffusion, and rotation of cells, in a culture medium with quenched disorder. The medium is composed of sites that inhibit any possible change in the internal state of the cells, representing the disorder, as well as by active medium sites that do not interfere with the cell dynamics. By means of Monte Carlo simulations we find that the velocity of the growing interface, which is taken as the order parameter of the model, strongly depends on the density of active medium sites (ρA). In fact, the model presents a (continuous) second-order pinning-depinning transition at a certain critical value of ρAcrit, such as, for ρA>ρAcrit , the interface moves freely across the disordered medium, but for ρA<ρAcrit the interface becomes irreversible pinned by the disorder. By determining the relevant critical exponents, our study reveals that within the depinned phase the interface can be rationalized in terms of the Kardar-Parisi-Zhang universality class, but when approaching the critical threshold, the nonlinear term of the Kardar-Parisi-Zhang equation tends to vanish and then the pinned interface belongs to the quenched Edwards-Wilkinson universality class.

  7. Pinning-depinning transition in a stochastic growth model for the evolution of cell colony fronts in a disordered medium.

    PubMed

    Moglia, Belén; Albano, Ezequiel V; Guisoni, Nara

    2016-11-01

    We study a stochastic lattice model for cell colony growth, which takes into account proliferation, diffusion, and rotation of cells, in a culture medium with quenched disorder. The medium is composed of sites that inhibit any possible change in the internal state of the cells, representing the disorder, as well as by active medium sites that do not interfere with the cell dynamics. By means of Monte Carlo simulations we find that the velocity of the growing interface, which is taken as the order parameter of the model, strongly depends on the density of active medium sites (ρ_{A}). In fact, the model presents a (continuous) second-order pinning-depinning transition at a certain critical value of ρ_{A}^{crit}, such as, for ρ_{A}>ρ_{A}^{crit}, the interface moves freely across the disordered medium, but for ρ_{A}<ρ_{A}^{crit} the interface becomes irreversible pinned by the disorder. By determining the relevant critical exponents, our study reveals that within the depinned phase the interface can be rationalized in terms of the Kardar-Parisi-Zhang universality class, but when approaching the critical threshold, the nonlinear term of the Kardar-Parisi-Zhang equation tends to vanish and then the pinned interface belongs to the quenched Edwards-Wilkinson universality class.

  8. An optimized colony forming assay for low-dose-radiation cell survival measurement

    SciTech Connect

    Zhu J.; Sutherland B.; Hu W.; Ding N.; Ye C.; Usikalu M.; Li S.; Hu B.; Zhou G.

    2011-11-01

    The aim of this study is to develop a simple and reliable method to quantify the cell survival of low-dose irradiations. Two crucial factors were considered, the same number of cells plated in each flask and an appropriate interval between cell plating and irradiation. For the former, we optimized cell harvest with trypsin, diluted cells in one container, and directly seeded cells on the bottom of flasks in a low density before irradiation. Reproducible plating efficiency was obtained. For the latter, we plated cells on the bottom of flasks and then monitored the processing of attachment, cell cycle variations, and the plating efficiency after exposure to 20 cGy of X-rays. The results showed that a period of 4.5 h to 7.5 h after plating was suitable for further treatment. In order to confirm the reliability and feasibility of our method, we also measured the survival curves of these M059K and M059J glioma cell lines by following the optimized protocol and obtained consistent results reported by others with cell sorting system. In conclusion, we successfully developed a reliable and simple way to measure the survival fractions of human cells exposed to low dose irradiation, which might be helpful for the studies on low-dose radiation biology.

  9. Novel solar cells in a wire format.

    PubMed

    Chen, Tao; Qiu, Longbin; Yang, Zhibin; Peng, Huisheng

    2013-06-21

    Photovoltaic devices in a wire format have recently attracted increasing attention as, compared with the conventional planar structure, they show unique and promising advantages. For instance, they are light-weight and can be easily woven into clothes or integrated into other structures, which enable applications in electronic textiles and various complex devices. In this tutorial review, the recent advancement in photovoltaic wires including both dye-sensitized and polymer solar cells are described. Two main architectures based on a single core-sheath fiber and twisted fibers are carefully illustrated with an emphasis on the comparison of various substrates which have been focused in past development. The current challenge including low energy conversion efficiency and low stability and future direction of the wire-shaped cell have been finally summarized.

  10. Vaccination with Irradiated Autologous Melanoma Cells Engineered to Secrete Human Granulocyte--Macrophage Colony-Stimulating Factor Generates Potent Antitumor Immunity in Patients with Metastatic Melanoma

    NASA Astrophysics Data System (ADS)

    Soiffer, Robert; Lynch, Thomas; Mihm, Martin; Jung, Ken; Rhuda, Catherine; Schmollinger, Jan C.; Hodi, F. Stephen; Liebster, Laura; Lam, Prudence; Mentzer, Steven; Singer, Samuel; Tanabe, Kenneth K.; Benedict Cosimi, A.; Duda, Rosemary; Sober, Arthur; Bhan, Atul; Daley, John; Neuberg, Donna; Parry, Gordon; Rokovich, Joseph; Richards, Laurie; Drayer, Jan; Berns, Anton; Clift, Shirley; Cohen, Lawrence K.; Mulligan, Richard C.; Dranoff, Glenn

    1998-10-01

    We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte--macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte--macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.

  11. Stem cell factor and granulocyte colony-stimulating factor exhibit therapeutic effects in a mouse model of CADASIL.

    PubMed

    Liu, Xiao-Yun; Gonzalez-Toledo, Maria E; Fagan, Austin; Duan, Wei-Ming; Liu, Yanying; Zhang, Siyuan; Li, Bin; Piao, Chun-Shu; Nelson, Lila; Zhao, Li-Ru

    2015-01-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a Notch3 dominant mutation-induced cerebral small vascular disease, is characterized by progressive degeneration of vascular smooth muscle cells (vSMCs) of small arteries in the brain, leading to recurrent ischemic stroke, vascular dementia and death. To date, no treatment can stop or delay the progression of this disease. Herein, we determined the therapeutic effects of stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in a mouse model of CADASIL carrying the human mutant Notch3 gene. SCF+G-CSF was subcutaneously administered for 5 days and repeated 4 times with 1-4 month intervals. We found through water maze testing that SCF+G-CSF treatment improved cognitive function. SCF+G-CSF also attenuated vSMC degeneration in small arteries, increased cerebral blood vascular density, and inhibited apoptosis in CADASIL mice. We also discovered that loss of cerebral capillary endothelial cells and neural stem cells/neural progenitor cells (NSCs/NPCs) occurred in CADASIL mice. SCF+G-CSF treatment inhibited the CADASIL-induced cell loss in the endothelia and NSCs/NPCs and promoted neurogenesis. In an in vitro model of apoptosis, SCF+G-CSF prevented apoptotic cell death in vSMCs through AKT signaling and by inhibiting caspase-3 activity. These data suggest that SCF+G-CSF restricts the pathological progression of CADASIL. This study offers new insights into developing therapeutic strategies for CADASIL.

  12. Reduced proliferation of endothelial colony-forming cells in unprovoked venous thromboembolic disease as a consequence of endothelial dysfunction

    PubMed Central

    Hernandez-Lopez, Rubicel; Chavez-Gonzalez, Antonieta; Torres-Barrera, Patricia; Moreno-Lorenzana, Dafne; Lopez-DiazGuerrero, Norma; Santiago-German, David; Isordia-Salas, Irma; Smadja, David; C. Yoder, Mervin; Majluf-Cruz, Abraham

    2017-01-01

    Background Venous thromboembolic disease (VTD) is a public health problem. We recently reported that endothelial colony-forming cells (ECFCs) derived from endothelial cells (EC) (ECFC-ECs) from patients with VTD have a dysfunctional state. For this study, we proposed that a dysfunctional status of these cells generates a reduction of its proliferative ability, which is also associated with senescence and reactive oxygen species (ROS). Methods and results Human mononuclear cells (MNCs) were obtained from peripheral blood from 40 healthy human volunteers (controls) and 50 patients with VTD matched by age (20−50 years) and sex to obtain ECFCs. We assayed their proliferative ability with plasma of patients and controls and supernatants of cultures from ECFC-ECs, senescence-associated β-galactosidase (SA-β-gal), ROS, and expression of ephrin-B2/Eph-B4 receptor. Compared with cells from controls, cells from VTD patients showed an 8-fold increase of ECFCs that emerged 1 week earlier, reduced proliferation at long term (39%) and, in passages 4 and 10, a highly senescent rate (30±1.05% vs. 91.3±15.07%, respectively) with an increase of ROS and impaired expression of ephrin-B2/Eph-4 genes. Proliferation potential of cells from VTD patients was reduced in endothelial medium [1.4±0.22 doubling population (DP)], control plasma (1.18±0.31 DP), or plasma from VTD patients (1.65±0.27 DP). Conclusions As compared with controls, ECFC-ECs from individuals with VTD have higher oxidative stress, proliferation stress, cellular senescence, and low proliferative potential. These findings suggest that patients with a history of VTD are ECFC-ECs dysfunctional that could be associated to permanent risk for new thrombotic events. PMID:28910333

  13. Reduced proliferation of endothelial colony-forming cells in unprovoked venous thromboembolic disease as a consequence of endothelial dysfunction.

    PubMed

    Hernandez-Lopez, Rubicel; Chavez-Gonzalez, Antonieta; Torres-Barrera, Patricia; Moreno-Lorenzana, Dafne; Lopez-DiazGuerrero, Norma; Santiago-German, David; Isordia-Salas, Irma; Smadja, David; C Yoder, Mervin; Majluf-Cruz, Abraham; Alvarado-Moreno, J Antonio

    2017-01-01

    Venous thromboembolic disease (VTD) is a public health problem. We recently reported that endothelial colony-forming cells (ECFCs) derived from endothelial cells (EC) (ECFC-ECs) from patients with VTD have a dysfunctional state. For this study, we proposed that a dysfunctional status of these cells generates a reduction of its proliferative ability, which is also associated with senescence and reactive oxygen species (ROS). Human mononuclear cells (MNCs) were obtained from peripheral blood from 40 healthy human volunteers (controls) and 50 patients with VTD matched by age (20-50 years) and sex to obtain ECFCs. We assayed their proliferative ability with plasma of patients and controls and supernatants of cultures from ECFC-ECs, senescence-associated β-galactosidase (SA-β-gal), ROS, and expression of ephrin-B2/Eph-B4 receptor. Compared with cells from controls, cells from VTD patients showed an 8-fold increase of ECFCs that emerged 1 week earlier, reduced proliferation at long term (39%) and, in passages 4 and 10, a highly senescent rate (30±1.05% vs. 91.3±15.07%, respectively) with an increase of ROS and impaired expression of ephrin-B2/Eph-4 genes. Proliferation potential of cells from VTD patients was reduced in endothelial medium [1.4±0.22 doubling population (DP)], control plasma (1.18±0.31 DP), or plasma from VTD patients (1.65±0.27 DP). As compared with controls, ECFC-ECs from individuals with VTD have higher oxidative stress, proliferation stress, cellular senescence, and low proliferative potential. These findings suggest that patients with a history of VTD are ECFC-ECs dysfunctional that could be associated to permanent risk for new thrombotic events.

  14. Induction of retinoic acid receptor-alpha by granulocyte macrophage colony-stimulating factor in human myeloid leukemia cell lines.

    PubMed

    Shimizu, T; Takeda, K

    2000-08-15

    We reported previously that treatment with all-trans retinoic acid (ATRA) and granulocyte macrophage colony-stimulating factor (GM-CSF) induces differentiation of human myeloblastic leukemia ML-1 cells to granulocytes, whereas treatment with ATRA alone induces practically no differentiation of these cells. To investigate the mechanism of the synergistic effect of these factors, we examined the effect of GM-CSF on retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in ML-1 cells. We reveal that GM-CSF induces the expression of RAR alpha mRNA and protein and stimulates the binding of nuclear proteins to direct repeat 5, a consensus sequence with high affinity for RAR-RXR heterodimers. Furthermore, expression of CD38 mRNA mediated through RAR alpha is induced synergistically by treatment with ATRA + GM-CSF. These results suggest that GM-CSF stimulates transcriptional activity mediated via RAR alpha in ML-1 cells. The induction of RAR alpha by GM-CSF may therefore be a mechanism for stimulation by GM-CSF. The induction of RAR alpha by GM-CSF was also detected in other myeloid leukemia cell lines (THP-1 and KG-1) that showed a synergistic effect similar to that seen in ML-1 cells in response to ATRA + GM-CSF. We also found that GM-CSF induced the expression of RAR alpha in blood cells obtained from patients with acute myeloid leukemia. This activity of GM-CSF may serve as a useful adjunct to differentiation therapy for retinoic acid-nonresponsive leukemias.

  15. Spontaneous emergence of large-scale cell cycle synchronization in amoeba colonies

    NASA Astrophysics Data System (ADS)

    Segota, Igor; Boulet, Laurent; Franck, David; Franck, Carl

    2014-06-01

    Unicellular eukaryotic amoebae Dictyostelium discoideum are generally believed to grow in their vegetative state as single cells until starvation, when their collective aspect emerges and they differentiate to form a multicellular slime mold. While major efforts continue to be aimed at their starvation-induced social aspect, our understanding of population dynamics and cell cycle in the vegetative growth phase has remained incomplete. Here we show that cell populations grown on a substrate spontaneously synchronize their cell cycles within several hours. These collective population-wide cell cycle oscillations span millimeter length scales and can be completely suppressed by washing away putative cell-secreted signals, implying signaling by means of a diffusible growth factor or mitogen. These observations give strong evidence for collective proliferation behavior in the vegetative state.

  16. T Cell Expression of Granulocyte–Macrophage Colony-Stimulating Factor in Juvenile Arthritis Is Contingent Upon Th17 Plasticity

    PubMed Central

    Piper, Christopher; Pesenacker, Anne M; Bending, David; Thirugnanabalan, Balathas; Varsani, Hemlata; Wedderburn, Lucy R; Nistala, Kiran

    2014-01-01

    Objective Granulocyte–macrophage colony stimulating factor (GM-CSF) is a potent inflammatory mediator that is responsible for recruitment and activation of innate immune cells. Recent data from murine studies have identified Th17 cells as a key source of GM-CSF and suggest that T cell–derived GM-CSF is instrumental in the induction of autoimmune disease. The present study was undertaken to analyze the expression of T cell–derived GM-CSF in the joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the differentiation of Th17 cells and how this relates to GM-CSF+ T helper cells. Methods Synovial fluid (SF) and peripheral blood (PB) samples from 24 patients with JIA were analyzed, by flow cytometry and reverse transcription–polymerase chain reaction, for expression of GM-CSF and the Th17 marker CD161. A cytokine capture assay was used to purify Th17 cells and test the plasticity of cytokine production in response to interleukin-12 (IL-12) and IL-23. Results The frequency of GM-CSF–producing T helper cells was significantly enriched in SF mononuclear cells compared to PB mononuclear cells from the patients with JIA (24.1% of CD4+ T cells versus 2.9%) and closely correlated with the erythrocyte sedimentation rate (r2 = 0.91, P < 0.001). Synovial GM-CSF+ T cells were predominantly CD161+ and coexpressed interferon-γ (IFNγ), but not IL-17. Culture of Th17 cells in the presence of IL-12 led to rapid up-regulation of GM-CSF and IFNγ, recapitulating the phenotype of GM-CSF–expressing cells within the joint. Conclusion Our results identify a novel outcome of Th17 plasticity in humans that may account for the enrichment of GM-CSF–expressing T cells in the joints of patients with JIA. The association of GM-CSF expression with systemic inflammation highlights the potential role of Th17-related cytokines in the pathology of JIA. PMID:24692225

  17. CORONETTE KERATINOCYTE COLONY FORMATION IS SUPPORTED BY EPIDERMAL-DERMAL CELL INTERACTIONS IN THE BOVINE CLAW

    USDA-ARS?s Scientific Manuscript database

    Delineating factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronette keratinocyt...

  18. Treatment of complete spinal cord injury patients by autologous bone marrow cell transplantation and administration of granulocyte-macrophage colony stimulating factor.

    PubMed

    Park, Hyung Chun; Shim, Yoo Shik; Ha, Yoon; Yoon, Seung Hwan; Park, So Ra; Choi, Byung Hyune; Park, Hyun Seon

    2005-01-01

    Transplantation of bone marrow cells into the injured spinal cord has been found to improve neurologic functions in experimental animal studies. However, it is unclear whether bone marrow cells can similarly improve the neurologic functions of complete spinal cord injury (SCI) in human patients. To address this issue, we evaluated the therapeutic effects of autologous bone marrow cell transplantation (BMT) in conjunction with the administration of granulocyte macrophage-colony stimulating factor (GM-CSF) in six complete SCI patients. BMT in the injury site (1.1 x 10(6) cells/microL in a total of 1.8 mL) and subcutaneous GM-CSF administration were performed on five patients. One patient was treated with GM-CSF only. The follow-up periods were from 6 to 18 months, depending on the patients. Sensory improvements were noted immediately after the operations. Sensory recovery in the sacral segment was noted mainly 3 weeks to 7 months postoperatively. Significant motor improvements were noted 3 to 7 months postoperatively. Four patients showed neurologic improvements in their American Spiral Injury Association Impairment Scale (AIS) grades (from A to C). One patient improved to AIS grade B from A and the last patient remained in AIS grade A. No immediate worsening of neurologic symptoms was found. Side effects of GMCSF treatment such as a fever (>38 degrees C) and myalgia were noted. Serious complications increasing mortality and morbidity were not found. The follow-up study with magnetic resonance imaging 4-6 months after injury showed slight enhancement within the zone of BMT. Syrinx formation was not definitely found. BMT and GM-CSF administration represent a safe protocol to efficiently manage SCI patients, especially those with acute complete injury. To demonstrate the full therapeutic value of this protocol, long-term and more comprehensive case-control clinical studies are required.

  19. Radioprotection of mice with interleukin-1: Relationship to the number of erythroid and granulocyte-macrophage colony-forming cells

    SciTech Connect

    Schwartz, G.N.; Patchen, M.L.; Neta, R.; MacVittie, T.J.

    1990-01-01

    This report presents the results of an investigation of changes in the number of erythroid and granulocyte-macrophage colony forming cells (GM-CFC) that had occurred in tissues of normal B6D2F1 mice 20 h after administration of a radioprotective dose (150 ng) of human recombinant interleukin-1 (rIL-1). Neutrophilia in the peripheral blood and changes in the tissue distribution of GM-CFC demonstrated that cells were mobilized from the bone marrow in response to rIL-1 injection. For example, 20 h after rIL-1 injection marrow GM-CFC numbers were 80% of the numbers in bone marrow from saline-injected mice. Associated with this decrease there was a twofold increase in the number of peripheral blood and splenic GM-CFC. Also, as determined by hydroxyurea injection, there was an increase in the number of GM-CFC in S phase of the cell cycle in the spleen, but not in the bone marrow. Data in this report suggest that when compared to the spleen, stimulation of granulopoiesis after rIL-1 injection is delayed in the bone marrow.

  20. Radioprotection of mice with interleukin-1: Relationship to the number of erythroid and granulocyte-macrophage colony-forming cells

    SciTech Connect

    Schwartz, G.N.; Patchen, M.L.; Neta, R.; MacVittie, T.J. )

    1990-02-01

    This report presents the results of an investigation of changes in the number of erythroid and granulocyte-macrophage colony-forming cells (GM-CFC) that had occurred in tissues of normal B6D2F1 mice 20 h after administration of a radioprotective dose (150 ng) of human recombinant interleukin-1 (rIL-1). Neutrophilia in the peripheral blood and changes in the tissue distribution of GM-CFC demonstrated that cells were mobilized from the bone marrow in response to rIL-1 injection. For example, 20 h after rIL-1 injection marrow GM-CFC numbers were 80% of the numbers in bone marrow from saline-injected mice. Associated with this decrease there was a twofold increase in the number of peripheral blood and splenic GM-CFC. Also, as determined by hydroxyurea injection, there was an increase in the number of GM-CFC in S phase of the cell cycle in the spleen, but not in the bone marrow. Data in this report suggest that when compared to the spleen, stimulation of granulopoiesis after rIL-1 injection is delayed in the bone marrow. Also, the earlier recovery of GM-CFC in the bone marrow of irradiated mice is not dependent upon an increase in the number of GM-CFC at the time of irradiation.

  1. Granulocyte colony-stimulating factor (G-CSF) transiently suppresses mitogen-stimulated T-cell proliferative response

    PubMed Central

    Reyes, E; García-Castro, I; Esquivel, F; Hornedo, J; Cortes-Funes, H; Solovera, J; Alvarez-Mon, M

    1999-01-01

    Granulocyte colony-stimulation factor (G-CSF) is a cytokine that selectively promotes growth and maturation of neutrophils and may modulate the cytokine response to inflammatory stimuli. The purpose of this study was to examine the effect of G-CSF on ex vivo peripheral blood mononuclear cell (PBMC) functions. Ten patients with breast cancer were included in a clinical trial in which r-metHuG-CSF was administrered daily for 5 days to mobilize peripheral blood stem cells. Ten healthy women were also included as controls. Our data show that G-CSF treatment induces an increase in peripheral blood leucocyte, neutrophil, lymphocyte and monocyte counts. We have found a modulation in the percentages of CD19+, CD45+CD14+, CD4+CD45RA+ and CD4+CD45RO+ cells in PBMC fractions during G-CSF treatment. We have also found a significant reduction in the proliferative response of PBMC to mitogenic stimulation that reverted 14 days after the fifth and the last dose of G-CSF. Furthermore, it was not associated with significant changes in the pattern of cytokine production. The mechanism of this immunoregulatory effect is probably indirect since G-CSF receptor has not been found in T lymphocytes. This mechanism and its potential clinical applications remain to be elucidated. © 1999 Cancer Research Campaign PMID:10390001

  2. Development of Aggressive Pancreatic Ductal Adenocarcinomas Depends on Granulocyte Colony Stimulating Factor Secretion in Carcinoma Cells.

    PubMed

    Pickup, Michael W; Owens, Philip; Gorska, Agnieszka E; Chytil, Anna; Ye, Fei; Shi, Chanjuan; Weaver, Valerie M; Kalluri, Raghu; Moses, Harold L; Novitskiy, Sergey V

    2017-09-01

    The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48(TGFβRIIKO)), we discovered an effect of TGFβ signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G(+) cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b(+) cells. Deletion of csf3 in PDAC cells or use of a G-CSF-blocking antibody decreased tumor growth. Anti-G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G(+) cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. Cancer Immunol Res; 5(9); 718-29. ©2017 AACR. ©2017 American Association for Cancer Research.

  3. Isolation and characterization of squamous cell carcinoma-derived stem-like cells: role in tumor formation.

    PubMed

    Dallaglio, Katiuscia; Petrachi, Tiziana; Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Saltari, Annalisa; Morandi, Paolo; Puviani, Mario; Maiorana, Antonino; Roop, Dennis R; Pincelli, Carlo

    2013-09-26

    In human epidermis, keratinocyte stem cells (KSC) are characterized by high levels of β1-integrin, resulting in the rapid adhesion to type IV collagen. Since epithelial tumors originate from KSC, we evaluated the features of rapidly adhering (RAD) keratinocytes derived from primary human squamous cell carcinoma of the skin (cSCC). RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells. Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells. RAD cells also migrated significantly better than NRAD cells. When seeded in a silicone chamber and grafted onto the back skin of NOD SCID mice, RAD cells formed tumors 2-4 fold bigger than those derived from NRAD cells. In tumors derived from RAD cells, the mitotic index was significantly higher than in those derived from NRAD cells, while Ki-67 and survivin expression were more pronounced in RAD tumors. This study suggests that SCC RAD stem cells play a critical role in the formation and development of epithelial tumors.

  4. Isolation and Characterization of Squamous Cell Carcinoma-Derived Stem-like Cells: Role in Tumor Formation

    PubMed Central

    Dallaglio, Katiuscia; Petrachi, Tiziana; Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Saltari, Annalisa; Morandi, Paolo; Puviani, Mario; Maiorana, Antonino; Roop, Dennis R.; Pincelli, Carlo

    2013-01-01

    In human epidermis, keratinocyte stem cells (KSC) are characterized by high levels of β1-integrin, resulting in the rapid adhesion to type IV collagen. Since epithelial tumors originate from KSC, we evaluated the features of rapidly adhering (RAD) keratinocytes derived from primary human squamous cell carcinoma of the skin (cSCC). RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells. Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells. RAD cells also migrated significantly better than NRAD cells. When seeded in a silicone chamber and grafted onto the back skin of NOD SCID mice, RAD cells formed tumors 2–4 fold bigger than those derived from NRAD cells. In tumors derived from RAD cells, the mitotic index was significantly higher than in those derived from NRAD cells, while Ki-67 and survivin expression were more pronounced in RAD tumors. This study suggests that SCC RAD stem cells play a critical role in the formation and development of epithelial tumors. PMID:24077125

  5. Organic Tandem Solar Cells: Design and Formation

    NASA Astrophysics Data System (ADS)

    Chen, Chun-Chao

    polyelectrolyte layer functioning as the surface dipole formation layer to provide better electrical contact with the photoactive layer. Due to the effectiveness of the conjugated polyelectrolyte layer, performance improvement was also observed. Furthermore, other issues regarding the semi-transparent tandem solar cells (e.g., photocurrent matching, exterior color tuning, and transparency tuning) are all explored to optimize best performance. In Chapter 5 and 6, the architectures of double- and triple-junction tandem solar cells are explored. Theoretically, triple-junction tandem solar cells with three photoactive absorbers with cascaded energy bandgaps have the potential to achieve higher performance, in comparison with double-junction tandem solar cells. Such expectations can be ascribed to the minimized carrier thermalization loss and further improved light absorption. However, the design of triple-junction solar cells often involves sophisticated multiple layer deposition as well as substantial optimization. Therefore, there is a lack of successful demonstrations of triple-junction solar cells outperforming the double-junction counterparts. To solve the incompatible issues related to the layer deposition in the fabrication, we proposed a novel architecture of inverted-structure tandem solar cells with newly designed interconnecting layers. Our design of interconnecting layers does not only focus on maintaining the orthogonal solution processing advantages, but also provides an excellent compatibility in the energy level alignment to allow different absorber materials to be used. Furthermore, we also explored the light management inside the double- and triple-junction tandem solar cells. The study of light management was carried out through optical simulation method based transfer matrix formalism. The intention is to obtain a balanced photocurrent output from each subcells inside the tandem solar cell, thus the minimal recombination loss at the contact of interconnecting

  6. Autologous cell sources in therapeutic vasculogenesis: In vitro and in vivo comparison of endothelial colony-forming cells from peripheral blood and endothelial cells isolated from adipose tissue.

    PubMed

    Szöke, Krisztina; Reinisch, Andreas; Østrup, Esben; Reinholt, Finn P; Brinchmann, Jan E

    2016-02-01

    Autologous endothelial cells are promising alternative angiogenic cell sources in trials of therapeutic vasculogenesis, in the treatment of vascular diseases and in the field of tissue engineering. A population of endothelial cells (ECs) with long-term proliferative capability, endothelial colony-forming cells (ECFCs), can be isolated from human peripheral blood. ECFCs are considered an endothelial precursor population. They can be expanded in cell factories in sufficient numbers for clinical applications, but because the number of isolated primary ECs is low, the culture period required may be long. Another EC population that is easily available in the autologous setting and may be expanded in vitro through several population doublings are ECs from adipose tissue (AT-ECs). Through extensive comparisons using whole-genome microarray analysis, morphology, phenotype and functional assays, we wanted to evaluate the potential of these EC populations for use in clinical neovascularization. Global gene expression profiling of ECFCs, AT-ECs and the classical EC population, human umbilical vein ECs, showed that the EC populations clustered as unique populations, but very close to each other. By cell surface phenotype and vasculogenic potential in vitro and in vivo, we also found the ECFCs to be extremely similar to AT-ECs. These properties, together with easy access in the autologous setting, suggest that both AT-ECs and ECFCs may be useful in trials of therapeutic neovascularization. However, AT-ECs may be a more practical alternative for obtaining large quantities of autologous ECs. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. Branching instability in expanding bacterial colonies

    PubMed Central

    Giverso, Chiara; Verani, Marco; Ciarletta, Pasquale

    2015-01-01

    Self-organization in developing living organisms relies on the capability of cells to duplicate and perform a collective motion inside the surrounding environment. Chemical and mechanical interactions coordinate such a cooperative behaviour, driving the dynamical evolution of the macroscopic system. In this work, we perform an analytical and computational analysis to study pattern formation during the spreading of an initially circular bacterial colony on a Petri dish. The continuous mathematical model addresses the growth and the chemotactic migration of the living monolayer, together with the diffusion and consumption of nutrients in the agar. The governing equations contain four dimensionless parameters, accounting for the interplay among the chemotactic response, the bacteria–substrate interaction and the experimental geometry. The spreading colony is found to be always linearly unstable to perturbations of the interface, whereas branching instability arises in finite-element numerical simulations. The typical length scales of such fingers, which align in the radial direction and later undergo further branching, are controlled by the size parameters of the problem, whereas the emergence of branching is favoured if the diffusion is dominant on the chemotaxis. The model is able to predict the experimental morphologies, confirming that compact (resp. branched) patterns arise for fast (resp. slow) expanding colonies. Such results, while providing new insights into pattern selection in bacterial colonies, may finally have important applications for designing controlled patterns. PMID:25652464

  8. Genistein Promotes Endothelial Colony-Forming Cell (ECFC) Bioactivities and Cardiac Regeneration in Myocardial Infarction

    PubMed Central

    Lee, Sang Hun; Lee, Jun Hee; Asahara, Takayuki; Kim, Yong Sook; Jeong, Hae Chang; Ahn, Youngkeun; Jung, Jin Sup; Kwon, Sang-Mo

    2014-01-01

    Although stem cell-mediated treatment of ischemic diseases offers significant therapeutic promise, the limitation in the therapeutic efficacy of transplanted stem cells in vivo because of poor engraftment remains a challenge. Several strategies aimed at improving survival and engraftment of stem cells in the ischemic myocardium have been developed, such as cell transplantation in combination with growth factor delivery, genetic modification of stem cells, and/or cell therapy using scaffolds. To improve therapeutic efficacy, we investigated the effects of genistein on the engraftment of transplanted ECFCs in an acute myocardial ischemia model. Results: We found that genistein treatment enhanced ECFCs' migration and proliferation, which was accompanied by increases in the expression of ILK, α-parvin, F-actin, and phospholylation of ERK 1/2 signaling. Transplantation of genistein-stimulates ECFCs (GS-ECFCs) into myocardial ischemic sites in vivo induced cellular proliferation and secretion of angiogenic cytokines at the ischemic sites and thereby enhanced neovascularization and decreased myocardial fibrosis as well as improved cardiac function, as shown by echocardiography. Taken together, these data suggest that pretreatment of ECFCs with genistein prior to transplantation can improve the regenerative potential in ischemic tissues, providing a novel strategy in adult stem cell therapy for ischemic diseases. PMID:24830850

  9. Mobilization and collection of CD34+ cells for autologous transplantation of peripheral blood hematopoietic progenitor cells in children: analysis of two different granulocyte-colony stimulating factor doses

    PubMed Central

    Eid, Kátia Aparecida de Brito; Miranda, Eliana Cristina Martins; Aguiar, Simone dos Santos

    2015-01-01

    Introduction The use of peripheral hematopoietic progenitor cells (HPCs) is the cell choice in autologous transplantation. The classic dose of granulocyte-colony stimulating factor (G-CSF) for mobilization is a single daily dose of 10 μg/kg of patient body weight. There is a theory that higher doses of granulocyte-colony stimulating factor applied twice daily could increase the number of CD34+ cells collected in fewer leukapheresis procedures. Objective The aim of this study was to compare a fractionated dose of 15 μg G-CSF/kg of body weight and the conventional dose of granulocyte-colony stimulating factor in respect to the number of leukapheresis procedures required to achieve a minimum collection of 3 × 106 CD34+ cells/kg body weight. Methods Patients were divided into two groups: Group 10 – patients who received a single daily dose of 10 μg G-CSF/kg body weight and Group 15 – patients who received a fractioned dose of 15 μg G-CSF/kg body weight daily. The leukapheresis procedure was carried out in an automated cell separator. The autologous transplantation was carried out when a minimum number of 3 × 106 CD34+ cells/kg body weight was achieved. Results Group 10 comprised 39 patients and Group 15 comprised 26 patients. A total of 146 apheresis procedures were performed: 110 (75.3%) for Group 10 and 36 (24.7%) for Group 15. For Group 10, a median of three (range: 1–7) leukapheresis procedures and a mean of 8.89 × 106 CD34+ cells/kg body weight (±9.59) were collected whereas for Group 15 the corresponding values were one (range: 1–3) and 5.29 × 106 cells/kg body weight (±4.95). A statistically significant difference was found in relation to the number of apheresis procedures (p-value <0.0001). Conclusions To collect a minimum target of 3 × 106 CD34+ cells/kg body weight, the administration of a fractionated dose of 15 μg G-CSF/kg body weight significantly decreased the number of leukapheresis procedures performed. PMID:26041417

  10. Spatio-temporal morphology changes in and quenching effects on the 2D spreading dynamics of cell colonies in both plain and methylcellulose-containing culture media.

    PubMed

    Muzzio, N E; Pasquale, M A; Huergo, M A C; Bolzán, A E; González, P H; Arvia, A J

    2016-06-01

    To deal with complex systems, microscopic and global approaches become of particular interest. Our previous results from the dynamics of large cell colonies indicated that their 2D front roughness dynamics is compatible with the standard Kardar-Parisi-Zhang (KPZ) or the quenched KPZ equations either in plain or methylcellulose (MC)-containing gel culture media, respectively. In both cases, the influence of a non-uniform distribution of the colony constituents was significant. These results encouraged us to investigate the overall dynamics of those systems considering the morphology and size, the duplication rate, and the motility of single cells. For this purpose, colonies with different cell populations (N) exhibiting quasi-circular and quasi-linear growth fronts in plain and MC-containing culture media are investigated. For small N, the average radial front velocity and its change with time depend on MC concentration. MC in the medium interferes with cell mitosis, contributes to the local enlargement of cells, and increases the distribution of spatio-temporal cell density heterogeneities. Colony spreading in MC-containing media proceeds under two main quenching effects, I and II; the former mainly depending on the culture medium composition and structure and the latter caused by the distribution of enlarged local cell domains. For large N, colony spreading occurs at constant velocity. The characteristics of cell motility, assessed by measuring their trajectories and the corresponding velocity field, reflect the effect of enlarged, slow-moving cells and the structure of the medium. Local average cell size distribution and individual cell motility data from plain and MC-containing media are qualitatively consistent with the predictions of both the extended cellular Potts models and the observed transition of the front roughness dynamics from a standard KPZ to a quenched KPZ. In this case, quenching effects I and II cooperate and give rise to the quenched

  11. Effects of 50 Hz magnetic field on cell cycle kinetics and the colony forming ability of budding yeast exposed to ultraviolet radiation.

    PubMed

    Markkanen, A; Juutilainen, J; Lang, S; Pelkonen, J; Rytömaa, T; Naarala, J

    2001-07-01

    To investigate the effects of extremely low frequency magnetic fields on ultraviolet radiation (UV) exposed budding yeast, haploid yeast (Saccharomyces cerevisiae) cells of the strain SEy2101a were exposed to 50 Hz sine wave magnetic field (MF) of 120 microT with simultaneous exposure to UV radiation. Most of the UV energy was in the UVB range (280-320 nm). The biologically weighted (CIE action spectrum) dose level for the UV radiation was 175 J/m2. We examined whether 50 Hz MF affected the ability of UV irradiated yeast cells to form colonies (Colony Forming Units, CFUs). In addition, the effect of coexposure on cell cycle kinetics was investigated. Although the significant effect of MF on the cell cycle phases of UV exposed yeast cells was seen only at one time point, the overall results showed that MF exposure may influence the cell cycle kinetics at the first cycle after UV irradiation. The effect of our particular MF exposure on the colony forming ability of the UV irradiated yeast cells was statistically significant 420 min after UV irradiation. Moreover, at 240, 360, and 420 min after UV irradiation, there were fewer CFUs in every experiment in (UV+MF) exposed populations than in only UV exposed yeast populations. These results could indicate that MF exposure in conjunction with UV may have some effects on yeast cell survival or growth. Copyright 2001 Wiley-Liss, Inc.

  12. Formative cell divisions: principal determinants of plant morphogenesis.

    PubMed

    Smolarkiewicz, Michalina; Dhonukshe, Pankaj

    2013-03-01

    Formative cell divisions utilizing precise rotations of cell division planes generate and spatially place asymmetric daughters to produce different cell layers. Therefore, by shaping tissues and organs, formative cell divisions dictate multicellular morphogenesis. In animal formative cell divisions, the orientation of the mitotic spindle and cell division planes relies on intrinsic and extrinsic cortical polarity cues. Plants lack known key players from animals, and cell division planes are determined prior to the mitotic spindle stage. Therefore, it appears that plants have evolved specialized mechanisms to execute formative cell divisions. Despite their profound influence on plant architecture, molecular players and cellular mechanisms regulating formative divisions in plants are not well understood. This is because formative cell divisions in plants have been difficult to track owing to their submerged positions and imprecise timings of occurrence. However, by identifying a spatiotemporally inducible cell division plane switch system applicable for advanced microscopy techniques, recent studies have begun to uncover molecular modules and mechanisms for formative cell divisions. The identified molecular modules comprise developmentally triggered transcriptional cascades feeding onto microtubule regulators that now allow dissection of the hierarchy of the events at better spatiotemporal resolutions. Here, we survey the current advances in understanding of formative cell divisions in plants in the context of embryogenesis, stem cell functionality and post-embryonic organ formation.

  13. Contact formation in gallium arsenide solar cells

    NASA Technical Reports Server (NTRS)

    Weizer, Victor G.; Fatemi, Navid S.

    1988-01-01

    Gold and gold-based alloys, commonly used as solar cell contact materials, are known to react readily with gallium arsenide. Experiments were performed to identify the mechanisms involved in these GaAs-metal interactions. It is shown that the reaction of GaAs with gold takes place via a dissociative diffusion process. It is shown further that the GaAs-metal reaction rate is controlled to a very great extent by the condition of the free surface of the contact metal, an interesting example of which is the previously unexplained increase in the reaction rate that has been observed for samples annealed in a vacuum environment as compared to those annealed in a gaseous ambient. A number of other hard-to-explain observations, such as the low-temperature formation of voids in the gold lattice and crystallite growth on the gold surface, are explained by invoking this mechanism.

  14. Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia

    PubMed Central

    Wang, Xiaowan; Li, Hailong; Ding, Shinghua

    2016-01-01

    We previously demonstrated that Pre-B-cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis pathway, plays a brain and neuronal protective role in ischemic stroke. In this study, we further investigated the mechanism of its neuroprotective effect after ischemia in the primary cultured mouse cortical neurons. Using apoptotic cell death assay, fluorescent imaging, molecular biology, mitochondrial biogenesis measurements and Western blotting analysis, our results show that the overexpression of PBEF in neurons can significantly promote neuronal survival, reduce the translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei and inhibit the activation of capase-3 after glutamate-induced excitotoxicity. We further found that the overexpression of PBEF can suppress glutamate-induced mitochondrial fragmentation, the loss of mitochondrial DNA (mtDNA) content and the reduction of PGC-1 and NRF-1 expressions. Furthermore, these beneficial effects by PBEF are dependent on its enzymatic activity of NAD+ synthesis. In summary, our study demonstrated that PBEF ameliorates ischemia-induced neuronal death through inhibiting caspase-dependent and independent apoptotic signaling pathways and suppressing mitochondrial damage and dysfunction. Our study provides novel insights into the mechanisms underlying the neuroprotective effect of PBEF, and helps to identify potential targets for ischemic stroke therapy. PMID:27576732

  15. X-ray-induced chromosome damage in live mammalian cells, and improved measurements of its effects on their colony-forming ability.

    PubMed

    Joshi, G P; Nelson, W J; Revell, S H; Shaw, C A

    1982-02-01

    We have improved the precision of the technique described by Grote et al. (1981 a,b) for the observation of the radiation responses of live cultured mammalian cells with an incubated phase-contrast microscope: the colony-forming abilities of single cells obtained by selective detachment of mitoses (instead of cell pairs as previously) may now be followed individually and may be directly compared with chromosome damage detected after post-radiation mitosis (M1). An X-ray dose of 1.4 Gy to diploid Syrian hamster cells (BHK 21 C13) in G1 had no effect on cell ability to reach M1. If chromosome fragment loss was then detected (as micronuclei) in the daughter-cell pair then colony-forming ability nearly always deteriorated, and either a stop-growth (79 per cent) or a slow-growth (21 per cent) colony resulted; but chromosomal bridges which persisted beyond M1 broke during interphase 1 and themselves caused no detectable cell damage additional to that attributable to the micronuclei which accompanied them.

  16. Flow cytometric method for in situ preparation of standard materials of a small defined number of microbial cells with colony-forming potentiality.

    PubMed

    Matsuoka, Hideaki; Nakano, Koichiro; Takatani, Norimasa; Yoshida, Tomonori; Igimi, Shizunobu; Saito, Mikako

    2014-01-01

    Standard materials of a small defined number of cells with colony-forming potentiality are essential for the rational validation of food microbiological methods. An in situ flow cytometric method using viable staining with 6-carboxyfluorescein diacetate (CFDA) and tryptic soy agar (TSA) was previously proposed and its feasibility was demonstrated with five strains. In this study, this method was applied to 16 strains to support its broad applicability. The cell sorting gate was previously determined based on the CFDA stainability alone. Now the structural properties of cells designated by forward and side-scattering intensities have been introduced as the second gating criteria. Under the optimum gate condition, 100 cells have been selected and sorted on TSA. Consequently, a 95% or higher colony-forming rate has been attained for every strain. A successful application to microaerophilic Campylobacter spp. is especially of great importance because it suggests further broader applicability.

  17. Carboxy-terminal domain phosphatase 1 silencing results in the inhibition of tumor formation ability in gastric cancer cells

    PubMed Central

    FU, HONGBING; YANG, DEJUN; WANG, CHANGMING; XU, JIAPENG; WANG, WEIMIN; YAN, RONGLIN; CAI, QINGPING

    2015-01-01

    Gastric cancer (GC), one of the most malignant types of cancer, is the second greatest cause of cancer-associated mortality worldwide. Novel therapeutic targets for GC treatment are therefore urgently required. Carboxy-terminal domain phosphatase 1 (CTDP1) has a crucial role in the regulation of gene expression. However, to the best of our knowledge, the role of CTDP1 in GC has not previously been explored. In the present study, reverse transcription-quantitative polymerase chain reaction analysis was used to detect CTDP1 messenger RNA expression in various GC cell lines. CTDP1 was subsequently silenced in GC cells by lentivirus-mediated small interfering RNA (siRNA) infection, and the effects of CTDP1 inhibition on cell proliferation were evaluated by cell number counting, cell cycle analysis with propidium iodide staining and fluorescence-activated cell sorting (FACS) analysis, apoptotic rate with Annexin V staining and FACS analysis, as well as colony formation assay in GC cells. The results revealed that CTDP1 was highly expressed in certain GC cell lines and lentivirus-mediated siRNA infection was able to effectively silence CTDP1 expression in GC cells. CTDP1 inhibition decreased cell proliferation, arrested the cell cycle at G0/G1 phase and increased cell apoptosis in GC cells. Furthermore, the colony formation ability of GC cells was also suppressed by silencing CTDP1. Taken together these results indicated that CTDP1 has a significant role in the tumor formation ability of GC cells and is a novel and promising therapeutic target for the treatment of GC. PMID:26722269

  18. The effects of granulocyte-macrophage colony-stimulating factor on tumour-infiltrating lymphocytes from renal cell carcinoma.

    PubMed Central

    Steger, G. G.; Kaboo, R.; deKernion, J. B.; Figlin, R.; Belldegrun, A.

    1995-01-01

    It has been shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce specific and non-specific anti-tumour cytotoxicity and also stimulates the proliferation and function of peripheral lymphocytes and thymocytes. GM-CSF and interleukin 2 (IL-2) act synergistically on peripheral lymphocytes for the induction of a highly effective cytotoxic cell population. Thus, the goal of our investigation was to study the effects of GM-CSF upon expansion, proliferation and in vitro killing activity of tumour-infiltrating lymphocytes (TILs) from renal cell carcinoma (RCC). TILs from seven consecutive tumours were cultured with GM-CSF (500 or 1000 nmol ml-1) without IL-2 supplementation, with suboptimal doses of IL-2 (8 and 40 U ml-1) plus GM-CSF (1000 nmol ml-1), and with a dose of IL-2 (400 U ml-1) which sufficed alone to induce TIL development plus GM-CSF (500 or 1000 nmol ml-1). GM-CSF alone or together with suboptimal doses of IL-2 was not able to induce or facilitate TIL development in these cultures. When GM-CSF at both concentrations studied was added to optimal doses of IL-2 the resulting TIL populations proliferated significantly better and faster (+66%), resulting in a higher cell yield (+24%) at the time of maximal expansion of the TIL cultures. The length of the culture periods of TILs was not affected by GM-CSF when compared with the control cultures supplemented with IL-2 alone. In vitro killing activity of TIL populations stimulated with IL-2 and GM-CSF remained unspecific, but lysis of the autologous tumour targets as well as the allogeneic renal tumour targets was significantly enhanced (+138%) as compared with the corresponding control TILs stimulated with IL-2 alone. Lysis of the natural killer (NK)-sensitive control cell line K562 and the NK-resistant Daudi cell line remained unchanged even though FACS analysis of TILs cultured with IL-2 and 1000 nmol of GM-CSF demonstrated a significantly higher proportion of cells expressing the CD56

  19. Formation of different abzymes in autoimmune-prone MRL-lpr/lpr mice is associated with changes in colony formation of haematopoietic progenitors

    PubMed Central

    Andryushkova, Alexandra A; Kuznetsova, Irina A; Bineva, Valentina N; Toporkova, Ludmila B; Sakhno, Ludmila V; Tikhonova, Marina A; Chernykh, Elena R; Orlovskaya, Irina A; Nevinsky, Georgy A

    2007-01-01

    Abstract It was shown that IgGs from the sera of 2–7-month-old control non-autoimmune (CBA x C57BL)F1 and BALB/c mice and 2–3-month-old autoimmune prone MRL-lpr/lpr mice (conditionally healthy mice) are catalytically inactive. During spontaneous development of deep systemic lupus erythematosus (SLE)-like pathology a specific reorganization of immune system of these mice leads to conditions associated with a production of IgGs hydrolyzing DNA, ATP and polysaccharides with low catalytic activities (conditionally pre-diseased mice).A significant increase in DNase, ATPase and amylase IgG relative activities associated with a transition from pre-diseased to deep diseased mice is correlated with additional changes in differentiation and proliferation of mice bone marrow haematopoietic stem cells (HSCs) and lymphocyte proliferation in different organs.The highest increase in all abzyme activities was found in mice immunized with DNA, which in comparison with pre-diseased and diseased mice are characterized by a different profile of HSC differentiation and by a suppression of cell apoptosis. Abzyme activities in the serum of pregnant females were comparable with those for pre-diseased mice, but the profile of HSC differentiation and cell apoptosis levels in pregnant and pre-diseased mice were quite different. Right after the beginning of lactation (4 days after delivery) and in a late time of lactation (14 days after delivery) there was an observed increase in cell apoptosis and two different stages of significant change in the HSC differentiation profiles; the first stage was accompanied with a significant increase and the second with a remarkable decrease in abzyme activities. Overall, all mouse groups investigated are characterized by a specific relationship between abzyme activities, HSC differentiation profiles, levels of lymphocyte proliferation, and cell apoptosis in different organs. From our point of view, the appearance of ATPase, DNase activities may be

  20. Stem cells are units of natural selection for tissue formation, for germline development, and in cancer development.

    PubMed

    Weissman, Irving L

    2015-07-21

    It is obvious that natural selection operates at the level of individuals and collections of individuals. Nearly two decades ago we showed that in multi-individual colonies of protochordate colonial tunicates sharing a blood circulation, there exists an exchange of somatic stem cells and germline stem cells, resulting in somatic chimeras and stem cell competitions for gonadal niches. Stem cells are unlike other cells in the body in that they alone self-renew, so that they form clones that are perpetuated for the life of the organism. Stem cell competitions have allowed the emergence of competitive somatic and germline stem cell clones. Highly successful germline stem cells usually outcompete less successful competitors both in the gonads of the genotype partner from which they arise and in the gonads of the natural parabiotic partners. Therefore, natural selection also operates at the level of germline stem cell clones. In the colonial tunicate Botryllus schlosseri the formation of natural parabionts is prevented by a single-locus highly polymorphic histocompatibility gene called Botryllus histocompatibility factor. This limits germline stem cell predation to kin, as the locus has hundreds of alleles. We show that in mice germline stem cells compete for gonad niches, and in mice and humans, blood-forming stem cells also compete for bone marrow niches. We show that the clonal progression from blood-forming stem cells to acute leukemias by successive genetic and epigenetic events in blood stem cells also involves competition and selection between clones and propose that this is a general theme in cancer.

  1. Stem cells are units of natural selection for tissue formation, for germline development, and in cancer development

    PubMed Central

    Weissman, Irving L.

    2015-01-01

    It is obvious that natural selection operates at the level of individuals and collections of individuals. Nearly two decades ago we showed that in multi-individual colonies of protochordate colonial tunicates sharing a blood circulation, there exists an exchange of somatic stem cells and germline stem cells, resulting in somatic chimeras and stem cell competitions for gonadal niches. Stem cells are unlike other cells in the body in that they alone self-renew, so that they form clones that are perpetuated for the life of the organism. Stem cell competitions have allowed the emergence of competitive somatic and germline stem cell clones. Highly successful germline stem cells usually outcompete less successful competitors both in the gonads of the genotype partner from which they arise and in the gonads of the natural parabiotic partners. Therefore, natural selection also operates at the level of germline stem cell clones. In the colonial tunicate Botryllus schlosseri the formation of natural parabionts is prevented by a single-locus highly polymorphic histocompatibility gene called Botryllus histocompatibility factor. This limits germline stem cell predation to kin, as the locus has hundreds of alleles. We show that in mice germline stem cells compete for gonad niches, and in mice and humans, blood-forming stem cells also compete for bone marrow niches. We show that the clonal progression from blood-forming stem cells to acute leukemias by successive genetic and epigenetic events in blood stem cells also involves competition and selection between clones and propose that this is a general theme in cancer. PMID:26195745

  2. Self-Organization of Stem Cell Colonies and of Early Mammalian Embryos: Recent Experiments Shed New Light on the Role of Autonomy vs. External Instructions in Basic Body Plan Development

    PubMed Central

    Denker, Hans-Werner

    2016-01-01

    “Organoids”, i.e., complex structures that can develop when pluripotent or multipotent stem cells are maintained in three-dimensional cultures, have become a new area of interest in stem cell research. Hopes have grown that when focussing experimentally on the mechanisms behind this type of in vitro morphogenesis, research aiming at tissue and organ replacements can be boosted. Processes leading to the formation of organoids in vitro are now often addressed as self-organization, a term referring to the formation of complex tissue architecture in groups of cells without depending on specific instruction provided by other cells or tissues. The present article focuses on recent reports using the term self-organization in the context of studies on embryogenesis, specifically addressing pattern formation processes in human blastocysts attaching in vitro, or in colonies of pluripotent stem cells (“gastruloids”). These morphogenetic processes are of particular interest because, during development in vivo, they lead to basic body plan formation and individuation. Since improved methodologies like those employed by the cited authors became available, early embryonic pattern formation/self-organization appears to evolve now as a research topic of its own. This review discusses concepts concerning the involved mechanisms, focussing on autonomy of basic body plan development vs. dependence on external signals, as possibly provided by implantation in the uterus, and it addresses biological differences between an early mammalian embryo, e.g., a morula, and a cluster of pluripotent stem cells. It is concluded that, apart from being of considerable biological interest, the described type of research needs to be contemplated carefully with regard to ethical implications when performed with human cells. PMID:27792143

  3. Self-Organization of Stem Cell Colonies and of Early Mammalian Embryos: Recent Experiments Shed New Light on the Role of Autonomy vs. External Instructions in Basic Body Plan Development.

    PubMed

    Denker, Hans-Werner

    2016-10-25

    "Organoids", i.e., complex structures that can develop when pluripotent or multipotent stem cells are maintained in three-dimensional cultures, have become a new area of interest in stem cell research. Hopes have grown that when focussing experimentally on the mechanisms behind this type of in vitro morphogenesis, research aiming at tissue and organ replacements can be boosted. Processes leading to the formation of organoids in vitro are now often addressed as self-organization, a term referring to the formation of complex tissue architecture in groups of cells without depending on specific instruction provided by other cells or tissues. The present article focuses on recent reports using the term self-organization in the context of studies on embryogenesis, specifically addressing pattern formation processes in human blastocysts attaching in vitro, or in colonies of pluripotent stem cells ("gastruloids"). These morphogenetic processes are of particular interest because, during development in vivo, they lead to basic body plan formation and individuation. Since improved methodologies like those employed by the cited authors became available, early embryonic pattern formation/self-organization appears to evolve now as a research topic of its own. This review discusses concepts concerning the involved mechanisms, focussing on autonomy of basic body plan development vs. dependence on external signals, as possibly provided by implantation in the uterus, and it addresses biological differences between an early mammalian embryo, e.g., a morula, and a cluster of pluripotent stem cells. It is concluded that, apart from being of considerable biological interest, the described type of research needs to be contemplated carefully with regard to ethical implications when performed with human cells.

  4. Theoretical size controls of the giant Phaeocystis globosa colonies

    NASA Astrophysics Data System (ADS)

    Liu, Xiao; Smith, Walker O.; Tang, Kam W.; Doan, Nhu Hai; Nguyen, Ngoc Lam

    2015-06-01

    An unusual characteristic of the cosmopolitan haptophyte Phaeocystis globosa is its ability to form colonies of strikingly large size-up to 3 cm in diameter. The large size and the presence of a mucoid envelope are believed to contribute to the formation of dense blooms in Southeast Asia. We collected colonies of different sizes in shallow coastal waters of Viet Nam and conducted a series of measurements and experiments on individual colonies. Using these empirical data, we developed a simple carbon-based model to predict the growth and maximal size of P. globosa colonies. Our model suggests that growth of a colony from 0.2 cm to 1.4 cm (the maximal size in our samples) would take 16 days. This number, however, is strongly influenced by the maximal photosynthetic rate and other physiological parameters used in the model. The model also returns a specific growth rate of 0.30 d-1 for colonial cells, comparable to satellite estimates, but lower than have been measured for unicellular P. globosa in batch culture at similar temperatures. We attribute this low growth rate to not only the model uncertainties, but factors such as self-shading and diffusive limitation of nutrient uptake.

  5. The hematopoietic factor GM-CSF (granulocyte-macrophage colony-stimulating factor) promotes neuronal differentiation of adult neural stem cells in vitro.

    PubMed

    Krüger, Carola; Laage, Rico; Pitzer, Claudia; Schäbitz, Wolf-Rüdiger; Schneider, Armin

    2007-10-22

    Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in the generation of granulocytes, macrophages, and dendritic cells from hematopoietic progenitor cells. We have recently demonstrated that GM-CSF has anti-apoptotic functions on neurons, and is neuroprotective in animal stroke models. The GM-CSF receptor alpha is expressed on adult neural stem cells in the rodent brain, and in culture. Addition of GM-CSF to NSCs in vitro increased neuronal differentiation in a dose-dependent manner as determined by quantitative PCR, reporter gene assays, and FACS analysis. Similar to the hematopoietic factor Granulocyte-colony stimulating factor (G-CSF), GM-CSF stimulates neuronal differentiation of adult NSCs. These data highlight the astonishingly similar functions of major hematopoietic factors in the brain, and raise the clinical attractiveness of GM-CSF as a novel drug for neurological disorders.

  6. Morula cells as key hemocytes of the lectin pathway of complement activation in the colonial tunicate Botryllus schlosseri.

    PubMed

    Nicola, Franchi; Loriano, Ballarin

    2017-04-01

    The complement system is deeply rooted in the evolution of humoral mechanism of innate immunity. In addition to the alternative pathway of complement activation, lectins and associated serine proteases exert important roles in the recognition of non-self and activation of the effectors. In the colonial tunicate Botryllus schlosseri, we identified, characterized and studied the expression of three orthologues of genes involved in the lectin pathway of complement activation of vertebrates, i.e., genes for a mannose-binding lectin (MBL), a ficolin and a mannose-associated serine protease 1 (MASP1). All the genes are transcribed by hemocytes, and specifically by morula cells, the same immunocytes responsible for the transcription of C3 and Bf orthologues. The transcription levels of MASP1 and ficolin orthologues are not affected by zymosan challenge, indicating a constitutive expression of complement system associated serine proteases, whereas the MBL orthologue is up-regulated after 15 min of zymosan exposure. Collectively, our data suggest the presence of a complete lectin activation pathway in Botryllus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Promoting the selection and maintenance of fetal liver stem/progenitor cell colonies by layer-by-layer polypeptide tethered supported lipid bilayer.

    PubMed

    Lee, I-Chi; Liu, Yung-Chiang; Tsai, Hsuan-Ang; Shen, Chia-Ning; Chang, Ying-Chih

    2014-12-10

    In this study, we designed and constructed a series of layer-by-layer polypeptide adsorbed supported lipid bilayer (SLB) films as a novel and label-free platform for the isolation and maintenance of rare populated stem cells. In particular, four alternative layers of anionic poly-l-glutamic acid and cationic poly-l-lysine were sequentially deposited on an anionic SLB. We found that the fetal liver stem/progenitor cells from the primary culture were selected and formed colonies on all layer-by-layer polypeptide adsorbed SLB surfaces, regardless of the number of alternative layers and the net charges on those layers. Interestingly, these isolated stem/progenitor cells formed colonies which were maintained for an 8 day observation period. Quartz crystal microbalance with dissipation measurements showed that all SLB-polypeptide films were protein resistant with serum levels significantly lower than those on the polypeptide multilayer films without an underlying SLB. We suggest the fluidic SLB promotes selective binding while minimizing the cell-surface interaction due to its nonfouling nature, thus limiting stem cell colonies from spreading.

  8. Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage

    PubMed Central

    Ushach, Irina; Zlotnik, Albert

    2016-01-01

    M-CSF and GM-CSF are 2 important cytokines that regulate macrophage numbers and function. Here, we review their known effects on cells of the macrophage-monocyte lineage. Important clues to their function come from their expression patterns. M-CSF exhibits a mostly homeostatic expression pattern, whereas GM-CSF is a product of cells activated during inflammatory or pathologic conditions. Accordingly, M-CSF regulates the numbers of various tissue macrophage and monocyte populations without altering their "activation" status. Conversely, GM-CSF induces activation of monocytes/macrophages and also mediates differentiation to other states that participate in immune responses [i.e., dendritic cells (DCs)]. Further insights into their function have come from analyses of mice deficient in either cytokine. M-CSF signals through its receptor (CSF-1R). Interestingly, mice deficient in CSF-1R expression exhibit a more significant phenotype than mice deficient in M-CSF. This observation was explained by the discovery of a novel cytokine (IL-34) that represents a second ligand of CSF-1R. Information about the function of these ligands/receptor system is still developing, but its complexity is intriguing and strongly suggests that more interesting biology remains to be elucidated. Based on our current knowledge, several therapeutic molecules targeting either the M-CSF or the GM-CSF pathways have been developed and are currently being tested in clinical trials targeting either autoimmune diseases or cancer. It is intriguing to consider how evolution has directed these pathways to develop; their complexity likely mirrors the multiple functions in which cells of the monocyte/macrophage system are involved. PMID:27354413

  9. Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage.

    PubMed

    Ushach, Irina; Zlotnik, Albert

    2016-09-01

    M-CSF and GM-CSF are 2 important cytokines that regulate macrophage numbers and function. Here, we review their known effects on cells of the macrophage-monocyte lineage. Important clues to their function come from their expression patterns. M-CSF exhibits a mostly homeostatic expression pattern, whereas GM-CSF is a product of cells activated during inflammatory or pathologic conditions. Accordingly, M-CSF regulates the numbers of various tissue macrophage and monocyte populations without altering their "activation" status. Conversely, GM-CSF induces activation of monocytes/macrophages and also mediates differentiation to other states that participate in immune responses [i.e., dendritic cells (DCs)]. Further insights into their function have come from analyses of mice deficient in either cytokine. M-CSF signals through its receptor (CSF-1R). Interestingly, mice deficient in CSF-1R expression exhibit a more significant phenotype than mice deficient in M-CSF. This observation was explained by the discovery of a novel cytokine (IL-34) that represents a second ligand of CSF-1R. Information about the function of these ligands/receptor system is still developing, but its complexity is intriguing and strongly suggests that more interesting biology remains to be elucidated. Based on our current knowledge, several therapeutic molecules targeting either the M-CSF or the GM-CSF pathways have been developed and are currently being tested in clinical trials targeting either autoimmune diseases or cancer. It is intriguing to consider how evolution has directed these pathways to develop; their complexity likely mirrors the multiple functions in which cells of the monocyte/macrophage system are involved. © Society for Leukocyte Biology.

  10. Vitrified canine testicular cells allow the formation of spermatogonial stem cells and seminiferous tubules following their xenotransplantation into nude mice

    PubMed Central

    Lee, Kyung Hoon; Lee, Won Young; Kim, Dong Hoon; Lee, Seung Hoon; Do, Jung Tae; Park, Chankyu; Kim, Jae Hwan; Choi, Young Suk; Song, Hyuk

    2016-01-01

    Belgian Malinois (BM), one of the excellent military dog breeds in South Korea, is usually castrated before sexual maturation. Therefore, the transfer of their genetic features to the next generation is difficult. To overcome this, testicular cells from 4-month-old BMs were frozen. Testicular cells were thawed after 3 months and cultured in StemPro-34 medium. Spermatogonial stem cell (SSC) characteristics were determined by the transplantation of the cultured germ cell-derived colonies (GDCs) into empty testes, containing only several endogenous SSCs and Sertoli cells, of immunodeficient mice, 4 weeks after busulfan treatment. Following the implantation, the transplanted cells localized in the basement membrane of the seminiferous tubules, and ultimately colonized the recipient testes. Xenotransplantation of GDCs together with testicular somatic cells conjugated with extracellular matrix (ECM), led to the formation of de novo seminiferous tubules. These seminiferous tubules were mostly composed of Sertoli cells. Some germ cells were localized in the basement membrane of seminiferous tubules. This study revealed that BM-derived SSCs, obtained from the castrated testes, might be a valuable tool for the transfer of BM genetic features to the next generation. PMID:26907750

  11. The combination of stem cell factor and granulocyte-colony stimulating factor for chronic stroke treatment in aged animals

    PubMed Central

    2012-01-01

    Background Stroke occurs more frequently in the elderly population and presents the number one leading cause of persistent disability worldwide. Lack of effective treatment to enhance brain repair and improve functional restoration in chronic stroke, the recovery phase of stroke, is a challenging medical problem to be solved in stroke research. Our early study has revealed the therapeutic effects of stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF) on chronic stroke in young animals. However, whether this treatment is effective and safe to the aged population remains to be determined. Methods Cortical brain ischemia was produced in aged C57BL mice or aged spontaneously hypertensive rats. SCF+G-CSF or equal volume of vehicle solution was subcutaneously injected for 7 days beginning at 3–4 months after induction of cortical brain ischemia. Using the approaches of biochemistry assays, flow cytometry, pathology, and evaluation of functional outcome, several doses of SCF+G-CSF have been examined for their safety and efficiency on chronic stroke in aged animals. Results All tested doses did not show acute or chronic toxicity in the aged animals. Additionally, SCF+G-CSF treatment in chronic stroke of aged animals mobilized bone marrow stem cells and improved functional outcome in a dose-dependent manner. Conclusions SCF+G-CSF treatment is a safe and effective approach to chronic stroke in the aged condition. This study provides important information needed for developing a new therapeutic strategy to improve the health of older adults with chronic stroke. PMID:23254113

  12. Design of Recombinant Stem Cell Factor macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

    NASA Astrophysics Data System (ADS)

    Chen, Tao; Yang, Jie; Wang, Yuelang; Zhan, Chenyang; Zang, Yuhui; Qin, Junchuan

    2005-05-01

    Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of 84 KD under non-reducing conditions and a monomer of 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

  13. Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients

    PubMed Central

    Chang, Ting-Yu; Tsai, Wei-Chi; Huang, Tse-Shun; Su, Shu-Han; Chang, Chih-Young; Ma, Hsiu-Yen; Wu, Chun-Hsien; Yang, Chih-Yung; Lin, Chi-Hung; Huang, Po-Hsun; Cheng, Cheng-Chung; Wang, Hsei-Wei

    2017-01-01

    Functional impairment of endothelial colony-forming cells (ECFCs), a specific cell lineage of endothelial progenitor cells (EPCs) is highly associated with the severity of coronary artery disease (CAD), the most common type of cardiovascular disease (CVD). Emerging evidence show that circulating microRNAs (miRNAs) in CAD patients’ body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq), we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson’s correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up

  14. Urban particulate matter suppresses priming of T helper type 1 cells by granulocyte/macrophage colony-stimulating factor-activated human dendritic cells.

    PubMed

    Matthews, Nick C; Faith, Alex; Pfeffer, Paul; Lu, Haw; Kelly, Frank J; Hawrylowicz, Catherine M; Lee, Tak H

    2014-02-01

    Urban particulate matter (UPM) exacerbates asthmatic lung inflammation and depresses lung immunity. Lung dendritic cells (DCs) react to airway particulates, and have a critical role in linking innate and adaptive immunity, but the direct effects of UPM on DCs, that have been activated by granulocyte/macrophage colony-stimulating factor (GM-CSF), a product of stimulated normal human bronchial epithelial cells, has not been investigated. Human blood CD1c(+) DCs were purified and activated with UPM in the presence or absence of GM-CSF with and without LPS, and DC maturation was assessed by flow cytometry. DC stimulatory capacity and priming of 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester-labeled naive CD4 T cells was investigated using the allogeneic mixed lymphocyte reaction. T cell proliferation and effector function were assessed using flow cytometry and secreted cytokines were measured by combined bead array. UPM enhanced DC maturation in an LPS-independent manner. DCs activated by UPM plus GM-CSF (UPM + GM-CSF DCs) induced higher naive CD4 T cell proliferation in the allogeneic mixed lymphocyte reaction than DCs pretreated by GM-CSF alone (GM-CSF DCs), and elicited both substantially lower levels of IFN-γ, IL-13, and IL-5 secretion, and lower frequencies of alloantigen-specific T helper (Th) type 1 effector cells than naive CD4 T cells primed by GM-CSF DCs. UPM-stimulated DCs produced IL-6 and TNF-α. Neutralization of IL-6 decreased naive CD4 T cell proliferation stimulated by UPM + GM-CSF DCs, and significantly increased the frequency of alloantigen-specific Th1 effector cells, but did not reverse UPM-induced inhibition of IFN-γ secretion. We conclude that UPM enhances GM-CSF-induced DC maturation and stimulatory capacity, but inhibits the generation of Th1 cells. Thus, UPM exposure may impair Th1 responses to pulmonary pathogens.

  15. Steroids and hematopoiesis. II. The effect of steroids on in vitro erythroid colony growth: evidence for different target cells for different classes of steroids.

    PubMed

    Singer, J W; Adamson, J W

    1976-06-01

    Androgenic steroids and their non-androgenic 5beta-H metabolites enhance the number of colonies of hemoglobin synthesizing cells grown from rat bone marrow in response to a standard (0.25 unit/ml) concentration of erythropoietin. The target cells for two steroids were found to be different. Cells influenced by the androgen, fluoxymesterone (fluoxy), resembled cells responding to erythropoietin in their cycle characteristics, as measured by tritiated thymidine suicide, and in their physical characteristics, as determined by velocity sedimentation gradient separation. Cells responding to etiocholanolone (etio) had a much lower tritiated thymidine suicide rate and different sedimentation velocities. Preincubation of marrow cells with etio for two hours was sufficient to enhance erythroid colony growth by 84%, whereas a similar incubation with fluoxy produced no increment. These studies demonstrate that different classes of steroids may influence in vitro erythropoiesis by acting on distinct populations of marrow cells. Fluoxymesterone appears to act through cells already committed to respond to erythropoietin, while etiocholanolone appears to act on a separate, perhaps more primitive population of marrow cells.

  16. Granulocyte-Colony-Stimulating Factor Stimulation of Bone Marrow Mesenchymal Stromal Cells Promotes CD34+ Cell Migration Via a Matrix Metalloproteinase-2-Dependent Mechanism

    PubMed Central

    Ponte, Adriana López; Ribeiro-Fleury, Tatiana; Chabot, Valérie; Gouilleux, Fabrice; Langonné, Alain; Hérault, Olivier; Charbord, Pierre

    2012-01-01

    Human hematopoietic stem/progenitor cells (HSPCs) can be mobilized into the circulation using granulocyte-colony stimulating factor (G-CSF), for graft collection in view of hematopoietic transplantation. This process has been related to bone marrow (BM) release of serine proteases and of the matrix metalloproteinase-9 (MMP-9). Yet, the role of these mediators in HSC egress from their niches remains questionable, because they are produced by nonstromal cells (mainly neutrophils and monocytes/macrophages) that are not a part of the niche. We show here that the G-CSF receptor (G-CSFR) is expressed by human BM mesenchymal stromal/stem cells (MSCs), and that G-CSF prestimulation of MSCs enhances the in vitro trans-stromal migration of CD34+ cells. Zymography analysis indicates that pro-MMP-2 (but not pro-MMP-9) is expressed in MSCs, and that G-CSF treatment increases its expression and induces its activation at the cell membrane. We further demonstrate that G-CSF-stimulated migration depends on G-CSFR expression and is mediated by a mechanism that involves MMPs. These results suggest a molecular model whereby G-CSF infusion may drive, by the direct action on MSCs, HSPC egress from BM niches via synthesis and activation of MMPs. In this model, MMP-2 instead of MMP-9 is implicated, which constitutes a major difference with mouse mobilization models. PMID:22651889

  17. Stage-dependent suppression of the formation of dentin-resorbing multinuclear cells with migration inhibitory factor in vitro

    PubMed Central

    KIKUIRI, TAKASHI; YOSHIMURA, YOSHITAKA; TABATA, FUTOSHI; HASEGAWA, TOMOKAZU; NISHIHIRA, JUN; SHIRAKAWA, TETSUO

    2012-01-01

    The macrophage migration inhibitory factor (MIF) is a crucial mediator of immune responses and is known to play a pivotal role in cell proliferation and differentiation. In this study, we assessed whether MIF exerts regulatory effects on osteoclast formation in bone marrow cells and, if so, by what mechanism. Bone marrow cells were either co-cultured with MC3T3-E1 cells or cultured with macrophage-colony stimulating factor (M-CSF) and the soluble form of the receptor activator of the nuclear factor-κB ligand (RANKL). Under the influence of MIF, the formation of osteoclastic multinuclear cells was examined. The number of multinuclear TRAP-positive cells formed in the co-culture was significantly reduced when MIF (≥0.1 μg/ml) was exogenously applied during the third and fourth days of the 6-day cultivation period. MIF affected neither the number of mononuclear TRAP-positive cells induced with M-CSF and RANKL, nor the expression of RANKL and osteoprotegerin in MC3T3-E1 cells. TRAP-positive cells cultured on dentin slices with MIF showed lower dentin-resorbing activity than those cultured without MIF. These results suggest that MIF has no regulatory roles in the differentiation of bone marrow cells to mononuclear TRAP-positive cells, but has inhibitory effects on the formation of mature osteoclasts by preventing cell fusion, which may eventually interfere with the osteoclast-mediated dentin resorption. PMID:22969841

  18. Pre-B cell colony enhancing factor induces Nampt-dependent translocation of the insulin receptor out of lipid microdomains in A549 lung epithelial cells.

    PubMed

    Peng, Qianyi; Jia, Song Hui; Parodo, Jean; Ai, Yuhang; Marshall, John C

    2015-02-15

    Pre-B cell colony-enhancing factor (PBEF) is a highly conserved pleiotropic protein reported to be an alternate ligand for the insulin receptor (IR). We sought to clarify the relationship between PBEF and insulin signaling by evaluating the effects of PBEF on the localization of the IRβ chain to lipid rafts in A549 epithelial cells. We isolated lipid rafts from A549 cells and detected the IR by immunoprecipitation from raft fractions or whole cell lysates. Cells were treated with rPBEF, its enzymatic product nicotinamide adenine dinucleotide (NAD), or the Nampt inhibitor daporinad to study the effect of PBEF on IRβ movement. We used coimmunoprecipitation studies in cells transfected with PBEF and IRβ constructs to detect interactions between PBEF, the IRβ, and caveolin-1 (Cav-1). PBEF was present in both lipid raft and nonraft fractions, whereas the IR was found only in lipid raft fractions of resting A549 cells. The IR-, PBEF-, and Cav-1-coimmunoprecipitated rPBEF treatment resulted in the movement of IRβ- and tyrosine-phosphorylated Cav-1 from lipid rafts to nonrafts, an effect that could be blocked by daporinad, suggesting that this effect was facilitated by the Nampt activity of PBEF. The addition of PBEF to insulin-treated cells resulted in reduced Akt phosphorylation of both Ser⁴⁷³ and Thr³⁰⁸. We conclude that PBEF can inhibit insulin signaling through the IR by Nampt-dependent promotion of IR translocation into the nonraft domains of A549 epithelial cells. PBEF-induced alterations in the spatial geometry of the IR provide a mechanistic explanation for insulin resistance in inflammatory states associated with upregulation of PBEF.

  19. In vitro assessment of bone marrow endothelial colonies (CFU-En) in non-Hodgkin's lymphoma patients undergoing peripheral blood stem cell transplantation.

    PubMed

    Lanza, F; Campioni, D; Punturieri, M; Moretti, S; Dabusti, M; Spanedda, R; Castoldi, G

    2003-12-01

    The distribution and functional characteristics of in vitro bone marrow (BM) endothelial colonies (CFU-En) were studied in 70 non-Hodgkin's lymphoma (NHL) patients in different phases of the disease to explore the association between CFU-En growth and angiogenesis, and between the number of CFU-En and the presence of hematopoietic and mesenchymal progenitor cells. The mean number of CFU-En/10(6) BM mononuclear cells seen in remission patients was significantly higher than that seen in newly diagnosed patients (P=0.04), and in normal subjects (P=0.008). Patients with low-grade NHL in remission displayed a higher CFU-En value compared with high-grade NHL (P=0.04). In the autograft group (40 patients), a significant reduction of CFU-En number was detected in the first 4-6 months after transplantation. In remission patients, the CFU-En number positively correlated with the incidence of BM colony-forming unit granulocyte-macrophage (CFU-GM) (P=0.013) and CFU-multilineage (CFU-GEMM) hematopoietic colonies (P=0.044). These in vitro data show that CFU-En numbers increase following standard-dose chemotherapy, thus providing a rationale for further investigating the effects of different cytostatic drugs on BM endothelial cells growth and function.

  20. Adrenaline administration promotes the efficiency of granulocyte colony stimulating factor-mediated hematopoietic stem and progenitor cell mobilization in mice.

    PubMed

    Chen, Chong; Cao, Jiang; Song, Xuguang; Zeng, Lingyu; Li, Zhenyu; Li, Yong; Xu, Kailin

    2013-01-01

    A high dose of granulocyte colony stimulating factor (G-CSF) is widely used to mobilize hematopoietic stem and progenitor cells (HSPC), but G-CSF is relatively inefficient and may cause adverse effects. Recently, adrenaline has been found to play important roles in HSPC mobilization. In this study, we explored whether adrenaline combined with G-CSF could induce HSPC mobilization in a mouse model. Mice were treated with adrenaline and either a high or low dose of G-CSF alone or in combination. Peripheral blood HSPC counts were evaluated by flow cytometry. Levels of bone marrow SDF-1 were measured by ELISA, the transcription of CXCR4 and SDF-1 was measured by real-time RT-PCR, and CXCR4 protein was detected by Western blot. Our results showed that adrenaline alone fails to mobilize HSPCs into the peripheral blood; however, when G-CSF and adrenaline are combined, the WBC counts and percentages of HSPCs are significantly higher compared to those in mice that received G-CSF alone. The combined use of adrenaline and G-CSF not only accelerated HSPC mobilization, but also enabled the efficient mobilization of HSPCs into the peripheral blood at lower doses of G-CSF. Adrenaline/G-CSF treatment also extensively downregulated levels of SDF-1 and CXCR4 in mouse bone marrow. These results demonstrated that adrenaline combined with G-CSF can induce HSPC mobilization by down-regulating the CXCR4/SDF-1 axis, indicating that the use of adrenaline may enable the use of reduced dosages or durations of G-CSF treatment, minimizing G-CSF-associated complications.

  1. Contribution of granulocyte colony-stimulating factor to the acute mobilization of endothelial precursor cells by vascular disrupting agents.

    PubMed

    Shaked, Yuval; Tang, Terence; Woloszynek, Jill; Daenen, Laura G; Man, Shan; Xu, Ping; Cai, Shi-Rong; Arbeit, Jeffrey M; Voest, Emile E; Chaplin, David J; Smythe, Jon; Harris, Adrian; Nathan, Paul; Judson, Ian; Rustin, Gordon; Bertolini, Francesco; Link, Daniel C; Kerbel, Robert S

    2009-10-01

    Vascular disrupting agents (VDA) cause acute shutdown of abnormal established tumor vasculature, followed by massive intratumoral hypoxia and necrosis. However, a viable rim of tumor tissue invariably remains from which tumor regrowth rapidly resumes. We have recently shown that an acute systemic mobilization and homing of bone marrow-derived circulating endothelial precursor (CEP) cells could promote tumor regrowth following treatment with either a VDA or certain chemotherapy drugs. The molecular mediators of this systemic reactive host process are unknown. Here, we show that following treatment of mice with OXi-4503, a second-generation potent prodrug derivative of combretastatin-A4 phosphate, rapid increases in circulating plasma vascular endothelial growth factor, stromal derived factor-1 (SDF-1), and granulocyte colony-stimulating factor (G-CSF) levels are detected. With the aim of determining whether G-CSF is involved in VDA-induced CEP mobilization, mutant G-CSF-R(-/-) mice were treated with OXi-4503. We found that as opposed to wild-type controls, G-CSF-R(-/-) mice failed to mobilize CEPs or show induction of SDF-1 plasma levels. Furthermore, Lewis lung carcinomas grown in such mice treated with OXi-4503 showed greater levels of necrosis compared with tumors treated in wild-type mice. Evidence for rapid elevations in circulating plasma G-CSF, vascular endothelial growth factor, and SDF-1 were also observed in patients with VDA (combretastatin-A4 phosphate)-treated cancer. These results highlight the possible effect of drug-induced G-CSF on tumor regrowth following certain cytotoxic drug therapies, in this case using a VDA, and hence G-CSF as a possible therapeutic target.

  2. Pre-B-cell colony-enhancing factor gene polymorphisms and risk of acute respiratory distress syndrome.

    PubMed

    Bajwa, Ednan K; Yu, Chu-Ling; Gong, Michelle N; Thompson, B Taylor; Christiani, David C

    2007-05-01

    Pre-B-cell colony-enhancing factor (PBEF) levels are elevated in bronchoalveolar lavage fluid and serum of patients with acute lung injury. There are several suspected functional polymorphisms of the corresponding PBEF gene. We hypothesized that variations in PBEF gene polymorphisms alter the risk of developing acute respiratory distress syndrome (ARDS). Nested case-control study. Tertiary academic medical center. We studied 375 patients with ARDS and 787 at-risk controls genotyped for the PBEF T-1001G and C-1543T polymorphisms. None. Patients with the -1001G (variant) allele had significantly greater odds of developing ARDS than wild-type homozygotes (odds ratio, 1.35; 95% confidence interval, 1.02-1.78). Patients with the -1543T (variant) allele did not have significantly different odds of developing ARDS than wild-type homozygotes (odds ratio, 0.86; 95% confidence interval, 0.65-1.13). When analysis was stratified by ARDS risk factor, -1543T was associated with decreased odds of developing ARDS in septic shock patients (odds ratio, 0.66; 95% confidence interval, 0.45-0.97). Also, -1001G was associated with increased hazard of intensive care unit mortality, whereas -1543T was associated with decreased hazard of 28-day and 60-day ARDS mortality, as well as shorter duration of mechanical ventilation. Similar results were found in analyses of the related GC (-1001G:-1543C) and TT (-1001T:-1543T) haplotypes. The PBEFT-1001G variant allele and related haplotype are associated with increased odds of developing ARDS and increased hazard of intensive care unit mortality among at-risk patients, whereas the C-1543T variant allele and related haplotype are associated with decreased odds of ARDS among patients with septic shock and better outcomes among patients with ARDS.

  3. Herbal medicine "sho-saiko-to" induces in vitro granulocyte colony-stimulating factor production on peripheral blood mononuclear cells.

    PubMed

    Yamashiki, M; Asakawa, M; Kayaba, Y; Kosaka, Y; Nishimura, A

    1992-01-01

    The herbal medicine "Sho-saiko-to (Xiao-Chai-Hu-Tang)" has been used in China for about 3000 years for the treatment of pyretic diseases. This medicine is now available as one of the prescribing drugs approved by the Ministry of Health and Welfare of Japan, and has also been widely used for patients with chronic viral liver disease as one of biological response modifiers in the field of Japan's Western Medicine. However, its mode of action has not been fully described. In the present in vitro study, we added "Sho-saiko-to" (TJ-9, Tsumura, Tokyo) to the culture of peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers, and observed a dose-dependent increase in the production of granulocyte colony-stimulating factor (G-CSF). The same experiment was conducted using other herbal medicines "Dai-saiko-to" (TJ-8) and "Saiko-keishi-to" (TJ-10) which showed similar effects, or "Sho-seiryu-to" (TJ-19) which consists of very different compounds and shows different efficacy. The increases of G-CSF production were similar when "Sho-saiko-to" (TJ-9) or one of the 2 reference drugs (TJ-8 and 10) was added, whereas the increase when the control drug "Sho-seiryu-to" (TJ-19) was added, was quite small. This result shows that G-CSF induction is not a common effect of herbal medicines, but a specific effect of TJ-8, 9, and 10. Among these 3 drugs the increase produced by "Sho-saiko-to" was the largest. Based on this result, we conclude that administration of "Sho-saiko-to" may be useful not only for the treatment of chronic liver disease, but also for malignant diseases and acute infectious diseases where G-CSF is efficacious.

  4. Antibodies binding granulocyte-macrophage colony stimulating factor produced by cord blood-derived B cell lines immortalized by Epstein-Barr virus in vitro.

    PubMed

    Revoltella, R P; Laricchia Robbio, L; Liberati, A M; Reato, G; Foa, R; Funaro, A; Vinante, F; Pizzolo, G

    2000-09-15

    We detected natural antibodies (auto-Abs) binding human granulocyte-macrophage colony stimulating factor (GM-CSF) in umbilical cord blood (CB) (23 of 94 samples screened) and peripheral blood of women at the end of pregnancy (6 of 42 samples tested). To demonstrate that Abs detected in CB were produced by the fetus, CB mononuclear cells were infected with Epstein-Barr virus in vitro. Ten cell lines producing constitutively anti-recombinant human GM-CSF (rhGM-CSF) Abs were isolated and characterized. These cells displayed a male karyotype, an early activated B cell phenotype, coexpressed surface IgM and IgD, and secreted only IgM with prevailing lambda clonal restriction. Specific cell surface binding of biotinylated rhGM-CSF and high-level anti-rhGM-CSF IgM Ab production were typical features of early cell cultures. In late cell passages the frequency of more undifferentiated B cells increased. Serum Abs of either maternal or fetal origin or Abs produced in culture did not affect the granulocyte and macrophage colony stimulating activity of rhGM-CSF from bone marrow progenitors in soft agar, suggesting that the Abs produced were nonneutralizing.

  5. Vimentin is necessary for colony growth of human diploid keratinocytes.

    PubMed

    Castro-Muñozledo, Federico; Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Hernández-Quintero, Miriam; Kuri-Harcuch, Walid

    2015-01-01

    The role of vimentin (Vim) in diploid epithelial cells is not well known. To understand its biological function, we cultured human epidermal keratinocytes under conditions that support migration, proliferation, stratification and terminal differentiation. We identified a keratinocyte subpopulation that shows a p63(+)/α5β1(bright) phenotype and displays Vim intermediate filaments (IFs) besides their keratin IF network. These cells were mainly located at the proliferative/migratory rim of the growing colonies; but also, they were scarce and scattered or formed small groups of basal cells in confluent stratified epithelia. Stimulation of cells with EGF and wounding experiments in confluent arrested epithelia increased the number of Vim(+) keratinocytes in an extent higher to the expected for a cell population doubling. BrdU labeling demonstrated that most of the proliferative cells located at the migratory border of the colony have Vim, in contrast with proliferative cells located at the basal layer at the center of big colonies which lacked of Vim IFs, suggesting that Vim expression was not solely linked to proliferation. Therefore, we silenced Vim mRNA in the cultured keratinocytes and observed an inhibition of colony growth. Such results, together with long-term cultivation assays which showed that Vim might be associated to pattern formation in cultured epithelia, suggest that Vim expression is essential for a highly motile phenotype, which is necessary for keratinocyte colony growth and possibly for development and wound healing. Vim(+)/p63(+)/α5β1(bright) epithelial cells may play a significant physiological role in embryonic morphogenetic movements; wound healing and other pathologies such as carcinomas and hyperproliferative diseases.

  6. Vibrio cholerae O1 El Tor: Identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation

    PubMed Central

    Yildiz, Fitnat H.; Schoolnik, Gary K.

    1999-01-01

    The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an exopolysaccharide, EPSETr, that confers chlorine resistance and biofilm-forming capacity. EPSETr production requires a chromosomal locus, vps, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-forming properties of EPSETr may enable the survival of V. cholerae O1 within environmental aquatic habitats between outbreaks of human disease. PMID:10097157

  7. Mechanical dissociation of human embryonic stem cell colonies by manual scraping after collagenase treatment is much more detrimental to cellular viability than is trypsinization with gentle pipetting.

    PubMed

    Heng, Boon Chin; Liu, Hua; Ge, Zigang; Cao, Tong

    2007-05-01

    Because hESC (human embryonic stem cells) are 'social cells' that require co-operative interactions and intimate physical contact with each other, it is absolutely essential to dissociate hESC colonies into cellular clumps rather than into a single-cell suspension during serial passage. The present study compared two commonly used protocols for dissociating hESC colonies. The first protocol involved mild enzymatic treatment with collagenase type IV (1 mg/ml) for approx. 5-10 min, prior to mechanical dissociation into cellular clumps through manual scraping with a plastic pipette tip. The second protocol involved a short duration of exposure (2-3 min) to low concentrations of trypsin (0.05%), followed by gentle pipetting. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay was used to compare the recovery of viable cells after dissociating hESC colonies with these two protocols, before and after conventional freeze-thawing with 10% (v/v) DMSO. Besides undifferentiated hESC, the randomly differentiated fibroblastic progenies of hESC at various passages (P0-P4), together with an immortalized cell line (CRL-1486), were also utilized to compare the two protocols. The results demonstrated that the second protocol (trypsinization with gentle pipetting) is much less detrimental to cellular viability than is the first protocol (collagenase treatment with scratching). This in turn translated to higher freeze-thaw survival rates. It is hypothesized that scratching after collagenase treatment (first protocol) somehow induces physical damage to the cells, thereby leading to a lower recovery of viable cells, both before and after freeze-thawing.

  8. Effects of western honey bee (Hymenoptera: Apidae) colony, cell type, and larval sex on host acquisition by female Varroa destructor (Acari: Varroidae).

    PubMed

    Calderone, N W; Kuenen, L P

    2001-10-01

    Female mites of the genus Varroa reproduce on the immature stages of Apis cerana F. and A. mellifera L. Mites are found more often in drone brood than worker brood, and while evolutionary explanations for this bias are well supported, the proximate mechanisms are not known. In one experiment, we verified that the proportion of hosts with one or more mites (MPV, mite prevalence value) was significantly greater for drones (0.763 +/- 0.043) (lsmean +/- SE) than for workers (0.253 +/- 0.043) in populations of mites and bees in the United States. Similar results were found for the average number of mites per host. In a second experiment, using a cross-fostering technique in which worker and drone larvae were reared in both worker and drone cells, we found that cell type, larval sex, colony and all interactions affected the level of mites on a host. Mite prevalence values were greatest in drone larvae reared in drone cells (0.907 +/- 0.025), followed by drone larvae reared in worker cells (0.751 +/- 0.025), worker larvae reared in worker cells (0.499 +/- 0.025), and worker larvae reared in drone cells (0.383 +/- 0.025). Similar results were found for the average number of mites per host. Our data show that mite levels are affected by environmental factors (cell type), by factors intrinsic to the host (sex), and by interactions between these factors. In addition, colony-to-colony variation is important to the expression of intrinsic and environmental factors.

  9. Formation of multilayer aggregates of mammalian cells by dielectrophoresis

    NASA Astrophysics Data System (ADS)

    Sebastian, Anil; Buckle, Anne-Marie; Markx, Gerard H.

    2006-09-01

    The formation of aggregates of mammalian cells at interdigitated oppositely castellated electrodes by positive dielectrophoresis was investigated. It is shown that, by using a constant small flow of fresh sorbitol iso-osmotic buffer through the chamber to remove ions leaking from the cells, a high positive DEP force can be maintained throughout the formation of the aggregates. Flow-rate dependent optima were found in the aggregate height as a function of the electrode size. It is shown that at low flow rates the creation of aggregates of mammalian cells with heights over 150 µm is feasible using relatively low voltages (20 Vpk-pk, 1 MHz). The formation of layered aggregates of two specialized cell types—stromal cells and Jurkat T lymphocytes—is demonstrated. The work confirms that dielectrophoresis can be reliably used for the formation of aggregates with three-dimensional architectures, which could be used as artificial microniches for the study of interactions between cells.

  10. Granulocyte macrophage colony stimulating factor produced in lesioned peripheral nerves induces the up-regulation of cell surface expression of MAC-2 by macrophages and Schwann cells

    PubMed Central

    1996-01-01

    Peripheral nerve injury is followed by Wallerian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose- specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes noneuronal cells, primarily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM- CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The proposed mechanism is based on the following novel observations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in intact nerves. The GM-CSF molecule was detected by ELISA in medium conditioned by in vitro and in vivo degenerating peripheral nerves as of the 4th h after injury. GM-CSF activity was demonstrated by two independent bioassays, and repressed by activity blocking antibodies. Significant levels of GM-CSF were produced by nerve derived fibroblasts, but neither by Schwann cells nor by nerve derived macrophages. Mouse rGM-CSF enhanced MAC-2 production in nerve explants, and up-regulated cell surface expression of MAC-2 by Schwann cells and macrophages. Interleukin-1 beta up-regulated GM-CSF production thus suggesting that injury induced GM-CSF production may be mediated by interleukin-1 beta. Our findings highlight the fact that fibroblasts, by producing GM-CSF and thereby affecting macrophage and Schwann function, play a significant role in the cascade of molecular events and cellular interactions of Wallerian degeneration. PMID:8601605

  11. Formation and cultivation of medaka primordial germ cells.

    PubMed

    Li, Zhendong; Li, Mingyou; Hong, Ni; Yi, Meisheng; Hong, Yunhan

    2014-07-01

    Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.

  12. Inhibition of osteoclast formation by 3-methylcholanthrene, a ligand for arylhydrocarbon receptor: suppression of osteoclast differentiation factor in osteogenic cells.

    PubMed

    Naruse, M; Otsuka, E; Naruse, M; Ishihara, Y; Miyagawa-Tomita, S; Hagiwara, H

    2004-01-01

    We investigated the effects of 3-methylcholanthrene (3MC), a ligand for arylhydrocarbon receptor (AhR), on osteoclastogenesis. Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells by a combination of the receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) produced by ST2 cells in response to 1alpha,25(OH)(2) Vitamin D(3). 3MC dose-dependently inhibited the formation of mono- and multinuclear osteoclast-like cells. However, 3MC did not inhibit the formation of osteoclast-like cells from mouse spleen cells which was supported by the exogenous soluble RANKL and M-CSF. 3MC did not affect the formation of an actin ring and pits on slices of dentine by osteoclast-like cells, both of which are typical indices of osteoclast activity. These results suggest that 3MC affects osteoclast-supporting cells such as ST2 cells but not osteoclast precursor cells and mature osteoclastic cells. When we measured the expression levels of RANKL mRNA in ST2 cells, 3MC dose-dependently decreased the level of this mRNA. However, 3MC did not affect levels of mRNAs for osteoprotegerin (OPG), M-CSF, and the receptor of 1alpha,25(OH)(2) Vitamin D(3) in ST2 cells. Furthermore, soluble RANKL was able to counteract the inhibitory effect of 3MC on the formation of osteoclast-like cells. Our findings indicate that 3MC inhibits osteoclastogenesis via the inhibition of RANKL expression in osteoblastic cells.

  13. Identification of a complex genetic network underlying Saccharomyces cerevisiae colony morphology.

    PubMed

    Voordeckers, Karin; De Maeyer, Dries; van der Zande, Elisa; Vinces, Marcelo D; Meert, Wim; Cloots, Lore; Ryan, Owen; Marchal, Kathleen; Verstrepen, Kevin J

    2012-10-01

    When grown on solid substrates, different microorganisms often form colonies with very specific morphologies. Whereas the pioneers of microbiology often used colony morphology to discriminate between species and strains, the phenomenon has not received much attention recently. In this study, we use a genome-wide assay in the model yeast Saccharomyces cerevisiae to identify all genes that affect colony morphology. We show that several major signalling cascades, including the MAPK, TORC, SNF1 and RIM101 pathways play a role, indicating that morphological changes are a reaction to changing environments. Other genes that affect colony morphology are involved in protein sorting and epigenetic regulation. Interestingly, the screen reveals only few genes that are likely to play a direct role in establishing colony morphology, with one notable example being FLO11, a gene encoding a cell-surface adhesin that has already been implicated in colony morphology, biofilm formation, and invasive and pseudohyphal growth. Using a series of modified promoters for fine-tuning FLO11 expression, we confirm the central role of Flo11 and show that differences in FLO11 expression result in distinct colony morphologies. Together, our results provide a first comprehensive look at the complex genetic network that underlies the diversity in the morphologies of yeast colonies. © 2012 Blackwell Publishing Ltd.

  14. Identification of a complex genetic network underlying Saccharomyces cerevisiae colony morphology

    PubMed Central

    Voordeckers, Karin; De Maeyer, Dries; Zande, Elisa; Vinces, Marcelo D; Meert, Wim; Cloots, Lore; Ryan, Owen; Marchal, Kathleen; Verstrepen, Kevin J

    2012-01-01

    When grown on solid substrates, different microorganisms often form colonies with very specific morphologies. Whereas the pioneers of microbiology often used colony morphology to discriminate between species and strains, the phenomenon has not received much attention recently. In this study, we use a genome-wide assay in the model yeast Saccharomyces cerevisiae to identify all genes that affect colony morphology. We show that several major signalling cascades, including the MAPK, TORC, SNF1 and RIM101 pathways play a role, indicating that morphological changes are a reaction to changing environments. Other genes that affect colony morphology are involved in protein sorting and epigenetic regulation. Interestingly, the screen reveals only few genes that are likely to play a direct role in establishing colony morphology, with one notable example being FLO11, a gene encoding a cell-surface adhesin that has already been implicated in colony morphology, biofilm formation, and invasive and pseudohyphal growth. Using a series of modified promoters for fine-tuning FLO11 expression, we confirm the central role of Flo11 and show that differences in FLO11 expression result in distinct colony morphologies. Together, our results provide a first comprehensive look at the complex genetic network that underlies the diversity in the morphologies of yeast colonies. PMID:22882838

  15. 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

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