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Sample records for affects cell expansion

  1. Culture materials affect ex vivo expansion of hematopoietic progenitor cells.

    PubMed

    LaIuppa, J A; McAdams, T A; Papoutsakis, E T; Miller, W M

    1997-09-01

    Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment

  2. The novel herbicide oxaziclomefone inhibits cell expansion in maize cell cultures without affecting turgor pressure or wall acidification.

    PubMed

    O'Looney, Nichola; Fry, Stephen C

    2005-11-01

    Oxaziclomefone [OAC; IUPAC name 3-(1-(3,5-dichlorophenyl)-1-methylethyl)-3,4-dihydro-6-methyl-5-phenyl-2H-1,3-oxazin-4-one] is a new herbicide that inhibits cell expansion in grass roots. Its effects on cell cultures and mode of action were unknown. In principle, cell expansion could be inhibited by a decrease in either turgor pressure or wall extensibility. Cell expansion was estimated as settled cell volume; cell division was estimated by cell counting. Membrane permeability to water was measured by a novel method involving simultaneous assay of the efflux of (3)H(2)O and [(14)C]mannitol from a 'bed' of cultured cells. Osmotic potential was measured by depression of freezing point. OAC inhibited cell expansion in cultures of maize (Zea mays), spinach (Spinacia oleracea) and rose (Rosa sp.), with an ID(50) of 5, 30 and 250 nm, respectively. In maize cultures, OAC did not affect cell division for the first 40 h. It did not affect the osmotic potential of cell sap or culture medium, nor did it impede water transport across cell membranes. It did not affect cells' ability to acidify the apoplast (medium), which may be necessary for 'acid growth'. As OAC did not diminish turgor pressure, its ability to inhibit cell expansion must depend on changes in wall extensibility. It could be a valuable tool for studies on cell expansion.

  3. The conflict between cell proliferation and expansion primarily affects stem organogenesis in Arabidopsis.

    PubMed

    Maeda, Saori; Gunji, Shizuka; Hanai, Kenya; Hirano, Tomonari; Kazama, Yusuke; Ohbayashi, Iwai; Abe, Tomoko; Sawa, Shinichiro; Tsukaya, Hirokazu; Ferjani, Ali

    2014-11-01

    Plant shoot organs such as stems, leaves and flowers are derived from specialized groups of stem cells organized at the shoot apical meristem (SAM). Organogenesis involves two major processes, namely cell proliferation and differentiation, whereby the former contributes to increasing the cell number and the latter involves substantial increases in cell volume through cell expansion. Co-ordination between the above processes in time and space is essential for proper organogenesis. To identify regulatory factors involved in proper organogenesis, heavy-ion beam-irradiated de-etiolated (det) 3-1 seeds have been used to identify striking phenotypes in the A#26-2; det3-1 mutant. In addition to the stunted plant stature mimicking det3-1, the A#26-2; det3-1 mutant exhibited stem thickening, increased floral organ number and a fruit shape reminiscent of clavata (clv) mutants. DNA sequencing analysis demonstrated that A#26-2; det3-1 harbors a mutation in the CLV3 gene. Importantly, A#26-2; det3-1 displayed cracks that randomly occurred on the main stem with a frequency of approximately 50%. Furthermore, the double mutants clv3-8 det3-1, clv1-4 det3-1 and clv2-1 det3-1 consistently showed stem cracks with frequencies of approximately 97, 38 and 35%, respectively. Cross-sections of stems further revealed an increase in vascular bundle number, cell number and size in the pith of clv3-8 det3-1 compared with det3-1. These findings suggest that the stem inner volume increase due to clv mutations exerts an outward mechanical stress; that in a det3-1 background (defective in cell expansion) resulted in cracking of the outermost layer of epidermal cells.

  4. BOLITA, an Arabidopsis AP2/ERF-like transcription factor that affects cell expansion and proliferation/differentiation pathways.

    PubMed

    Marsch-Martinez, Nayelli; Greco, Raffaella; Becker, Jörg D; Dixit, Shital; Bergervoet, Jan H W; Karaba, Aarati; de Folter, Stefan; Pereira, Andy

    2006-12-01

    The BOLITA (BOL) gene, an AP2/ERF transcription factor, was characterized with the help of an activation tag mutant and overexpression lines in Arabidopsis and tobacco. The leaf size of plants overexpressing BOL was smaller than wild type plants due to a reduction in both cell size and cell number. Moreover, severe overexpressors showed ectopic callus formation in roots. Accordingly, global gene expression analysis using the overexpression mutant reflected the alterations in cell proliferation, differentiation and growth through expression changes in RBR, CYCD, and TCP genes, as well as genes involved in cell expansion (i.e. expansins and the actin remodeling factor ADF5). Furthermore, the expression of hormone signaling (i.e. auxin and cytokinin), biosynthesis (i.e. ethylene and jasmonic acid) and regulatory genes was found to be perturbed in bol-D mutant leaves. PMID:17096212

  5. Feline foamy virus adversely affects feline mesenchymal stem cell culture and expansion: implications for animal model development.

    PubMed

    Arzi, Boaz; Kol, Amir; Murphy, Brian; Walker, Naomi J; Wood, Joshua A; Clark, Kaitlin; Verstraete, Frank J M; Borjesson, Dori L

    2015-04-01

    Mesenchymal stem cells (MSCs) are a promising therapeutic option for various immune-mediated and inflammatory disorders due to their potent immunomodulatory and trophic properties. Naturally occurring diseases in large animal species may serve as surrogate animal models of human disease, as they may better reflect the complex genetic, environmental, and physiologic variation present in outbred populations. We work with naturally occurring diseases in large animal species to better understand how MSCs work and to facilitate optimal translation of MSC-based therapies. We are investigating the use of MSC therapy for a chronic oral inflammatory disease in cats. During our efforts to expand fat-derived feline MSCs (fMSCs), we observed that∼50% of the cell lines developed giant foamy multinucleated cells in later passages. These morphologic alterations were associated with proliferation arrest. We hypothesized that the cytopathic effects were caused by infection with a retrovirus, feline foamy virus (FFV). Using transmission electron microscopy, polymerase chain reaction, and in vitro assays, we determined that syncytial cell formation and proliferation arrest in fMSCs were caused by FFV strains that were highly homologous to previously reported FFV strains. We determined that the antiretroviral drug, tenofovir, may be used to support ex vivo expansion and salvage of FFV-infected fMSC lines. MSC lines derived from specific pathogen-free cats do not appear to be infected with FFV and may be a source of allogeneic fMSCs for clinical application. FFV infection of fMSC lines may hinder large-scale expansion of autologous MSC for therapeutic use in feline patients.

  6. The ROCK inhibitor Y-27632 negatively affects the expansion/survival of both fresh and cryopreserved cord blood-derived CD34+ hematopoietic progenitor cells: Y-27632 negatively affects the expansion/survival of CD34+HSPCs.

    PubMed

    Bueno, Clara; Montes, Rosa; Menendez, Pablo

    2010-06-01

    Cord blood (CB) is an unlimited source of hematopoietic stem and progenitor cells (HSPC). The use of cryopreserved CB-derived CD34+ HSPCs is successful in children and usually leads to rapid hematopoietic recovery upon transplantation. However, current methods for ex vivo expansion of HSPCs still result in a loss of multilineage differentiation potential and current freeze-thawing protocols result in significant cell death and loss of CD34+ HSPCs. The major cause for the loss of viability after slow freezing is apoptosis induced directly by cryoinjury. Very recent reports have demonstrated that Y-27632, a selective and robust ROCK inhibitor is a potent inhibitor of the apoptosis and is efficient in enhancing the post-thaw survival and recovery of different human stem cells including human embryos, hESCs, induced pluripotent stem cells and mesenchymal stem cells. Here, we analyzed the effect of such an inhibitor in CB-derived CD34+ HSPCs. CB-derived CD34+ HSPCs were MACS-isolated and treated with or without 10 microM of Y-27632. The effect of Y-27632 on culture homeostasis was determined in both fresh and cryopreserved CB-derived CD34+ HSPCs. Our results indicate that the Y-27632 not only dramatically inhibits cell expansion of both fresh and cryopreserved CD34+ HSPCs but also impairs survival/recovery of CD34+ HSPCs upon thawing regardless whether Y-27632 is added to both the cryopreservation and the expansion media and or just to the expansion culture medium with or without hematopoietic cytokines. This study identifies for the first time a detrimental effect of Y-27632 on the expansion and survival of both fresh and cryopreserved CB-derived CD34+ HSPCs, suggesting that Y-27632 may have a differential impact on distinct lineage/tissue-specific stem cells. Our data suggest different functions of Y-27632 on human stem cells growing in suspension versus those growing attached to either treated tissue culture plastic or extracellular matrix. We discourage any clinical

  7. Anisotropic Cell Expansion Is Affected through the Bidirectional Mobility of Cellulose Synthase Complexes and Phosphorylation at Two Critical Residues on CESA31[OPEN

    PubMed Central

    Liu, Yanmei; Bauer, Stefan

    2016-01-01

    Here we report that phosphorylation status of S211 and T212 of the CESA3 component of Arabidopsis (Arabidopsis thaliana) cellulose synthase impacts the regulation of anisotropic cell expansion as well as cellulose synthesis and deposition and microtubule-dependent bidirectional mobility of CESA complexes. Mutation of S211 to Ala caused a significant decrease in the length of etiolated hypocotyls and primary roots, while root hairs were not significantly affected. By contrast, the S211E mutation stunted the growth of root hairs, but primary roots were not significantly affected. Similarly, T212E caused a decrease in the length of root hairs but not root length. However, T212E stunted the growth of etiolated hypocotyls. Live-cell imaging of fluorescently labeled CESA showed that the rate of movement of CESA particles was directionally asymmetric in etiolated hypocotyls of S211A and T212E mutants, while similar bidirectional velocities were observed with the wild-type control and S211E and T212A mutant lines. Analysis of cell wall composition and the innermost layer of cell wall suggests a role for phosphorylation of CESA3 S211 and T212 in cellulose aggregation into fibrillar bundles. These results suggest that microtubule-guided bidirectional mobility of CESA complexes is fine-tuned by phosphorylation of CESA3 S211 and T212, which may, in turn, modulate cellulose synthesis and organization, resulting in or contributing to the observed defects of anisotropic cell expansion. PMID:26969722

  8. Coupling of solute transport and cell expansion in pea stems

    NASA Technical Reports Server (NTRS)

    Schmalstig, J. G.; Cosgrove, D. J.

    1990-01-01

    As cells expand and are displaced through the elongation zone of the epicotyl of etiolated pea (Pisum sativum L. var Alaska) seedlings, there is little net dilution of the cell sap, implying a coordination between cell expansion and solute uptake from the phloem. Using [14C] sucrose as a phloem tracer (applied to the hypogeous cotyledons), the pattern of label accumulation along the stem closely matched the growth rate pattern: high accumulation in the growing zone, little accumulation in nongrowing regions. Several results suggest that a major portion of phloem contents enters elongating cells through the symplast. We propose that the coordination between phloem transport and cell expansion is accomplished via regulatory pathways affecting both plasmodesmata conductivity and cell expansion.

  9. Ex vivo expansion of hematopoietic stem cells.

    PubMed

    Xie, JingJing; Zhang, ChengCheng

    2015-09-01

    Ex vivo expansion of hematopoietic stem cells (HSCs) would benefit clinical applications in several aspects, to improve patient survival, utilize cord blood stem cells for adult applications, and selectively propagate stem cell populations after genetic manipulation. In this review we summarize and discuss recent advances in the culture systems of mouse and human HSCs, which include stroma/HSC co-culture, continuous perfusion and fed-batch cultures, and those supplemented with extrinsic ligands, membrane transportable transcription factors, complement components, protein modification enzymes, metabolites, or small molecule chemicals. Some of the expansion systems have been tested in clinical trials. The optimal condition for ex vivo expansion of the primitive and functional human HSCs is still under development. An improved understanding of the mechanisms for HSC cell fate determination and the HSC culture characteristics will guide development of new strategies to overcome difficulties. In the future, development of a combination treatment regimen with agents that enhance self-renewal, block differentiation, and improve homing will be critical. Methods to enhance yields and lower cost during collection and processing should be employed. The employment of an efficient system for ex vivo expansion of HSCs will facilitate the further development of novel strategies for cell and gene therapies including genome editing.

  10. Medicaid Expansion Affects Rural And Urban Hospitals Differently.

    PubMed

    Kaufman, Brystana G; Reiter, Kristin L; Pink, George H; Holmes, George M

    2016-09-01

    Rural hospitals differ from urban hospitals in many ways. For example, rural hospitals are more reliant on public payers and have lower operating margins. In addition, enrollment in the health insurance Marketplaces of the Affordable Care Act (ACA) has varied across rural and urban areas. This study employed a difference-in-differences approach to evaluate the average effect of Medicaid expansion in 2014 on payer mix and profitability for urban and rural hospitals, controlling for secular trends. For both types of hospitals, we found that Medicaid expansion was associated with increases in Medicaid-covered discharges. However, the increases in Medicaid revenue were greater among rural hospitals than urban hospitals, and the decrease in the proportion of costs for uncompensated care were greater among urban hospitals than rural hospitals. This preliminary analysis of the early effects of Medicaid expansion suggests that its financial impacts may be different for hospitals in urban and rural locations. PMID:27605649

  11. Cell wall, cytoskeleton, and cell expansion in higher plants.

    PubMed

    Bashline, Logan; Lei, Lei; Li, Shundai; Gu, Ying

    2014-04-01

    To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.

  12. [Feedback control mechanisms of plant cell expansion

    SciTech Connect

    Cosgrove, D.J.

    1992-01-01

    We have generated considerable evidence for the significance of wall stress relaxation in the control of plant growth and found that several agents (gibberellin, light, genetic loci for dwarf stature) influence growth rate via alteration of wall relaxation. We have refined our methods for measuring wall relaxation and, moreover, have found that wall relaxation properties bear only a distance relationship to wall mechanical properties. We have garnered novel insights into the nature of cell expansion mechanisms by analyzing spontaneous fluctuations of plant growth rate in seedlings. These experiments involved the application of mathematical techniques for analyzing growth rate fluctuations and the development of new instrumentation for measuring and forcing plant growth in a controlled fashion. These studies conclude that growth rate fluctuations generated by the plant as consequence of a feedback control system. This conclusion has important implications for the nature of wall loosening processes and demands a different framework for thinking about growth control. It also implies the existence of a growth rate sensor.

  13. Fetal liver stromal cells promote hematopoietic cell expansion

    SciTech Connect

    Zhou, Kun; Hu, Caihong; Zhou, Zhigang; Huang, Lifang; Liu, Wenli; Sun, Hanying

    2009-09-25

    Future application of hematopoietic stem and progenitor cells (HSPCs) in clinical therapies largely depends on their successful expansion in vitro. Fetal liver (FL) is a unique hematopoietic organ in which hematopoietic cells markedly expand in number, but the mechanisms involved remain unclear. Stromal cells (StroCs) have been suggested to provide a suitable cellular environment for in vitro expansion of HSPCs. In this study, murine StroCs derived from FL at E14.5, with a high level of Sonic hedgehog (Shh) and Wnt expression, were found to have an increased ability to support the proliferation of HSPCs. This effect was inhibited by blocking Shh signaling. Supplementation with soluble Shh-N promoted the proliferation of hematopoietic cells by activating Wnt signaling. Our findings suggest that FL-derived StroCs support proliferation of HSPCs via Shh inducing an autocrine Wnt signaling loop. The use of FL-derived StroCs and regulation of the Shh pathway might further enhance HPSC expansion.

  14. LECT2 drives haematopoietic stem cell expansion and mobilization via regulating the macrophages and osteolineage cells

    PubMed Central

    Lu, Xin-Jiang; Chen, Qiang; Rong, Ye-Jing; Yang, Guan-Jun; Li, Chang-Hong; Xu, Ning-Yi; Yu, Chao-Hui; Wang, Hui-Ying; Zhang, Shun; Shi, Yu-Hong; Chen, Jiong

    2016-01-01

    Haematopoietic stem cells (HSCs) can differentiate into cells of all lineages in the blood. However, the mechanisms by which cytokines in the blood affect HSC homeostasis remain largely unknown. Here we show that leukocyte cell-derived chemotaxin 2 (LECT2), a multifunctional cytokine, induces HSC expansion and mobilization. Recombinant LECT2 administration results in HSC expansion in the bone marrow and mobilization to the blood via CD209a. The effect of LECT2 on HSCs is reduced after specific depletion of macrophages or reduction of osteolineage cells. LECT2 treatment reduces the tumour necrosis factor (TNF) expression in macrophages and osteolineage cells. In TNF knockout mice, the effect of LECT2 on HSCs is reduced. Moreover, LECT2 induces HSC mobilization in irradiated mice, while granulocyte colony-stimulating factor does not. Our results illustrate that LECT2 is an extramedullar cytokine that contributes to HSC homeostasis and may be useful to induce HSC mobilization. PMID:27596364

  15. LECT2 drives haematopoietic stem cell expansion and mobilization via regulating the macrophages and osteolineage cells.

    PubMed

    Lu, Xin-Jiang; Chen, Qiang; Rong, Ye-Jing; Yang, Guan-Jun; Li, Chang-Hong; Xu, Ning-Yi; Yu, Chao-Hui; Wang, Hui-Ying; Zhang, Shun; Shi, Yu-Hong; Chen, Jiong

    2016-01-01

    Haematopoietic stem cells (HSCs) can differentiate into cells of all lineages in the blood. However, the mechanisms by which cytokines in the blood affect HSC homeostasis remain largely unknown. Here we show that leukocyte cell-derived chemotaxin 2 (LECT2), a multifunctional cytokine, induces HSC expansion and mobilization. Recombinant LECT2 administration results in HSC expansion in the bone marrow and mobilization to the blood via CD209a. The effect of LECT2 on HSCs is reduced after specific depletion of macrophages or reduction of osteolineage cells. LECT2 treatment reduces the tumour necrosis factor (TNF) expression in macrophages and osteolineage cells. In TNF knockout mice, the effect of LECT2 on HSCs is reduced. Moreover, LECT2 induces HSC mobilization in irradiated mice, while granulocyte colony-stimulating factor does not. Our results illustrate that LECT2 is an extramedullar cytokine that contributes to HSC homeostasis and may be useful to induce HSC mobilization. PMID:27596364

  16. Phase cell cluster expansion for Euclidean field theories

    SciTech Connect

    Battle, G.A. III; Federbush, P.

    1982-08-01

    We adapt the cluster expansion first used to treat infrared problems for lattice models (a mass zero cluster expansion) to the usual field theory situation. The field is expanded in terms of spectral block spin functions and the cluster expansion given in terms of the expansion coefficients (phase cell variables); the other cluster expansion expresses correlation functions in terms of contributions from finite coupled subsets of these variables. Most of the present work is carried through in d space time dimensions (for phi/sup 4//sub 2/ the details of the cluster expansion are pursued and convergence is proven). Thus most of the results in this present work will apply to a treatment of phi/sup 4//sub 3/ to which we hope to return in a succeeding paper. Of particular interest in this paper is a substitute for the stability of the vaccum bound appropriate to this cluster expansion (for d = 2 and d = 3), and the new method for performing estimates with tree graphs. The phase cell cluster expansions have the renormalization group incorporated intimately into their structure. We hope they will be useful ultimately in treating four dimensional field theories.

  17. (Rapid regulatory control of plant cell expansion and wall relaxation)

    SciTech Connect

    Cosgrove, D.J.

    1990-01-01

    This section presents a brief overview of accomplishments related to this project in the past 3-year period. Our work has focused on the basic mechanisms of plant cell expansion, particularly on the interrelations of water and solute transport with cell wall relaxation and expansion. To study these processes, we have developed new methods and used these methods to analyze the dynamic behavior of growth processes and to examine how various agents (GA, drought, light, genetic lesions) alter the growth machinery of the cell.

  18. Mammary stem cells: expansion and animal productivity

    PubMed Central

    2014-01-01

    Identification and characterization of mammary stem cells and progenitor cells from dairy animals is important in the understanding of mammogenesis, tissue turnover, lactation persistency and regenerative therapy. It has been realized by many investigators that altered lactation, long dry periods (non-milking period between two consecutive lactation cycles), abrupt cessation of lactation (common in water buffaloes) and disease conditions like mastitis, greatly reduce milk yield thus render huge financial losses within the dairy sector. Cellular manipulation of specialized cell types within the mammary gland, called mammary stem cells (MaSCs)/progenitor cells, might provide potential solutions to these problems and may improve milk production. In addition, MaSCs/progenitor cells could be used in regenerative therapy against tissue damage caused by mastitis. This review discusses methods of MaSC/progenitor cell manipulation and their mechanisms in bovine and caprine animals. Author believes that intervention of MaSCs/progenitor cells could lessen the huge financial losses to the dairy industry globally. PMID:25057352

  19. Mammary stem cells: expansion and animal productivity.

    PubMed

    Choudhary, Ratan K

    2014-01-01

    Identification and characterization of mammary stem cells and progenitor cells from dairy animals is important in the understanding of mammogenesis, tissue turnover, lactation persistency and regenerative therapy. It has been realized by many investigators that altered lactation, long dry periods (non-milking period between two consecutive lactation cycles), abrupt cessation of lactation (common in water buffaloes) and disease conditions like mastitis, greatly reduce milk yield thus render huge financial losses within the dairy sector. Cellular manipulation of specialized cell types within the mammary gland, called mammary stem cells (MaSCs)/progenitor cells, might provide potential solutions to these problems and may improve milk production. In addition, MaSCs/progenitor cells could be used in regenerative therapy against tissue damage caused by mastitis. This review discusses methods of MaSC/progenitor cell manipulation and their mechanisms in bovine and caprine animals. Author believes that intervention of MaSCs/progenitor cells could lessen the huge financial losses to the dairy industry globally.

  20. How did medicaid expansions affect labor supply and welfare enrollment? Evidence from the early 2000s.

    PubMed

    Agirdas, Cagdas

    2016-12-01

    In the early 2000s, Arizona, Maine, New Mexico, New York, Oregon, and Vermont expanded Medicaid to cover more low-income individuals, primarily childless adults. This change provides the researcher with an opportunity to analyze the effects of these expansions on labor supply and welfare enrollment. I use a large data set of 176 counties over 7 years, including 3 years of pre-expansion period, 1 year of implementation year, and 3 years of post-expansion period. Using a difference-in-differences approach, I find the most-affected counties had a 1.4 percentage-point more decline in labor force participation rate in comparison to other counties. Furthermore, I observe a 0.32 h decrease in average weekly hours and a 1.1 % increase in average weekly wages. This indicates labor supply was affected more than labor demand. I also observe a 0.49 % increase in Supplemental Nutrition Assistance Program (SNAP) enrollment after the Medicaid expansions. These results are robust to an alternative identification of the most-affected counties, inclusion of counties from comparison states, limiting the control group to only high-poverty counties from comparison states, exclusion of county-specific time trends, and different configuration of clustered errors. My findings provide early insights on the potential effects of new Medicaid expansions of the Affordable Care Act (ACA), since 82 % of those newly eligible are expected to be childless adults.

  1. The cell biology of fat expansion

    PubMed Central

    Rutkowski, Joseph M.; Stern, Jennifer H.

    2015-01-01

    Adipose tissue is a complex, multicellular organ that profoundly influences the function of nearly all other organ systems through its diverse metabolite and adipokine secretome. Adipocytes are the primary cell type of adipose tissue and play a key role in maintaining energy homeostasis. The efficiency with which adipose tissue responds to whole-body energetic demands reflects the ability of adipocytes to adapt to an altered nutrient environment, and has profound systemic implications. Deciphering adipocyte cell biology is an important component of understanding how the aberrant physiology of expanding adipose tissue contributes to the metabolic dysregulation associated with obesity. PMID:25733711

  2. Testicular Niche Required for Human Spermatogonial Stem Cell Expansion

    PubMed Central

    Smith, James F.; Yango, Pamela; Altman, Eran; Choudhry, Shweta; Poelzl, Andrea; Zamah, Alberuni M.; Rosen, Mitchell; Klatsky, Peter C.

    2014-01-01

    Prepubertal boys treated with high-dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence-activated cell sorting using SSEA-4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA-4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys. PMID:25038247

  3. Methodology for Optimal In Vitro Cell Expansion in Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Melero-Martin, J. M.; Santhalingam, S.; Al-Rubeai, M.

    Expansion of the cell population in vitro has become an essential step in the process of tissue engineering and also the systematic optimization of culture conditions is now a fundamental problem that needs to be addressed. Herein, we provide a rational methodology for searching culture conditions that optimize the acquisition of large quantities of cells following a sequential expansion process. In particular, the analysis of both seeding density and passage length was consid ered crucial, and their correct selection should be taken as a requisite to establish culture conditions for monolayer systems. This methodology also introduces addi tional considerations concerning the running cost of the expansion process. The selection of culture conditions will be a compromise between optimal cell expan sion and acceptable running cost. This compromise will normally translate into an increase of passage length further away from the optimal value dictated by the growth kinetic of the cells. Finally, the importance of incorporating functional assays to validate the phenotypical and functional characteristics of the expanded cells has been highlighted. The optimization approach presented will contribute to the development of feasible large scale expansion of cells required by the tissue engineering industry.

  4. Stirred bioreactors for the expansion of adult pancreatic stem cells.

    PubMed

    Serra, Margarida; Brito, Catarina; Leite, Sofia B; Gorjup, Erwin; von Briesen, Hagen; Carrondo, Manuel J T; Alves, Paula M

    2009-01-01

    Adult pluripotent stem cells are a cellular resource representing unprecedented potential for cell therapy and tissue engineering. Complementary to this promise, there is a need for efficient bioprocesses for their large scale expansion and/or differentiation. With this goal in mind, our work focused on the development of three-dimensional (3-D) culture systems for controlled expansion of adult pancreatic stem cells (PSCs). For this purpose, two different culturing strategies were evaluated, using spinner vessels: cell aggregated cultures versus microcarrier technology. The use of microcarrier supports (Cytodex 1 and Cytodex 3) rendered expanded cell populations which retained their self-renewal ability, cell marker, and the potential to differentiate into adipocytes. This strategy surmounted the drawbacks of aggregates in culture which were demonstrably unfeasible as cells clumped together did not proliferate and lost PSC marker expression. Furthermore, the results obtained showed that although both microcarriers tested here were suitable for sustaining cell expansion, Cytodex 3 provided a better substrate for the promotion of cell adherence and growth. For the latter approach, the potential of bioreactor technology was combined with the efficient Cytodex 3 strategy under controlled environmental conditions (pH-7.2, pO2-30% and temperature-37 degrees C); cell growth was more efficient, as shown by faster doubling time, higher growth rate and higher fold increase in cell concentration, when compared to spinner cultures. This study describes a robust bioprocess for the controlled expansion of adult PSC, representing an efficient starting point for the development of novel technologies for cell therapy.

  5. Wall relaxation and the driving forces for cell expansive growth

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1987-01-01

    When water uptake by growing cells is prevented, the turgor pressure and the tensile stress in the cell wall are reduced by continued wall loosening. This process, termed in vivo stress relaxation, provides a new way to study the dynamics of wall loosening and to measure the wall yield threshold and the physiological wall extensibility. Stress relaxation experiments indicate that wall stress supplies the mechanical driving force for wall yielding. Cell expansion also requires water absorption. The driving force for water uptake during growth is created by wall relaxation, which lowers the water potential of the expanding cells. New techniques for measuring this driving force show that it is smaller than believed previously; in elongating stems it is only 0.3 to 0.5 bar. This means that the hydraulic resistance of the water transport pathway is small and that rate of cell expansion is controlled primarily by wall loosening and yielding.

  6. Genetic control of polar cell expansion in Arabidopsis thaliana

    SciTech Connect

    Schiefelbein, J.; Ford, S. ); Somerville, C. )

    1990-05-01

    Certain plant cells, like root hairs and pollen tubes, exhibit polar cell growth, with expansion limited to the tip of the growing cell. In order to understand the mechanisms regulating polar cell expansion, we are studying the process of root hair elongation in Arabidopsis thaliana. By visually screening roots from 12,000 mutagenized Arabidopsis seedlings on Petri dishes, more than 40 root hair mutants have been identified. We have focused our attention on mutants that possess nuclear recessive mutations in three genes (RHD2, RHD3, and RDH4) that appear to be involved in controlling polar cell growth in root hairs. We are currently using cellular, genetic, and molecular approaches to understand these genes' normal roles in root hair elongation.

  7. In Vitro Expansion of Corneal Endothelial Cells on Biomimetic Substrates

    PubMed Central

    Palchesko, Rachelle N.; Lathrop, Kira L.; Funderburgh, James L.; Feinberg, Adam W.

    2015-01-01

    Corneal endothelial (CE) cells do not divide in vivo, leading to edema, corneal clouding and vision loss when the density drops below a critical level. The endothelium can be replaced by transplanting allogeneic tissue; however, access to donated tissue is limited worldwide resulting in critical need for new sources of corneal grafts. In vitro expansion of CE cells is a potential solution, but is challenging due to limited proliferation and loss of phenotype in vitro via endothelial to mesenchymal transformation (EMT) and senescence. We hypothesized that a bioengineered substrate recapitulating chemo-mechanical properties of Descemet's membrane would improve the in vitro expansion of CE cells while maintaining phenotype. Results show that bovine CE cells cultured on a polydimethylsiloxane surface with elastic modulus of 50 kPa and collagen IV coating achieved >3000-fold expansion. Cells grew in higher-density monolayers with polygonal morphology and ZO-1 localization at cell-cell junctions in contrast to control cells on polystyrene that lost these phenotypic markers coupled with increased α-smooth muscle actin expression and fibronectin fibril assembly. In total, these results demonstrate that a biomimetic substrate presenting native basement membrane ECM proteins and mechanical environment may be a key element in bioengineering functional CE layers for potential therapeutic applications. PMID:25609008

  8. In vitro expansion of corneal endothelial cells on biomimetic substrates.

    PubMed

    Palchesko, Rachelle N; Lathrop, Kira L; Funderburgh, James L; Feinberg, Adam W

    2015-01-01

    Corneal endothelial (CE) cells do not divide in vivo, leading to edema, corneal clouding and vision loss when the density drops below a critical level. The endothelium can be replaced by transplanting allogeneic tissue; however, access to donated tissue is limited worldwide resulting in critical need for new sources of corneal grafts. In vitro expansion of CE cells is a potential solution, but is challenging due to limited proliferation and loss of phenotype in vitro via endothelial to mesenchymal transformation (EMT) and senescence. We hypothesized that a bioengineered substrate recapitulating chemo-mechanical properties of Descemet's membrane would improve the in vitro expansion of CE cells while maintaining phenotype. Results show that bovine CE cells cultured on a polydimethylsiloxane surface with elastic modulus of 50 kPa and collagen IV coating achieved >3000-fold expansion. Cells grew in higher-density monolayers with polygonal morphology and ZO-1 localization at cell-cell junctions in contrast to control cells on polystyrene that lost these phenotypic markers coupled with increased α-smooth muscle actin expression and fibronectin fibril assembly. In total, these results demonstrate that a biomimetic substrate presenting native basement membrane ECM proteins and mechanical environment may be a key element in bioengineering functional CE layers for potential therapeutic applications. PMID:25609008

  9. Expansion of human cord blood hematopoietic stem cells for transplantation.

    PubMed

    Chou, Song; Chu, Pat; Hwang, William; Lodish, Harvey

    2010-10-01

    A recent Science paper reported a purine derivative that expands human cord blood hematopoietic stem cells in culture (Boitano et al., 2010) by antagonizing the aryl hydrocarbon receptor. Major problems need to be overcome before ex vivo HSC expansion can be used clinically.

  10. Integrating human stem cell expansion and neuronal differentiation in bioreactors

    PubMed Central

    Serra, Margarida; Brito, Catarina; Costa, Eunice M; Sousa, Marcos FQ; Alves, Paula M

    2009-01-01

    Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line) as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors. PMID:19772662

  11. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

    SciTech Connect

    Wei, Yan; Li, Yuan; Chen, Chao; Stoelzel, Katharina; Kaufmann, Andreas M.

    2011-04-15

    Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined using reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.

  12. Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor.

    PubMed

    Tsai, Ang-Chen; Ma, Teng

    2016-01-01

    Human mesenchymal stem cells (hMSCs) are considered as a primary candidate in cell therapy owing to their self-renewability, high differentiation capabilities, and secretions of trophic factors. In clinical application, a large quantity of therapeutically competent hMSCs is required that cannot be produced in conventional petri dish culture. Bioreactors are scalable and have the capacity to meet the production demand. Microcarrier suspension culture in stirred-tank bioreactors is the most widely used method to expand anchorage dependent cells in a large scale. Stirred-tank bioreactors have the potential to scale up and microcarriers provide the high surface-volume ratio. As a result, a spinner flask bioreactor with microcarriers has been commonly used in large scale expansion of adherent cells. This chapter describes a detailed culture protocol for hMSC expansion in a 125 mL spinner flask using microcarriers, Cytodex I, and a procedure for cell seeding, expansion, metabolic sampling, and quantification and visualization using microculture tetrazolium (MTT) reagent. PMID:27032950

  13. Expansion of Multipotent Stem Cells from the Adult Human Brain

    PubMed Central

    Murrell, Wayne; Palmero, Emily; Bianco, John; Stangeland, Biljana; Joel, Mrinal; Paulson, Linda; Thiede, Bernd; Grieg, Zanina; Ramsnes, Ingunn; Skjellegrind, Håvard K.; Nygård, Ståle; Brandal, Petter; Sandberg, Cecilie; Vik-Mo, Einar; Palmero, Sheryl; Langmoen, Iver A.

    2013-01-01

    The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells. PMID:23967194

  14. Expansion of beta-cell mass in response to pregnancy.

    PubMed

    Rieck, Sebastian; Kaestner, Klaus H

    2010-03-01

    Inadequate beta-cell mass can lead to insulin insufficiency and diabetes. During times of prolonged metabolic demand for insulin, the endocrine pancreas can respond by increasing beta-cell mass, both by increasing cell size and by changing the balance between beta-cell proliferation and apoptosis. In this paper, we review recent advances in our understanding of the mechanisms that control the adaptive expansion of beta-cell mass, focusing on the islet's response to pregnancy, a physiological state of insulin resistance. Functional characterization of factors controlling both beta-cell proliferation and survival might not only lead to the development of successful therapeutic strategies to enhance the response of the beta-cell to increased metabolic loads, but also improve islet transplantation regimens.

  15. Differential response of cell-cycle and cell-expansion regulators to heat stress in apple (Malus domestica) fruitlets.

    PubMed

    Flaishman, Moshe A; Peles, Yuval; Dahan, Yardena; Milo-Cochavi, Shira; Frieman, Aviad; Naor, Amos

    2015-04-01

    Temperature is one of the most significant factors affecting physiological and biochemical aspects of fruit development. Current and progressing global warming is expected to change climate in the traditional deciduous fruit tree cultivation regions. In this study, 'Golden Delicious' trees, grown in a controlled environment or commercial orchard, were exposed to different periods of heat treatment. Early fruitlet development was documented by evaluating cell number, cell size and fruit diameter for 5-70 days after full bloom. Normal activities of molecular developmental and growth processes in apple fruitlets were disrupted under daytime air temperatures of 29°C and higher as a result of significant temporary declines in cell-production and cell-expansion rates, respectively. Expression screening of selected cell cycle and cell expansion genes revealed the influence of high temperature on genetic regulation of apple fruitlet development. Several core cell-cycle and cell-expansion genes were differentially expressed under high temperatures. While expression levels of B-type cyclin-dependent kinases and A- and B-type cyclins declined moderately in response to elevated temperatures, expression of several cell-cycle inhibitors, such as Mdwee1, Mdrbr and Mdkrps was sharply enhanced as the temperature rose, blocking the cell-cycle cascade at the G1/S and G2/M transition points. Moreover, expression of several expansin genes was associated with high temperatures, making them potentially useful as molecular platforms to enhance cell-expansion processes under high-temperature regimes. Understanding the molecular mechanisms of heat tolerance associated with genes controlling cell cycle and cell expansion may lead to the development of novel strategies for improving apple fruit productivity under global warming.

  16. Bioreactor design for clinical-grade expansion of stem cells.

    PubMed

    dos Santos, Francisco F; Andrade, Pedro Z; da Silva, Cláudia Lobato; Cabral, Joaquim M S

    2013-06-01

    The many clinical trials currently in progress will likely lead to the widespread use of stem cell-based therapies for an extensive variety of diseases, either in autologous or allogeneic settings. With the current pace of progress, in a few years' time, the field of stem cell-based therapy should be able to respond to the market demand for safe, robust and clinically efficient stem cell-based therapeutics. Due to the limited number of stem cells that can be obtained from a single donor, one of the major challenges on the roadmap for regulatory approval of such medicinal products is the expansion of stem cells using Good Manufacturing Practices (GMP)-compliant culture systems. In fact, manufacturing costs, which include production and quality control procedures, may be the main hurdle for developing cost-effective stem cell therapies. Bioreactors provide a viable alternative to the traditional static culture systems in that bioreactors provide the required scalability, incorporate monitoring and control tools, and possess the operational flexibility to be adapted to the differing requirements imposed by various clinical applications. Bioreactor systems face a number of issues when incorporated into stem cell expansion protocols, both during development at the research level and when bioreactors are used in on-going clinical trials. This review provides an overview of the issues that must be confronted during the development of GMP-compliant bioreactors systems used to support the various clinical applications employing stem cells. PMID:23625834

  17. Selection and expansion of natural killer cells for NK cell-based immunotherapy.

    PubMed

    Becker, Petra S A; Suck, Garnet; Nowakowska, Paulina; Ullrich, Evelyn; Seifried, Erhard; Bader, Peter; Tonn, Torsten; Seidl, Christian

    2016-04-01

    Natural killer (NK) cells have been used in several clinical trials as adaptive immunotherapy. The low numbers of these cells in peripheral blood mononuclear cells (PBMC) have resulted in various approaches to preferentially expand primary NK cells from PBMC. While some clinical trials have used the addition of interleukin 2 (IL-2) to co-stimulate the expansion of purified NK cells from allogeneic donors, recent studies have shown promising results in achieving in vitro expansion of NK cells to large numbers for adoptive immunotherapy. NK cell expansion requires multiple cell signals for survival, proliferation and activation. Thus, expansion strategies have been focused either to substitute these factors using autologous feeder cells or to use genetically modified allogeneic feeder cells. Recent developments in the clinical use of genetically modified NK cell lines with chimeric antigen receptors, the development of expansion protocols for the clinical use of NK cell from human embryonic stem cells and induced pluripotent stem cells are challenging improvements for NK cell-based immunotherapy. Transfer of several of these protocols to clinical-grade production of NK cells necessitates adaptation of good manufacturing practice conditions, and the development of freezing conditions to establish NK cell stocks will require some effort and, however, should enhance the therapeutic options of NK cells in clinical medicine.

  18. Expansion of Regulatory T Cells in Patients with Langerhans Cell Histiocytosis

    PubMed Central

    Senechal, Brigitte; Elain, Gaelle; Jeziorski, Eric; Grondin, Virginie; Patey-Mariaud de Serre, Natacha; Jaubert, Francis; Beldjord, Kheira; Lellouch, Arielle; Glorion, Christophe; Zerah, Michel; Mary, Pierre; Barkaoui, Mohammed; Emile, Jean Francois; Boccon-Gibod, Liliane; Josset, Patrice; Debré, Marianne; Fischer, Alain; Donadieu, Jean; Geissmann, Frederic

    2007-01-01

    Background Langerhans cell histiocytosis (LCH) is a rare clonal granulomatous disease that affects mainly children. LCH can involve various tissues such as bone, skin, lung, bone marrow, lymph nodes, and the central nervous system, and is frequently responsible for functional sequelae. The pathophysiology of LCH is unclear, but the uncontrolled proliferation of Langerhans cells (LCs) is believed to be the primary event in the formation of granulomas. The present study was designed to further investigate the nature of proliferating cells and the immune mechanisms involved in the LCH granulomas. Methods and Findings Biopsies (n = 24) and/or blood samples (n = 25) from 40 patients aged 0.25 to 13 y (mean 7.8 y), were studied to identify cells that proliferate in blood and granulomas. We found that the proliferating index of LCs was low (∼1.9%), and we did not observe expansion of a monocyte or dendritic cell compartment in patients. We found that LCH lesions were a site of active inflammation, tissue remodeling, and neo-angiogenesis, and the majority of proliferating cells were endothelial cells, fibroblasts, and polyclonal T lymphocytes. Within granulomas, interleukin 10 was abundant, LCs expressed the TNF receptor family member RANK, and CD4+ CD25high FoxP3high regulatory T cells (T-regs) represented 20% of T cells, and were found in close contact with LCs. FoxP3+ T-regs were also expanded compared to controls, in the blood of LCH patients with active disease, among whom seven out of seven tested exhibited an impaired skin delayed-type hypersensitivity response. In contrast, the number of blood T-regs were normal after remission of LCH. Conclusions These findings indicate that LC accumulation in LCH results from survival rather than uncontrolled proliferation, and is associated with the expansion of T-regs. These data suggest that LCs may be involved in the expansion of T-regs in vivo, resulting in the failure of the host immune system to eliminate LCH cells. Thus

  19. Genetic Code Expansion of Mammalian Cells with Unnatural Amino Acids.

    PubMed

    Brown, Kalyn A; Deiters, Alexander

    2015-09-01

    The expansion of the genetic code of mammalian cells enables the incorporation of unnatural amino acids into proteins. This is achieved by adding components to the protein biosynthetic machinery, specifically an engineered aminoacyl-tRNA synthetase/tRNA pair. The unnatural amino acids are chemically synthesized and supplemented to the growth medium. Using this methodology, fundamental new chemistries can be added to the functional repertoire of the genetic code of mammalian cells. This protocol outlines the steps necessary to incorporate a photocaged lysine into proteins and showcases its application in the optical triggering of protein translocation to the nucleus.

  20. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2014-04-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2-4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2-4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:24717634

  1. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

    PubMed Central

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2014-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone’s expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:24717634

  2. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

    PubMed Central

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2015-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:26042727

  3. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2015-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2-4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2-4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:26042727

  4. Pectate chemistry links cell expansion to wall deposition in Chara corallina.

    PubMed

    Proseus, Timothy E; Boyer, John S

    2012-11-01

    Pectate (polygalacturonic acid) acts as a chelator to bind calcium and form cross-links that hold adjacent pectate polymers and thus plant cell walls together. When under tension from turgor pressure in the cell, the cross-links appear to distort and weaken. New pectate supplied by the cytoplasm is undistorted and removes wall calcium preferentially from the weakened bonds, loosening the wall and accelerating cell expansion. The new pectate now containing the removed calcium can bind to the wall, strengthening it and linking expansion to wall deposition. But new calcium needs to be added as well to replenish the calcium lost from the vacated wall pectate.  A recent report demonstrated that growth was disrupted if new calcium was unavailable.  The present addendum highlights this conclusion by reviewing an experiment from before the chelation chemistry was understood. Using cell wall labeling, a direct link appeared between wall expansion and wall deposition. Together, these experiments support the concept that newly supplied pectate has growth activity on its way to deposition in the wall. Growth rate is thus controlled by signals affecting the rate of pectate release. After release, the coordination of expansion and deposition arises naturally from chelation chemistry when polymers are under tension from turgor pressure. 

  5. CD101 inhibits the expansion of colitogenic T cells

    PubMed Central

    Schey, Regina; Dornhoff, Heike; Baier, Julia L.C.; Purtak, Martin; Opoka, Robert; Koller, Anna Katharina; Atreya, Raya; Rau, Tilman T.; Daniel, Christoph; Amann, Kerstin; Bogdan, Christian; Mattner, Jochen

    2015-01-01

    CD101 exerts negative-costimulatory effects in vitro, but its function in vivo remains poorly defined. CD101 is abundantly expressed on lymphoid and myeloid cells in intestinal tissues, but absent from naïve splenic T cells. Here, we assessed the impact of CD101 on the course of inflammatory bowel disease (IBD). Using a T cell transfer model of chronic colitis, we found that in recipients of naïve T cells from CD101+/+ donors up to 30% of the recovered lymphocytes expressed CD101, correlating with an increased IL-2-mediated FoxP3-expression. Transfer of CD101−/− T cells caused more severe colitis and was associated with an expansion of IL-17-producing T cells and an enhanced expression of IL-2Rα/β independently of FoxP3. The co-transfer of naïve and regulatory T cells (Treg) protected most effectively from colitis, when both donor and recipient mice expressed CD101. While the expression of CD101 on T cells was sufficient for Treg-function and the inhibition of T cell proliferation, sustained IL-10-production required additional CD101-expression by myeloid cells. Finally, in patients with IBD a reduced CD101-expression on peripheral and intestinal monocytes and CD4+ T cells correlated with enhanced IL-17-production and disease activity. Thus, CD101-deficiency is a novel marker for progressive colitis and potential target for therapeutic intervention. PMID:26813346

  6. Alginate Microfiber System for Expansion and Direct Differentiation of Human Embryonic Stem Cells.

    PubMed

    Leong, Meng Fatt; Lu, Hong Fang; Lim, Tze Chiun; Narayanan, Karthikeyan; Gao, Shujun; Wang, Luna Yue; Toh, Rebecca P K; Funke, Henrik; Abdul Samad, Muhammad Hamizan; Wan, Andrew C A; Ying, Jackie Y

    2016-09-01

    Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications, there is a need to develop scalable cell culture platforms to propagate hESCs. In this study, we encapsulated hESCs in calcium alginate microfibers as single cells, for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (>10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM), which did not affect hESCs viability, was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical, as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line, HUES7, for up to five passages while maintaining pluripotency. Immunohistochemistry, polymerase chain reaction, and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells, possessed normal karyotype, and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively, our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions. PMID:27484909

  7. Morphogenesis of the Fission Yeast Cell through Cell Wall Expansion.

    PubMed

    Atilgan, Erdinc; Magidson, Valentin; Khodjakov, Alexey; Chang, Fred

    2015-08-17

    The shape of walled cells such as fungi, bacteria, and plants are determined by the cell wall. Models for cell morphogenesis postulate that the effects of turgor pressure and mechanical properties of the cell wall can explain the shapes of these diverse cell types. However, in general, these models await validation through quantitative experiments. Fission yeast Schizosaccharomyces pombe are rod-shaped cells that grow by tip extension and then divide medially through formation of a cell wall septum. Upon cell separation after cytokinesis, the new cell ends adopt a rounded morphology. Here, we show that this shape is generated by a very simple mechanical-based mechanism in which turgor pressure inflates the elastic cell wall in the absence of cell growth. This process is independent of actin and new cell wall synthesis. To model this morphological change, we first estimate the mechanical properties of the cell wall using several approaches. The lateral cell wall behaves as an isotropic elastic material with a Young's modulus of 50 ± 10 MPa inflated by a turgor pressure estimated to be 1.5 ± 0.2 MPa. Based upon these parameters, we develop a quantitative mechanical-based model for new end formation that reveals that the cell wall at the new end expands into its characteristic rounded shape in part because it is softer than the mature lateral wall. These studies provide a simple example of how turgor pressure expands the elastic cell wall to generate a particular cell shape.

  8. Variables that affect the expansion rate and outcome of small abdominal aortic aneurysms.

    PubMed

    Cronenwett, J L; Sargent, S K; Wall, M H; Hawkes, M L; Freeman, D H; Dain, B J; Curé, J K; Walsh, D B; Zwolak, R M; McDaniel, M D

    1990-02-01

    Seventy-three patients with small (less than 6 cm in diameter) abdominal aortic aneurysms (AAAs) were selected for nonoperative management and followed up with sequential ultrasound size measurements. Fifty-four men and 19 women, 51 to 89 years of age (mean 70 years), had an initial mean AAA size of 4.1 cm (anteroposterior) x 4.3 cm (lateral) diameter, with a calculated elliptic cross-sectional area of 14.3 cm2. After a mean of 37 months of follow-up, AAA area increased at a mean rate of 20% per year (3 cm2 yr; 0.4 to 0.5 cm/yr diameter). Expansion rate was not affected by initial aneurysm size. During follow-up, only 3 patients (4%) required urgent operation (1 died), 26 patients (36%) died of non-AAA causes, and 26 patients (36%) underwent elective AAA repair because of progressive size increase (1 died). Elective operations were performed at the rate of 10% per year, when mean AAA size had increased to 22 cm2 (5.1 cm in diameter). Multiple regression analysis of clinical parameters available at presentation indicated that subsequent elective AAA repair was predicted by younger age at diagnosis and larger initial aneurysm size. As anticipated, patients who underwent surgery had more rapid aneurysm expansion (5.3 cm2/yr) compared with patients who did not undergo surgery (1.6 cm2/yr; p less than 0.05). This difference was caused by more rapid expansion during later follow-up intervals among patients selected for operation and was not predicted by the change in aneurysm size observed during initial ultrasonographic follow-up. Final aneurysm size was predicted by initial size, duration of follow-up, and both systolic and diastolic pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Rapid regulatory control of plant cell expansion and wall relaxation

    SciTech Connect

    Cosgrove, D.J.

    1991-08-14

    The aim of this project is to elucidate the biophysical and cellular mechanisms that control plant cell expansion. At present we are attempting to characterize the kinetics of the system(s) responsible for regulatory and compensatory behavior of growing cells and tissues. This work is significantly because it indicates that biochemical loosening and biophysical stress relaxation of the wall are part of a feedback loop controlling growth. This report briefly summarizes the efforts and results of the past 12 months. In large part, we have been trying to analyze the nature of growth rate noise,'' i.e. spontaneous and often erratic variations in growth rate. We are obtaining evidence that such noise'' is not random, but rather reveals an underlying growth mechanism with complex dynamics.

  10. Argonaute2 Mediates Compensatory Expansion of the Pancreatic β Cell

    PubMed Central

    Tattikota, Sudhir G.; Rathjen, Thomas; McAnulty, Sarah J.; Wessels, Hans-Hermann; Akerman, Ildem; van de Bunt, Martijn; Hausser, Jean; Esguerra, Jonathan L.S.; Musahl, Anne; Pandey, Amit K.; You, Xintian; Chen, Wei; Herrera, Pedro L.; Johnson, Paul R.; O’Carroll, Donal; Eliasson, Lena; Zavolan, Mihaela; Gloyn, Anna L.; Ferrer, Jorge; Shalom-Feuerstein, Ruby; Aberdam, Daniel; Poy, Matthew N.

    2014-01-01

    Summary Pancreatic β cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in β cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory β cell expansion. Loss of Ago2 during insulin resistance blocked β cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and β cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity. PMID:24361012

  11. IGFBP2 promotes glioma tumor stem cell expansion and survival

    SciTech Connect

    Hsieh, David; Hsieh, Antony; Stea, Baldassarre; Ellsworth, Ron

    2010-06-25

    IGFBP2 is overexpressed in the most common brain tumor, glioblastoma (GBM), and its expression is inversely correlated to GBM patient survival. Previous reports have demonstrated a role for IGFBP2 in glioma cell invasion and astrocytoma development. However, the function of IGFBP2 in the restricted, self-renewing, and tumorigenic GBM cell population comprised of tumor-initiating stem cells has yet to be determined. Herein we demonstrate that IGFBP2 is overexpressed within the stem cell compartment of GBMs and is integral for the clonal expansion and proliferative properties of glioma stem cells (GSCs). In addition, IGFBP2 inhibition reduced Akt-dependent GSC genotoxic and drug resistance. These results suggest that IGFBP2 is a selective malignant factor that may contribute significantly to GBM pathogenesis by enriching for GSCs and mediating their survival. Given the current dearth of selective molecular targets against GSCs, we anticipate our results to be of high therapeutic relevance in combating the rapid and lethal course of GBM.

  12. Phloem unloading and cell expansion in pea stems

    SciTech Connect

    Schmalstig, J.G.; Cosgrove, D.J. )

    1989-04-01

    Phloem unloading into elongating stems of dark-grown pea seedlings was greater in regions with higher relative growth rates. Phloem transport was monitored over 1 h by measuring accumulation of radiolabel from {sup 14}C-sucrose added between the cotyledons. The apical hook and plumule and 8 mm of the growing region of an intact plant were sealed in a pressure chamber and the pressure was raised to stop elongation. Phloem unloading was inhibited in the pressurized zone of elongation and accelerated in the apical hook and plumule, with the result that the magnitude of phloem transport into the stem was unchanged. The results demonstrate a coupling between cell expansion and phloem unloading.

  13. Importance of the GDF9 signaling pathway on cumulus cell expansion and oocyte competency in sheep.

    PubMed

    Varnosfaderani, Sh Rouhollahi; Ostadhosseini, S; Hajian, M; Hosseini, S M; Khashouei, E Asadi; Abbasi, H; Hosseinnia, P; Nasr-Esfahani, M H

    2013-09-15

    Acquisition of developmental competency in cumulus oocyte complexes (COCs) is derived from endocrine hormones and oocyte secreted factors. The contribution of these factors in oocyte maturation and development is an active area of research. The objective of this research was to investigate whether growth differentiation factor 9 (GDF9) that is secreted by oocyte affects cumulus expansion and oocyte development in sheep. Immature ovine COCs were cultured in the presence of recombinant human GDF9 (rhGDF9), denuded oocytes, SB-431542, a specific inhibitor of activin-like kinase 4/5/7; or a combination of these factors. Routine in vitro maturation of COCs and denuded oocytes were used as external control samples. Cultured COCs were used for assessment of (1) cumulus expansion; (2) expression of cumulus-related transcripts including pentraxin 3, hyaluronan synthase 2 (HAS2), tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, B-cell lymphoma 2 (BCL2), and Bcl2-associated X (BAX); and (3) yield and quality of embryo development. It was observed that cumulus expansion was not affected by any of these treatments. HAS2 mRNA expression confirmed this observation. In the presence of exogenous GDF9, cleavage rate was reduced, blastocyst rate did not differ from other groups, and trophectoderm cell number significantly increased. This suggests that exogenous GDF9 could improve embryo quality. It was also observed that oocyte secreted factors reduced proapoptotic BAX mRNA, and BCL2 mRNA expression was not significantly different from other groups. This study provides evidence that GDF9 signaling might have a minor influence on ovine cumulus expansion and oocyte development and that other signaling pathway(s) might have a dominant role.

  14. Using the quantum cell expansion system for the automated expansion of clinical-grade bone marrow-derived human mesenchymal stromal cells.

    PubMed

    Martin-Manso, Gema; Hanley, Patrick J

    2015-01-01

    Bone marrow-derived human mesenchymal stromal cells (hMSCs) constitute a promising therapeutic approach. However, the extremely low frequency of hMSCs in bone marrow makes the translation of these regulatory cells to clinical therapies difficult for large patient populations. Here, we describe a good manufacturing practices-compliant procedure for the expansion of hMSCs using the Quantum Cell Expansion System. This closed and automated system allows the large-scale expansion of hMSCs while maintaining their multipotency, immunophenotype, morphology, and karyotype. PMID:25523809

  15. Three-dimensional patterns of cell division and expansion throughout the development of Arabidopsis thaliana leaves.

    PubMed

    Kalve, Shweta; Fotschki, Joanna; Beeckman, Tom; Vissenberg, Kris; Beemster, Gerrit T S

    2014-12-01

    Variations in size and shape of multicellular organs depend on spatio-temporal regulation of cell division and expansion. Here, cell division and expansion rates were quantified relative to the three spatial axes in the first leaf pair of Arabidopsis thaliana. The results show striking differences in expansion rates: the expansion rate in the petiole is higher than in the leaf blade; expansion rates in the lateral direction are higher than longitudinal rates between 5 and 10 days after stratification, but become equal at later stages of leaf blade development; and anticlinal expansion co-occurs with, but is an order of magnitude slower than periclinal expansion. Anticlinal expansion rates also differed greatly between tissues: the highest rates occurred in the spongy mesophyll and the lowest in the epidermis. Cell division rates were higher and continued for longer in the epidermis compared with the palisade mesophyll, causing a larger increase of palisade than epidermal cell area over the course of leaf development. The cellular dynamics underlying the effect of shading on petiole length and leaf thickness were then investigated. Low light reduced leaf expansion rates, which was partly compensated by increased duration of the growth phase. Inversely, shading enhanced expansion rates in the petiole, so that the blade to petiole ratio was reduced by 50%. Low light reduced leaf thickness by inhibiting anticlinal cell expansion rates. This effect on cell expansion was preceded by an effect on cell division, leading to one less layer of palisade cells. The two effects could be uncoupled by shifting plants to contrasting light conditions immediately after germination. This extended kinematic analysis maps the spatial and temporal heterogeneity of cell division and expansion, providing a framework for further research to understand the molecular regulatory mechanisms involved.

  16. Fetal hepatic progenitors support long-term expansion of hematopoietic stem cells.

    PubMed

    Chou, Song; Flygare, Johan; Lodish, Harvey F

    2013-05-01

    We have developed a coculture system that establishes DLK(+) fetal hepatic progenitors as the authentic supportive cells for expansion of hematopoietic stem (HSCs) and progenitor cells. In 1-week cultures supplemented with serum and supportive cytokines, both cocultured DLK(+) fetal hepatic progenitors and their conditioned medium supported rapid expansion of hematopoietic progenitors and a small increase in HSC numbers. In 2- and 3-week cultures DLK(+) cells, but not their conditioned medium, continuously and significantly (>20-fold) expanded both hematopoietic stem and progenitor cells. Physical contact between HSCs and DLK(+) cells was crucial to maintaining this long-term expansion. Similar HSC expansion (approximately sevenfold) was achieved in cocultures using a serum-free, low cytokine- containing medium. In contrast, DLK(-) cells are incapable of expanding hematopoietic cells, demonstrating that hepatic progenitors are the principle supportive cells for HSC expansion in the fetal liver.

  17. Sex-dependent mechanisms for expansions and contractions of the CAG repeat on affected Huntington disease chromosomes

    SciTech Connect

    Kremer, B.; Theilmann, J.; Spence, N.

    1995-08-01

    A total of 254 affected parent-child pairs with Huntington disease (HD) and 440 parent-child pairs with CAG size in the normal range were assessed to determine the nature and frequency of intergenerational CAG changes in the HD gene. Intergenerational CAG changes are extremely rare (3/440 [0.68%]) on normal chromosomes. In contrast, on HD chromosomes, changes in CAG size occur in {approximately}70% of meioses on HD chromosomes, with expansions accounting for 73% of these changes. These intergenerational CAG changes make a significant but minor contribution to changes in age at onset (r{sup 2}=.19). The size of the CAG repeat influenced larger intergenerational expansions (>7 CAG repeats), but the likelihood of smaller expansions or contractions was not influenced by CAG size. Large expansions (>7 CAG repeats) occur almost exclusively through paternal transmission (0.96%; P<10{sub -7}), while offspring of affected mothers are more likely to show no change (P=.01) or contractions in CAG size (P=.002). This study demonstrates that sex of the transmitting parent is the major determinant for CAG intergenerational changes in the HD gene. Similar paternal sex effects are seen in the evolution of new mutations for HD from intermediate alleles and for large expansions on affected chromosomes. Affected mothers almost never transmit a significantly expanded CAG repeat, despite the fact that many have similar large-sized alleles, compared with affected fathers. The sex-dependent effects of major expansion and contractions of the CAG repeat in the HD gene implicate different effects of gametogenesis, in males versus females, on intergenerational CAG repeat stability. 22 refs., 4 figs., 3 tabs.

  18. In vitro expansion of human cardiac progenitor cells: exploring 'omics tools for characterization of cell-based allogeneic products.

    PubMed

    Gomes-Alves, P; Serra, M; Brito, C; Ricardo, C P; Cunha, R; Sousa, M F; Sanchez, B; Bernad, A; Carrondo, M J T; Rodriguez-Borlado, L; Alves, P M

    2016-05-01

    Human cardiac stem/progenitor cells (hCPCs) have been shown to be capable to regenerate contractile myocardium. However, because of their relative low abundance in the heart, in vitro expansion of hCPC is mandatory to achieve necessary quantities for allogeneic or autologous cardiac regeneration therapy applications (10(6)-10(9) cells/patient). Up to now, cell number requirements of ongoing phase I/IIa trials have been fulfilled with production in static monolayer cultures. However, this manufacturing process poses critical limitations when moving to the following clinical phases where hundreds of patients will be enrolled. For this, increased process yield is required, while guaranteeing the quality of the cell-based products. In this work, we developed and validated a robust, scalable, and good manufacturing practice (GMP)-compatible bioprocess for the expansion of high-quality hCPC. We applied platforms extensively used by the biopharmaceutical industry, such as microcarrier technology and stirred systems, and assessed culture conditions' impact on hCPC's quality and potency, as required by regulatory agencies. Complementary analytical assays including gene expression microarrays and mass spectrometry-based approaches were explored to compare transcriptome, proteome, surface markers, and secretion profiles of hCPC cultured in static monolayers and in stirred microcarrier-based systems. Our results show that stirred microcarrier-based culture systems enabled achieving more than 3-fold increase in hCPC expansion, when compared with traditional static monolayers, while retaining cell's phenotype and similar "omics" profiles. These findings demonstrate that this change in the production process does not affect cell's identity and quality, with potential to be translated into a transversal production platform for clinical development of stem-cell therapies. PMID:26924043

  19. The endoplasmic reticulum exerts control over organelle streaming during cell expansion.

    PubMed

    Stefano, Giovanni; Renna, Luciana; Brandizzi, Federica

    2014-03-01

    Cytoplasmic streaming is crucial for cell homeostasis and expansion but the precise driving forces are largely unknown. In plants, partial loss of cytoplasmic streaming due to chemical and genetic ablation of myosins supports the existence of yet-unknown motors for organelle movement. Here we tested a role of the endoplasmic reticulum (ER) as propelling force for cytoplasmic streaming during cell expansion. Through quantitative live-cell analyses in wild-type Arabidopsis thaliana cells and mutants with compromised ER structure and streaming, we demonstrate that cytoplasmic streaming undergoes profound changes during cell expansion and that it depends on motor forces co-exerted by the ER and the cytoskeleton.

  20. Short-term expansion of breast circulating cancer cells predicts response to anti-cancer therapy.

    PubMed

    Khoo, Bee Luan; Lee, Soo Chin; Kumar, Prashant; Tan, Tuan Zea; Warkiani, Majid Ebrahimi; Ow, Samuel G W; Nandi, Sayantani; Lim, Chwee Teck; Thiery, Jean Paul

    2015-06-20

    Circulating tumor cells (CTCs) are considered as surrogate markers for prognosticating and evaluating patient treatment responses. Here, 226 blood samples from 92 patients with breast cancer, including patients with newly diagnosed or metastatic refractory cancer, and 16 blood samples from healthy subjects were cultured in laser-ablated microwells. Clusters containing an increasing number of cytokeratin-positive (CK+) cells appeared after 2 weeks, while most blood cells disappeared with time. Cultures were heterogeneous and exhibited two distinct sub-populations of cells: 'Small' (≤ 25 μm; high nuclear/cytoplasmic ratio; CD45-) cells, comprising CTCs, and 'Large' (> 25 μm; low nuclear/cytoplasmic ratio; CD68+ or CD56+) cells, corresponding to macrophage and natural killer-like cells. The Small cell fraction also showed copy number increases in six target genes (FGFR1, Myc, CCND1, HER2, TOP2A and ZNF217) associated with breast cancer. These expanded CTCs exhibited different proportions of epithelial-mesenchymal phenotypes and were transferable for further expansion as spheroids in serum-free suspension or 3D cultures. Cluster formation was affected by the presence and duration of systemic therapy, and its persistence may reflect therapeutic resistance. This novel and advanced method estimates CTC clonal heterogeneity and can predict, within a relatively short time frame, patient responses to therapy. PMID:26008969

  1. Delta-Like 4 Differentially Regulates Murine CD4+ T Cell Expansion via BMI1

    PubMed Central

    Schaller, Matthew A.; Logue, Hannah; Mukherjee, Sumanta; Lindell, Dennis M.; Coelho, Ana Lucia; Lincoln, Pamela; Carson, William F.; Ito, Toshihiro; Cavassani, Karen A.; Chensue, Stephen W.; Hogaboam, Cory M.; Lukacs, Nicholas W.; Kunkel, Steven L.

    2010-01-01

    Background Studies have shown that Notch is essential for the maintenance of a T cell Th2 phenotype in vivo. It has also been shown that Notch ligands have diverse functions during T cell activation. We chose to investigate the role of Notch ligands during the Th2 response. Principal Findings We studied the relationship of two Notch ligands, delta-like 4 and jagged-1, to T cell proliferation in C57 Bl/6 mice. Our findings indicate that jagged-1 does not affect the rate of T cell proliferation in any subset examined. However, delta-like 4 causes an increase in the expansion of Th2 memory cells and a decrease in effector cell proliferation. Our in vivo studies indicate that the Notch system is dynamically regulated, and that blocking one Notch ligand increases the effective concentration of other Notch ligands, thus altering the response. Examination of genes related to the Notch pathway revealed that the Notch receptors were increased in memory T cells. Expression of BMI1, a gene involved in T cell proliferation, was also higher in memory T cells. Further experiments demonstrated that Notch directly regulates the expression of the BMI1 gene in T cells and may govern T cell proliferation through this pathway. Conclusions From these experiments we can make several novel conclusions about the role of Notch ligands in T cell biology. The first is that delta-like 4 suppresses effector cell proliferation and enhances Th2 memory cell proliferation. The second is that blocking one Notch ligand in vivo effectively increases the concentration of other Notch ligands, which can then alter the response. PMID:20808960

  2. Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour.

    PubMed

    Hernández-Hernández, Oscar; Guiraud-Dogan, Céline; Sicot, Géraldine; Huguet, Aline; Luilier, Sabrina; Steidl, Esther; Saenger, Stefanie; Marciniak, Elodie; Obriot, Hélène; Chevarin, Caroline; Nicole, Annie; Revillod, Lucile; Charizanis, Konstantinos; Lee, Kuang-Yung; Suzuki, Yasuhiro; Kimura, Takashi; Matsuura, Tohru; Cisneros, Bulmaro; Swanson, Maurice S; Trovero, Fabrice; Buisson, Bruno; Bizot, Jean-Charles; Hamon, Michel; Humez, Sandrine; Bassez, Guillaume; Metzger, Friedrich; Buée, Luc; Munnich, Arnold; Sergeant, Nicolas; Gourdon, Geneviève; Gomes-Pereira, Mário

    2013-03-01

    Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

  3. Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour

    PubMed Central

    Hernández-Hernández, Oscar; Guiraud-Dogan, Céline; Sicot, Géraldine; Huguet, Aline; Luilier, Sabrina; Steidl, Esther; Saenger, Stefanie; Marciniak, Elodie; Obriot, Hélène; Chevarin, Caroline; Nicole, Annie; Revillod, Lucile; Charizanis, Konstantinos; Lee, Kuang-Yung; Suzuki, Yasuhiro; Kimura, Takashi; Matsuura, Tohru; Cisneros, Bulmaro; Swanson, Maurice S.; Trovero, Fabrice; Buisson, Bruno; Bizot, Jean-Charles; Hamon, Michel; Humez, Sandrine; Bassez, Guillaume; Metzger, Friedrich; Buée, Luc; Munnich, Arnold; Sergeant, Nicolas; Gourdon, Geneviève

    2013-01-01

    Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology. PMID:23404338

  4. Low-density subculture: a technical note on the importance of avoiding cell-to-cell contact during mesenchymal stromal cell expansion.

    PubMed

    Balint, Richard; Richardson, Stephen M; Cartmell, Sarah H

    2015-10-01

    Numerous scientific studies and clinical trials are carried out each year exploring the use of mesenchymal stromal cells in regenerative medicine and tissue engineering. However, the effective and reliable expansion of this very important cell type remains a challenge. In this study the importance of cell-to-cell contact during expansion has been explored on the proliferation and differentiation potential of the produced cells. Cells were cultured up to passage 5 under conditions where cell-to-cell contact was either probable (40-70% confluence; see supporting information, Protocol A) or where it was unlikely (10-50% confluence; see supporting information, Protocol B). The effect of the two different conditions on expansion efficiency; proliferation rate and tri-lineage differentiation potential was assessed. Differences in immunophenotype, cell size and senescence were also investigated. Protocol B cultures expanded twice as fast as those cultured with Protocol A. In passage 5 experiments low confluence expanded cells displayed a 10% higher overall proliferation rate, and produced 23% more cells in growth, 12% more in osteogenic, 77% more in adipogenic, but 27% less in chondrogenic medium. Differentiation potential wasn't decisively affected at the mRNA level. However, Protocol B favoured bone and cartilage differentiation at the secretional level. Protocol A populations showed reduced purity, expressing CD105 in only 76% compared to the 96.7% in Protocol B cultures. Protocol A populations also contained significantly more (+4.2%) senescent cells, however, no difference was found in cell size between the two protocols. The findings of this study suggest that cell-to-cell contact, and therefore high confluence levels, is detrimental to MSC quality.

  5. Reversible expansion of primate mast cell populations in vivo by stem cell factor.

    PubMed Central

    Galli, S J; Iemura, A; Garlick, D S; Gamba-Vitalo, C; Zsebo, K M; Andrews, R G

    1993-01-01

    Mast cell development in mice is critically regulated by stem cell factor (SCF), the term used here to designate a product of fibroblasts and other cell types that is a ligand for the tyrosine kinase receptor protein encoded by the proto-oncogene c-kit. However, the factors which regulate the size of mast cell populations in primates are poorly understood. Here we report that the subcutaneous administration of recombinant human SCF (rhSCF) to baboons (Papio cynocephalus) or cynomolgus monkeys (Macaca fascicularis) produced a striking expansion of mast cell populations in many anatomical sites, with numbers of mast cells in some organs of rhSCF-treated monkeys exceeding the corresponding values in control monkeys by more than 100-fold. Animals treated with rhSCF did not exhibit clinical evidence of mast cell activation, and discontinuation of treatment with rhSCF resulted in a rapid decline of mast cell numbers nearly to baseline levels. These findings are the first to demonstrate that a specific cytokine can regulate mast cell development in primates in vivo. They also provide the first evidence, in any mammalian species, to indicate that the cytokine-dependent expansion of tissue mast cell populations can be reversed when administration of the cytokine is discontinued. Images PMID:7678600

  6. LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis

    PubMed Central

    Odena, Gemma; Chen, Jiegen; Lozano, Juan Jose; Altamirano, Jose; Rodrigo-Torres, Daniel; Affo, Silvia; Morales-Ibanez, Oriol; Matsushita, Hiroshi; Zou, Jian; Dumitru, Raluca; Caballeria, Juan; Gines, Pere; Arroyo, Vicente; You, Min; Rautou, Pierre-Emmanuel; Valla, Dominique; Crews, Fulton; Seki, Ekihiro; Sancho-Bru, Pau; Bataller, Ramon

    2016-01-01

    Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development. PMID:27752144

  7. An epilepsy-related ARX polyalanine expansion modifies glutamatergic neurons excitability and morphology without affecting GABAergic neurons development.

    PubMed

    Beguin, Shirley; Crépel, Valérie; Aniksztejn, Laurent; Becq, Hélène; Pelosi, Barbara; Pallesi-Pocachard, Emilie; Bouamrane, Lamine; Pasqualetti, Massimo; Kitamura, Kunio; Cardoso, Carlos; Represa, Alfonso

    2013-06-01

    Epileptic encephalopathies comprise a heterogeneous group of severe infantile disorders for which the pathophysiological basis of epilepsy is inaccurately clarified by genotype-phenotype analysis. Because a deficit of GABA neurons has been found in some of these syndromes, notably in patients with X-linked lissencephaly with abnormal genitalia, epilepsy was suggested to result from an imbalance in GABAergic inhibition, and the notion of "interneuronopathy" was proposed. Here, we studied the impact of a polyalanine expansion of aristaless-related homeobox (ARX) gene, a mutation notably found in West and Ohtahara syndromes. Analysis of Arx((GCG)7/Y) knock-in mice revealed that GABA neuron development is not affected. Moreover, pyramidal cell migration and cortical layering are unaltered in these mice. Interestingly, electrophysiological recordings show that hippocampal pyramidal neurons displayed a frequency of inhibitory postsynaptic currents similar to wild-type (WT) mice. However, these neurons show a dramatic increase in the frequency of excitatory inputs associated with a remodeling of their axonal arborization, suggesting that epilepsy in Arx((GCG)7/Y)mice would result from a glutamate network remodeling. We therefore propose that secondary alterations are instrumental for the development of disease-specific phenotypes and should be considered to explain the phenotypic diversity associated with epileptogenic mutations. PMID:22628459

  8. Microcarrier Culture for Efficient Expansion and Osteogenic Differentiation of Human Fetal Mesenchymal Stem Cells

    PubMed Central

    Goh, Tony Kwang-Poh; Zhang, Zhi-Yong; Chen, Allen Kuan-Liang; Reuveny, Shaul; Choolani, Mahesh

    2013-01-01

    Abstract Stirred microcarrier (MC) culture has been suggested as the method of choice for supplying large volumes of mesenchymal stem cells (MSCs) for bone tissue engineering. In this study, we show that in addition to the improvement in cell expansion capacity, MSCs propagated and harvested from MC culture also demonstrate higher osteogenic potency when differentiated in vivo or in vitro in three-dimensional (3D) scaffold cultures as compared with traditional monolayer (MNL) cultures. Cytodex 3 microcarrier-expanded human fetal MSC (hfMSC) cultures (MC-hfMSCs) achieved 12- to 16-fold expansion efficiency (6×105–8×105 cells/mL) compared to 4- to 6-fold (1.2×105–1.8×105 cells/mL) achieved by traditional MNL-expanded hfMSC culture (MNL-hfMSCs; p<0.05). Both MC-hfMSCs and MNL-hfMSCs maintained similar colony-forming capacity, doubling times, and immunophenotype postexpansion. However, when differentiated under in vitro two-dimensional (2D) osteogenic conditions, MC-hfMSCs exhibited a 45-fold reduction in alkaline phosphatase level and a 37.5% decrease in calcium deposition compared with MNL-hfMSCs (p<0.05). Surprisingly, when MC-hfMSCs and MNL-hfMSCs were seeded on 3D macroporous scaffold culture or subcutaneously implanted into nonobese diabetic/severe combined immunodeficient mice, MC-hfMSCs deposited 63.5% (p<0.05) more calcium and formed 47.2% (p<0.05) more bone volume, respectively. These results suggest that the mode of hfMSC growth in the expansion phase affects the osteogenic potential of hfMSCs differently in various differentiation platforms. In conclusion, MC cultures are advantageous over MNL cultures in bone tissue engineering because MC-hfMSCs have improved cell expansion capacity and exhibit higher osteogenic potential than MNL-hfMSCs when seeded in vitro into 3D scaffolds or implanted in vivo. PMID:23593561

  9. Balanced cell proliferation and expansion is essential for flowering stem growth control.

    PubMed

    Ferjani, Ali; Hanai, Kenya; Gunji, Shizuka; Maeda, Saori; Sawa, Shinichiro; Tsukaya, Hirokazu

    2015-01-01

    The postembryonic development of aboveground plant organs relies on a continuous supply of cells from the shoot apical meristem. Previous studies of developmental regulation in leaves and flowers have revealed the crucial role of coordinated cell proliferation and differentiation during organogenesis. However, the importance of this coordination has not been examined in flowering stems. Very recently, we attempted to identify regulatory factors that maintain flowering stem integrity. We found that the increased cell number in clavata (clv) mutants and the decreased cell size in de-etiolated (det)3-1 resulted in flowering stems that were thicker and thinner, respectively, than in wild-type (WT) plants. Interestingly, in the cell proliferation- and cell expansion-defective double mutant clv det3-1, the flowering stems often exhibited severe cracking, resulting in exposure of their inner tissues. In this study, further quantification of the cellular phenotypes in the cotyledons and leaves revealed no differences between det3-1 and clv3 det3-1. Together, the above findings suggest that the clv3 mutation in a det3-1 background primarily affects flowering stems, while its effect on other organs is likely negligible. We propose that the coordination between cell proliferation and differentiation is not only important during leaf development, but also plays a role in the growth control of Arabidopsis flowering stems.

  10. T cell clonal expansions detected in patients with primary biliary cirrhosis express CX3CR1

    PubMed Central

    Zhang, Weici; Ono, Yoko; Miyamura, Yoshinori; Bowlus, Christopher L.; Gershwin, M. Eric; Maverakis, Emanual

    2014-01-01

    The intrahepatic biliary destruction of primary biliary cirrhosis (PBC) appears secondary to a multi-lineage response that includes autoantibodies, biliary apotopes, and cellular responses. Although there has been considerable effort in defining the role and specificity of anti-mitochondrial autoantibodies, a major challenge has been the characterization of T effector pathways. This difficulty is due in part to the limitation of current technologies for directly isolating and characterizing autoreactive T cells from patients. Herein, we successfully demonstrate a novel technology for characterizing the surface phenotype of T cell oligoclonal expansions directly ex vivo. Using PBC as a prototypic disease we were able to detect clonal T cell expansions in 15/15 patients examined. Although the T cell expansions from different patients expressed different TCR Vβ gene segments, the surface phenotype of the cells was the same. The clonal T cell expansions in PBC patients are CX3CR1+ Fas+ effector-memory T cells, a finding of particular importance given the known up-regulation of fractalkine on injured biliary epithelial cells (BEC). In contrast to the persistent aberrantly expanded T cells observed in the PBC patients, T cell expansions detected in response to a herpes viral infection were very dynamic and resolved over time. This protocol can be used to characterize T cell expansions in other autoimmune diseases. PMID:21636249

  11. Does Schooling Affect Health Behavior? Evidence from the Educational Expansion in Western Germany

    ERIC Educational Resources Information Center

    Jurges, Hendrik; Reinhold, Steffen; Salm, Martin

    2011-01-01

    During the postwar period German states pursued policies to increase the share of young Germans obtaining a university entrance diploma (Abitur) by building more academic track schools, but the timing of educational expansion differed between states. This creates exogenous variation in the availability of upper secondary schooling, which allows…

  12. Endothelial progenitor cells: characterization, in vitro expansion, and prospects for autologous cell therapy.

    PubMed

    Smadja, D M; Cornet, A; Emmerich, J; Aiach, M; Gaussem, P

    2007-07-01

    Injection of hematopoietic stem cells or endothelial progenitor cells (EPCs) expanded ex vivo has been shown to augment neovascularization in adult patients, but the precise origin and identity of the cell population responsible for these clinical benefits are controversial. The limited quantity of EPCs in the circulation has been the main obstacle to clinical trials. Several authors have therefore attempted to expand these cells ex vivo in order to obtain a homogeneous cell therapy product. One possible means of expanding EPCs ex vivo is to activate the thrombin receptor PAR-1 with the specific peptide SFLLRN. Indeed, PAR-1 activation promotes cell proliferation and C-X-C chemokine receptor type 4 (CXCR4) dependent migration and differentiation, with an overall angiogenic effect. This review summarizes the results and rationale of clinical trials of angiogenic therapy, the nature of EPCs, the different methods of ex vivo expansion, and current methods of quantification. PMID:17370127

  13. Optimising Cell Aggregate Expansion in a Perfused Hollow Fibre Bioreactor via Mathematical Modelling

    PubMed Central

    Chapman, Lloyd A. C.; Shipley, Rebecca J.; Whiteley, Jonathan P.; Ellis, Marianne J.; Byrne, Helen M.; Waters, Sarah L.

    2014-01-01

    The need for efficient and controlled expansion of cell populations is paramount in tissue engineering. Hollow fibre bioreactors (HFBs) have the potential to meet this need, but only with improved understanding of how operating conditions and cell seeding strategy affect cell proliferation in the bioreactor. This study is designed to assess the effects of two key operating parameters (the flow rate of culture medium into the fibre lumen and the fluid pressure imposed at the lumen outlet), together with the cell seeding distribution, on cell population growth in a single-fibre HFB. This is achieved using mathematical modelling and numerical methods to simulate the growth of cell aggregates along the outer surface of the fibre in response to the local oxygen concentration and fluid shear stress. The oxygen delivery to the cell aggregates and the fluid shear stress increase as the flow rate and pressure imposed at the lumen outlet are increased. Although the increased oxygen delivery promotes growth, the higher fluid shear stress can lead to cell death. For a given cell type and initial aggregate distribution, the operating parameters that give the most rapid overall growth can be identified from simulations. For example, when aggregates of rat cardiomyocytes that can tolerate shear stresses of up to are evenly distributed along the fibre, the inlet flow rate and outlet pressure that maximise the overall growth rate are predicted to be in the ranges to (equivalent to to ) and to (or 15.6 psi to 15.7 psi) respectively. The combined effects of the seeding distribution and flow on the growth are also investigated and the optimal conditions for growth found to depend on the shear tolerance and oxygen demands of the cells. PMID:25157635

  14. Paracrine WNT5A Signaling Inhibits Expansion of Tumor-Initiating Cells.

    PubMed

    Borcherding, Nicholas; Kusner, David; Kolb, Ryan; Xie, Qing; Li, Wei; Yuan, Fang; Velez, Gabriel; Askeland, Ryan; Weigel, Ronald J; Zhang, Weizhou

    2015-05-15

    It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ-SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal cross-talk in normal mammary tissue.

  15. Paracrine WNT5A signaling inhibits expansion of tumor-initiating cells

    PubMed Central

    Borcherding, Nicholas; Kusner, David; Kolb, Ryan; Xie, Qing; Li, Wei; Yuan, Fang; Velez, Gabriel; Askeland, Ryan; Weigel, Ronald J.; Zhang, Weizhou

    2015-01-01

    It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared to the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of TGFβ/SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal crosstalk in normal mammary tissue. PMID:25769722

  16. Allogeneic Cell Therapy Bioprocess Economics and Optimization: Single-Use Cell Expansion Technologies

    PubMed Central

    Simaria, Ana S; Hassan, Sally; Varadaraju, Hemanthram; Rowley, Jon; Warren, Kim; Vanek, Philip; Farid, Suzanne S

    2014-01-01

    For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot-sizes capable of meeting commercial demands of up to 109 cells/dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost-effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and microcarrier-based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost-effective and where microcarrier-based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier-based systems. These data are presented using a technology S-curve as well as windows of operation to identify the combination of cell productivities and scale of single-use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future R&D investment should be focused to improve the performance of the most promising technologies so that they become a robust and scalable option that enables the cell therapy industry reach commercially relevant lot sizes. The tool outputs can facilitate decision-making very early on in development and be used to predict, and better manage, the risk of process changes needed as products proceed through the development pathway. Biotechnol. Bioeng. 2014;111: 69–83. © 2013 Wiley Periodicals, Inc. PMID:23893544

  17. Chemical expansion affected oxygen vacancy stability in different oxide structures from first principles calculations

    DOE PAGES

    Aidhy, Dilpuneet S.; Liu, Bin; Zhang, Yanwen; Weber, William J.

    2015-01-21

    We study the chemical expansion for neutral and charged oxygen vacancies in fluorite, rocksalt, perovskite and pyrochlores materials using first principles calculations. We show that the neutral oxygen vacancy leads to lattice expansion whereas the charged vacancy leads to lattice contraction. In addition, we show that there is a window of strain within which an oxygen vacancy is stable; beyond that range, the vacancy can become unstable. Using CeO2|ZrO2 interface structure as an example, we show that the concentration of oxygen vacancies can be manipulated via strain, and the vacancies can be preferentially stabilized. Furthermore, these results could serve asmore » guiding principles in predicting oxygen vacancy stability in strained systems and in the design of vacancy stabilized materials.« less

  18. Chemical expansion affected oxygen vacancy stability in different oxide structures from first principles calculations

    SciTech Connect

    Aidhy, Dilpuneet S.; Liu, Bin; Zhang, Yanwen; Weber, William J.

    2015-01-21

    We study the chemical expansion for neutral and charged oxygen vacancies in fluorite, rocksalt, perovskite and pyrochlores materials using first principles calculations. We show that the neutral oxygen vacancy leads to lattice expansion whereas the charged vacancy leads to lattice contraction. In addition, we show that there is a window of strain within which an oxygen vacancy is stable; beyond that range, the vacancy can become unstable. Using CeO2|ZrO2 interface structure as an example, we show that the concentration of oxygen vacancies can be manipulated via strain, and the vacancies can be preferentially stabilized. Furthermore, these results could serve as guiding principles in predicting oxygen vacancy stability in strained systems and in the design of vacancy stabilized materials.

  19. Chemical expansion affected oxygen vacancy stability in different oxide structures from first principles calculations

    SciTech Connect

    Aidhy, Dilpuneet S.; Liu, Bin; Zhang, Yanwen; Weber, William J.

    2015-03-01

    We study the chemical expansion for neutral and charged oxygen vacancies in fluorite, rocksalt, perovskite and pyrochlores materials using first principles calculations. We show that the neutral oxygen vacancy leads to lattice expansion whereas the charged vacancy leads to lattice contraction. In addition, we show that there is a window of strain within which an oxygen vacancy is stable; beyond that range, the vacancy can become unstable. Using CeO2|ZrO2 interface structure as an example, we show that the concentration of oxygen vacancies can be manipulated via strain, and the vacancies can be preferentially stabilized. These results could serve as guiding principles in predicting oxygen vacancy stability in strained systems and in the design of vacancy stabilized materials.

  20. Fluoride inhibits root water transport and affects leaf expansion and gas exchange in aspen (Populus tremuloides) seedlings.

    PubMed

    Kamaluddin, Mohammed; Zwiazek, Janusz J.

    2003-03-01

    The effects of sodium fluoride (0.3, 5 and 10 mM NaF) on root hydraulic conductivity, and gas exchange processes were examined in aspen (Populus tremuloides Michx.) seedlings grown in solution culture. A long-term exposure of roots to NaF significantly decreased root hydraulic conductivity (Lp) and stomatal conductance ( gs). Root absorbed NaF significantly affected electrolyte leakage in leaf tissues and substantially restricted leaf expansion. NaF did not significantly affect leaf chlorophyll contents but decreased net photosynthesis (Pn). A short-term exposure of excised roots to 5 mM NaF and KF significantly decreased root water flow (Qv) with a concomitant decline in root respiration and reduced gs when applied through intact roots or excised stems. The same molar concentration of NaCl also decreased Qv and gs in intact seedlings, but to a lesser extent than NaF or KF, and did not significantly affect root respiration. The results suggest that fluoride metabolically inhibited Qv or Lp, probably by affecting water channel activity. We suggest that the metabolic inhibition of Lp by root-absorbed fluoride affected gas exchange and leaf expansion in aspen seedlings.

  1. Antigen Presenting Cell-Mediated Expansion of Human Umbilical Cord Blood Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity

    PubMed Central

    Shah, Nina; Martin-Antonio, Beatriz; Yang, Hong; Ku, Stephanie; Lee, Dean A.; Cooper, Laurence J. N.; Decker, William K.; Li, Sufang; Robinson, Simon N.; Sekine, Takuya; Parmar, Simrit; Gribben, John; Wang, Michael; Rezvani, Katy; Yvon, Eric; Najjar, Amer; Burks, Jared; Kaur, Indreshpal; Champlin, Richard E.; Bollard, Catherine M.; Shpall, Elizabeth J.

    2013-01-01

    Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56+/CD3−) and less than 1% CD3+ cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM. PMID:24204673

  2. 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

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

    PubMed

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

    2016-06-01

    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.

  4. Human Head and Neck Squamous Cell Carcinoma–Associated Semaphorin 4D Induces Expansion of Myeloid-Derived Suppressor Cells

    PubMed Central

    Han, Kyu Lee; Webb, Tonya J.

    2016-01-01

    One of the mechanisms by which malignancies can induce immune suppression is through the production of cytokines that affect the maturation and differentiation of inflammatory cells in the tumor microenvironment. Semaphorin 4D (Sema4D) is a proangiogenic cytokine produced by several malignancies, which has been described in the regulation of the immune system. In the present study, we examined the role of human head and neck squamous cell carcinoma (HNSCC)–secreted Sema4D on myeloid cell differentiation. CD33+ cells cultured in HNSCC cell line–derived conditioned medium differentiated into myeloid derived suppressor cells (MDSC) (CD33+CD11b+HLA-DR−/low). The addition of anti-Sema4D Ab to HNSCC conditioned medium significantly reduced the expansion of the MDSC population. Similarly, knockdown of Sema4D in an HNSCC cell line resulted in a loss of MDSC function as shown by a decrease in the production of the immune-suppressive cytokines arginase-1, TGF-β, and IL-10 by MDSC, concomitant with recovery of T cell proliferation and IFN-γ production following stimulation of CD3/CD28. Importantly, CD33+ myeloid and T cells cultured in conditioned medium of HNSCC cells in which Sema4D was knocked down promoted antitumor inflammatory profile, through recovery of the effector T cells (CD4+T-bet+ and CD8+T-bet+), as well as a decrease in regulatory T cells (CD4+CD25+FOXP3+). We also showed that Sema4D was comparable to GM-CSF in its induction of MDSC. Collectively, this study describes a novel immunosuppressive role for Sema4D in HNSCC through induction of MDSC, and it highlights Sema4D as a therapeutic target for future studies to enhance the antitumorigenic inflammatory response in HNSCC and other epithelial malignancies. PMID:26740106

  5. Substrates for Expansion of Corneal Endothelial Cells towards Bioengineering of Human Corneal Endothelium

    PubMed Central

    Navaratnam, Jesintha; Utheim, Tor P.; Rajasekhar, Vinagolu K.; Shahdadfar, Aboulghassem

    2015-01-01

    Corneal endothelium is a single layer of specialized cells that lines the posterior surface of cornea and maintains corneal hydration and corneal transparency essential for vision. Currently, transplantation is the only therapeutic option for diseases affecting the corneal endothelium. Transplantation of corneal endothelium, called endothelial keratoplasty, is widely used for corneal endothelial diseases. However, corneal transplantation is limited by global donor shortage. Therefore, there is a need to overcome the deficiency of sufficient donor corneal tissue. New approaches are being explored to engineer corneal tissues such that sufficient amount of corneal endothelium becomes available to offset the present shortage of functional cornea. Although human corneal endothelial cells have limited proliferative capacity in vivo, several laboratories have been successful in in vitro expansion of human corneal endothelial cells. Here we provide a comprehensive analysis of different substrates employed for in vitro cultivation of human corneal endothelial cells. Advances and emerging challenges with ex vivo cultured corneal endothelial layer for the ultimate goal of therapeutic replacement of dysfunctional corneal endothelium in humans with functional corneal endothelium are also presented. PMID:26378588

  6. Activated CD8+ T cells induce expansion of Vβ5+ regulatory T cells via TNFR2 signaling

    PubMed Central

    Joedicke, Jara J; Myers, Lara; Carmody, Aaron B; Messer, Ronald J; Wajant, Harald; Lang, Karl S; Lang, Philipp A; Mak, Tak W; Hasenkrug, Kim J; Dittmer, Ulf

    2014-01-01

    Vβ5+ regulatory T cells (Tregs), which are specific for a mouse endogenous retroviral superantigen, become activated and proliferate in response to Friend retrovirus (FV) infection. We previously reported that FV-induced expansion of this Treg subset was dependent on CD8+ T cells and TNFα, but independent of IL-2. We now show that the inflammatory milieu associated with FV infection is not necessary for induction of Vβ5+ Treg expansion. Rather, it is the presence of activated CD8+ T cells that is critical for their expansion. The data indicate that the mechanism involves signaling between the membrane-bound form of TNFα (memTNFα) on activated CD8+ T cells and TNF receptor 2 (TNFR2) on Tregs. CD8+ T cells expressing memTNFα but no soluble TNFα (solTNFα) remained competent to induce strong Vβ5+ Treg expansion in vivo. In addition, Vβ5+ Tregs expressing only TNFR2 but no TNFR1 were still responsive to expansion. Finally, treatment of naïve mice with solTNFα did not induce Vβ5+ Treg expansion, but treatment with a TNFR2-specific agonist did. These results reveal a new mechanism of intercellular communication between activated CD8+ T cell effectors and Tregs that results in the activation and expansion of a Treg subset that subsequently suppresses CD8+ T cell functions. PMID:25098294

  7. Polyclonal Expansion of NKG2C+ NK Cells in TAP-Deficient Patients

    PubMed Central

    Béziat, Vivien; Sleiman, Marwan; Goodridge, Jodie P.; Kaarbø, Mari; Liu, Lisa L.; Rollag, Halvor; Ljunggren, Hans-Gustaf; Zimmer, Jacques; Malmberg, Karl-Johan

    2015-01-01

    Adaptive natural killer (NK) cell responses to human cytomegalovirus infection are characterized by the expansion of NKG2C+ NK cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs). Here, we set out to study the HLA class I dependency of such NKG2C+ NK cell expansions. We demonstrate the expansion of NKG2C+ NK cells in patients with transporter associated with antigen presentation (TAP) deficiency, who express less than 10% of normal HLA class I levels. In contrast to normal individuals, expanded NKG2C+ NK cell populations in TAP-deficient patients display a polyclonal KIR profile and remain hyporesponsive to HLA class I-negative target cells. Nonetheless, agonistic stimulation of NKG2C on NK cells from TAP-deficient patients yielded significant responses in terms of degranulation and cytokine production. Thus, while interactions with self-HLA class I molecules likely shape the KIR repertoire of expanding NKG2C+ NK cells during adaptive NK cell responses in normal individuals, they are not a prerequisite for NKG2C+ NK cell expansions to occur. The emergence of NKG2C-responsive adaptive NK cells in TAP-deficient patients may contribute to antiviral immunity and potentially explain these patients’ low incidence of severe viral infections. PMID:26500647

  8. Stalled DNA Replication Forks at the Endogenous GAA Repeats Drive Repeat Expansion in Friedreich's Ataxia Cells.

    PubMed

    Gerhardt, Jeannine; Bhalla, Angela D; Butler, Jill Sergesketter; Puckett, James W; Dervan, Peter B; Rosenwaks, Zev; Napierala, Marek

    2016-08-01

    Friedreich's ataxia (FRDA) is caused by the expansion of GAA repeats located in the Frataxin (FXN) gene. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism leading to repeat expansion and decreased FXN transcription remains unclear. Using single-molecule analysis of replicated DNA, we detected that expanded GAA repeats present a substantial obstacle for the replication machinery at the FXN locus in FRDA cells. Furthermore, aberrant origin activation and lack of a proper stress response to rescue the stalled forks in FRDA cells cause an increase in 3'-5' progressing forks, which could enhance repeat expansion and hinder FXN transcription by head-on collision with RNA polymerases. Treatment of FRDA cells with GAA-specific polyamides rescues DNA replication fork stalling and alleviates expansion of the GAA repeats, implicating DNA triplexes as a replication impediment and suggesting that fork stalling might be a therapeutic target for FRDA.

  9. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells.

    PubMed

    Zhang, Cheng Cheng; Kaba, Megan; Ge, Guangtao; Xie, Kathleen; Tong, Wei; Hug, Christopher; Lodish, Harvey F

    2006-02-01

    Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3(+) cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.

  10. Stalled DNA Replication Forks at the Endogenous GAA Repeats Drive Repeat Expansion in Friedreich's Ataxia Cells.

    PubMed

    Gerhardt, Jeannine; Bhalla, Angela D; Butler, Jill Sergesketter; Puckett, James W; Dervan, Peter B; Rosenwaks, Zev; Napierala, Marek

    2016-08-01

    Friedreich's ataxia (FRDA) is caused by the expansion of GAA repeats located in the Frataxin (FXN) gene. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism leading to repeat expansion and decreased FXN transcription remains unclear. Using single-molecule analysis of replicated DNA, we detected that expanded GAA repeats present a substantial obstacle for the replication machinery at the FXN locus in FRDA cells. Furthermore, aberrant origin activation and lack of a proper stress response to rescue the stalled forks in FRDA cells cause an increase in 3'-5' progressing forks, which could enhance repeat expansion and hinder FXN transcription by head-on collision with RNA polymerases. Treatment of FRDA cells with GAA-specific polyamides rescues DNA replication fork stalling and alleviates expansion of the GAA repeats, implicating DNA triplexes as a replication impediment and suggesting that fork stalling might be a therapeutic target for FRDA. PMID:27425605

  11. Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.

    PubMed

    Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-Ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

    2013-11-19

    Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.

  12. Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells

    PubMed Central

    Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

    2013-01-01

    Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001 PMID:24252877

  13. Mechanism of rapid suppression of cell expansion in cucumber hypocotyls after blue-light irradiation

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1988-01-01

    Rapid suppression of hypocotyl elongation by blue light in cucumber (Cucumis sativus L.) was studied to examine possible hydraulic and wall changes responsible for diminished growth. Cell-sap osmotic pressure, measured by vapor-pressure osmometry, was not decreased by blue light; turgor pressure, measured by the pressure-probe technique, remained constant during the growth inhibition; and stem hydraulic conductance, measured by dynamic and static methods, was likewise unaffected by blue light. Wall yielding properties were assessed by the pressure-block technique for in-vivo stress relaxation. Blue light reduced the initial rate of relaxation by 77%, but had little effect on the final amount of relaxation. The results demonstrate that blue irradiation acts to decrease the wall yielding coefficient, but not the yield threshold. Stress-strain (Instron) analysis showed that irradiation of the seedlings had little effect on the mechanical extensibilities of the isolated wall. The results indicate that blue light can reduce cell-wall loosening without affecting bulk viscoelastic properties, and indicate a chemorheological mechanism of cell-wall expansion.

  14. Engineering Cell-Material Interfaces for Long-term Expansion of Human Pluripotent Stem Cells

    PubMed Central

    Chang, Chien-Wen; Hwang, Yongsung; Brafman, Dave; Hagan, Thomas; Phung, Catherine; Varghese, Shyni

    2014-01-01

    Cost-effective and scalable synthetic matrices that support long-term expansion of human pluripotent stem cells (hPSCs) have many applications, ranging from drug screening platforms to regenerative medicine. Here, we report the development of a hydrogel-based matrix containing synthetic heparin-mimicking moieties that supports the long-term expansion of hPSCs (≥20 passages) in a chemically defined medium. HPSCs expanded on this synthetic matrix maintained their characteristic morphology, colony forming ability, karyotypic stability, and differentiation potential. We also used the synthetic matrix as a platform to investigate the effects of various physicochemical properties of the extracellular environment on the adhesion, growth, and self-renewal of hPSCs. The observed cellular responses can be explained in terms of matrix interface-mediated binding of extracellular matrix proteins, growth factors, and other cell secreted factors, which create an instructive microenvironment to support self-renewal of hPSCs. These synthetic matrices, which comprise of “off-the-shelf” components and are easy to synthesize, provide an ideal tool to elucidate the molecular mechanisms that control stem cell fate. PMID:23131532

  15. Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells.

    PubMed

    Mou, Hongmei; Vinarsky, Vladimir; Tata, Purushothama Rao; Brazauskas, Karissa; Choi, Soon H; Crooke, Adrianne K; Zhang, Bing; Solomon, George M; Turner, Brett; Bihler, Hermann; Harrington, Jan; Lapey, Allen; Channick, Colleen; Keyes, Colleen; Freund, Adam; Artandi, Steven; Mense, Martin; Rowe, Steven; Engelhardt, John F; Hsu, Ya-Chieh; Rajagopal, Jayaraj

    2016-08-01

    Functional modeling of many adult epithelia is limited by the difficulty in maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways enables robust expansion of primary epithelial basal cell populations. We find that TGFβ/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD signaling inhibition in a feeder-free culture system, we have been able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium physiologically responsive to clinically relevant drugs, such as CFTR modulators. This approach is effective for the clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers and therefore may be broadly applicable for modeling of epithelia. PMID:27320041

  16. SH2B1 in β-Cells Regulates Glucose Metabolism by Promoting β-Cell Survival and Islet Expansion

    PubMed Central

    Chen, Zheng; Morris, David L.; Jiang, Lin; Liu, Yong; Rui, Liangyou

    2014-01-01

    IGF-1 and insulin promote β-cell expansion by inhibiting β-cell death and stimulating β-cell proliferation, and the phosphatidylinositol (PI) 3-kinase/Akt pathway mediates insulin and IGF-1 action. Impaired β-cell expansion is a risk factor for type 2 diabetes. Here, we identified SH2B1, which is highly expressed in β-cells, as a novel regulator of β-cell expansion. Silencing of SH2B1 in INS-1 832/13 β-cells attenuated insulin- and IGF-1–stimulated activation of the PI 3-kinase/Akt pathway and increased streptozotocin (STZ)-induced apoptosis; conversely, overexpression of SH2B1 had the opposite effects. Activation of the PI 3-kinase/Akt pathway in β-cells was impaired in pancreas-specific SH2B1 knockout (PKO) mice fed a high-fat diet (HFD). HFD-fed PKO mice also had increased β-cell apoptosis, decreased β-cell proliferation, decreased β-cell mass, decreased pancreatic insulin content, impaired insulin secretion, and exacerbated glucose intolerance. Furthermore, PKO mice were more susceptible to STZ-induced β-cell destruction, insulin deficiency, and hyperglycemia. These data indicate that SH2B1 in β-cells is an important prosurvival and proproliferative protein and promotes compensatory β-cell expansion in the insulin-resistant state and in response to β-cell stress. PMID:24150605

  17. Model-based analysis of Arabidopsis leaf epidermal cells reveals distinct division and expansion patterns for pavement and guard cells.

    PubMed

    Asl, Leila Kheibarshekan; Dhondt, Stijn; Boudolf, Véronique; Beemster, Gerrit T S; Beeckman, Tom; Inzé, Dirk; Govaerts, Willy; De Veylder, Lieven

    2011-08-01

    To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery.

  18. Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells.

    PubMed

    Stier, Sebastian; Cheng, Tao; Forkert, Randolf; Lutz, Christoph; Dombkowski, David M; Zhang, Jie Lin; Scadden, David T

    2003-08-15

    Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling, we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor, p21Cip1/Waf1 (p21), have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling, we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control, manipulated cells. Further, we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore, lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion. PMID:12702511

  19. Apparent expansion of CD34+ cells during the evolution of myelodysplastic syndromes to acute myeloid leukemia.

    PubMed

    Span, L F; Dar, S E; Shetty, V; Mundle, S D; Broady-Robinson, L; Alvi, S; Raymakers, R A; de Witte, T; Raza, A

    1998-11-01

    Myelodysplastic syndromes (MDS) are highly proliferative bone marrow (BM) disorders where the primary lesion presumably affects a CD34+ early progenitor or stem cell. We investigated the proliferative characteristics of CD34+ cells of 33 untreated MDS patients (19 RA, 5 RARS, 7 RAEB, 2 RAEBt) and five patients with acute myeloid leukemia after MDS (sAML). All patients received a 1-h infusion of the thymidine analogue iodoor bromodeoxyuridine intravenously before a BM aspirate and biopsy was taken. A double-labeling immunohistochemistry technique by monoclonal anti-CD34 (QBend/10) and anti-IUdR/BrdU antibodies was developed and performed. By this technique we recognised CD34+ and CD34- cells actively engaged in DNA synthesis or not. As MDS evolves a significant increase occurred in the percentage of CD34+ cells of all myeloid cells (mean value: RA/RARS 1.67%; RAEB(t) 8.68%; sAML 23.83%) as well as in the percentage of proliferating CD34+ cells of all myeloid cells (RA/RARS 0.19%; RAEB(t) 0.43%; and sAML 3.30%). This was associated with a decreasing trend in the overall myeloid labeling index (LI: RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5%). This decrease in overall myeloid LI is due to an exponential increase in the proportion of CD34+ cells of the proliferating compartment during MDS evolution (RA/RARS 0.35%, RAEB(t) 1.44% and sAML 11.98% of all S-phase cells). These CD34+ cells appeared to proliferate more slowly than their more mature CD34 negative counterparts, since we found a progressive increment in the mean total cell cycling time (Tc) of all myeloid cells during MDS progression (RA/RARS 39.8, RAEB(t) 45.2 and sAML 65.8 h). This study showed that during MDS evolution to sAML the CD34+ compartment develops a growth advantage leading to apparent expansion.

  20. Suppression of cell expansion by ectopic expression of the Arabidopsis SUPERMAN gene in transgenic petunia and tobacco.

    PubMed

    Kater, M M; Franken, J; van Aelst, A; Angenent, G C

    2000-08-01

    Molecular and genetic analyses have shown that the Arabidopsis thaliana gene SUPERMAN (SUP) has at least two functions in Arabidopsis flower development. SUP is necessary to control the correct distribution of cells with either a stamen or carpel fate, and is essential for proper outgrowth of the ovule outer integument. Both these functions indicate a role for SUP in cell proliferation. To study the function of the Arabidopsis SUP gene in more detail, we over-expressed the SUP gene in petunia and tobacco in a tissue-specific manner. The petunia FLORAL BINDING PROTEIN 1 (FBP1) gene promoter was used to restrict the expression of SUP to petals and stamens. The development of petals and stamens was severely affected in both petunia and tobacco plants over-expressing SUP. Petals remained small and did not unfold, resulting in closed flowers. Stamen filaments were thin and very short. Detailed analysis of these floral organs from the petunia transformants showed that cell expansion was dramatically reduced without affecting cell division. These results reveal a novel activity for SUP as a regulator of cell expansion. PMID:10929133

  1. Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

    NASA Astrophysics Data System (ADS)

    Weber, C.; Pohl, S.; Poertner, R.; Pino-Grace, Pablo; Freimark, D.; Wallrapp, C.; Geigle, P.; Czermak, P.

    Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

  2. HOT CELL BUILDING, TRA632. FLOOR PLAN OF EXPANSION SHOWS LOCATION ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    HOT CELL BUILDING, TRA-632. FLOOR PLAN OF EXPANSION SHOWS LOCATION OF NEW CELLS, "HEAVY" CELL AT WEST END, "LIGHT" CELLS AT EAST. MOCK-UP AND STORAGE AREAS IN SOUTH HALF OF FLOOR. H.K. FERGUSON 895-MTR-ETR-632-A1, 12/1958. INL INDEX NO. 531-0632-00-279-101924, REV. 4. - Idaho National Engineering Laboratory, Test Reactor Area, Materials & Engineering Test Reactors, Scoville, Butte County, ID

  3. Suitability of human mesenchymal stem cells for gene therapy depends on the expansion medium

    SciTech Connect

    Apel, Anja; Groth, Ariane; Schlesinger, Sabine; Bruns, Helge; Schemmer, Peter; Buechler, Markus W.; Herr, Ingrid

    2009-02-01

    Great hope is set in the use of mesenchymal stem cells for gene therapy and regenerative medicine. Since the frequency of this subpopulation of stem cells in bone marrow is low, mesenchymal stem cells are expanded ex vivo and manipulated prior to experimental or clinical use. Different methods for isolation and expansion are available, but the particular effect on the stem cell character is unclear. While the isolation of mesenchymal stem cells by density centrifugation followed by selection of the plastic adherent fraction is frequently used, the composition of expansion media differs. Thus, in the present study we cultured mesenchymal stem cells isolated from five healthy young volunteers in three widely used expansion media and performed a detailed analysis of the effect on morphology, proliferation, clonogenicity, passaging, differentiation and senescence. By this way we clearly show that the type of expansion medium used determines the stem cell character and time of senescence which is critical for future gene therapeutic and regenerative approaches using mesenchymal stem cells.

  4. Augmented lymphocyte expansion from solid tumors with engineered cells for costimulatory enhancement.

    PubMed

    Friedman, Kevin M; Devillier, Laura E; Feldman, Steven A; Rosenberg, Steven A; Dudley, Mark E

    2011-01-01

    Treatment of patients with adoptive T-cell therapy requires expansion of unique tumor-infiltrating lymphocyte (TIL) cultures from single-cell suspensions processed from melanoma biopsies. Strategies which increase the expansion and reliability of TIL generation from tumor digests are necessary to improve access to TIL therapy. Previous studies have evaluated artificial antigen presenting cells for their antigen-specific and costimulatory properties. We investigated engineered cells for costimulatory enhancement (ECCE) consisting of K562 cells that express 4-1BBL in the absence of artificial antigen stimulation. ECCE accelerated TIL expansion and significantly improved TIL numbers (P=0.001) from single-cell melanoma suspensions. TIL generated with ECCE contain significantly more CD8CD62L and CD8CD27 T cells then comparable interleukin-2-expanded TIL and maintained antitumor reactivity. Moreover, ECCE improved TIL expansion from nonmelanoma-cell suspensions similar to that seen with melanoma tumors. These data demonstrate that the addition of ECCE to TIL production will enable the treatment of patients that are ineligible using current methods. PMID:21989413

  5. Physical parameters affecting living cells in space

    NASA Astrophysics Data System (ADS)

    Langbein, Dieter

    The question is posed: Why does a living cell react to the absence of gravity? What sensors may it have? Does it note pressure, sedimentation, convection, or other parameters? If somewhere in a liquid volume sodium ions are replaced by potassium ions, the density of the liquid changes locally: the heavier regions sink, the lighter regions rise. This may contribute to species transport, to the metabolism. Under microgravity this mechanism is strongly reduced. On the other hand, other reasons for convection like thermal and solutal interface convection are left. Do they affect species transport? Another important effect of gravity is the hydrostatic pressure. On the macroscopic side, the pressure between our head and feet changes by 0.35 atmospheres. On the microscopic level the hydrostatic pressure on the upper half of a cell membrane is lower than on the lower half. This, by affecting the ion transport through the membrane, may change the surrounding electric potential. It has been suggested to be one of the reasons for graviperception. Following the discussion of these and other effects possibly important in life sciences in space, an order of magnitude analysis of the residual accelerations tolerable during experiments in materials sciences is outlined. In the field of life sciences only rough estimates are available at present.

  6. Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells.

    PubMed

    Holst, Jeff; Watson, Sarah; Lord, Megan S; Eamegdool, Steven S; Bax, Daniel V; Nivison-Smith, Lisa B; Kondyurin, Alexey; Ma, Liang; Oberhauser, Andres F; Weiss, Anthony S; Rasko, John E J

    2010-10-01

    Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture. PMID:20890282

  7. Enrichment and Expansion with Nanoscale Artificial Antigen Presenting Cells for Adoptive Immunotherapy.

    PubMed

    Perica, Karlo; Bieler, Joan Glick; Schütz, Christian; Varela, Juan Carlos; Douglass, Jacqueline; Skora, Andrew; Chiu, Yen Ling; Oelke, Mathias; Kinzler, Kenneth; Zhou, Shibin; Vogelstein, Bert; Schneck, Jonathan P

    2015-07-28

    Adoptive immunotherapy (AIT) can mediate durable regression of cancer, but widespread adoption of AIT is limited by the cost and complexity of generating tumor-specific T cells. Here we develop an Enrichment + Expansion strategy using paramagnetic, nanoscale artificial antigen presenting cells (aAPC) to rapidly expand tumor-specific T cells from rare naïve precursors and predicted neo-epitope responses. Nano-aAPC are capable of enriching rare tumor-specific T cells in a magnetic column and subsequently activating them to induce proliferation. Enrichment + Expansion resulted in greater than 1000-fold expansion of both mouse and human tumor-specific T cells in 1 week, with nano-aAPC based enrichment conferring a proliferation advantage during both in vitro culture and after adoptive transfer in vivo. Robust T cell responses were seen not only for shared tumor antigens, but also for computationally predicted neo-epitopes. Streamlining the rapid generation of large numbers of tumor-specific T cells in a cost-effective fashion through Enrichment + Expansion can be a powerful tool for immunotherapy. PMID:26171764

  8. Expansion of NK Cells and Reduction of NKG2D Expression in Chronic Lymphocytic Leukemia. Correlation with Progressive Disease

    PubMed Central

    Huergo-Zapico, Leticia; Acebes-Huerta, Andrea; Gonzalez-Rodriguez, Ana Pilar; Contesti, Juan; Gonzalez-García, Esther; Payer, Angel R.; Villa-Alvarez, Monica; Fernández-Guizán, Azahara; López-Soto, Alejandro; Gonzalez, Segundo

    2014-01-01

    The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL. PMID:25286418

  9. Sorting and Expansion of Murine Embryonic Stem Cell Colonies using Micropallet Arrays

    PubMed Central

    Shadpour, Hamed; Sims, Christopher E.; Thresher, Randy J.; Allbritton, Nancy L.

    2008-01-01

    Background Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption. Methods Murine embryonic stem cells were cultured on arrays composed of releasable elements termed micropallets created from a biocompatible photoresist. Micropallets containing undifferentiated colonies were released using a laser-based technique followed by cell collection and expansion in culture. Results The micropallet arrays provided a biocompatible substrate for maintaining undifferentiated murine stem cells in culture. A surface coating of 0.025% gelatin was shown to be optimal for cell culture and collection. Arrays composed of surface roughened micropallets provided further improvements in culture and isolation. Colonies of viable stem cells were efficiently isolated and collected. Colonies sorted in this manner were shown to remain undifferentiated even after collection and further expansion in culture. Conclusions Qualitative and quantitative analyses of sorting, collection efficiency and cell viability after release and expansion of stem cell colonies demonstrated that the micropallet array technology is a promising alternative to conventional sorting methods for stem cell applications. PMID:19012319

  10. Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

    PubMed Central

    Malpique, Rita; Brito, Catarina; Jensen, Janne; Bjorquist, Petter; Carrondo, Manuel J. T.; Alves, Paula M.

    2011-01-01

    The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications. PMID:21850261

  11. Safety and efficient ex vivo expansion of stem cells using platelet-rich plasma technology.

    PubMed

    Anitua, Eduardo; Prado, Roberto; Orive, Gorka

    2013-09-01

    The goal of this Review is to provide an overview of the cell culture media supplements used in the ex vivo expansion of stem cells intended for cell therapy. Currently, the gold standard is the culture supplemented with fetal bovine serum, however, their use in cell therapy raises many concerns. The alternatives to its use are presented, ranging from the use of human serum to platelet-rich plasma (PRP), to serum-free media or extracellular matrix components. Finally, various growth factors present in PRP are described, which make it a safe and effective stem cell expansion supplement. These growth factors could be responsible for their efficiency, as they increase both stem cell proliferation and survival. The different PRP formulations are also discussed, as well as the need for protocol standardization.

  12. Aberrant epigenetic regulators control expansion of human CD34+ hematopoietic stem/progenitor cells

    PubMed Central

    Faridi, Farnaz; Ponnusamy, Kanagaraju; Quagliano-Lo Coco, Isabell; Chen-Wichmann, Linping; Grez, Manuel; Henschler, Reinhard; Wichmann, Christian

    2013-01-01

    Transcription is a tightly regulated process ensuring the proper expression of numerous genes regulating all aspects of cellular behavior. Transcription factors regulate multiple genes including other transcription factors that together control a highly complex gene network. The transcriptional machinery can be “hijacked” by oncogenic transcription factors, thereby leading to malignant cell transformation. Oncogenic transcription factors manipulate a variety of epigenetic control mechanisms to fulfill gene regulatory and cell transforming functions. These factors assemble epigenetic regulators at target gene promoter sequences, thereby disturbing physiological gene expression patterns. Retroviral vector technology and the availability of “healthy” human hematopoietic CD34+ progenitor cells enable the generation of pre-leukemic cell models for the analysis of aberrant human hematopoietic progenitor cell expansion mediated by leukemogenic transcription factors. This review summarizes recent findings regarding the mechanism by which leukemogenic gene products control human hematopoietic CD34+ progenitor cell expansion by disrupting the normal epigenetic program. PMID:24348510

  13. Expansion of Human Induced Pluripotent Stem Cells in Stirred Suspension Bioreactors.

    PubMed

    Almutawaa, Walaa; Rohani, Leili; Rancourt, Derrick E

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) hold great promise as a cell source for therapeutic applications and regenerative medicine. Traditionally, hiPSCs are expanded in two-dimensional static culture as colonies in the presence or absence of feeder cells. However, this expansion procedure is associated with lack of reproducibility and low cell yields. To fulfill the large cell number demand for clinical use, robust large-scale production of these cells under defined conditions is needed. Herein, we describe a scalable, low-cost protocol for expanding hiPSCs as aggregates in a lab-scale bioreactor.

  14. Affective agnosia: Expansion of the alexithymia construct and a new opportunity to integrate and extend Freud's legacy.

    PubMed

    Lane, Richard D; Weihs, Karen L; Herring, Anne; Hishaw, Alex; Smith, Ryan

    2015-08-01

    We describe a new type of agnosia consisting of an impairment in the ability to mentally represent or know what one is feeling. Freud the neurologist coined the term "agnosia" in 1891 before creating psychoanalysis in 1895 but the term has not been previously applied to the domain of affective processing. We propose that the concept of "affective agnosia" advances the theory, measurement and treatment of what is now called "alexithymia," meaning "lack of words for emotion." We trace the origin of the alexithymia construct and discuss the strengths and limitations of extant research. We review evidence that the ability to represent and put emotions into words is a developmental achievement that strongly influences one's ability to experience, recognize, understand and use one's own emotional responses. We describe the neural substrates of emotional awareness and affective agnosia and compare and contrast these with related conditions. We then describe how this expansion of the conceptualization and measurement of affective processing deficits has important implications for basic emotion research and clinical practice. PMID:26054794

  15. Affective agnosia: Expansion of the alexithymia construct and a new opportunity to integrate and extend Freud's legacy.

    PubMed

    Lane, Richard D; Weihs, Karen L; Herring, Anne; Hishaw, Alex; Smith, Ryan

    2015-08-01

    We describe a new type of agnosia consisting of an impairment in the ability to mentally represent or know what one is feeling. Freud the neurologist coined the term "agnosia" in 1891 before creating psychoanalysis in 1895 but the term has not been previously applied to the domain of affective processing. We propose that the concept of "affective agnosia" advances the theory, measurement and treatment of what is now called "alexithymia," meaning "lack of words for emotion." We trace the origin of the alexithymia construct and discuss the strengths and limitations of extant research. We review evidence that the ability to represent and put emotions into words is a developmental achievement that strongly influences one's ability to experience, recognize, understand and use one's own emotional responses. We describe the neural substrates of emotional awareness and affective agnosia and compare and contrast these with related conditions. We then describe how this expansion of the conceptualization and measurement of affective processing deficits has important implications for basic emotion research and clinical practice.

  16. A TNFR2-Agonist Facilitates High Purity Expansion of Human Low Purity Treg Cells.

    PubMed

    He, Xuehui; Landman, Sija; Bauland, Stijn C G; van den Dolder, Juliette; Koenen, Hans J P M; Joosten, Irma

    2016-01-01

    Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for ex vivo expansion. Although therapeutic Treg flow-sorting is feasible, most centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFNγ and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize ex vivo Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that ex vivo expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the FOXP3 gene. These cells reveal a low IL-17A and IFNγ producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation in vivo. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the ex vivo expansion of Treg for clinical immunotherapy. PMID:27224512

  17. A TNFR2-Agonist Facilitates High Purity Expansion of Human Low Purity Treg Cells

    PubMed Central

    Landman, Sija; Bauland, Stijn C. G.; van den Dolder, Juliette

    2016-01-01

    Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for ex vivo expansion. Although therapeutic Treg flow-sorting is feasible, most centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFNγ and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize ex vivo Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that ex vivo expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the FOXP3 gene. These cells reveal a low IL-17A and IFNγ producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation in vivo. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the ex vivo expansion of Treg for clinical immunotherapy. PMID:27224512

  18. Advances in umbilical cord blood stem cell expansion and clinical translation.

    PubMed

    Pineault, Nicolas; Abu-Khader, Ahmad

    2015-07-01

    Umbilical cord blood (CB) is a rich source of hematopoietic stem cells (HSCs) with important applications in allogeneic stem cell transplantation. However, the low numbers of hematopoietic stem and progenitor cells (HSPCs) in banked units remain a major limitation. Protocols developed for HSPC expansion ex vivo or to improve HSPC homing to the marrow represent solutions to overcome this shortcoming. In recent decades, wide arrays of functionally divergent approaches were developed for the amplification of HSPCs. These include optimization of cytokine cocktails, coculture systems, small molecules, and delivery systems for HSPC-expansion genes. Herein, we review past and current strategies, focusing on studies that characterize the contribution of expanded CB HSPC to short- and long-term engraftment in transplantation models or in clinical trials. Also discussed are homing effectors used to promote engraftment. In summary, these studies underscore that early-acting cytokines alone can expand HSPC with short-term engraftment activity, but that robust expansion of HSPCs with long-term engraftment necessitates the synergistic action of multiple HSC-expansion agonists. In support of this, early clinical trials based on cytokine-driven HSPC-expansion protocols delivered disappointing results, whereas recent trials based on the synergistic action of cytokines and HSPC-expansion agonists reported significant improvements in engraftment and therapeutic outcomes. Conversely, molecules that enhance homing of HSPC may represent a complementary approach to improve and perhaps accelerate engraftment. Optimization of the next generation of HSPC-expansion and priming strategies should support a paradigm shift in CB transplantation in which smaller, better matched units may preferentially be used.

  19. Expansion of embryonic stem cells in suspension and fibrous bed bioreactors.

    PubMed

    Liu, Ning; Li, Yan; Yang, Shang-Tian

    2014-05-20

    Applications of embryonic stem (ES) cells in cellular transplantation and tissue engineering require scalable processes for mass production of these cells with controlled qualities. The main objective of this work was to evaluate two cell culture processes for long-term expansion of murine embryonic stem (mES) cells. With serial passaging, suspension cultures in spinner flasks were able to expand mES cells as aggregates for 12.5-fold in each passage of 4 days. However, extending the culturing time to 6 days in each passage caused significant loss in cell viability and induced differentiation as indicated by the reduced expression levels of SSEA-1 and Oct-4. Long-term expansion of mES cells in a fibrous bed bioreactor (FBB) was also studied for 30 days in 2 passages, 15 days in each passage. With periodically refreshing the culture medium, a high expansion fold of 60-77 was achieved in each passage. Flow cytometry and RT-PCR were used to analyze key pluripotency and differentiation markers. The results showed that the expanded cells in both suspension and FBB cultures remained in a highly pluripotent state, which was also confirmed with the embryoid body (EB) forming efficiency test. It is concluded that both the suspension and FBB cultures are suitable to support long-term expansion of undifferentiated mES cells. However, the FBB culture can sustain cell growth for a longer period without frequent passaging, requires less media and labor, and is thus more economical to use for mass production of ES cells.

  20. Dendritic cells control fibroblastic reticular network tension and lymph node expansion.

    PubMed

    Acton, Sophie E; Farrugia, Aaron J; Astarita, Jillian L; Mourão-Sá, Diego; Jenkins, Robert P; Nye, Emma; Hooper, Steven; van Blijswijk, Janneke; Rogers, Neil C; Snelgrove, Kathryn J; Rosewell, Ian; Moita, Luis F; Stamp, Gordon; Turley, Shannon J; Sahai, Erik; Reis e Sousa, Caetano

    2014-10-23

    After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity.

  1. High efficacy of clonal growth and expansion of adult neural stem cells.

    PubMed

    Wachs, Frank-Peter; Couillard-Despres, Sebastien; Engelhardt, Maren; Wilhelm, Daniel; Ploetz, Sonja; Vroemen, Maurice; Kaesbauer, Johanna; Uyanik, Goekhan; Klucken, Jochen; Karl, Claudia; Tebbing, Johanna; Svendsen, Clive; Weidner, Norbert; Kuhn, Hans-Georg; Winkler, Juergen; Aigner, Ludwig

    2003-07-01

    Neural stem cells (NSCs) from the adult central nervous system are currently being investigated for their potential use in autologous cell replacement strategies. High expansion rates of NSCs in culture are crucial for the generation of a sufficient amount of cells needed for transplantation. Here, we describe efficient growth of adult NSCs in Neurobasal medium containing B27 supplement under clonal and low-density conditions in the absence of serum or conditioned medium. Expansion of up to 15-fold within 1 week was achieved on low-density NSC cultures derived from the lateral ventricle wall, the hippocampal formation, and the spinal cord of adult rats. A 27% single-cell cloning efficiency in Neurobasal/B27 combination further demonstrates its growth-promoting ability. Multipotency and nontumorgenicity of NSCs were retained despite the high rate of culture expansion. In addition, increased cell survival was obtained when Accutase, instead of trypsin, was used for enzymatic dissociation of NSC cultures. This work provides an important step toward the development of standardized protocols for highly efficient in vitro expansion of NSCs from the adult central nervous system to move more closely to the clinical use of NSCs. PMID:12861035

  2. Single-Pass, Closed-System Rapid Expansion of Lymphocyte Cultures for Adoptive Cell Therapy

    PubMed Central

    Klapper, Jacob A.; Thomasian, Armen A.; Smith, Douglas M.; Gorgas, Gayle C.; Wunderlich, John R.; Smith, Franz O.; Hampson, Brian S.; Rosenberg, Steven A.; Dudley, Mark E.

    2009-01-01

    Adoptive cell therapy (ACT) for metastatic melanoma involves the ex vivo expansion and re-infusion of tumor infiltrating lymphocytes (TIL) obtained from resected specimens. With an overall objective response rate of fifty-six percent, this T-cell immunotherapy provides an appealing alternative to other therapies, including conventional therapies with lower response rates. However, there are significant regulatory and logistical concerns associated with the ex vivo activation and large scale expansion of these cells. The best current practice uses a rapid expansion protocol (REP) consisting of an ex vivo process that occurs in tissue culture flasks (T-flasks) and gas-permeable bags, utilizes OKT3 (anti-CD3 monoclonal antibody), recombinant human interleukin-2, and irradiated peripheral blood mononuclear cells to initiate rapid lymphocyte growth. A major limitation to the widespread delivery of therapy to large numbers of melanoma patients is the open system in which a REP is initiated. To address this problem, we have investigated the initiation, expansion and harvest at clinical scale of TIL in a closed-system continuous perfusion bioreactor. Each cell product met all safety criteria for patient treatment and by head-to-head comparison had a similar potency and phenotype as cells grown in control T-flasks and gas-permeable bags. However, the currently available bioreactor cassettes were limited in the total cell numbers that could be generated. This bioreactor may simplify the process of the rapid expansion of TIL under stringent regulatory conditions thereby enabling other institutions to pursue this form of ACT. PMID:19389403

  3. Enhancing Post-Expansion Chondrogenic Potential of Costochondral Cells in Self-Assembled Neocartilage

    PubMed Central

    Murphy, Meghan K.; Huey, Daniel J.; Reimer, Andrew J.; Hu, Jerry C.; Athanasiou, Kyriacos A.

    2013-01-01

    The insufficient healing capacity of articular cartilage necessitates mechanically functional biologic tissue replacements. Using cells to form biomimetic cartilage implants is met with the challenges of cell scarcity and donor site morbidity, requiring expanded cells that possess the ability to generate robust neocartilage. To address this, this study assesses the effects of expansion medium supplementation (bFGF, TFP, FBS) and self-assembled construct seeding density (2, 3, 4 million cells/5 mm dia. construct) on the ability of costochondral cells to generate biochemically and biomechanically robust neocartilage. Results show TFP (1 ng/mL TGF-β1, 5 ng/mL bFGF, 10 ng/mL PDGF) supplementation of serum-free chondrogenic expansion medium enhances the post-expansion chondrogenic potential of costochondral cells, evidenced by increased glycosaminoglycan content, decreased type I/II collagen ratio, and enhanced compressive properties. Low density (2 million cells/construct) enhances matrix synthesis and tensile and compressive mechanical properties. Combined, TFP and Low density interact to further enhance construct properties. That is, with TFP, Low density increases type II collagen content by over 100%, tensile stiffness by over 300%, and compressive moduli by over 140%, compared with High density. In conclusion, the interaction of TFP and Low density seeding enhances construct material properties, allowing for a mechanically functional, biomimetic cartilage to be formed using clinically relevant costochondral cells. PMID:23437288

  4. Satellite cell activity, without expansion, after nonhypertrophic stimuli.

    PubMed

    Joanisse, Sophie; McKay, Bryon R; Nederveen, Joshua P; Scribbans, Trisha D; Gurd, Brendon J; Gillen, Jenna B; Gibala, Martin J; Tarnopolsky, Mark; Parise, Gianni

    2015-11-01

    The purpose of the present studies was to determine the effect of various nonhypertrophic exercise stimuli on satellite cell (SC) pool activity in human skeletal muscle. Previously untrained men and women (men: 29 ± 9 yr and women: 29 ± 2 yr, n = 7 each) completed 6 wk of very low-volume high-intensity sprint interval training. In a separate study, recreationally active men (n = 16) and women (n = 3) completed 6 wk of either traditional moderate-intensity continuous exercise (n = 9, 21 ± 4 yr) or low-volume sprint interval training (n = 10, 21 ± 2 yr). Muscle biopsies were obtained from the vastus lateralis before and after training. The fiber type-specific SC response to training was determined, as was the activity of the SC pool using immunofluorescent microscopy of muscle cross sections. Training did not induce hypertrophy, as assessed by muscle cross-sectional area, nor did the SC pool expand in any group. However, there was an increase in the number of active SCs after each intervention. Specifically, the number of activated (Pax7(+)/MyoD(+), P ≤ 0.05) and differentiating (Pax7(-)/MyoD(+), P ≤ 0.05) SCs increased after each training intervention. Here, we report evidence of activated and cycling SCs that may or may not contribute to exercise-induced adaptations while the SC pool remains constant after three nonhypertrophic exercise training protocols.

  5. Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion.

    PubMed

    Chopra, Martin; Biehl, Marlene; Steinfatt, Tim; Brandl, Andreas; Kums, Juliane; Amich, Jorge; Vaeth, Martin; Kuen, Janina; Holtappels, Rafaela; Podlech, Jürgen; Mottok, Anja; Kraus, Sabrina; Jordán-Garrote, Ana-Laura; Bäuerlein, Carina A; Brede, Christian; Ribechini, Eliana; Fick, Andrea; Seher, Axel; Polz, Johannes; Ottmüller, Katja J; Baker, Jeanette; Nishikii, Hidekazu; Ritz, Miriam; Mattenheimer, Katharina; Schwinn, Stefanie; Winter, Thorsten; Schäfer, Viktoria; Krappmann, Sven; Einsele, Hermann; Müller, Thomas D; Reddehase, Matthias J; Lutz, Manfred B; Männel, Daniela N; Berberich-Siebelt, Friederike; Wajant, Harald; Beilhack, Andreas

    2016-08-22

    Donor CD4(+)Foxp3(+) regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell-dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo. PMID:27526711

  6. Expansion of prostate epithelial progenitor cells after inflammation of the mouse prostate

    PubMed Central

    Wang, Liang; Zoetemelk, Marloes; Chitteti, Brahmananda R.; Ratliff, Timothy L.; Myers, Jason D.; Srour, Edward F.; Broxmeyer, Hal

    2015-01-01

    Prostatic inflammation is a nearly ubiquitous pathological feature observed in specimens from benign prostate hyperplasia and prostate cancer patients. The microenvironment of the inflamed prostate is highly reactive, and epithelial hyperplasia is a hallmark feature of inflamed prostates. How inflammation orchestrates epithelial proliferation as part of its repair and recovery action is not well understood. Here, we report that a novel epithelial progenitor cell population is induced to expand during inflammation. We used sphere culture assays, immunofluorescence, and flow cytometry to show that this population is increased in bacterially induced inflamed mouse prostates relative to naïve control prostates. We confirmed from previous reports that this population exclusively possesses the ability to regrow entire prostatic structures from single cell culture using renal grafts. In addition, putative progenitor cells harvested from inflamed animals have greater aggregation capacity than those isolated from naïve control prostates. Expansion of this critical cell population requires IL-1 signaling, as IL-1 receptor 1-null mice exhibit inflammation similar to wild-type inflamed animals but exhibit significantly reduced progenitor cell proliferation and hyperplasia. These data demonstrate that inflammation promotes hyperplasia in the mouse prostatic epithelium by inducing the expansion of a selected epithelial progenitor cell population in an IL-1 receptor-dependent manner. These findings may have significant impact on our understanding of how inflammation promotes proliferative diseases such as benign prostatic hyperplasia and prostate cancer, both of which depend on expansion of cells that exhibit a progenitor-like nature. PMID:25925259

  7. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    PubMed

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  8. Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system.

    PubMed

    Mizukami, Amanda; Orellana, Maristela D; Caruso, Sâmia R; de Lima Prata, Karen; Covas, Dimas T; Swiech, Kamilla

    2013-01-01

    The need for efficient and reliable technologies for clinical-scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood-derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10(7) cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10(8) cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra-Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier-based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical-scale production system.

  9. Substrate properties affect collective cell motion

    NASA Astrophysics Data System (ADS)

    Pegoraro, Adrian; Guo, Ming; Ehrlicher, Allen; Weitz, David

    2013-03-01

    When cells move collectively, cooperative motion, which is characterized by long range correlations in cell movement, is necessary for migration. This collective cell motion is influenced by cell-cell interactions as well as by cell-substrate coupling. Furthermore, on soft substrates it is possible for cells to mechanically couple over long distances through the substrate itself. By changing the properties of the substrate, it is possible to decouple some of these contributions and better understand the role they play in collective cell motion. We vary both the substrate stiffness and adhesion protein concentration and find changes in the collective cell motion of the cells despite only small differences in total cell density and average cell size in the confluent layers. We test these changes on polyacrylamide and PDMS substrates as well as on structured substrates made of PDMS posts that prevent mechanical coupling through the substrate while still allowing stiffness to be varied.

  10. Expansion of CD8+CD57+ T Cells in an Immunocompetent Patient with Acute Toxoplasmosis

    PubMed Central

    García-Muñoz, R.; Rodríguez-Otero, P.; Galar, A.; Merino, J.; Beunza, J. J.; Páramo, J. A.; Lecumberri, R.

    2009-01-01

    CD57+ T cells increase in several viral infections like cytomegalovirus, herpesvirus, parvovirus, HIV and hepatitis C virus and are associated with several clinical conditions related to immune dysfunction and ageing. We report for the first time an expansion of CD8+ CD57+ T cells in a young patient with an acute infection with Toxoplasma gondii. Our report supports the concept that CD8+ CD57+ T cells could be important in the control of chronic phase of intracellular microorganisms and that the high numbers of these cells may reflect the continuing survey of the immune system, searching for parasite proliferation in the tissues. PMID:19946421

  11. Expansion of CD8+CD57+ T Cells in an Immunocompetent Patient with Acute Toxoplasmosis.

    PubMed

    García-Muñoz, R; Rodríguez-Otero, P; Galar, A; Merino, J; Beunza, J J; Páramo, J A; Lecumberri, R

    2009-01-01

    CD57+ T cells increase in several viral infections like cytomegalovirus, herpesvirus, parvovirus, HIV and hepatitis C virus and are associated with several clinical conditions related to immune dysfunction and ageing. We report for the first time an expansion of CD8+ CD57+ T cells in a young patient with an acute infection with Toxoplasma gondii. Our report supports the concept that CD8+ CD57+ T cells could be important in the control of chronic phase of intracellular microorganisms and that the high numbers of these cells may reflect the continuing survey of the immune system, searching for parasite proliferation in the tissues.

  12. Ex vivo expansion of circulating CD34(+) cells enhances the regenerative effect on rat liver cirrhosis.

    PubMed

    Nakamura, Toru; Koga, Hironori; Iwamoto, Hideki; Tsutsumi, Victor; Imamura, Yasuko; Naitou, Masako; Masuda, Atsutaka; Ikezono, Yu; Abe, Mitsuhiko; Wada, Fumitaka; Sakaue, Takahiko; Ueno, Takato; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Asahara, Takayuki; Torimura, Takuji

    2016-01-01

    Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with liver cirrhosis. The aim of this study was to investigate the efficacy of human ex vivo-expanded CD34(+) cells for treatment of cirrhotic rat liver. Recipient rats were intraperitoneally injected with CCl4 twice weekly for 3 weeks before administration of CD34(+) cells. CCl4 was then re-administered twice weekly for 3 more weeks, and the rats were sacrificed. Saline, nonexpanded or expanded CD34(+) cells were injected via the spleen. After 7 days, CD34(+) cells were effectively expanded in a serum-free culture medium. Expanded CD34(+) cells were also increasingly positive for cell surface markers of VE-cadherin, VEGF receptor-2, and Tie-2. The expression of proangiogenic growth factors and adhesion molecules in expanded CD34(+) cells increased compared with nonexpanded CD34(+) cells. Expanded CD34(+) cell transplantation reduced liver fibrosis, with a decrease of αSMA(+) cells. Assessments of hepatocyte and sinusoidal endothelial cell proliferative activity indicated the superior potency of expanded CD34(+) cells over non-expanded CD34(+) cells. The inhibition of integrin αvβ3 and αvβ5 disturbed the engraftment of transplanted CD34(+) cells and aggravated liver fibrosis. These findings suggest that expanded CD34(+) cells enhanced the preventive efficacy of cell transplantation in a cirrhotic model. PMID:27162932

  13. Ex vivo expansion of circulating CD34+ cells enhances the regenerative effect on rat liver cirrhosis

    PubMed Central

    Nakamura, Toru; Koga, Hironori; Iwamoto, Hideki; Tsutsumi, Victor; Imamura, Yasuko; Naitou, Masako; Masuda, Atsutaka; Ikezono, Yu; Abe, Mitsuhiko; Wada, Fumitaka; Sakaue, Takahiko; Ueno, Takato; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Asahara, Takayuki; Torimura, Takuji

    2016-01-01

    Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with liver cirrhosis. The aim of this study was to investigate the efficacy of human ex vivo-expanded CD34+ cells for treatment of cirrhotic rat liver. Recipient rats were intraperitoneally injected with CCl4 twice weekly for 3 weeks before administration of CD34+ cells. CCl4 was then re-administered twice weekly for 3 more weeks, and the rats were sacrificed. Saline, nonexpanded or expanded CD34+ cells were injected via the spleen. After 7 days, CD34+ cells were effectively expanded in a serum-free culture medium. Expanded CD34+ cells were also increasingly positive for cell surface markers of VE-cadherin, VEGF receptor-2, and Tie-2. The expression of proangiogenic growth factors and adhesion molecules in expanded CD34+ cells increased compared with nonexpanded CD34+ cells. Expanded CD34+ cell transplantation reduced liver fibrosis, with a decrease of αSMA+ cells. Assessments of hepatocyte and sinusoidal endothelial cell proliferative activity indicated the superior potency of expanded CD34+ cells over non-expanded CD34+ cells. The inhibition of integrin αvβ3 and αvβ5 disturbed the engraftment of transplanted CD34+ cells and aggravated liver fibrosis. These findings suggest that expanded CD34+ cells enhanced the preventive efficacy of cell transplantation in a cirrhotic model. PMID:27162932

  14. IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection

    PubMed Central

    Younes, Souheil-Antoine; Freeman, Michael L.; Mudd, Joseph C.; Shive, Carey L.; Reynaldi, Arnold; Estes, Jacob D.; Deleage, Claire; Lucero, Carissa; Anderson, Jodi; Schacker, Timothy W.; Davenport, Miles P.; McCune, Joseph M.; Hunt, Peter W.; Lee, Sulggi A.; Debernardo, Robert L.; Jacobson, Jeffrey M.; Canaday, David H.; Sekaly, Rafick-Pierre; Sieg, Scott F.; Lederman, Michael M.

    2016-01-01

    In HIV-1–infected patients, increased numbers of circulating CD8+ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B+, CD8+ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B+ phenotype in CD8+ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8+ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8+ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8+ T cell expansion that is linked to increased morbidity and mortality in treated patients. PMID:27322062

  15. Area Expansivity Moduli of Regenerating Plant Protoplast Cell Walls Exposed to Shear Flows

    NASA Astrophysics Data System (ADS)

    Fujimura, Yuu; Iino, Masaaki; Watanabe, Ugai

    2005-05-01

    To control the elasticity of the plant cell wall, protoplasts isolated from cultured Catharanthus roseus cells were regenerated in shear flows of 115 s-1 (high shear) and 19.2 s-1 (low shear, as a control). The surface area expansivity modulus and the surface breaking strength of these regenerating protoplasts were measured by a micropipette aspiration technique. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye. High shear exposure for 3 h doubled both the surface area modulus and breaking strength observed under low shear, significantly decreased cell wall synthesis, and roughly quadrupled the moduli of the cell wall. Based on the cell wall synthesis data, we estimated the three-dimensional modulus of the cell wall to be 4.1± 1.2 GPa for the high shear, and 0.35± 0.2 GPa for the low shear condition, using the surface area expansivity modulus divided by the cell wall thickness, which is identical with the Young’s modulus divided by 2(1-σ), where σ is Poisson's ratio. We concluded that high shear exposure considerably strengthens the newly synthesized cell wall.

  16. Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway.

    PubMed

    Mah, In Kyoung; Soloff, Rachel; Hedrick, Stephen M; Mariani, Francesca V

    2015-11-10

    The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.

  17. With respect to coefficient of linear thermal expansion, bacterial vegetative cells and spores resemble plastics and metals, respectively

    PubMed Central

    2013-01-01

    Background If a fixed stress is applied to the three-dimensional z-axis of a solid material, followed by heating, the amount of thermal expansion increases according to a fixed coefficient of thermal expansion. When expansion is plotted against temperature, the transition temperature at which the physical properties of the material change is at the apex of the curve. The composition of a microbial cell depends on the species and condition of the cell; consequently, the rate of thermal expansion and the transition temperature also depend on the species and condition of the cell. We have developed a method for measuring the coefficient of thermal expansion and the transition temperature of cells using a nano thermal analysis system in order to study the physical nature of the cells. Results The tendency was seen that among vegetative cells, the Gram-negative Escherichia coli and Pseudomonas aeruginosa have higher coefficients of linear expansion and lower transition temperatures than the Gram-positive Staphylococcus aureus and Bacillus subtilis. On the other hand, spores, which have low water content, overall showed lower coefficients of linear expansion and higher transition temperatures than vegetative cells. Comparing these trends to non-microbial materials, vegetative cells showed phenomenon similar to plastics and spores showed behaviour similar to metals with regards to the coefficient of liner thermal expansion. Conclusions We show that vegetative cells occur phenomenon of similar to plastics and spores to metals with regard to the coefficient of liner thermal expansion. Cells may be characterized by the coefficient of linear expansion as a physical index; the coefficient of linear expansion may also characterize cells structurally since it relates to volumetric changes, surface area changes, the degree of expansion of water contained within the cell, and the intensity of the internal stress on the cellular membrane. The coefficient of linear expansion holds

  18. Fallopian tube secretory cell expansion: a sensitive biomarker for ovarian serous carcinogenesis

    PubMed Central

    Wang, Yiying; Li, Li; Wang, Yue; Tang, Sarah Ngocvi; Zheng, Wenxin

    2016-01-01

    Recent advances suggest that precancerous lesions of pelvic serous carcinoma originate from tubal secretory cells. The purpose of our study was to determine if an increased number of secretory cells vary with age or location in the fallopian tube and to examine its association with serous neoplasia. Three groups (benign control, high-risk, and pelvic serous carcinoma) of age-matched patients were studied. The age data were stratified into 10-year intervals ranging from 20-29 to older than 80. The number of secretory and ciliated cells from both tubal fimbria and ampulla segments was counted by microscopy and immunohistochemical staining methods. The data were analyzed by standard contingency table and Poisson distribution methods after age justification. We found that the absolute number of tubal secretory cells increased significantly with age in all three groups. But a more dramatic increase of secretory cells was observed in high-risk and pelvic serous carcinoma patients. Secretory cell expansion is more prevalent than secretory cell outgrowth in both fimbria and ampulla tubal segments and is significantly associated with serous neoplasia (p < 0.001). Furthermore, age remained a significant risk factor for serous neoplasia after age adjustment. These findings suggest that secretory cell expansion could serve as a potential sensitive biomarker for early serous carcinogenesis within the fallopian tube. The study also supports a relationship between serous neoplasia and increased secretory to ciliated cell ratios, and the relationship between frequency of secretory cell expansion within the fallopian tube and increasing age and-more significantly-presence of high-risk factors or co-existing serous cancers. PMID:27069556

  19. Notch signaling: from stem cell expansion to improving cord blood transplantation.

    PubMed

    Mayani, Hector

    2010-08-01

    Ex vivo expansion of hematopoietic stem and progenitor cells has been a major goal for experimental hematologists and stem cell biologists during the last two decades. The clinical implications of such a procedure are obvious, considering the increasing interest in cell therapy protocols. This is particularly true in the setting of cord blood transplants, in which increased numbers of such primitive cells are needed. The study analyzed in this article indicates that by stimulating the Notch signal transduction pathway in primitive cord blood cells it is possible to significantly increase the numbers of both hematopoietic stem and progenitor cells. Furthermore, infusion of such expanded cells in patients receiving a cord blood transplant results in a significant reduction in the time to myeloid engraftment. The relevance of this study is twofold--on the one hand, it shows that the Notch pathway is involved in the expansion capacity of primitive hematopoietic cells in culture, and on the other hand, it indicates that ex vivo-expanded stem/progenitor cells can have a role in hematopoietic transplantation settings. PMID:21083031

  20. Antigen-specific activation and cytokine-facilitated expansion of naive, human CD8+ T cells

    PubMed Central

    Wölfl, Matthias; Greenberg, Philip D

    2014-01-01

    Antigen-specific priming of human, naïve T-cells has been difficult to assess. Due to the low initial frequency in the naïve cell pool of specific T-cell precursors, such an analysis has been obscured by the requirements for repeated stimulations and prolonged culture time. In this protocol, we describe how to rapidly evaluate antigen-specific priming of CD8+ -cells following a single stimulation. The assay provides reference conditions, which result in the expansion of a significant population of antigen-specific T-cells from the naïve repertoire. Various conditions and modifications during the priming process (e.g. testing new cytokines, costimulators, etc.) can now be directly compared to the reference conditions. Factors relevant to achieving effective priming include the dendritic cell preparation, the T-cell preparation, the cell ratio at the time of priming, the serum source used for the experiment, and the timing of addition and concentration of the cytokines used for expansion. This protocol is relevant for human immunology, vaccine biology and drug development. PMID:24675735

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

    PubMed

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

    2014-06-01

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

  2. Modeling diseases of noncoding unstable repeat expansions using mutant pluripotent stem cells

    PubMed Central

    Yanovsky-Dagan, Shira; Mor-Shaked, Hagar; Eiges, Rachel

    2015-01-01

    Pathogenic mutations involving DNA repeat expansions are responsible for over 20 different neuronal and neuromuscular diseases. All result from expanded tracts of repetitive DNA sequences (mostly microsatellites) that become unstable beyond a critical length when transmitted across generations. Nearly all are inherited as autosomal dominant conditions and are typically associated with anticipation. Pathologic unstable repeat expansions can be classified according to their length, repeat sequence, gene location and underlying pathologic mechanisms. This review summarizes the current contribution of mutant pluripotent stem cells (diseased human embryonic stem cells and patient-derived induced pluripotent stem cells) to the research of unstable repeat pathologies by focusing on particularly large unstable noncoding expansions. Among this class of disorders are Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome, myotonic dystrophy type 1 and myotonic dystrophy type 2, Friedreich ataxia and C9 related amyotrophic lateral sclerosis and/or frontotemporal dementia, Facioscapulohumeral Muscular Dystrophy and potentially more. Common features that are typical to this subclass of conditions are RNA toxic gain-of-function, epigenetic loss-of-function, toxic repeat-associated non-ATG translation and somatic instability. For each mechanism we summarize the currently available stem cell based models, highlight how they contributed to better understanding of the related mechanism, and discuss how they may be utilized in future investigations. PMID:26131313

  3. Enrichment and Expansion of Cells using Antibody-Coated Micropallet Arrays

    PubMed Central

    Shadpour, Hamed; Sims, Christopher E.; Allbritton, Nancy L.

    2009-01-01

    Background Positive selection, sorting, and collection of single cells from within a heterogeneous population are required for many biological studies. We recently demonstrated a miniaturized cell array for this purpose; however, on-chip pre-enrichment and isolation of specific target cells would provide significant value for cell isolation. Methods In the current work, mixed cell samples of fewer than 30,000 cells were used for panning by means of on-array antibody-capture to pre-enrich the target population. The cell surface receptors FcεR1, c-Kit, and ErbB2 were used for positive selection of RBL, RBL, and SK-BR-3 cells, respectively, from the mixed population. The capture efficiency, selectivity, and enrichment for the target cells were calculated and compared to fibronectin-coated controls. Results As expected, the capture efficiency depended upon the frequency of the target cell in the mixed population over the range of 0.3% to 33%. For a frequency of 5% target cells, the capture efficiency was 39 – 53% for the three conditions, while the selectivity varied between 78 – 98% with 16 – 20 fold enrichment. Furthermore, single-cell cloning studies demonstrated a high cloning efficiency of target cells selectively isolated from the array. Conclusions Antibody-based pre-enrichment in combination with micropallet-based cell selection will be a valuable tool for isolation and expansion of rare cells from small heterogeneous populations. PMID:19504569

  4. TiO2-doped phosphate glass microcarriers: A stable bioactive substrate for expansion of adherent mammalian cells

    PubMed Central

    Guedes, Joana C; Park, Jeong-Hui; Lakhkar, Nilay J; Kim, Hae-Won; Knowles, Jonathan C

    2013-01-01

    Scalable expansion of cells for regenerative cell therapy or to produce large quantities for high-throughput screening remains a challenge for bioprocess engineers. Laboratory scale cell expansion using t-flasks requires frequent passaging that exposes cells to many poorly defined bioprocess forces that can cause damage or alter their phenotype. Microcarriers offer a potential solution to scalable production, lending themselves to cell culture processes more akin to fermentation, removing the need for frequent passaging throughout the expansion period. One main problem with microcarrier expansion, however, is the difficulty in harvesting cells at the end of the process. Therefore, therapies that rely on cell delivery using biomaterial scaffolds could benefit from a microcarrier expansion system whereby the cells and microcarriers are transplanted together. In the current study, we used bioactive glass microcarriers doped with 5% TiO2 that display a controlled rate of degradation and conducted experiments to assess biocompatibility and growth of primary fibroblast cells as a model for cell therapy products. We found that the microcarriers are highly biocompatible and facilitate cell growth in a gradual controlled manner. Therefore, even without additional biofunctionalization methods, Ti-doped bioactive glass microcarriers offer potential as a cell expansion platform. PMID:22935537

  5. ABA Inhibits Embryo Cell Expansion and Early Cell Division Events During Coffee (Coffea arabica ‘Rubi’) Seed Germination

    PubMed Central

    Da Silva, E. A. Amaral; Toorop, Peter E.; Van Lammeren, André A. M.; Hilhorst, Henk W. M.

    2008-01-01

    Background and Aims Coffee seed germination represents an interplay between the embryo and the surrounding endosperm. A sequence of events in both parts of the seed determines whether germination will be successful or not. Following previous studies, the aim here was to further characterize the morphology of endosperm degradation and embryo growth with respect to morphology and cell cycle, and the influence of abscisic acid on these processes. Methods Growth of cells in a fixed region of the axis was quantified from light micrographs. Cell cycle events were measured by flow cytometry and by immunocytochemistry, using antibodies against β-tubulin. Aspects of the endosperm were visualized by light and scanning electron microscopy. Key Results The embryonic axis cells grew initially by isodiametric expansion. This event coincided with reorientation and increase in abundance of microtubules and with accumulation of β-tubulin. Radicle protrusion was characterized by a shift from isodiametric expansion to elongation of radicle cells and further accumulation of β-tubulin. Early cell division events started prior to radicle protrusion. Abscisic acid decreased the abundance of microtubules and inhibited the growth of the embryo cells, the reorganization of the microtubules, DNA replication in the embryonic axis, the formation of a protuberance and the completion of germination. The endosperm cap cells had smaller and thinner cell walls than the rest of the endosperm. Cells in the endosperm cap displayed compression followed by loss of cell integrity and the appearance of a protuberance prior to radicle protrusion. Conclusions Coffee seed germination is the result of isodiametric growth of the embryo followed by elongation, at the expense of integrity of endosperm cap cells. The cell cycle, including cell division, is initiated prior to radicle protrusion. ABA inhibits expansion of the embryo, and hence subsequent events, including germination. PMID:18617534

  6. A comprehensive platform for ex vivo T-cell expansion based on biodegradable polymeric artificial antigen-presenting cells.

    PubMed

    Steenblock, Erin R; Fahmy, Tarek M

    2008-04-01

    Efficient T-cell stimulation and proliferation in response to specific antigens is a goal of immunotherapy against infectious disease and cancer. Manipulation of this response can be accomplished by adoptive immunotherapy involving the infusion of antigen-specific T-cell populations expanded ex vivo with antigen presenting cells. We mimicked physiological antigen presentation on a biodegradable microparticle constructed from poly(lactide-co-glycolide) (PLGA), a polymer system whose safety has been established for use in humans. These particles present a high density of adaptor elements for attaching both recognition ligands and co-stimulatory ligands to a biodegradable core encapsulating the cytokine interleukin-2 (IL-2). We demonstrate the utility of this system in efficient polyclonal and antigen-specific T-cell stimulation and expansion, showing that sustained release of IL-2 in the vicinity of T-cell contacts dramatically improves the stimulatory capacity of these acellular systems, as compared to the effect of exogenous addition of cytokine. This results in a 45-fold enhancement in T-cell expansion. In addition, this mode of antigen presentation skews the expansion toward the CD8(+) T-cell phenotype. This comprehensive acellular platform, capable of delivering recognition, co-stimulatory, and cytokine signals, represents a promising new technology for artificial antigen presentation. PMID:18334990

  7. Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+ Cells

    PubMed Central

    Xie, Hui; Sun, Li; Zhang, Liming; Liu, Teng; Chen, Li; Zhao, Aiqi; Lei, Qian; Gao, Fei; Zou, Ping; Li, Qiubai; Guo, An-yuan; Chen, Zhichao; Wang, Hongxiang

    2016-01-01

    Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+ cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation. PMID:27042183

  8. Clinical grade purification and expansion of NK cell products for an optimized manufacturing protocol.

    PubMed

    Koehl, Ulrike; Brehm, Claudia; Huenecke, Sabine; Zimmermann, Stefanie-Yvonne; Kloess, Stephan; Bremm, Melanie; Ullrich, Evelyn; Soerensen, Jan; Quaiser, Andrea; Erben, Stephanie; Wunram, Claudia; Gardlowski, Tanja; Auth, Eileen; Tonn, Torsten; Seidl, Christian; Meyer-Monard, Sandrine; Stern, Martin; Passweg, Jakob; Klingebiel, Thomas; Bader, Peter; Schwabe, Dirk; Esser, Ruth

    2013-01-01

    Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 10(8) CD56(+)CD3(-) cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56(+)CD3(-) NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.

  9. Thermoresponsive worms for expansion and release of human embryonic stem cells.

    PubMed

    Chen, Xiaoli; Prowse, Andrew B J; Jia, Zhongfan; Tellier, Helena; Munro, Trent P; Gray, Peter P; Monteiro, Michael J

    2014-03-10

    The development of robust suspension cultures of human embryonic stem cells (hESCs) without the use of cell membrane disrupting enzymes or inhibitors is critical for future clinical applications in regenerative medicine. We have achieved this by using long, flexible, and thermoresponsive polymer worms decorated with a recombinant vitronectin subdomain that bridge hESCs, aiding in hESC's natural ability to form embryoid bodies (EBs) and satisfying their inherent requirement for cell-cell and cell-extracellular matrix contact. When the EBs reached an optimal upper size where cytokine and nutrient penetration becomes limiting, these long and flexible polymer worms facilitated EB breakdown via a temperature shift from 37 to 25 °C. The thermoresponsive nature of the worms enabled a cyclical dissociation and propagation of the cells. Repeating the process for three cycles (over eighteen days) provided a >30-fold expansion in cell number while maintaining pluripotency, thereby providing a simple, nondestructive process for the 3D expansion of hESC. PMID:24571238

  10. Large scale industrialized cell expansion: producing the critical raw material for biofabrication processes.

    PubMed

    Kumar, Arun; Starly, Binil

    2015-01-01

    Cellular biomanufacturing technologies are a critical link to the successful application of cell and scaffold based regenerative therapies, organs-on-chip devices, disease models and any products with living cells contained in them. How do we achieve production level quantities of the key ingredient-'the living cells' for all biofabrication processes, including bioprinting and biopatterning? We review key cell expansion based bioreactor operating principles and how 3D culture will play an important role in achieving production quantities of billions to even trillions of anchorage dependent cells. Furthermore, we highlight some of the challenges in the field of cellular biomanufacturing that must be addressed to achieve desired cellular yields while adhering to the key pillars of good manufacturing practices-safety, purity, stability, potency and identity. Biofabrication technologies are uniquely positioned to provide improved 3D culture surfaces for the industrialized production of living cells. PMID:26539629

  11. Functional properties of bone marrow derived multipotent mesenchymal stromal cells are altered in heart failure patients, and could be corrected by adjustment of expansion strategies

    PubMed Central

    Dmitrieva, Renata I.; Revittser, Alla V.; Klukina, Maria A.; Sviryaev, Yuri V.; Korostovtseva, Ludmila S.; Kostareva, Anna A.; Zaritskey, Andrey Yu.; Shlyakhto, Evgeny V.

    2015-01-01

    Background: Bone marrow multipotent mesenchymal stromal cells (BM-MMSC) considered as a prospective substrate for cell therapy applications, however adult stem cells could be affected by donor-specific factors: age, gender, medical history. Our aim was to investigate how HF affects the functional properties of BM-MMSC. Materials and methods: BM-MMSC from 10 healthy donors (HD), and 16 donors with chronic HF were evaluated for proliferative activity, ability to differentiate, replicative senescence, expression of genes that affect regeneration and fibrosis. The effect of culturing conditions on efficiency of BM-MMSC expansion was determined. Results: HF-derived BM-MMSC demonstrated early decrease of proliferative activity and upregulation of genes that control both, regeneration and fibrosis: Tgf-β pathway, synthesis of ECM, remodeling enzymes, adhesion molecules. We assume that these effects were related to increase of frequency of myofibroblast-like CD146+/SMAα+ CFU-F in HF samples; (ii) low seeding density and hypoxia resulted in predominant purification and expansion of CD146+/SMAα- CFU-Fs. (iii) the activity of NPs system was downregulated in HF BM-MMSC; Conclusions: downregulation of NP signaling in combination with upregulation of Tgf-β pathway in BM-MMSC would result in pro-fibrotic phenotype and make these cells non-effective for therapeutic applications; the corrections in culturing strategy resulted in 23-27 increase of expansion efficiency. PMID:25606985

  12. Potential role of 20S proteasome in maintaining stem cell integrity of human bone marrow stromal cells in prolonged culture expansion

    SciTech Connect

    Lu, Li; Song, Hui-Fang; Zhang, Wei-Guo; Liu, Xue-Qin; Zhu, Qian; Cheng, Xiao-Long; Yang, Gui-Jiao; Li, Ang; Xiao, Zhi-Cheng

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Prolonged culture expansion retards proliferation and induces senescence of hBMSCs. Black-Right-Pointing-Pointer Reduced 20S proteasomal activity and expression potentially contribute to cell aging. Black-Right-Pointing-Pointer MG132-mediated 20S proteasomal inhibition induces senescence-like phenotype. Black-Right-Pointing-Pointer 18{alpha}-GA stimulates proteasomal activity and restores replicative senescence. Black-Right-Pointing-Pointer 18{alpha}-GA retains differentiation without affecting stem cell characterizations. -- Abstract: Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18{alpha}-glycyrrhetinic acid (18{alpha}-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.

  13. The inhibitory effect of hypoxic cytotoxin on the expansion of cancer stem cells in ovarian cancer.

    PubMed

    Nozawa-Suzuki, Noriko; Nagasawa, Hideko; Ohnishi, Ken; Morishige, Ken-Ichirou

    2015-02-20

    While an increase in progression free survival time is seen when an angiogenesis inhibitor is used in the treatment of high-relapse rate ovarian cancer, it has little effect on overall survival. A possible cause of treatment-resistance to angiogenesis inhibitors is the growth of stem cells in a hypoxic microenvironment built inside the tumor tissue by angiogenesis inhibition. In this study, we examined the possible suppression of stem cell and cancer stem cell (CSC) expansion by hypoxic cytotoxin, TX-402. TX-402, an analogue of tirapazamine, has been developed as a hypoxia selective prodrug with inhibitory effects of HIF-1 and angiogenesis. We considered TX-402 as a possible molecular-target drug candidate for ovarian cancer due to its inhibition of CSC expansion. In this study, we found that the expressions of HIF-1α and HIF-2α were increased under hypoxia in serous ovarian cancer cell lines. The expressions of HIF-1α and HIF-2α induced under hypoxia were repressed by TX-402 in a dose-dependent manner. Next, we investigated the effects of hypoxia on the expression levels of stem cell factors, Oct4, Nanog, Sox2 and Lin28, and showed that their expressions were induced by hypoxia. It was also observed that the expressions of putative ovarian cancer stem cell markers, CD133 and CD44 were induced under hypoxia. Furthermore, TX-402 was found to dose-dependently inhibit the expressions of CSC markers and stem cell factors. Oct4 expression was repressed by HIF-2α silencing, but not by HIF-1α silencing, indicating that TX-402 may repress the expression of Oct4 by inhibiting HIF-2α. We constructed CaOV3 spheroids as a 3-dimensional hypoxia model, in which the internal hypoxic region contained CSC-like cells expressing Oct4. The internal hypoxic region, which contained Oct4 expressing cells, disappeared following TX-402 treatment. In conclusion, hypoxia promoted the expansion of CSCs expressing CD133 and CD44 accompanied by an increase of stem cell factors. Its

  14. Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells

    PubMed Central

    2014-01-01

    Background Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. Methods PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation. Results PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain

  15. Peptidoglycan-linked protein A promotes T cell-dependent antibody expansion during Staphylococcus aureus infection

    PubMed Central

    Kim, Hwan Keun; Falugi, Fabiana; Missiakas, Dominique M.; Schneewind, Olaf

    2016-01-01

    A hallmark of Staphylococcus aureus disease in humans is persistent infections without development of protective immune responses. Infected patients generate VH3 plasmablast expansions and increased VH3 idiotype Ig; however, the mechanisms for staphylococcal modification of immune responses are not known. We report here that S. aureus-infected mice generate VH3 antibody expansions via a mechanism requiring MHC-restricted antigen presentation to CD4+ T cells and staphylococcal protein A (SpA), a cell wall-anchored surface molecule that binds Fcγ and VH3 variant heavy chains of Ig. VH3 expansion occurred with peptidoglycan-linked SpA from the bacterial envelope but not with recombinant SpA, and optimally required five tandem repeats of its Ig-binding domains. Signaling via receptor-interacting serine/threonine protein kinase 2 (RIPK2) was essential for implementing peptidoglycan-linked SpA superantigen activity. VH3 clan IgG from S. aureus-infected or SpA-treated animals was not pathogen-specific, suggesting that SpA cross-linking of VH3 idiotype B-cell receptors and activation via attached peptidoglycan are the determinants of staphylococcal escape from adaptive immune responses. PMID:27140614

  16. Novel role for tumor-induced expansion of myeloid-derived cells in cancer cachexia.

    PubMed

    Cuenca, Alex G; Cuenca, Angela L; Winfield, Robert D; Joiner, Dallas N; Gentile, Lori; Delano, Matthew J; Kelly-Scumpia, Kindra M; Scumpia, Philip O; Matheny, Michael K; Scarpace, Philip J; Vila, Lizette; Efron, Philip A; LaFace, Drake M; Moldawer, Lyle L

    2014-06-15

    Cancer progression is associated with inflammation, increased metabolic demand, infection, cachexia, and eventually death. Myeloid-derived suppressor cells (MDSCs) commonly expand during cancer and are associated with adaptive immune suppression and inflammatory metabolite production. We propose that cancer-induced cachexia is driven at least in part by the expansion of MDSCs. MDSC expansion in 4T1 mammary carcinoma-bearing hosts is associated with induction of a hepatic acute-phase protein response and altered host energy and fat metabolism, and eventually reduced survival to polymicrobial sepsis and endotoxemia. Similar results are also seen in mice bearing a Lewis lung carcinoma and a C26 colon adenocarcinoma. However, a similar cachexia response is not seen with equivalent growth of the 66C4 subclone of 4T1, in which MDSC expansion does not occur. Importantly, reducing MDSC numbers in 4T1-bearing animals can ameliorate some of these late responses and reduce susceptibility to inflammation-induced organ injury and death. In addition, administering MDSCs from both tumor- and nontumor-bearing mice can produce an acute-phase response. Thus, we propose a previously undescribed mechanism for the development of cancer cachexia, whereby progressive MDSC expansion contributes to changes in host protein and energy metabolism and reduced resistance to infection.

  17. Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells.

    PubMed

    Breems, D A; Blokland, E A; Siebel, K E; Mayen, A E; Engels, L J; Ploemacher, R E

    1998-01-01

    Stroma-supported long-term cultures (LTC) allow estimation of stem cell quality by simultaneous enumeration of hematopoietic stem cell (HSC) frequencies in a graft using the cobblestone area forming cell (CAFC) assay, and the ability of the graft to generate progenitors in flask LTC (LTC-CFC). We have recently observed that the number and quality of mobilized peripheral blood stem cells (PBSC) was low in patients having received multiple rounds of chemotherapy. Moreover, grafts with low numbers of HSC and poor HSC quality had a high probability to cause graft failure upon their autologous infusion. Because ex vivo culture of stem cells has been suggested to present an attractive tool to improve hematological recovery or reduce graft size, we have studied the possibility that such propagation may affect stem cell quality. In order to do so, we have assessed the recovery of different stem cell subsets in CD34+ PBSC after a 7-day serum-free liquid culture using CAFC and LTC-CFC assays. A numerical expansion of stem cell subsets was observed in the presence of interleukin-3 (IL-3), stem cell factor, and IL-6, while stroma-contact, stromal soluble factors, or combined addition of FLT3-ligand and thrombopoietin improved this parameter. In contrast, ex vivo culture severely reduced the ability of the graft to produce progenitors in LTC while stromal soluble factors partly abrogated this quality loss. The best conservation of graft quality was observed when the PBSC were cultured in stroma-contact. These data suggest that ex vivo propagation of PBSC may allow numerical expansion of various stem cell subsets, however, at the expense of their quality. In addition, cytokine-driven PBSC cultures require stroma for optimal maintenance of graft quality. PMID:9414274

  18. MicroRNA-203 represses selection and expansion of oncogenic Hras transformed tumor initiating cells.

    PubMed

    Riemondy, Kent; Wang, Xiao-jing; Torchia, Enrique C; Roop, Dennis R; Yi, Rui

    2015-07-23

    In many mouse models of skin cancer, only a few tumors typically form even though many cells competent for tumorigenesis receive the same oncogenic stimuli. These observations suggest an active selection process for tumor-initiating cells. Here, we use quantitative mRNA- and miR-Seq to determine the impact of Hras(G12V) on the transcriptome of keratinocytes. We discover that microRNA-203 is downregulated by Hras(G12V). Using a knockout mouse model, we demonstrate that loss of microRNA-203 promotes selection and expansion of tumor-initiating cells. Conversely, restoration of microRNA-203 using an inducible model potently inhibits proliferation of these cells. We comprehensively identify microRNA-203 targets required for Hras-initiated tumorigenesis. These targets include critical regulators of the Ras pathway and essential genes required for cell division. This study establishes a role for the loss of microRNA-203 in promoting selection and expansion of Hras mutated cells and identifies a mechanism through which microRNA-203 antagonizes Hras-mediated tumorigenesis.

  19. Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control.

    PubMed

    Serra, Margarida; Brito, Catarina; Sousa, Marcos F Q; Jensen, Janne; Tostões, Rui; Clemente, João; Strehl, Raimund; Hyllner, Johan; Carrondo, Manuel J T; Alves, Paula M

    2010-08-01

    The successful transfer of human embryonic stem cell (hESC) technology and cellular products into clinical and industrial applications needs to address issues of automation, standardization and the generation of relevant cell numbers of high quality. In this study, we combined microcarrier technology and controlled stirred tank bioreactors, to develop an efficient and scalable system for expansion of pluripotent hESCs. We demonstrate the importance of controlling pO(2) at 30% air saturation to improve hESCs growth. This concentration allowed for a higher energetic cell metabolism, increased growth rate and maximum cell concentration in contrast to 5% pO(2) where a shift to anaerobic metabolism was observed, decreasing cell expansion 3-fold. Importantly, the incorporation of an automated perfusion system in the bioreactor enhanced culture performance and allowed the continuous addition of small molecules assuring higher cell concentrations for a longer time period. The expanded hESCs retained their undifferentiated phenotype and pluripotency. Our results show, for the first time, that the use of controlled bioreactors is critical to ensure the production of high quality hESCs. When compared to the standard colony culture, our strategy improves the final yield of hESCs by 12-fold, providing a potential bioprocess to be transferred to clinical and industrial applications.

  20. The viral context instructs the redundancy of costimulatory pathways in driving CD8(+) T cell expansion.

    PubMed

    Welten, Suzanne P M; Redeker, Anke; Franken, Kees L M C; Oduro, Jennifer D; Ossendorp, Ferry; Čičin-Šain, Luka; Melief, Cornelis J M; Aichele, Peter; Arens, Ramon

    2015-01-01

    Signals delivered by costimulatory molecules are implicated in driving T cell expansion. The requirements for these signals, however, vary from dispensable to essential in different infections. We examined the underlying mechanisms of this differential T cell costimulation dependence and found that the viral context determined the dependence on CD28/B7-mediated costimulation for expansion of naive and memory CD8(+) T cells, indicating that the requirement for costimulatory signals is not imprinted. Notably, related to the high-level costimulatory molecule expression induced by lymphocytic choriomeningitis virus (LCMV), CD28/B7-mediated costimulation was dispensable for accumulation of LCMV-specific CD8(+) T cells because of redundancy with the costimulatory pathways induced by TNF receptor family members (i.e., CD27, OX40, and 4-1BB). Type I IFN signaling in viral-specific CD8(+) T cells is slightly redundant with costimulatory signals. These results highlight that pathogen-specific conditions differentially and uniquely dictate the utilization of costimulatory pathways allowing shaping of effector and memory antigen-specific CD8(+) T cell responses. PMID:26263500

  1. MicroRNA-203 represses selection and expansion of oncogenic Hras transformed tumor initiating cells

    PubMed Central

    Riemondy, Kent; Wang, Xiao-jing; Torchia, Enrique C; Roop, Dennis R; Yi, Rui

    2015-01-01

    In many mouse models of skin cancer, only a few tumors typically form even though many cells competent for tumorigenesis receive the same oncogenic stimuli. These observations suggest an active selection process for tumor-initiating cells. Here, we use quantitative mRNA- and miR-Seq to determine the impact of HrasG12V on the transcriptome of keratinocytes. We discover that microRNA-203 is downregulated by HrasG12V. Using a knockout mouse model, we demonstrate that loss of microRNA-203 promotes selection and expansion of tumor-initiating cells. Conversely, restoration of microRNA-203 using an inducible model potently inhibits proliferation of these cells. We comprehensively identify microRNA-203 targets required for Hras-initiated tumorigenesis. These targets include critical regulators of the Ras pathway and essential genes required for cell division. This study establishes a role for the loss of microRNA-203 in promoting selection and expansion of Hras mutated cells and identifies a mechanism through which microRNA-203 antagonizes Hras-mediated tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.07004.001 PMID:26203562

  2. Expansion and long-term maintenance of induced pluripotent stem cells in stirred suspension bioreactors.

    PubMed

    Shafa, Mehdi; Sjonnesen, Kirsten; Yamashita, Akihiro; Liu, Shiying; Michalak, Marek; Kallos, Michael S; Rancourt, Derrick E

    2012-06-01

    Induced pluripotent stem cells (iPSCs) can provide an important source of cells for the next-generation of cell therapies in regenerative medicine, in part due to their similarity to embryonic stem cells (ESCs). Patient-specific iPSCs represent an opportunity for autologous cell therapies that are not restricted by immunological, ethical and technical obstacles. One of the technical hurdles that must be overcome before iPSCs can be clinically implemented is the scalable, reproducible production of iPSCs and their differentiated progeny. All of the iPSC lines established thus far have been generated and expanded with static tissue culture protocols, which are time-consuming and suffer from batch-to-batch variability. Alternatively, stirred suspension bioreactors propose several benefits and their homogeneous culture environment facilitates the large-scale expansion required for clinical studies at less cost. We have previously developed protocols for expanding murine and human ESCs as undifferentiated aggregates in stirred suspension bioreactors. The resulting cells were karyotypically normal, expressed pluripotency markers and could be differentiated into all three germ lineages, both in vitro and in vivo. In this study, we demonstrate that stirred suspension bioreactors yield 58-fold expansion of undifferentiated pluripotent iPSCs over 4 days. In vitro differentiation into cartilage, bone and cardiomyocytes lineages, in addition to in vivo teratoma formation, further confirmed the existence of fully functional and undifferentiated pluripotent iPSC aggregates following long-term passaging. Stirred suspension bioreactor culture represents an efficient process for the large-scale expansion and maintenance of iPSCs, which is an important first step in their clinical application.

  3. ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression

    NASA Astrophysics Data System (ADS)

    Aryal, Binod; Rotllan, Noemi; Araldi, Elisa; Ramírez, Cristina M.; He, Shun; Chousterman, Benjamin G.; Fenn, Ashley M.; Wanschel, Amarylis; Madrigal-Matute, Julio; Warrier, Nikhil; Martín-Ventura, Jose L.; Swirski, Filip K.; Suárez, Yajaira; Fernández-Hernando, Carlos

    2016-07-01

    Lipid accumulation in macrophages has profound effects on macrophage gene expression and contributes to the development of atherosclerosis. Here, we report that angiopoietin-like protein 4 (ANGPTL4) is the most highly upregulated gene in foamy macrophages and it's absence in haematopoietic cells results in larger atherosclerotic plaques, characterized by bigger necrotic core areas and increased macrophage apoptosis. Furthermore, hyperlipidemic mice deficient in haematopoietic ANGPTL4 have higher blood leukocyte counts, which is associated with an increase in the common myeloid progenitor (CMP) population. ANGPTL4-deficient CMPs have higher lipid raft content, are more proliferative and less apoptotic compared with the wild-type (WT) CMPs. Finally, we observe that ANGPTL4 deficiency in macrophages promotes foam cell formation by enhancing CD36 expression and reducing ABCA1 localization in the cell surface. Altogether, these findings demonstrate that haematopoietic ANGPTL4 deficiency increases atherogenesis through regulating myeloid progenitor cell expansion and differentiation, foam cell formation and vascular inflammation.

  4. ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression

    PubMed Central

    Aryal, Binod; Rotllan, Noemi; Araldi, Elisa; Ramírez, Cristina M.; He, Shun; Chousterman, Benjamin G.; Fenn, Ashley M.; Wanschel, Amarylis; Madrigal-Matute, Julio; Warrier, Nikhil; Martín-Ventura, Jose L.; Swirski, Filip K.; Suárez, Yajaira; Fernández-Hernando, Carlos

    2016-01-01

    Lipid accumulation in macrophages has profound effects on macrophage gene expression and contributes to the development of atherosclerosis. Here, we report that angiopoietin-like protein 4 (ANGPTL4) is the most highly upregulated gene in foamy macrophages and it's absence in haematopoietic cells results in larger atherosclerotic plaques, characterized by bigger necrotic core areas and increased macrophage apoptosis. Furthermore, hyperlipidemic mice deficient in haematopoietic ANGPTL4 have higher blood leukocyte counts, which is associated with an increase in the common myeloid progenitor (CMP) population. ANGPTL4-deficient CMPs have higher lipid raft content, are more proliferative and less apoptotic compared with the wild-type (WT) CMPs. Finally, we observe that ANGPTL4 deficiency in macrophages promotes foam cell formation by enhancing CD36 expression and reducing ABCA1 localization in the cell surface. Altogether, these findings demonstrate that haematopoietic ANGPTL4 deficiency increases atherogenesis through regulating myeloid progenitor cell expansion and differentiation, foam cell formation and vascular inflammation. PMID:27460411

  5. Regulation of cell division and expansion by sugar and auxin signaling

    PubMed Central

    Wang, Lu; Ruan, Yong-Ling

    2013-01-01

    Plant growth and development are modulated by concerted actions of a variety of signaling molecules. In recent years, evidence has emerged on the roles of sugar and auxin signals network in diverse aspects of plant growth and development. Here, based on recent progress of genetic analyses and gene expression profiling studies, we summarize the functional similarities, diversities, and their interactions of sugar and auxin signals in regulating two major processes of plant development: cell division and cell expansion. We focus on roles of sugar and auxin signaling in both vegetative and reproductive tissues including developing seed. PMID:23755057

  6. Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner*

    PubMed Central

    Åhsberg, Josefine; Ungerbäck, Jonas; Strid, Tobias; Welinder, Eva; Stjernberg, Jenny; Larsson, Malin; Qian, Hong; Sigvardsson, Mikael

    2013-01-01

    Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1+/− pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development. PMID:24078629

  7. Dyrk1A induces pancreatic β cell mass expansion and improves glucose tolerance.

    PubMed

    Rachdi, Latif; Kariyawasam, Dulanjalee; Aïello, Virginie; Herault, Yann; Janel, Nathalie; Delabar, Jean-Maurice; Polak, Michel; Scharfmann, Raphaël

    2014-01-01

    Type 2 diabetes is caused by a limited capacity of insulin-producing pancreatic β cells to increase their mass and function in response to insulin resistance. The signaling pathways that positively regulate functional β cell mass have not been fully elucidated. DYRK1A (also called minibrain/MNB) is a member of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family. A significant amount of data implicates DYRK1A in brain growth and Down syndrome, and recent data indicate that Dyrk1A haploinsufficient mice have a low functional β cell mass. Here we ask whether Dyrk1A upregulation could be a way to increase functional β cell mass. We used mice overexpressing Dyrk1A under the control of its own regulatory sequences (mBACTgDyrk1A). These mice exhibit decreased glucose levels and hyperinsulinemia in the fasting state. Improved glucose tolerance is observed in these mice as early as 4 weeks of age. Upregulation of Dyrk1A in β cells induces expansion of β cell mass through increased proliferation and cell size. Importantly, mBACTgDyrk1A mice are protected against high-fat-diet-induced β cell failure through increase in β cell mass and insulin sensitivity. These studies show the crucial role of the DYRK1A pathway in the regulation of β cell mass and carbohydrate metabolism in vivo. Activating the DYRK1A pathway could thus represent an innovative way to increase functional β cell mass. PMID:24870561

  8. Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells.

    PubMed

    Birket, Matthew J; Ribeiro, Marcelo C; Verkerk, Arie O; Ward, Dorien; Leitoguinho, Ana Rita; den Hartogh, Sabine C; Orlova, Valeria V; Devalla, Harsha D; Schwach, Verena; Bellin, Milena; Passier, Robert; Mummery, Christine L

    2015-09-01

    The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of cardiomyocytes and vascular cells. This contrasts with neural progenitors, which can be expanded robustly and are a renewable source of their derivatives. Here we use human pluripotent stem cells bearing a cardiac lineage reporter to show that regulated MYC expression enables robust expansion of CPCs with insulin-like growth factor-1 (IGF-1) and a hedgehog pathway agonist. The CPCs can be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes. The cells are clonogenic and can be expanded for >40 population doublings while retaining the ability to differentiate into cardiomyocytes and vascular cells. Access to CPCs will allow precise recreation of elements of heart development in vitro and facilitate investigation of the molecular basis of cardiac fate determination. This technology is applicable for cardiac disease modeling, toxicology studies and tissue engineering. PMID:26192318

  9. RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development.

    PubMed

    Verma, Rakesh; Su, Su; McCrann, Donald J; Green, Jennifer M; Leu, Karen; Young, Peter R; Schatz, Peter J; Silva, Jeffrey C; Stokes, Matthew P; Wojchowski, Don M

    2014-08-25

    Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.

  10. RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development.

    PubMed

    Verma, Rakesh; Su, Su; McCrann, Donald J; Green, Jennifer M; Leu, Karen; Young, Peter R; Schatz, Peter J; Silva, Jeffrey C; Stokes, Matthew P; Wojchowski, Don M

    2014-08-25

    Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development. PMID:25092874

  11. Manipulating the tumor microenvironment ex vivo for enhanced expansion of tumor-infiltrating lymphocytes for adoptive cell therapy

    PubMed Central

    Chacon, Jessica Ann; Sarnaik, Amod A; Chen, Jie Qing; Creasy, Caitlin; Kale, Charuta; Robinson, John; Weber, Jeffrey; Hwu, Patrick; Pilon-Thomas, Shari; Radvanyi, Laszlo

    2014-01-01

    Purpose Cultured tumor fragments from melanoma metastases have been used for years as a source of tumor-infiltrating lymphocytes (TIL) for adoptive cell therapy. The expansion of tumor-reactive CD8+ T cells with IL-2 in these early cultures is critical in generating clinically active TIL infusion products, with a population of activated 4-1BB CD8+ T cells recently found to constitute the majority of tumor-specific T cells. Experimental Design We used an agonistic anti-4-1BB antibody added during the initial tumor fragment cultures to provide in situ 4-1BB co-stimulation. Results We found that addition of an agonistic anti-4-1BB antibody could activate 4-1BB signaling within early cultured tumor fragments and accelerated the rate of memory CD8+ TIL outgrowth that were highly enriched for melanoma antigen specificity. This was associated with NFκB activation and the induction of T-cell survival and memory genes, as well as enhanced IL-2 responsiveness, in the CD8+ T cells in the fragments and emerging from the fragments. Early provision of 4-1BB co-stimulation also affected the dendritic cells (DC) by activating NFκB in DC and promoting their maturation inside the tumor fragments. Blocking HLA class I prevented the enhanced outgrowth of CD8+ T cells with anti-4-1BB, suggesting that an ongoing HLA class I-mediated antigen presentation in early tumor fragment cultures plays a role in mediating tumor-specific CD8+ TIL outgrowth. Conclusions Our results highlight a previously unrecognized concept in TIL adoptive cell therapy that the tumor microenvironment can be dynamically regulated in the initial tumor fragment cultures to regulate the types of T cells expanded and their functional characteristics. PMID:25472998

  12. Effects of mutations in the Arabidopsis Cold Shock Domain Protein 3 (AtCSP3) gene on leaf cell expansion.

    PubMed

    Yang, Yongil; Karlson, Dale

    2012-08-01

    The cold shock domain is among the most evolutionarily conserved nucleic acid binding domains from prokaryotes to higher eukaryotes, including plants. Although eukaryotic cold shock domain proteins have been extensively studied as transcriptional and post-transcriptional regulators during various developmental processes, their functional roles in plants remains poorly understood. In this study, AtCSP3 (At2g17870), which is one of four Arabidopsis thaliana c old s hock domain proteins (AtCSPs), was functionally characterized. Quantitative RT-PCR analysis confirmed high expression of AtCSP3 in reproductive and meristematic tissues. A homozygous atcsp3 loss-of-function mutant exhibits an overall reduced seedling size, stunted and orbicular rosette leaves, reduced petiole length, and curled leaf blades. Palisade mesophyll cells are smaller and more circular in atcsp3 leaves. Cell size analysis indicated that the reduced size of the circular mesophyll cells appears to be generated by a reduction of cell length along the leaf-length axis, resulting in an orbicular leaf shape. It was also determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss-of-function mutant, but leaf cell proliferation is not affected. AtCSP3 loss-of-function resulted in a dramatic reduction of LNG1 transcript, a gene that is involved in two-dimensional leaf polarity regulation. Transient subcellular localization of AtCSP3 in onion epidermal cells confirmed a nucleocytoplasmic localization pattern. Collectively, these data suggest that AtCSP3 is functionally linked to the regulation of leaf length by affecting LNG1 transcript accumulation during leaf development. A putative function of AtCSP3 as an RNA binding protein is also discussed in relation to leaf development.

  13. Large-Scale Expansion and Differentiation of Mesenchymal Stem Cells in Microcarrier-Based Stirred Bioreactors.

    PubMed

    Sart, Sébastien; Agathos, Spiros N

    2016-01-01

    Mesenchymal stem cells (MSCs) have emerged as an important tool for tissue engineering, thanks to their differentiation potential and their broad trophic activities. However, for clinical purposes or for relevant in vitro applications, large quantities of MSCs are required, which could hardly be reached using conventional cultivation in plastic dishes. Microcarriers have high surface to volume ratio, which enables the easy scale-up of the expansion and differentiation of MSCs. In addition, the agitation in stirred tank bioreactors limits the diffusion gradient of nutrients or morphogens, thus providing a physiologically relevant environment to favor MSC production at large scale. This work describes a simple method for the mass expansion and differentiation of MSCs, including the procedures to monitor the proliferation, metabolic status and phenotype of MSCs during suspension culture. Moreover, this work proposes suitable materials for cGMP compliant culture conditions enabling the clinical grade production of MSCs.

  14. Mannosylated self-peptide inhibits the development of experimental autoimmune encephalomyelitis via expansion of nonencephalitogenic T cells.

    PubMed

    Kel, Junda M; Slütter, Bram; Drijfhout, Jan Wouter; Koning, Frits; Nagelkerken, Lex

    2008-07-01

    Tolerance to experimental autoimmune encephalomyelitis (EAE) in SJL mice can be induced by immunization with a mannosylated form of the proteolipid protein (M-PLP139-151), despite the presence of CFA. The state of tolerance is characterized by poor delayed-type hypersensitivity responses and the absence of clinical EAE symptoms. In vivo monitoring of CFSE-labeled PLP139-151-specific TCR-transgenic (5B6) T cells revealed that immunization with M-PLP139-151 increases the clonal expansion of 5B6 T cells that do not develop full effector functions. Moreover, nonfunctional T cells obtained from M-PLP139-151-immunized mice showed poor blastogenesis and were unable to transfer EAE to naïve recipients. Nevertheless, the in vitro production of cytokines and chemokines associated with EAE was unaffected. Importantly, tolerance induced by M-PLP139-151 was abrogated by the administration of pertussis toxin, resulting in EAE development. Our results suggest that M-PLP139-151 inhibits EAE development by affecting the differentiation of T cells into encephalitogenic effector cells.

  15. Continuum Model of Collective Cell Migration in Wound Healing and Colony Expansion

    PubMed Central

    Arciero, Julia C.; Mi, Qi; Branca, Maria F.; Hackam, David J.; Swigon, David

    2011-01-01

    Collective cell migration plays an important role during wound healing and embryo development. Although the exact mechanisms that coordinate such migration are still unknown, experimental studies of moving cell layers have shown that the primary interactions governing the motion of the layer are the force of lamellipodia, the adhesion of cells to the substrate, and the adhesion of cells to each other. Here, we derive a two-dimensional continuum mechanical model of cell-layer migration that is based on a novel assumption of elastic deformation of the layer and incorporates basic mechanical interactions of cells as well as cell proliferation and apoptosis. The evolution equations are solved numerically using a level set method. The model successfully reproduces data from two types of experiments: 1), the contraction of an enterocyte cell layer during wound healing; and 2), the expansion of a radially symmetric colony of MDCK cells, both in the edge migration velocity and in cell-layer density. In accord with experimental observations, and in contrast to reaction-diffusion models, this model predicts a partial wound closure if lamellipod formation is inhibited at the wound edge and gives implications of the effect of spatially restricted proliferation. PMID:21281567

  16. ROCK Inhibition Facilitates In Vitro Expansion of Glioblastoma Stem-Like Cells

    PubMed Central

    Tilson, Samantha G.; Haley, Elizabeth M.; Triantafillu, Ursula L.; Dozier, David A.; Langford, Catherine P.; Gillespie, G. Yancey; Kim, Yonghyun

    2015-01-01

    Due to their stem-like characteristics and their resistance to existing chemo- and radiation therapies, there is a growing appreciation that cancer stem cells (CSCs) are the root cause behind cancer metastasis and recurrence. However, these cells represent a small subpopulation of cancer cells and are difficult to propagate in vitro. Glioblastoma is an extremely deadly form of brain cancer that is hypothesized to have a subpopulation of CSCs called glioblastoma stem cells (GSCs; also called brain tumor initiating cells, BTICs). We propose the use of selective Rho-kinase (ROCK) inhibitors, Y-27632 and fasudil, to promote GSC/BTIC-like cell survival and propagation in vitro. ROCK inhibitors have been implicated in suppressing apoptosis, and it was hypothesized that they would increase the number of GSC/BTIC-like cells grown in vitro and improve cloning efficiencies. Indeed, our data demonstrate that transient and continuous supplementation of non-toxic concentrations of Y-27632 and fasudil inhibited apoptosis, enhanced the cells’ ability to form spheres, and increased stem cell marker expressing GSC/BTIC-like cell subpopulation. Our data indicated that pharmacological and genetic (siRNA) inhibitions of the ROCK pathway facilitates in vitro expansion of GSC/BTIC-like cells. Thus, ROCK pathway inhibition shows promise for future optimization of CSC culture media. PMID:26167936

  17. Human fibrocytic myeloid-derived suppressor cells express IDO and promote tolerance via Treg-cell expansion.

    PubMed

    Zoso, Alessia; Mazza, Emilia M C; Bicciato, Silvio; Mandruzzato, Susanna; Bronte, Vincenzo; Serafini, Paolo; Inverardi, Luca

    2014-11-01

    By restraining T-cell activation and promoting Treg-cell expansion, myeloid-derived suppressor cells (MDSCs) and tolerogenic DCs can control self-reactive and antigraft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC, which are differentiated from umbilical cord blood precursors by 4-day culture with FDA-approved cytokines (recombinant human-GM-CSF and recombinant human-G-CSF). This MDSC subset, characterized by the expression of MDSC-, DC-, and fibrocyte-associated markers, promotes Treg-cell expansion and induces normoglycemia in a xenogeneic mouse model of Type 1 diabetes. In order to exert their protolerogenic function, fibrocytic MDSCs require direct contact with activated T cells, which leads to the production and secretion of IDO. This new myeloid subset may have an important role in the in vitro and in vivo production of Treg cells for the treatment of autoimmune diseases, and in either the prevention or control of allograft rejection.

  18. Improving Embryonic Stem Cell Expansion through the Combination of Perfusion and Bioprocess Model Design

    PubMed Central

    Yeo, David; Kiparissides, Alexandros; Cha, Jae Min; Aguilar-Gallardo, Cristobal; Polak, Julia M.; Tsiridis, Elefterios; Pistikopoulos, Efstratios N.; Mantalaris, Athanasios

    2013-01-01

    Background High proliferative and differentiation capacity renders embryonic stem cells (ESCs) a promising cell source for tissue engineering and cell-based therapies. Harnessing their potential, however, requires well-designed, efficient and reproducible expansion and differentiation protocols as well as avoiding hazardous by-products, such as teratoma formation. Traditional, standard culture methodologies are fragmented and limited in their fed-batch feeding strategies that afford a sub-optimal environment for cellular metabolism. Herein, we investigate the impact of metabolic stress as a result of inefficient feeding utilizing a novel perfusion bioreactor and a mathematical model to achieve bioprocess improvement. Methodology/Principal Findings To characterize nutritional requirements, the expansion of undifferentiated murine ESCs (mESCs) encapsulated in hydrogels was performed in batch and perfusion cultures using bioreactors. Despite sufficient nutrient and growth factor provision, the accumulation of inhibitory metabolites resulted in the unscheduled differentiation of mESCs and a decline in their cell numbers in the batch cultures. In contrast, perfusion cultures maintained metabolite concentration below toxic levels, resulting in the robust expansion (>16-fold) of high quality ‘naïve’ mESCs within 4 days. A multi-scale mathematical model describing population segregated growth kinetics, metabolism and the expression of selected pluripotency (‘stemness’) genes was implemented to maximize information from available experimental data. A global sensitivity analysis (GSA) was employed that identified significant (6/29) model parameters and enabled model validation. Predicting the preferential propagation of undifferentiated ESCs in perfusion culture conditions demonstrates synchrony between theory and experiment. Conclusions/Significance The limitations of batch culture highlight the importance of cellular metabolism in maintaining pluripotency, which

  19. Ex Vivo Expansion and In Vivo Self-Renewal of Human Muscle Stem Cells.

    PubMed

    Charville, Gregory W; Cheung, Tom H; Yoo, Bryan; Santos, Pauline J; Lee, Gordon K; Shrager, Joseph B; Rando, Thomas A

    2015-10-13

    Adult skeletal muscle stem cells, or satellite cells (SCs), regenerate functional muscle following transplantation into injured or diseased tissue. To gain insight into human SC (huSC) biology, we analyzed transcriptome dynamics by RNA sequencing of prospectively isolated quiescent and activated huSCs. This analysis indicated that huSCs differentiate and lose proliferative potential when maintained in high-mitogen conditions ex vivo. Further analysis of gene expression revealed that p38 MAPK acts in a transcriptional network underlying huSC self-renewal. Activation of p38 signaling correlated with huSC differentiation, while inhibition of p38 reversibly prevented differentiation, enabling expansion of huSCs. When transplanted, expanded huSCs differentiated to generate chimeric muscle and engrafted as SCs in the sublaminar niche with a greater frequency than freshly isolated cells or cells cultured without p38 inhibition. These studies indicate characteristics of the huSC transcriptome that promote expansion ex vivo to allow enhanced functional engraftment of a defined population of self-renewing huSCs. PMID:26344908

  20. Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer.

    PubMed

    Dubeykovskaya, Zina; Si, Yiling; Chen, Xiaowei; Worthley, Daniel L; Renz, Bernhard W; Urbanska, Aleksandra M; Hayakawa, Yoku; Xu, Ting; Westphalen, C Benedikt; Dubeykovskiy, Alexander; Chen, Duan; Friedman, Richard A; Asfaha, Samuel; Nagar, Karan; Tailor, Yagnesh; Muthupalani, Sureshkumar; Fox, James G; Kitajewski, Jan; Wang, Timothy C

    2016-01-01

    CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) expand in the spleen during cancer and promote progression through suppression of cytotoxic T cells. An anti-inflammatory reflex arc involving the vagus nerve and memory T cells is necessary for resolution of acute inflammation. Failure of this neural circuit could promote procarcinogenic inflammation and altered tumour immunity. Here we show that splenic TFF2, a secreted anti-inflammatory peptide, is released by vagally modulated memory T cells to suppress the expansion of MDSCs through CXCR4. Splenic denervation interrupts the anti-inflammatory neural arc, resulting in the expansion of MDSCs and colorectal cancer. Deletion of Tff2 recapitulates splenic denervation to promote carcinogenesis. Colorectal carcinogenesis could be suppressed through transgenic overexpression of TFF2, adenoviral transfer of TFF2 or transplantation of TFF2-expressing bone marrow. TFF2 is important to the anti-inflammatory reflex arc and plays an essential role in arresting MDSC proliferation. TFF2 offers a potential approach to prevent and to treat cancer. PMID:26841680

  1. Intramuscular injection of bone marrow mononuclear cells contributes to bone repair following midpalatal expansion in rats

    PubMed Central

    CHE, XIAOXIA; GUO, JIE; LI, XIANGDONG; WANG, LVE; WEI, SILONG

    2016-01-01

    Healing from injury requires the activation and proliferation of stem cells for tissue repair. Previous studies have demonstrated that bone marrow is a central pool of stem cells. The present study aimed to investigate the route undertaken by bone marrow mononuclear cells (BMMCs) following BMMC transplantation by masseter injection in a rat model of midpalatal expansion. The rats were divided into five groups according to the types of midpalatal expansion, incision and BMMC transplantation. Samples of midpalatal bone from the rats in each group were used for histological and immunohistochemical assessments to track and evaluate the differential potentials of the transplanted BMMCs in the masseter muscle and midpalatal bone. Bromodeoxyuridine was used as a BMMC tracing label, and M-cadherin was used to detect muscle satellite cells. The BMMCs injected into the masseter were observed, not only in the masseter, but also in the blood vessels and oral mucosa, and enveloped the midpalatal bone. A number of the BMMCs transformed into osteoblasts at the boundary of the neuromuscular bundle, and were embedded in the newly formed bone during midpalatal bone regeneration. The results of the present study suggested that BMMCs entered the circulation and migrated from muscle to the bone tissue, where they were involved in bone repair. Therefore, BMMCs may prove useful in the treatment of various types of cancer. PMID:26648442

  2. Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer

    PubMed Central

    Dubeykovskaya, Zina; Si, Yiling; Chen, Xiaowei; Worthley, Daniel L.; Renz, Bernhard W.; Urbanska, Aleksandra M.; Hayakawa, Yoku; Xu, Ting; Westphalen, C. Benedikt; Dubeykovskiy, Alexander; Chen, Duan; Friedman, Richard A.; Asfaha, Samuel; Nagar, Karan; Tailor, Yagnesh; Muthupalani, Sureshkumar; Fox, James G.; Kitajewski, Jan; Wang, Timothy C.

    2016-01-01

    CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) expand in the spleen during cancer and promote progression through suppression of cytotoxic T cells. An anti-inflammatory reflex arc involving the vagus nerve and memory T cells is necessary for resolution of acute inflammation. Failure of this neural circuit could promote procarcinogenic inflammation and altered tumour immunity. Here we show that splenic TFF2, a secreted anti-inflammatory peptide, is released by vagally modulated memory T cells to suppress the expansion of MDSCs through CXCR4. Splenic denervation interrupts the anti-inflammatory neural arc, resulting in the expansion of MDSCs and colorectal cancer. Deletion of Tff2 recapitulates splenic denervation to promote carcinogenesis. Colorectal carcinogenesis could be suppressed through transgenic overexpression of TFF2, adenoviral transfer of TFF2 or transplantation of TFF2-expressing bone marrow. TFF2 is important to the anti-inflammatory reflex arc and plays an essential role in arresting MDSC proliferation. TFF2 offers a potential approach to prevent and to treat cancer. PMID:26841680

  3. Stem cell factor (SCF) can regulate the activation and expansion of murine intraepithelial lymphocytes.

    PubMed

    Wang, T; Langley, K E; Gourley, W K; Klimpel, G R

    2000-03-01

    Murine intraepithelial lymphocytes (IEL) express c-kit, the receptor for stem cell factor (SCF). SCF induced a low but significant proliferative response in IEL, but not in splenic T cells. SCF stimulation of IEL resulted in an expansion of the c-kit(+), TCRgammadelta(+)cell population. SCF-induced proliferation was dependent upon SCF-c-kit interactions, since antibody to c-kit blocked this response, and IEL obtained from c-kit mutant (W/W(v)) mice failed to respond to SCF. SCF acted synergistically with anti-TCRgammadelta and with concavalin A (Con A) to induce proliferation and interferon gamma (IFN-gamma) production in IEL. Finally, mice injected with SCF had a significant increase in the number of IEL in the small intestine. SCF-treated mice had increased numbers of TCRalphabeta(+)and TCRgammadelta(+)cell populations, as well as increased numbers of c-kit(+)and c-kit(-)IEL. These data suggest that SCF-c-kit interactions play an important role in regulating IEL expansion and activation.

  4. Evaluating Clonal Expansion of HIV-Infected Cells: Optimization of PCR Strategies to Predict Clonality

    PubMed Central

    Laskey, Sarah B.; Pohlmeyer, Christopher W.; Bruner, Katherine M.; Siliciano, Robert F.

    2016-01-01

    In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings. PMID:27494508

  5. Expansion of engrafting human hematopoietic stem/progenitor cells in three-dimensional scaffolds with surface-immobilized fibronectin

    PubMed Central

    Feng, Qi; Chai, Chou; Jiang, Xue-Song; Leong, Kam W.; Mao, Hai-Quan

    2008-01-01

    An efficient and practical ex vivo expansion methodology for human hematopoietic stem/progenitor cells (HSPCs) is critical in realizing the potential of HSPC transplantation in treating a variety of hematologic disorders and as a supportive therapy for malignant diseases. We report here an expansion strategy using a three-dimensional (3D) scaffold conjugated with an extracellular matrix molecule, fibronectin (FN), to partially mimic the hematopoietic stem cell niche. FN-immobilized 3D polyethylene terephthalate (PET) scaffold was synthesized and evaluated for HSPC expansion efficiency, in comparison with a FN-immobilized 2D PET substrate and a 3D scaffold with FN supplemented in the medium. Covalent conjugation of FN produced substrate and scaffold with higher cell expansion efficiency than that on their unmodified counterparts. After 10 days of culture in serum-free medium, human umbilical cord blood CD34+ cells cultured in FN-conjugated scaffold yielded the highest expansion of CD34+ cells (∼100 fold) and long-term culture initiating cells (∼47-fold). The expanded human CD34+ cells successfully reconstituted hematopoiesis in NOD/SCID mice. This study demonstrated the synergistic effect between the three-dimensionality of the scaffold and surface-conjugated FN, and the potential of this FN-conjugated 3D scaffold for ex vivo expansion of HSPCs. PMID:16739181

  6. Nutrient Regulation by Continuous Feeding Removes Limitations on Cell Yield in the Large-Scale Expansion of Mammalian Cell Spheroids

    PubMed Central

    Weegman, Bradley P.; Nash, Peter; Carlson, Alexandra L.; Voltzke, Kristin J.; Geng, Zhaohui; Jahani, Marjan; Becker, Benjamin B.; Papas, Klearchos K.; Firpo, Meri T.

    2013-01-01

    Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods. Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed. Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and SSB culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products. Continuously fed bioreactor systems combined with nutrient regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies and will facilitate the translation of cell culture from the research lab to clinical applications. PMID:24204645

  7. Identification, isolation and expansion of myoendothelial cells involved in leech muscle regeneration.

    PubMed

    Grimaldi, Annalisa; Banfi, Serena; Gerosa, Laura; Tettamanti, Gianluca; Noonan, Douglas M; Valvassori, Roberto; de Eguileor, Magda

    2009-01-01

    Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells. PMID:19876402

  8. Thymic epithelial cell expansion through matricellular protein CYR61 boosts progenitor homing and T-cell output.

    PubMed

    Emre, Yalin; Irla, Magali; Dunand-Sauthier, Isabelle; Ballet, Romain; Meguenani, Mehdi; Jemelin, Stephane; Vesin, Christian; Reith, Walter; Imhof, Beat A

    2013-01-01

    Thymic epithelial cells (TEC) are heterogeneous stromal cells that generate microenvironments required for the formation of T cells within the thymus. Defects in TEC lead to immunodeficiency or autoimmunity. Here we identify TEC as the major source of cysteine-rich protein 61 (CYR61), a matricellular protein implicated in cell proliferation and migration. Binding of CYR61 to LFA-1, ICAM-1 and integrin α6 supports the adhesion of TEC and thymocytes as well as their interaction. Treatment of thymic lobes with recombinant CYR61 expands the stromal compartment by inducing the proliferation of TEC and activates Akt signalling. Engraftment of CYR61-overexpressing thymic lobes into athymic nude mice drastically boosts the yield of thymic output via expansion of TEC. This increases the space for the recruitment of circulating hematopoietic progenitors and the development of T cells. Our discovery paves the way for therapeutic interventions designed to restore thymus stroma and T-cell generation.

  9. Thymic epithelial cell expansion through matricellular protein CYR61 boosts progenitor homing and T-cell output

    NASA Astrophysics Data System (ADS)

    Emre, Yalin; Irla, Magali; Dunand-Sauthier, Isabelle; Ballet, Romain; Meguenani, Mehdi; Jemelin, Stephane; Vesin, Christian; Reith, Walter; Imhof, Beat A.

    2013-11-01

    Thymic epithelial cells (TEC) are heterogeneous stromal cells that generate microenvironments required for the formation of T cells within the thymus. Defects in TEC lead to immunodeficiency or autoimmunity. Here we identify TEC as the major source of cysteine-rich protein 61 (CYR61), a matricellular protein implicated in cell proliferation and migration. Binding of CYR61 to LFA-1, ICAM-1 and integrin α6 supports the adhesion of TEC and thymocytes as well as their interaction. Treatment of thymic lobes with recombinant CYR61 expands the stromal compartment by inducing the proliferation of TEC and activates Akt signalling. Engraftment of CYR61-overexpressing thymic lobes into athymic nude mice drastically boosts the yield of thymic output via expansion of TEC. This increases the space for the recruitment of circulating hematopoietic progenitors and the development of T cells. Our discovery paves the way for therapeutic interventions designed to restore thymus stroma and T-cell generation.

  10. Expansion of Inefficient HIV-Specific CD8 T Cells during Acute Infection

    PubMed Central

    Eller, Michael A.; Goonetilleke, Nilu; Tassaneetrithep, Boonrat; Eller, Leigh Anne; Costanzo, Margaret C.; Johnson, Susan; Betts, Michael R.; Krebs, Shelly J.; Slike, Bonnie M.; Nitayaphan, Sorachai; Rono, Kathleen; Tovanabutra, Sodsai; Maganga, Lucas; Kibuuka, Hannah; Jagodzinski, Linda; Peel, Sheila; Rolland, Morgane; Marovich, Mary A.; Kim, Jerome H.; Michael, Nelson L.; Robb, Merlin L.

    2016-01-01

    ABSTRACT Attrition within the CD4+ T cell compartment, high viremia, and a cytokine storm characterize the early days after HIV infection. When the first emerging HIV-specific CD8+ T cell responses gain control over viral replication it is incomplete, and clearance of HIV infection is not achieved even in the rare cases of individuals who spontaneously control viral replication to nearly immeasurably low levels. Thus, despite their partial ability to control viremia, HIV-specific CD8+ T cell responses are insufficient to clear HIV infection. Studying individuals in the first few days of acute HIV infection, we detected the emergence of a unique population of CD38+ CD27− CD8+ T cells characterized by the low expression of the CD8 receptor (CD8dim). Interestingly, while high frequencies of HIV-specific CD8+ T cell responses occur within the CD38+ CD27− CD8dim T cell population, the minority populations of CD8bright T cells are significantly more effective in inhibiting HIV replication. Furthermore, the frequency of CD8dim T cells directly correlates with viral load and clinical predictors of more rapid disease progression. We found that a canonical burst of proliferative cytokines coincides with the emergence of CD8dim T cells, and the size of this population inversely correlates with the acute loss of CD4+ T cells. These data indicate, for the first time, that early CD4+ T cell loss coincides with the expansion of a functionally impaired HIV-specific CD8dim T cell population less efficient in controlling HIV viremia. IMPORTANCE A distinct population of activated CD8+ T cells appears during acute HIV infection with diminished capacity to inhibit HIV replication and is predictive of viral set point, offering the first immunologic evidence of CD8+ T cell dysfunction during acute infection. PMID:26842474

  11. Matrix Expansion and Syncytial Aggregation of Syndecan-1+ Cells Underpin Villous Atrophy in Coeliac Disease

    PubMed Central

    Salvestrini, Camilla; Lucas, Mark; Lionetti, Paolo; Torrente, Franco; James, Sean; Phillips, Alan D.; Murch, Simon H.

    2014-01-01

    Background We studied the expression of sulphated glycosaminoglycans (GAGs) in coeliac disease (CD) mucosa, as they are critical determinants of tissue volume, which increases in active disease. We also examined mucosal expression of IL-6, which stimulates excess GAG synthesis in disorders such as Grave's ophthalmopathy. Methods We stained archival jejunal biopsies from 5 children with CD at diagnosis, on gluten-free diet and challenge for sulphated GAGs. We then examined duodenal biopsies from 9 children with CD compared to 9 histological normal controls, staining for sulphated GAGs, heparan sulphate proteoglycans (HSPG), short-chain HSPG (Δ-HSPG) and the proteoglycan syndecan-1 (CD138), which is expressed on epithelium and plasma cells. We confirmed findings with a second monoclonal in another 12 coeliac children. We determined mucosal IL-6 expression by immunohistochemistry and PCR in 9 further cases and controls, and used quantitative real time PCR for other Th17 pathway cytokines in an additional 10 cases and controls. Results In CD, HSPG expression was lost in the epithelial compartment but contrastingly maintained within an expanded lamina propria. Within the upper lamina propria, clusters of syndecan-1+ plasma cells formed extensive syncytial sheets, comprising adherent plasma cells, lysed cells with punctate cytoplasmic staining and shed syndecan ectodomains. A dense infiltrate of IL-6+ mononuclear cells was detected in active coeliac disease, also localised to the upper lamina propria, with significantly increased mRNA expression of IL-6 and IL-17A but not IL-23 p19. Conclusions Matrix expansion, through syndecan-1+ cell recruitment and lamina propria GAG increase, underpins villous atrophy in coeliac disease. The syndecan-1+ cell syncytia and excess GAG production recapitulate elements of the invertebrate encapsulation reaction, itself dependent on insect transglutaminase and glutaminated early response proteins. As in other matrix expansion disorders

  12. IL-10 Conditioning of Human Skin Affects the Distribution of Migratory Dendritic Cell Subsets and Functional T Cell Differentiation

    PubMed Central

    Lindenberg, Jelle J.; Oosterhoff, Dinja; Sombroek, Claudia C.; Lougheed, Sinéad M.; Hooijberg, Erik; Stam, Anita G. M.; Santegoets, Saskia J. A. M.; Tijssen, Henk J.; Buter, Jan; Pinedo, Herbert M.; van den Eertwegh, Alfons J. M.; Scheper, Rik J.; Koenen, Hans J. P. M.; van de Ven, Rieneke; de Gruijl, Tanja D.

    2013-01-01

    In cancer patients pervasive systemic suppression of Dendritic Cell (DC) differentiation and maturation can hinder vaccination efficacy. In this study we have extensively characterized migratory DC subsets from human skin and studied how their migration and T cell-stimulatory abilities were affected by conditioning of the dermal microenvironment through cancer-related suppressive cytokines. To assess effects in the context of a complex tissue structure, we made use of a near-physiological skin explant model. By 4-color flow cytometry, we identified migrated Langerhans Cells (LC) and five dermis-derived DC populations in differential states of maturation. From a panel of known tumor-associated suppressive cytokines, IL-10 showed a unique ability to induce predominant migration of an immature CD14+CD141+DC-SIGN+ DC subset with low levels of co-stimulatory molecules, up-regulated expression of the co-inhibitory molecule PD-L1 and the M2-associated macrophage marker CD163. A similarly immature subset composition was observed for DC migrating from explants taken from skin overlying breast tumors. Whereas predominant migration of mature CD1a+ subsets was associated with release of IL-12p70, efficient Th cell expansion with a Th1 profile, and expansion of functional MART-1-specific CD8+ T cells, migration of immature CD14+ DDC was accompanied by increased release of IL-10, poor expansion of CD4+ and CD8+ T cells, and skewing of Th responses to favor coordinated FoxP3 and IL-10 expression and regulatory T cell differentiation and outgrowth. Thus, high levels of IL-10 impact the composition of skin-emigrated DC subsets and appear to favor migration of M2-like immature DC with functional qualities conducive to T cell tolerance. PMID:23875023

  13. How does cancer cell metabolism affect tumor migration and invasion?

    PubMed

    Han, Tianyu; Kang, De; Ji, Daokun; Wang, Xiaoyu; Zhan, Weihua; Fu, Minggui; Xin, Hong-Bo; Wang, Jian-Bin

    2013-01-01

    Cancer metastasis is the major cause of cancer-associated death. Accordingly, identification of the regulatory mechanisms that control whether or not tumor cells become "directed walkers" is a crucial issue of cancer research. The deregulation of cell migration during cancer progression determines the capacity of tumor cells to escape from the primary tumors and invade adjacent tissues to finally form metastases. The ability to switch from a predominantly oxidative metabolism to glycolysis and the production of lactate even when oxygen is plentiful is a key characteristic of cancer cells. This metabolic switch, known as the Warburg effect, was first described in 1920s, and affected not only tumor cell growth but also tumor cell migration. In this review, we will focus on the recent studies on how cancer cell metabolism affects tumor cell migration and invasion. Understanding the new aspects on molecular mechanisms and signaling pathways controlling tumor cell migration is critical for development of therapeutic strategies for cancer patients.

  14. Development of a novel automated cell isolation, expansion, and characterization platform.

    PubMed

    Franscini, Nicola; Wuertz, Karin; Patocchi-Tenzer, Isabel; Durner, Roland; Boos, Norbert; Graf-Hausner, Ursula

    2011-06-01

    Implementation of regenerative medicine in the clinical setting requires not only biological inventions, but also the development of reproducible and safe method for cell isolation and expansion. As the currently used manual techniques do not fulfill these requirements, there is a clear need to develop an adequate robotic platform for automated, large-scale production of cells or cell-based products. Here, we demonstrate an automated liquid-handling cell-culture platform that can be used to isolate, expand, and characterize human primary cells (e.g., from intervertebral disc tissue) with results that are comparable to the manual procedure. Specifically, no differences could be observed for cell yield, viability, aggregation rate, growth rate, and phenotype. Importantly, all steps-from the enzymatic isolation of cells through the biopsy to the final quality control-can be performed completely by the automated system because of novel tools that were incorporated into the platform. This automated cell-culture platform can therefore replace entirely manual processes in areas that require high throughput while maintaining stability and safety, such as clinical or industrial settings.

  15. Neocortex expansion in development and evolution - from cell biology to single genes.

    PubMed

    Wilsch-Bräuninger, Michaela; Florio, Marta; Huttner, Wieland B

    2016-08-01

    Neocortex expansion in development and evolution reflects an increased and prolonged activity of neural progenitor cells. Insight into key aspects of the underlying cell biology has recently been obtained. First, the restriction of apical progenitors to undergo mitosis at the ventricular surface is overcome by generation of basal progenitors, which are free to undergo mitosis at abventricular location, typically the subventricular zone. This process involves basolateral ciliogenesis, delamination from the apical adherens junction belt, and loss of apical cell polarity. Second, proliferative capacity of basal progenitors is supported by self-produced extracellular matrix constituents, which in turn promote growth factor signalling. Humans amplify these processes by characteristic alterations in expression of key regulatory genes (PAX6), and via human-specific genes (ARHGAP11B).

  16. Human Pluripotent Stem Cell Culture: Considerations for Maintenance, Expansion, and Therapeutics

    PubMed Central

    Chen, Kevin G.; Mallon, Barbara S.; McKay, Ronald D.G.; Robey, Pamela G.

    2014-01-01

    Summary Human pluripotent stem cells (hPSCs) provide powerful resources for application in regenerative medicine and pharmaceutical development. In the past decade, various methods have been developed for large-scale hPSC culture that rely on combined use of multiple growth components, including media containing various growth factors, extracellular matrices, three-dimensional environmental (3D) cues and modes of multicellular association. In this review, we dissect these growth components by comparing cell culture methods and identifying the benefits and pitfalls associated with each one. We further provide criteria, considerations, and suggestions to achieve optimal cell growth for hPSC expansion, differentiation, and use in future therapeutic applications. PMID:24388173

  17. Synthesis of Eupalinilide E, a Promoter of Human Hematopoietic Stem and Progenitor Cell Expansion.

    PubMed

    Johnson, Trevor C; Chin, Matthew R; Han, Tianxu; Shen, John Paul; Rana, Tariq; Siegel, Dionicio

    2016-05-11

    Improving the ex vivo and in vivo production of hematopoietic stem and progenitor cells (HSPCs) has the potential to address the short supply of these cells that are used in the treatment of various blood diseases and disorders. Eupalinilide E promotes the expansion of human HSPCs and inhibits subsequent differentiation, leading to increased numbers of clinically useful cells. This natural product represents an important tool to uncover new methods to drive expansion while inhibiting differentiation. However, in the process of examining these effects, which occur through a novel mechanism, the natural product was consumed, which limited additional investigation. To provide renewed and improved access to eupalinilide E, a laboratory synthesis has been developed and is reported herein. The synthetic route can access >400 mg in a single batch, employing reactions conducted on useful scales in a single vessel. Key transformations enabling the approach include a diastereoselective borylative enyne cyclization and a late-stage double allylic C-H oxidation as well as adapted Luche reduction and aluminum-mediated epoxidation reactions to maximize the synthetic efficiency. Retesting of the synthetic eupalinilide E confirmed the compound's ability to expand HSPCs and inhibit differentiation. PMID:27096704

  18. CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

    PubMed Central

    Zurawel, Ashley A.; Kabeche, Ruth; DiGregorio, Sonja E.; Deng, Lin; Menon, Kartikeya M.; Opalko, Hannah

    2016-01-01

    ABSTRACT Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms. PMID:27677791

  19. Current Injection Provokes Rapid Expansion of the Guard Cell Cytosolic Volume and Triggers Ca(2+) Signals.

    PubMed

    Voss, Lena J; Hedrich, Rainer; Roelfsema, M Rob G

    2016-03-01

    High-resolution microscopy opens the door for detailed single-cell studies with fluorescent reporter dyes and proteins. We used a confocal spinning disc microscope to monitor fluorescent dyes and the fluorescent protein Venus in tobacco and Arabidopsis guard cells. Multi-barreled microelectrodes were used to inject dyes and apply voltage pulses, which provoke transient rises in the cytosolic Ca(2+) level. Voltage pulses also caused changes in the distribution of Lucifer Yellow and Venus, which pointed to a reversible increase of guard cell cytosolic volume. The dynamic cytosolic volume changes turned out to be provoked by current injection of ions. A reduction of the clamp current, by blocking K(+) uptake channels with Cs(+), strongly suppressed the cytosolic volume changes. Cs(+) not only inhibited the expansion of the cytosol, but also inhibited hyperpolarization-induced elevations of the cytosolic Ca(2+) concentration. A complete loss of voltage-induced Ca(2+) signals occurred when Ca(2+)-permeable plasma membrane channels were simultaneously blocked with La(3+). This shows that two mechanisms cause hyperpolarization-induced elevation of the cytosolic Ca(2+)-concentration: (i) activation of voltage-dependent Ca(2+)-permeable channels, (ii) osmotically induced expansion of the cytosol, which leads to a release of Ca(2+) from intracellular stores.

  20. Klebsiella pneumoniae alleviates influenza-induced acute lung injury via limiting NK cell expansion.

    PubMed

    Wang, Jian; Li, Fengqi; Sun, Rui; Gao, Xiang; Wei, Haiming; Tian, Zhigang

    2014-08-01

    A protective effect induced by bacterial preinfection upon a subsequent lethal influenza virus infection has been observed, but the underlying immune mechanisms have not yet been fully elucidated. In this study, we used a mouse model of Klebsiella pneumoniae preinfection to gain insight into how bacterial preinfection influences the subsequent lethal influenza virus infection. We found that K. pneumoniae preinfection significantly attenuated lung immune injury and decreased mortality during influenza virus infection, but K. pneumoniae-specific immunity was not involved in this cross-protection against influenza virus. K. pneumoniae preinfection limited NK cell expansion, which was involved in influenza-induced immune injury and death. Furthermore, K. pneumoniae preinfection could not control NK cell expansion and death during influenza virus infection in Rag1(-/-) mice, but adoptive transfer of T cells from wild-type mice was able to restore this protective effect. Our data suggest that the adaptive immune response activated by bacterial infection limits the excessive innate immune response induced by a subsequent influenza infection, ultimately protecting mice from death.

  1. Fork stalling and template switching as a mechanism for polyalanine tract expansion affecting the DYC mutant of HOXD13, a new murine model of synpolydactyly.

    PubMed

    Cocquempot, Olivier; Brault, Véronique; Babinet, Charles; Herault, Yann

    2009-09-01

    Polyalanine expansion diseases are proposed to result from unequal crossover of sister chromatids that increases the number of repeats. In this report we suggest an alternative mechanism we put forward while we investigated a new spontaneous mutant that we named "Dyc" for "Digit in Y and Carpe" phenotype. Phenotypic analysis revealed an abnormal limb patterning similar to that of the human inherited congenital disease synpolydactyly (SPD) and to the mouse mutant model Spdh. Both human SPD and mouse Spdh mutations affect the Hoxd13 gene within a 15-residue polyalanine-encoding repeat in the first exon of the gene, leading to a dominant negative HOXD13. Genetic analysis of the Dyc mutant revealed a trinucleotide expansion in the polyalanine-encoding region of the Hoxd13 gene resulting in a 7-alanine expansion. However, unlike the Spdh mutation, this expansion cannot result from a simple duplication of a short segment. Instead, we propose the fork stalling and template switching (FosTeS) described for generation of nonrecurrent genomic rearrangements as a possible mechanism for the Dyc polyalanine extension, as well as for other polyalanine expansions described in the literature and that could not be explained by unequal crossing over.

  2. Clonal expansion of antitumor T cells in breast cancer correlates with response to neoadjuvant chemotherapy

    PubMed Central

    Park, Jae-Hyun; Jang, Miran; Tarhan, Yunus Emre; Katagiri, Toyomasa; Sasa, Mitsunori; Miyoshi, Yasuo; Kalari, Krishna R.; Suman, Vera J.; Weinshilboum, Richard; Wang, Liewei; Boughey, Judy C.; Goetz, Matthew P.; Nakamura, Yusuke

    2016-01-01

    The immune microenvironment of tumor plays a critical role in therapeutic responses to chemotherapy. Cancer tissues are composed of a complex network between anti-tumor and pro-tumor immune cells and molecules; therefore a comprehensive analysis of the tumor immune condition is imperative for better understanding of the roles of the immune microenvironment in anticancer treatment response. In this study, we performed T cell receptor (TCR) repertoire analysis of tumor infiltrating T cells (TILs) in cancer tissues of pre- and post-neoadjuvant chemotherapy (NAC) from 19 breast cancer patients; five cases showed CR (complete response), ten showed PR (partial response), and four showed SD/PD (stable disease/progressive disease) to the treatment. From the TCR sequencing results, we calculated the diversity index of the TCRβ chain and found that clonal expansion of TILs could be detected in patients who showed CR or PR to NAC. Noteworthy, the diversity of TCR was further reduced in the post-NAC tumors of CR patients. Our quantitative RT-PCR also showed that expression ratio of CD8/Foxp3 was significantly elevated in the post-NAC tumors of CR cases (p=0.0032), indicating that antitumor T cells were activated and enriched in these tumors. Collectively, our findings suggest that the clonal expansion of antitumor T cells may be a critical factor associated with response to chemotherapy and that their TCR sequences might be applicable for the development of TCR-engineered T cells treatment for individual breast cancer patients when their tumors relapse. PMID:27278091

  3. Chemically crosslinked alginate porous microcarriers modified with bioactive molecule for expansion of human hepatocellular carcinoma cells.

    PubMed

    Li, Chunge; Zhao, Shuang; Zhao, Yanyan; Qian, Yufeng; Li, Junjie; Yin, Yuji

    2014-11-01

    Microcarrier is an essential matrix for the large-scale culture of anchorage-dependent cells. In this study, chemical cross-linked alginate porous microcarriers (AMC) were prepared using microemulsion and freeze-drying technology. Moreover, chitosan was coated on the surface of microcarriers (AMC-CS) via electrostatic interactions to improve the mechanical strength. The size of AMC can be modulated through adjusting the concentration of alginate, amount of dispersant and stirring rate. The surface chemical characteristics and morphology of AMC-CS were evaluated by Fourier transformed infrared, X-ray photoelectron spectroscopy, and scanning electron microscope. Fibronectin (Fn) or heparin/basic fibroblast growth factor (bFGF) was then immobilized on the surface of microcarriers via layer-by-layer technology to improve the cytocompatibility. Our data suggested that the size of AMC can be accurately modulated from 90 μm to 900 μm with a narrow size distribution. Micropore structures of AMC-CS were relatively disordered and the pore size ranged between 20 μm and 100 μm. Using AMC after modified with Fn or bFGF as the cell expansion microcarriers, we showed that the proliferation rates of HepG2 cells increased significantly, reaching to more than 30-fold of cell expansion after 10 days of culture, with minor cellular damage caused by the microcarriers. Moreover, the AMC microcarriers modified with Fn or bFGF can increase albumin secretion of HepG2. We suggest that our new modified AMC-based microcarriers will be an attractive candidate for the large-scale cell culture of therapeutic cells.

  4. Histone deacetylase 11: A novel epigenetic regulator of myeloid derived suppressor cell expansion and function.

    PubMed

    Sahakian, Eva; Powers, John J; Chen, Jie; Deng, Susan L; Cheng, Fengdong; Distler, Allison; Woods, David M; Rock-Klotz, Jennifer; Sodre, Andressa L; Youn, Je-In; Woan, Karrune V; Villagra, Alejandro; Gabrilovich, Dmitry; Sotomayor, Eduardo M; Pinilla-Ibarz, Javier

    2015-02-01

    Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells capable of suppressing anti-tumor T cell function in the tumor microenvironment, represent an imposing obstacle in the development of cancer immunotherapeutics. Thus, identifying elements essential to the development and perpetuation of these cells will undoubtedly improve our ability to circumvent their suppressive impact. HDAC11 has emerged as a key regulator of IL-10 gene expression in myeloid cells, suggesting that this may represent an important targetable axis through which to dampen MDSC formation. Using a murine transgenic reporter model system where eGFP expression is controlled by the HDAC11 promoter (Tg-HDAC11-eGFP), we provide evidence that HDAC11 appears to function as a negative regulator of MDSC expansion/function in vivo. MDSCs isolated from EL4 tumor-bearing Tg-HDAC11-eGFP display high expression of eGFP, indicative of HDAC11 transcriptional activation at steady state. In striking contrast, immature myeloid cells in tumor-bearing mice display a diminished eGFP expression, implying that the transition of IMC to MDSC's require a decrease in the expression of HDAC11, where we postulate that it acts as a gate-keeper of myeloid differentiation. Indeed, tumor-bearing HDAC11-knockout mice (HDAC11-KO) demonstrate a more suppressive MDSC population as compared to wild-type (WT) tumor-bearing control. Notably, the HDAC11-KO tumor-bearing mice exhibit enhanced tumor growth kinetics when compare to the WT control mice. Thus, through a better understanding of this previously unknown role of HDAC11 in MDSC expansion and function, rational development of targeted epigenetic modifiers may allow us to thwart a powerful barrier to efficacious immunotherapies.

  5. Differential Responsiveness of Cortical Microtubule Orientation to Suppression of Cell Expansion among the Developmental Zones of Arabidopsis thaliana Root Apex

    PubMed Central

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S.; Daras, Gerasimos; Hatzopoulos, Polydefkis; Rigas, Stamatis

    2013-01-01

    Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone. PMID:24324790

  6. Differential responsiveness of cortical microtubule orientation to suppression of cell expansion among the developmental zones of Arabidopsis thaliana root apex.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Hatzopoulos, Polydefkis; Rigas, Stamatis

    2013-01-01

    Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone. PMID:24324790

  7. Narrow-Band Ultraviolet B Phototherapy Ameliorates Acute Graft-Versus-Host Disease of the Intestine by Expansion of Regulatory T Cells.

    PubMed

    Hashimoto, Akari; Sato, Tsutomu; Iyama, Satoshi; Yoshida, Masahiro; Ibata, Soushi; Tatekoshi, Ayumi; Kamihara, Yusuke; Horiguchi, Hiroto; Murase, Kazuyuki; Kawano, Yutaka; Takada, Kohichi; Miyanishi, Koji; Kobune, Masayoshi; Ichimiya, Shingo; Kato, Junji

    2016-01-01

    Narrowband ultraviolet B (NB-UVB) has been widely used in dermatological phototherapy. As for the application of NB-UVB phototherapy to graft-versus-host disease (GVHD), we previously reported that it was highly efficacious for cutaneous lesions of acute GVHD (aGVHD) and that expansion of regulatory T (Treg) cells induced by NB-UVB might be one of the mechanisms. In order to examine whether NB-UVB irradiation through expansion of Treg cells is effective for the treatment of not only cutaneous aGVHD but also aGVHD of inner organs such as the intestine or liver, we conducted experiments in which a murine lethal aGVHD model, characterized by severe involvement of the intestine, was irradiated with NB-UVB. We found that NB-UVB irradiation improved the clinical score and survival rate. The pathological score of aGVHD was improved in all affected organs: intestine, liver, and skin. In the serum of mice irradiated with NB-UVB, the levels of Treg cells-associated cytokines such as transforming growth factor beta (TGFβ) and interleukin-10 (IL-10) were elevated. The numbers of infiltrating Treg cells in inflamed tissue of the intestine and those in spleen were increased in mice treated with NB-UVB. This is the first report demonstrating that NB-UVB phototherapy has the ability to ameliorate intestinal aGVHD through the expansion of Treg cells.

  8. Narrow-Band Ultraviolet B Phototherapy Ameliorates Acute Graft-Versus-Host Disease of the Intestine by Expansion of Regulatory T Cells

    PubMed Central

    Iyama, Satoshi; Yoshida, Masahiro; Ibata, Soushi; Tatekoshi, Ayumi; Kamihara, Yusuke; Horiguchi, Hiroto; Murase, Kazuyuki; Kawano, Yutaka; Takada, Kohichi; Miyanishi, Koji; Kobune, Masayoshi; Ichimiya, Shingo; Kato, Junji

    2016-01-01

    Narrowband ultraviolet B (NB-UVB) has been widely used in dermatological phototherapy. As for the application of NB-UVB phototherapy to graft-versus-host disease (GVHD), we previously reported that it was highly efficacious for cutaneous lesions of acute GVHD (aGVHD) and that expansion of regulatory T (Treg) cells induced by NB-UVB might be one of the mechanisms. In order to examine whether NB-UVB irradiation through expansion of Treg cells is effective for the treatment of not only cutaneous aGVHD but also aGVHD of inner organs such as the intestine or liver, we conducted experiments in which a murine lethal aGVHD model, characterized by severe involvement of the intestine, was irradiated with NB-UVB. We found that NB-UVB irradiation improved the clinical score and survival rate. The pathological score of aGVHD was improved in all affected organs: intestine, liver, and skin. In the serum of mice irradiated with NB-UVB, the levels of Treg cells-associated cytokines such as transforming growth factor beta (TGFβ) and interleukin-10 (IL-10) were elevated. The numbers of infiltrating Treg cells in inflamed tissue of the intestine and those in spleen were increased in mice treated with NB-UVB. This is the first report demonstrating that NB-UVB phototherapy has the ability to ameliorate intestinal aGVHD through the expansion of Treg cells. PMID:27031239

  9. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration

    PubMed Central

    Huch, Meritxell; Dorrell, Craig; Boj, Sylvia F.; van Es, Johan H.; van de Wetering, Marc; Li, Vivian S.W.; Hamer, Karien; Sasaki, Nobuo; Finegold, Milton J.; Haft, Annelise; Grompe, Markus; Clevers, Hans

    2013-01-01

    The Wnt target gene Lgr5 marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon1, stomach2 and hair follicles3. A 3D culture system allows long-term clonal expansion of single Lgr5+ stem cells into transplantable organoids that retain many characteristics of the original epithelial architecture2, 4, 5. A crucial component of the culture medium is the Wnt agonist Rspo16, the recently discovered ligand of Lgr57, 8. Here we show that Lgr5-LacZ is not expressed in healthy adult liver, yet that small Lgr5-LacZ+ cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signaling. As shown by lineage tracing using a novel Lgr5-ires-CreERT2 knock-in allele, damage-induced Lgr5+ cells generate hepatocytes and bile ducts in vivo. Single Lgr5+ cells from damaged liver can be clonally expanded as organoids in Rspo1-based culture medium over multiple months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into FAH−/− mice. These findings imply that previous observations on Lgr5+ stem cells in actively self-renewing tissues extend to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation. PMID:23354049

  10. TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms

    PubMed Central

    Aichberger, Karl J.; Luty, Samuel B.; Bumm, Thomas G.; Petersen, Curtis L.; Doratotaj, Shirin; Vasudevan, Kavin B.; LaTocha, Dorian H.; Yang, Fei; Press, Richard D.; Loriaux, Marc M.; Pahl, Heike L.; Silver, Richard T.; Agarwal, Anupriya; O'Hare, Thomas; Druker, Brian J.; Bagby, Grover C.

    2011-01-01

    Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2V617F expressing cells in MPN. We show that JAK2V617F kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2V617F allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2V617F-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2V617F expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2V617F-positive MPN. Altogether our data are consistent with a model where JAK2V617F promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN. PMID:21860020

  11. FICTION-TSA analysis of the B-cell compartment in myeloma shows no significant expansion of myeloma precursor cells.

    PubMed

    Davies, F E; Rawstron, A C; Pratt, G; O'Connor, S; Su'ut, L; Blythe, D; Fenton, J; Claydon, D; Child, J A; Jack, A S; Morgan, G J

    1999-07-01

    Studies utilizing flow cytometry and PCR have shown that the B-cell compartment in myeloma contains cells which are clonally related to the myelomatous plasma cells. Current data, however, remains inconclusive regarding the extent of this involvement. By combining fluorescent immunophenotyping, tyramine signal amplification and fluorescence in-situ hybridization (FICTION-TSA), we have used the presence of numerical chromosomal abnormalities within plasma cells as a clonal marker to examine the CD20+ B-cell compartment for the presence of aneuploidy. A series of 54 cases of myeloma were screened for the presence of numerical abnormalities of chromosomes 3 and 11. FICTION-TSA was performed on 13 cases with either trisomy 3 or 11 and on a control group of six cases known to be disomic for the two chromosomes. B-cell numbers were reduced in the myeloma cases compared to the normal controls (median 1.8% v 3.0%, P = 0.05). In the cases with a chromosomal marker, three signals were seen in a median of 1.88% of CD20+ B cells compared to 2.58% within the control group. Comparison of the two groups using a Wilcoxon-Mann-Whitney U test showed no statistical significant difference. Using this data set, it was possible to exclude a 3.03% expansion of clonally related B cells (95% confidence level). We conclude that the B-cell compartment in myeloma does not represent the major site of clonal expansion, and if clonally related cells are present then the numbers are few. PMID:10444161

  12. Suppression of IL-7-dependent Effector T-cell Expansion by Multipotent Adult Progenitor Cells and PGE2.

    PubMed

    Reading, James L; Vaes, Bart; Hull, Caroline; Sabbah, Shereen; Hayday, Thomas; Wang, Nancy S; DiPiero, Anthony; Lehman, Nicholas A; Taggart, Jen M; Carty, Fiona; English, Karen; Pinxteren, Jef; Deans, Robert; Ting, Anthony E; Tree, Timothy I M

    2015-11-01

    T-cell depletion therapy is used to prevent acute allograft rejection, treat autoimmunity and create space for bone marrow or hematopoietic cell transplantation. The evolved response to T-cell loss is a transient increase in IL-7 that drives compensatory homeostatic proliferation (HP) of mature T cells. Paradoxically, the exaggerated form of this process that occurs following lymphodepletion expands effector T-cells, often causing loss of immunological tolerance that results in rapid graft rejection, autoimmunity, and exacerbated graft-versus-host disease (GVHD). While standard immune suppression is unable to treat these pathologies, growing evidence suggests that manipulating the incipient process of HP increases allograft survival, prevents autoimmunity, and markedly reduces GVHD. Multipotent adult progenitor cells (MAPC) are a clinical grade immunomodulatory cell therapy known to alter γ-chain cytokine responses in T-cells. Herein, we demonstrate that MAPC regulate HP of human T-cells, prevent the expansion of Th1, Th17, and Th22 effectors, and block the development of pathogenic allograft responses. This occurs via IL-1β-primed secretion of PGE2 and activates T-cell intrinsic regulatory mechanisms (SOCS2, GADD45A). These data provide proof-of-principle that HP of human T-cells can be targeted by cellular and molecular therapies and lays a basis for the development of novel strategies to prevent immunopathology in lymphodepleted patients. PMID:26216515

  13. Brain and muscle Arnt-like 1 promotes skeletal muscle regeneration through satellite cell expansion

    SciTech Connect

    Chatterjee, Somik; Yin, Hongshan; Nam, Deokhwa; Li, Yong; Ma, Ke

    2015-02-01

    Circadian clock is an evolutionarily conserved timing mechanism governing diverse biological processes and the skeletal muscle possesses intrinsic functional clocks. Interestingly, although the essential clock transcription activator, Brain and muscle Arnt-like 1 (Bmal1), participates in maintenance of muscle mass, little is known regarding its role in muscle growth and repair. In this report, we investigate the in vivo function of Bmal1 in skeletal muscle regeneration using two muscle injury models. Bmal1 is highly up-regulated by cardiotoxin injury, and its genetic ablation significantly impairs regeneration with markedly suppressed new myofiber formation and attenuated myogenic induction. A similarly defective regenerative response is observed in Bmal1-null mice as compared to wild-type controls upon freeze injury. Lack of satellite cell expansion accounts for the regeneration defect, as Bmal1{sup −/−} mice display significantly lower satellite cell number with nearly abolished induction of the satellite cell marker, Pax7. Furthermore, satellite cell-derived primary myoblasts devoid of Bmal1 display reduced growth and proliferation ex vivo. Collectively, our results demonstrate, for the first time, that Bmal1 is an integral component of the pro-myogenic response that is required for muscle repair. This mechanism may underlie its role in preserving adult muscle mass and could be targeted therapeutically to prevent muscle-wasting diseases. - Highlights: • Bmal1 is highly inducible by muscle injury and myogenic stimuli. • Genetic ablation of Bmal1 significantly impairs muscle regeneration. • Bmal1 promotes satellite cell expansion during muscle regeneration. • Bmal1-deficient primary myoblasts display attenuated growth and proliferation.

  14. GMP-Compliant Expansion of Clinical-Grade Human Mesenchymal Stromal/Stem Cells Using a Closed Hollow Fiber Bioreactor.

    PubMed

    Barckhausen, Christina; Rice, Brent; Baila, Stefano; Sensebé, Luc; Schrezenmeier, Hubert; Nold, Philipp; Hackstein, Holger; Rojewski, Markus Thomas

    2016-01-01

    This chapter describes a method for GMP-compliant expansion of human mesenchymal stromal/stem cells (hMSC) from bone marrow aspirates, using the Quantum(®) Cell Expansion System from Terumo BCT. The Quantum system is a functionally closed, automated hollow fiber bioreactor system designed to reproducibly grow cells in either GMP or research laboratory environments. The chapter includes protocols for preparation of media, setup of the Quantum system, coating of the hollow fiber bioreactor, as well as loading, feeding, and harvesting of cells. We suggest a panel of quality controls for the starting material, the interim product, as well as the final product. PMID:27236685

  15. Prostaglandin E2-EP4 signaling promotes immune inflammation through Th1 cell differentiation and Th17 cell expansion.

    PubMed

    Yao, Chengcan; Sakata, Daiji; Esaki, Yoshiyasu; Li, Youxian; Matsuoka, Toshiyuki; Kuroiwa, Kenji; Sugimoto, Yukihiko; Narumiya, Shuh

    2009-06-01

    Two distinct helper T (TH) subsets, TH1 and TH17, mediate tissue damage and inflammation in animal models of various immune diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases and allergic skin disorders. These experimental findings, and the implication of these TH subsets in human diseases, suggest the need for pharmacological measures to manipulate these TH subsets. Here we show that prostaglandin E2 (PGE2) acting on its receptor EP4 on T cells and dendritic cells not only facilitates TH1 cell differentiation but also amplifies interleukin-23-mediated TH17 cell expansion in vitro. Administration of an EP4-selective antagonist in vivo decreases accumulation of both TH1 and TH17 cells in regional lymph nodes and suppresses the disease progression in mice subjected to experimental autoimmune encephalomyelitis or contact hypersensitivity. Thus, PGE2-EP4 signaling promotes immune inflammation through TH1 differentiation and TH17 expansion, and EP4 antagonism may be therapeutically useful for various immune diseases.

  16. Middle-term expansion of hematopoietic cord blood cells with new human stromal cell line feeder-layers and different cytokine cocktails.

    PubMed

    De Angeli, S; Baiguera, S; Del Pup, L; Pavan, E; Gajo, G B; Di Liddo, R; Conconi, M T; Grandi, C; Schiavon, O; Parnigotto, P P

    2009-12-01

    Cord blood (CB) is a source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow for allogenic transplantation in patients with hematological disorders. The improvement of HSC in vitro expansion is one of the main challenges in cell therapy. Stromal components and soluble factors, such as cytokines, can be useful to induce in vitro cell expansion. Hence, we investigated whether feeder-layers from new stromal cell lines and different exogenous cytokine cocktails induce HSC expansion in middle-term cultures. CB HSC middle-term expansion was carried out in co-cultures on different feeder-layers exposed to three different cytokine cocktails. CB HSC expansion was also carried out in stroma-free cultures in the presence of different cytokine cocktails. Clonogenic tests were performed, and cell growth levels were evaluated. Moreover, the presence of VCAM-1 mRNA was assessed, and the mesenchymal cell-like phenotype expression was detected. All feeder-layers were able to induce a significant clonogenic growth with respect to the control culture, and all of the cytokine cocktails induced a significant increase in CB cell expansion indexes, even though no potential variation dependent on their composition was noted. The modulative effects of the different cocktails, exerted on each cell line used, was dependent on their composition. Finally, all cell lines were positive for CD73, CD117 and CD309, similar to mesenchymal stem cells present in adult bone marrow and in other human tissues, and negative for the hematopoietic markers. These data indicate that our cell lines have, not only a stromal cell-like phenotype, but also a mesenchymal cell-like phenotype, and they have the potential to support in vitro expansion of CB HSCs. Moreover, exogenous cytokines can be used in synergism with feeder-layers to improve the expansion levels of CB HSCs in preparation for their clinical use in allogenic transplantation. PMID:19885627

  17. Modeling of thermal expansion coefficient of perovskite oxide for solid oxide fuel cell cathode

    NASA Astrophysics Data System (ADS)

    Heydari, F.; Maghsoudipour, A.; Alizadeh, M.; Khakpour, Z.; Javaheri, M.

    2015-09-01

    Artificial intelligence models have the capacity to eliminate the need for expensive experimental investigation in various areas of manufacturing processes, including the material science. This study investigates the applicability of adaptive neuro-fuzzy inference system (ANFIS) approach for modeling the performance parameters of thermal expansion coefficient (TEC) of perovskite oxide for solid oxide fuel cell cathode. Oxides (Ln = La, Nd, Sm and M = Fe, Ni, Mn) have been prepared and characterized to study the influence of the different cations on TEC. Experimental results have shown TEC decreases favorably with substitution of Nd3+ and Mn3+ ions in the lattice. Structural parameters of compounds have been determined by X-ray diffraction, and field emission scanning electron microscopy has been used for the morphological study. Comparison results indicated that the ANFIS technique could be employed successfully in modeling thermal expansion coefficient of perovskite oxide for solid oxide fuel cell cathode, and considerable savings in terms of cost and time could be obtained by using ANFIS technique.

  18. Making the Switch: Alternatives to Fetal Bovine Serum for Adipose-Derived Stromal Cell Expansion

    PubMed Central

    Dessels, Carla; Potgieter, Marnie; Pepper, Michael S.

    2016-01-01

    Adipose-derived stromal cells (ASCs) are being used extensively in clinical trials. These trials require that ASCs are prepared using good manufacturing practices (GMPs) and are safe for use in humans. The majority of clinical trials in which ASCs are expanded make use of fetal bovine serum (FBS). While FBS is used traditionally in the research setting for in vitro expansion, it does carry the risk of xenoimmunization and zoonotic transmission when used for expanding cells destined for therapeutic purposes. In order to ensure a GMP quality product for cellular therapy, in vitro expansion of ASCs has been undertaken using xeno-free (XF), chemically-defined, and human blood-derived alternatives. These investigations usually include the criteria proposed by the International Society of Cellular Therapy (ISCT) and International Fat Applied Technology Society (IFATS). The majority of studies use these criteria to compare plastic-adherence, morphology, the immunophenotype and the trilineage differentiation of ASCs under the different medium supplemented conditions. Based on these studies, all of the alternatives to FBS seem to be suitable replacements; however, each has its own advantages and drawbacks. Very few studies have investigated the effects of the supplements on the immunomodulation of ASCs; the transcriptome, proteome and secretome; and the ultimate effects in appropriate animal models. The selection of medium supplementation will depend on the downstream application of the ASCs and their efficacy and safety in preclinical studies. PMID:27800478

  19. Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells.

    PubMed

    Soh, Boon-Seng; Ng, Shi-Yan; Wu, Hao; Buac, Kristina; Park, Joo-Hye C; Lian, Xiaojun; Xu, Jiejia; Foo, Kylie S; Felldin, Ulrika; He, Xiaobing; Nichane, Massimo; Yang, Henry; Bu, Lei; Li, Ronald A; Lim, Bing; Chien, Kenneth R

    2016-03-08

    Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo.

  20. Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells

    PubMed Central

    Soh, Boon-Seng; Ng, Shi-Yan; Wu, Hao; Buac, Kristina; Park, Joo-Hye C.; Lian, Xiaojun; Xu, Jiejia; Foo, Kylie S.; Felldin, Ulrika; He, Xiaobing; Nichane, Massimo; Yang, Henry; Bu, Lei; Li, Ronald A.; Lim, Bing; Chien, Kenneth R.

    2016-01-01

    Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human–mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1+ vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo. PMID:26952167

  1. Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells.

    PubMed

    Soh, Boon-Seng; Ng, Shi-Yan; Wu, Hao; Buac, Kristina; Park, Joo-Hye C; Lian, Xiaojun; Xu, Jiejia; Foo, Kylie S; Felldin, Ulrika; He, Xiaobing; Nichane, Massimo; Yang, Henry; Bu, Lei; Li, Ronald A; Lim, Bing; Chien, Kenneth R

    2016-01-01

    Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo. PMID:26952167

  2. Robust Pluripotent Stem Cell Expansion and Cardiomyocyte Differentiation via Geometric Patterning

    PubMed Central

    Myers, Frank B.; Silver, Jason S.; Zhuge, Yan; Beygui, Ramin E.; Zarins, Christopher K.; Lee, Luke P.; Abilez, Oscar J.

    2013-01-01

    Geometric factors including the size, shape, density, and spacing of pluripotent stem cell colonies play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such, there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Here, we demonstrate a simple, robust technique for pluripotent stem cell expansion and cardiomyocyte differentiation by patterning cell colonies with a silicone stencil. We have observed that patterning human induced pluripotent stem cell (hiPSC) colonies improves the uniformity and repeatability of their size, density, and shape. Uniformity of colony geometry leads to improved homogeneity in the expression of pluripotency markers SSEA4 and Nanog as compared with conventional clump passaging. Patterned cell colonies are capable of undergoing directed differentiation into spontaneously beating cardiomyocyte clusters with improved yield and repeatability over unpatterned cultures seeded either as cell clumps or uniform single cell suspensions. Circular patterns result in a highly repeatable 3D ring-shaped band of cardiomyocytes which electrically couple and lead to propagating contraction waves around the ring. Because of these advantages, geometrically patterning stem cells using stencils may offer greater repeatability from batch-to-batch and person-to-person, an increase in differentiation yield, a faster experimental workflow, and a simpler protocol to communicate and follow. Furthermore, the ability to control where cardiomyocytes arise across a culture well during differentiation could greatly aid the design of electrophysiological assays for drug-screening. PMID:24141327

  3. Robust pluripotent stem cell expansion and cardiomyocyte differentiation via geometric patterning.

    PubMed

    Myers, Frank B; Silver, Jason S; Zhuge, Yan; Beygui, Ramin E; Zarins, Christopher K; Lee, Luke P; Abilez, Oscar J

    2013-12-01

    Geometric factors including the size, shape, density, and spacing of pluripotent stem cell colonies play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such, there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Here, we demonstrate a simple, robust technique for pluripotent stem cell expansion and cardiomyocyte differentiation by patterning cell colonies with a silicone stencil. We have observed that patterning human induced pluripotent stem cell (hiPSC) colonies improves the uniformity and repeatability of their size, density, and shape. Uniformity of colony geometry leads to improved homogeneity in the expression of pluripotency markers SSEA4 and Nanog as compared with conventional clump passaging. Patterned cell colonies are capable of undergoing directed differentiation into spontaneously beating cardiomyocyte clusters with improved yield and repeatability over unpatterned cultures seeded either as cell clumps or uniform single cell suspensions. Circular patterns result in a highly repeatable 3D ring-shaped band of cardiomyocytes which electrically couple and lead to propagating contraction waves around the ring. Because of these advantages, geometrically patterning stem cells using stencils may offer greater repeatability from batch-to-batch and person-to-person, an increase in differentiation yield, a faster experimental workflow, and a simpler protocol to communicate and follow. Furthermore, the ability to control where cardiomyocytes arise across a culture well during differentiation could greatly aid the design of electrophysiological assays for drug-screening. PMID:24141327

  4. Generation and In Vitro Expansion of Hepatic Progenitor Cells from Human iPS Cells.

    PubMed

    Yanagida, Ayaka; Nakauchi, Hiromitsu; Kamiya, Akihide

    2016-01-01

    Stem cells have the unique properties of self-renewal and multipotency (producing progeny belonging to two or more lineages). Induced pluripotent stem (iPS) cells can be generated from somatic cells by simultaneous expression of pluripotent factors (Oct3/4, Klf4, Sox2, and c-Myc). They share the same properties as embryonic stem (ES) cells and can differentiate into several tissue cells, i.e., neurons, hematopoietic cells, and liver cells. Therefore, iPS cells are suitable candidate cells for regenerative medicine and analyses of disease mechanisms.The liver is the major organ that regulates a multitude of metabolic functions. Hepatocytes are the major cell type populating the liver parenchyma and express several metabolic enzymes that are necessary for liver functions. Although hepatocytes are essential for maintaining homeostasis, it is difficult to alter artificial and transplanted cells because of their multifunctionality, donor shortage, and immunorejection risk. During liver development, hepatic progenitor cells in the fetal liver differentiate into both mature hepatocytes and cholangiocytes. As hepatic progenitor cells have bipotency and high proliferation ability, they could present a potential source for generating transplantable cells or as a liver study model. Here we describe the induction and purification of hepatic progenitor cells derived from human iPS cells. These cells can proliferate for a long term under suitable culture conditions.

  5. Stem cell expansion during carcinogenesis in stem cell-depleted conditional telomeric repeat factor 2 null mutant mice.

    PubMed

    Bojovic, B; Ho, H-Y; Wu, J; Crowe, D L

    2013-10-24

    To examine the role of telomeric repeat-binding factor 2 (TRF2) in epithelial tumorigenesis, we characterized conditional loss of TRF2 expression in the basal layer of mouse epidermis. These mice exhibit some characteristics of dyskeratosis congenita, a human stem cell depletion syndrome caused by telomere dysfunction. The epidermis in conditional TRF2 null mice exhibited DNA damage response and apoptosis, which correlated with stem cell depletion. The stem cell population in conditional TRF2 null epidermis exhibited shorter telomeres than those in control mice. Squamous cell carcinomas induced in conditional TRF2 null mice developed with increased latency and slower growth due to reduced numbers of proliferating cells as the result of increased apoptosis. TRF2 null epidermal stem cells were found in both primary and metastatic tumors. Despite the low-grade phenotype of the conditional TRF2 null primary tumors, the number of metastatic lesions was similar to control cancers. Basal cells from TRF2 null tumors demonstrated extreme telomere shortening and dramatically increased numbers of telomeric signals by fluorescence in situ hybridization due to increased genomic instability and aneuploidy in these cancers. DNA damage response signals were detected at telomeres in TRF2 null tumor cells from these mice. The increased genomic instability in these tumors correlated with eightfold expansion of the transformed stem cell population compared with that in control cancers. We concluded that genomic instability resulting from loss of TRF2 expression provides biological advantages to the cancer stem cell population.

  6. FACKEL is a sterol C-14 reductase required for organized cell division and expansion in Arabidopsis embryogenesis

    PubMed Central

    Schrick, Kathrin; Mayer, Ulrike; Horrichs, Andrea; Kuhnt, Christine; Bellini, Catherine; Dangl, Jeff; Schmidt, Jürgen; Jürgens, Gerd

    2000-01-01

    In flowering plants, the developing embryo consists of growing populations of cells whose fates are determined in a position-dependent manner to form the adult organism. Mutations in the FACKEL (FK) gene affect body organization of the Arabidopsis seedling. We report that FK is required for cell division and expansion and is involved in proper organization of the embryo. We isolated FK by positional cloning. Expression analysis in embryos revealed that FK mRNA becomes localized to meristematic zones. FK encodes a predicted integral membrane protein related to the vertebrate lamin B receptor and sterol reductases across species, including yeast sterol C-14 reductase ERG24. We provide functional evidence that FK encodes a sterol C-14 reductase by complementation of erg24. GC/MS analysis confirmed that fk mutations lead to accumulation of intermediates in the biosynthetic pathway preceding the C-14 reductase step. Although fk represents a sterol biosynthetic mutant, the phenotype was not rescued by feeding with brassinosteroids (BRs), the only plant sterol signaling molecules known so far. We propose that synthesis of sterol signals in addition to BRs is important in mediating regulated cell growth and organization during embryonic development. Our results indicate a novel role for sterols in the embryogenesis of plants. PMID:10859166

  7. Rapid regulatory control of plant cell expansion and wall relaxation. Annual technical progress report

    SciTech Connect

    Cosgrove, D.J.

    1991-08-14

    The aim of this project is to elucidate the biophysical and cellular mechanisms that control plant cell expansion. At present we are attempting to characterize the kinetics of the system(s) responsible for regulatory and compensatory behavior of growing cells and tissues. This work is significantly because it indicates that biochemical loosening and biophysical stress relaxation of the wall are part of a feedback loop controlling growth. This report briefly summarizes the efforts and results of the past 12 months. In large part, we have been trying to analyze the nature of growth rate ``noise,`` i.e. spontaneous and often erratic variations in growth rate. We are obtaining evidence that such ``noise`` is not random, but rather reveals an underlying growth mechanism with complex dynamics.

  8. The isolation and in vitro expansion of hepatic Sca-1 progenitor cells

    SciTech Connect

    Clayton, Elizabeth

    2009-04-17

    The intra-hepatic population of liver progenitor cells expands during liver injury when hepatocyte proliferation is inhibited. These cells can be purified by density gradient centrifugation and cultured. Separated by size only this population contains small cells of hematopoietic, epithelial and endothelial lineages and is thought to contain liver stem cells. The identity of liver stem cells remains unknown although there is some evidence that tissue Sca1{sup +} CD45{sup -} cells display progenitor cell characteristics. We identified both intra-hepatic and gall bladder Sca1{sup +} cells following liver injury and expanded ex vivo Sca1 cells as part of heterogenous cell culture or as a purified population. We found significant difference between the proliferation of Sca-1 cells when plated on laminin or collagen I while proliferation of heterogenous population was not affected by the extracellular matrix indicating the necessity for culture of Sca1{sup +} cells with laminin matrix or laminin producing cells in long term liver progenitor cell cultures.

  9. Expansion of brain T cells in homeostatic conditions in lymphopenic Rag2(-/-) mice.

    PubMed

    Song, Chang; Nicholson, James D; Clark, Sarah M; Li, Xin; Keegan, Achsah D; Tonelli, Leonardo H

    2016-10-01

    The concept of the brain as an immune privileged organ is rapidly evolving in light of new findings outlining the sophisticated relationship between the central nervous and the immune systems. The role of T cells in brain development and function, as well as modulation of behavior has been demonstrated by an increasing number of studies. Moreover, recent studies have redefined the existence of a brain lymphatic system and the presence of T cells in specific brain structures, such as the meninges and choroid plexus. Nevertheless, much information is needed to further the understanding of brain T cells and their relationship with the central nervous system under non-inflammatory conditions. In the present study we employed the Rag2(-/-) mouse model of lymphocyte deficiency and reconstitution by adoptive transfer to study the temporal and anatomical expansion of T cells in the brain under homeostatic conditions. Lymphopenic Rag2(-/-) mice were reconstituted with 10 million lymphoid cells and studied at one, two and four weeks after transfer. Moreover, lymphoid cells and purified CD4(+) and CD8(+) T cells from transgenic GFP expressing mice were used to define the neuroanatomical localization of transferred cells. T cell numbers were very low in the brain of reconstituted mice up to one week after transfer and significantly increased by 2weeks, reaching wild type values at 4weeks after transfer. CD4(+) T cells were the most abundant lymphocyte subtype found in the brain followed by CD8(+) T cells and lastly B cells. Furthermore, proliferation studies showed that CD4(+) T cells expand more rapidly than CD8(+) T cells. Lymphoid cells localize abundantly in meningeal structures, choroid plexus, and circumventricular organs. Lymphocytes were also found in vascular and perivascular spaces and in the brain parenchyma across several regions of the brain, in particular in structures rich in white matter content. These results provide proof of concept that the brain meningeal

  10. Expansion of brain T cells in homeostatic conditions in lymphopenic Rag2(-/-) mice.

    PubMed

    Song, Chang; Nicholson, James D; Clark, Sarah M; Li, Xin; Keegan, Achsah D; Tonelli, Leonardo H

    2016-10-01

    The concept of the brain as an immune privileged organ is rapidly evolving in light of new findings outlining the sophisticated relationship between the central nervous and the immune systems. The role of T cells in brain development and function, as well as modulation of behavior has been demonstrated by an increasing number of studies. Moreover, recent studies have redefined the existence of a brain lymphatic system and the presence of T cells in specific brain structures, such as the meninges and choroid plexus. Nevertheless, much information is needed to further the understanding of brain T cells and their relationship with the central nervous system under non-inflammatory conditions. In the present study we employed the Rag2(-/-) mouse model of lymphocyte deficiency and reconstitution by adoptive transfer to study the temporal and anatomical expansion of T cells in the brain under homeostatic conditions. Lymphopenic Rag2(-/-) mice were reconstituted with 10 million lymphoid cells and studied at one, two and four weeks after transfer. Moreover, lymphoid cells and purified CD4(+) and CD8(+) T cells from transgenic GFP expressing mice were used to define the neuroanatomical localization of transferred cells. T cell numbers were very low in the brain of reconstituted mice up to one week after transfer and significantly increased by 2weeks, reaching wild type values at 4weeks after transfer. CD4(+) T cells were the most abundant lymphocyte subtype found in the brain followed by CD8(+) T cells and lastly B cells. Furthermore, proliferation studies showed that CD4(+) T cells expand more rapidly than CD8(+) T cells. Lymphoid cells localize abundantly in meningeal structures, choroid plexus, and circumventricular organs. Lymphocytes were also found in vascular and perivascular spaces and in the brain parenchyma across several regions of the brain, in particular in structures rich in white matter content. These results provide proof of concept that the brain meningeal

  11. WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis.

    PubMed

    Yuen, Christen Y L; Pearlman, Rebecca S; Silo-Suh, Laura; Hilson, Pierre; Carroll, Kathleen L; Masson, Patrick H

    2003-02-01

    Wild-type Arabidopsis roots develop a wavy pattern of growth on tilted agar surfaces. For many Arabidopsis ecotypes, roots also grow askew on such surfaces, typically slanting to the right of the gravity vector. We identified a mutant, wvd2-1, that displays suppressed root waving and leftward root slanting under these conditions. These phenotypes arise from transcriptional activation of the novel WAVE-DAMPENED2 (WVD2) gene by the cauliflower mosaic virus 35S promoter in mutant plants. Seedlings overexpressing WVD2 exhibit constitutive right-handed helical growth in both roots and etiolated hypocotyls, whereas the petioles of WVD2-overexpressing rosette leaves exhibit left-handed twisting. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs. In roots, the phenotype is accompanied by a change in the arrangement of cortical microtubules within peripheral cap cells and cells at the basal end of the elongation zone. WVD2 transcripts are detectable by reverse transcriptase-polymerase chain reaction in multiple organs of wild-type plants. Its predicted gene product contains a conserved region named "KLEEK," which is found only in plant proteins. The Arabidopsis genome possesses seven other genes predicted to encode KLEEK-containing products. Overexpression of one of these genes, WVD2-LIKE 1, which encodes a protein with regions of similarity to WVD2 extending beyond the KLEEK domain, results in phenotypes that are highly similar to wvd2-1. Silencing of WVD2 and its paralogs results in enhanced root skewing in the wild-type direction. Our observations suggest that at least two members of this gene family may modulate both rotational polarity and anisotropic cell expansion during organ growth.

  12. WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Yuen, Christen Y L.; Pearlman, Rebecca S.; Silo-Suh, Laura; Hilson, Pierre; Carroll, Kathleen L.; Masson, Patrick H.

    2003-01-01

    Wild-type Arabidopsis roots develop a wavy pattern of growth on tilted agar surfaces. For many Arabidopsis ecotypes, roots also grow askew on such surfaces, typically slanting to the right of the gravity vector. We identified a mutant, wvd2-1, that displays suppressed root waving and leftward root slanting under these conditions. These phenotypes arise from transcriptional activation of the novel WAVE-DAMPENED2 (WVD2) gene by the cauliflower mosaic virus 35S promoter in mutant plants. Seedlings overexpressing WVD2 exhibit constitutive right-handed helical growth in both roots and etiolated hypocotyls, whereas the petioles of WVD2-overexpressing rosette leaves exhibit left-handed twisting. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs. In roots, the phenotype is accompanied by a change in the arrangement of cortical microtubules within peripheral cap cells and cells at the basal end of the elongation zone. WVD2 transcripts are detectable by reverse transcriptase-polymerase chain reaction in multiple organs of wild-type plants. Its predicted gene product contains a conserved region named "KLEEK," which is found only in plant proteins. The Arabidopsis genome possesses seven other genes predicted to encode KLEEK-containing products. Overexpression of one of these genes, WVD2-LIKE 1, which encodes a protein with regions of similarity to WVD2 extending beyond the KLEEK domain, results in phenotypes that are highly similar to wvd2-1. Silencing of WVD2 and its paralogs results in enhanced root skewing in the wild-type direction. Our observations suggest that at least two members of this gene family may modulate both rotational polarity and anisotropic cell expansion during organ growth.

  13. Expansion of CD11b(+)Ly6G (+)Ly6C (int) cells driven by medroxyprogesterone acetate in mice bearing breast tumors restrains NK cell effector functions.

    PubMed

    Spallanzani, Raúl Germán; Dalotto-Moreno, Tomás; Raffo Iraolagoitia, Ximena Lucía; Ziblat, Andrea; Domaica, Carolina Inés; Avila, Damián Ezequiel; Rossi, Lucas Ezequiel; Fuertes, Mercedes Beatriz; Battistone, María Agustina; Rabinovich, Gabriel Adrián; Salatino, Mariana; Zwirner, Norberto Walter

    2013-12-01

    The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b(+)Gr-1(+), mostly CD11b(+)Ly6G(+)Ly6C(int) and CD11b(+)Ly6G(-)Ly6C(high) cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b(+)Ly6G(+)Ly6C(int) cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b(+)Ly6G(+)Ly6C(int) cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b(+)Gr-1(+) cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b(+)Gr-1(+) cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b(+)Ly6G(+)Ly6C(int) cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence. PMID:24114144

  14. Epicutaneous sensitization results in IgE-dependent intestinal mast cell expansion and food anaphylaxis

    PubMed Central

    Bartnikas, Lisa M.; Gurish, Michael F.; Burton, Oliver T.; Leisten, Sabine; Janssen, Erin; Oettgen, Hans C.; Beaupré, Jacqueline; Lewis, Christopher N.; Austen, K. Frank; Schulte, Stephanie; Hornick, Jason L.; Geha, Raif S.; Oyoshi, Michiko K.

    2013-01-01

    Background Sensitization to food antigen may occur through cutaneous exposure. Objective Test the hypothesis that epicutaneous (EC) sensitization with food antigen predisposes to IgE-mediated anaphylaxis upon oral allergen challenge. Methods BALB/c mice were EC sensitized by repeated application of ovalbumin (OVA) to tape-stripped skin over 7 weeks, or orally immunized with OVA and cholera toxin (CT) weekly for 8 weeks, then orally challenged with OVA. Body temperature was monitored and serum mouse mast cell protease 1 (mMCP-1) level was determined following challenge. Tissue mast cells (MCs) were examined by chloroacetate esterase (CAE) staining. Serum OVA-specific IgE and IgG1 antibodies, and cytokines in supernatants of OVA-stimulated splenocytes, were measured by ELISA. Serum interleukin-4 (IL-4) levels were measured using an in vivo cytokine capture assay (IVCCA). Results EC sensitized mice exhibited expansion of connective tissue MC in the jejunum, increased serum IL-4 levels, and systemic anaphylaxis following oral challenge, as evidenced by decreased body temperature and increased serum mMCP-1 level. Intestinal MC expansion and anaphylaxis were IgE-dependent, as they did not occur in EC sensitized IgE−/− mice. Mice orally immunized with OVA+CT failed to increase serum IL-4 levels, expand their intestinal MCs, or develop anaphylaxis following oral challenge, despite OVA-specific IgE levels and splenocyte cytokine production in response to OVA stimulation, which were comparable to those of EC sensitized mice. Conclusion EC sensitized mice, but not mice orally immunized with antigen+CT, develop expansion of intestinal MCs and IgE-mediated anaphylaxis following single oral antigen challenge. IgE is necessary but not sufficient for food anaphylaxis, and MC expansion in the gut may play an important role in the development of anaphylaxis. Clinical Implications The skin may be an important route of sensitization to food antigens. Avoidance of cutaneous

  15. Expansion and Homing of Adoptively Transferred Human NK Cells in Immunodeficient Mice Varies with Product Preparation and In Vivo Cytokine Administration: Implications for Clinical Therapy

    PubMed Central

    Miller, Jeffrey S.; Rooney, Cliona M; Curtsinger, Julie; McElmurry, Ron; McCullar, Valarie; Verneris, Michael R.; Lapteva, Natalia; McKenna, David; Wagner, John E.; Blazar, Bruce R.; Tolar, Jakub

    2014-01-01

    NK cell efficacy correlates with in vivo proliferation and we hypothesize that NK cell product manipulations may optimize this endpoint. Xenotransplantation was used to compare GMP grade freshly activated NK cells (FA-NK) and ex vivo expanded NK cells (Ex-NK). Cells were infused into NSG mice followed by IL-2, IL-15, or no cytokines. Evaluation of blood, spleen and marrow showed that persistence and expansion was cytokine dependent, IL-15 being superior to IL-2. Cryopreservation and immediate infusion resulted in less cytotoxicity and fewer NK cells in vivo and this could be rescued in FA-NK by overnight culture and testing the next day. Marked differences in the kinetics and homing of FA-NK versus Ex-NK were apparent: FA-NK cells preferentially homed to spleen, and persisted longer after cytokine withdrawal. These data would suggest that cryopreservation of FA-NK and Ex-NK is detrimental and that culture conditions profoundly affect homing, persistence and expansion of NK cells in vivo. The NSG mouse model is an adjuvant to in vitro assays prior to clinical testing. PMID:24816582

  16. Artificial antigen presenting cell (aAPC) mediated activation and expansion of natural killer T cells.

    PubMed

    East, James E; Sun, Wenji; Webb, Tonya J

    2012-01-01

    Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells(1). Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules(2). NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity(3-6), and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d (7). The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines(8). Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines(9,10). In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients(11). However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells(12, 13). Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is

  17. Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration.

    PubMed

    Rodrigo Albors, Aida; Tazaki, Akira; Rost, Fabian; Nowoshilow, Sergej; Chara, Osvaldo; Tanaka, Elly M

    2015-11-14

    Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue.

  18. Cutting Edge: Innate Lymphoid Cells Suppress Homeostatic T Cell Expansion in Neonatal Mice.

    PubMed

    Bank, Ute; Deiser, Katrin; Finke, Daniela; Hämmerling, Günter J; Arnold, Bernd; Schüler, Thomas

    2016-05-01

    In adult mice, lymphopenia-induced proliferation (LIP) leads to T cell activation, memory differentiation, tissue destruction, and a loss of TCR diversity. Neonatal mice are lymphopenic within the first week of life. This enables some recent thymic emigrants to undergo LIP and convert into long-lived memory T cells. Surprisingly, however, most neonatal T cells do not undergo LIP. We therefore asked whether neonate-specific mechanisms prevent lymphopenia-driven T cell activation. In this study, we show that IL-7R-dependent innate lymphoid cells (ILCs) block LIP of CD8(+) T cells in neonatal but not adult mice. Importantly, CD8(+) T cell responses against a foreign Ag are not inhibited by neonatal ILCs. This ILC-based inhibition of LIP ensures the generation of a diverse naive T cell pool in lymphopenic neonates that is mandatory for the maintenance of T cell homeostasis and immunological self-tolerance later in life.

  19. Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration.

    PubMed

    Rodrigo Albors, Aida; Tazaki, Akira; Rost, Fabian; Nowoshilow, Sergej; Chara, Osvaldo; Tanaka, Elly M

    2015-01-01

    Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue. PMID:26568310

  20. Pleural cavity type 2 innate lymphoid cells precede Th2 expansion in murine Litomosoides sigmodontis infection.

    PubMed

    Boyd, Alexis; Killoran, Kristin; Mitre, Edward; Nutman, Thomas B

    2015-12-01

    Recently, a family of innate cells has been identified that respond to IL-25 and IL-33 in murine intestinal helminths. Termed Type 2 innate lymphoid cells (ILC2s) they facilitate the development of Th2 responses responsible for helminth clearance. We evaluated these cells in a tissue-invasive helminth model. Using Litomosides sigmodontis (a strong Th2 polarizing filarial infection) we observed a robust Th2 response in the pleural cavity, where adult worms reside, marked by increased levels of IL-5 and IL-13 in infected mice. In parallel, ILC2s were expanded in the pleural cavity early in the infection, peaking during the pre-patent period. L. sigmodontis also elicits a strong systemic Th2 response, which includes significantly increased levels of IgG1, IgE and IL-5 in the plasma of infected mice. Although ILC2s were expanded locally, they were not expanded in the spleen, blood, or mediastinal lymph nodes in response to L. sigmodontis infection, suggesting that ILC2s function primarily at the site of infection. The increase in ILC2s in the pleural cavity and the expansion in Th2 responses indicates a probable role for these cells in initiating and maintaining the Th2 response and highlights the importance of these cells in helminth infections and their role in Th2 immunity.

  1. Pleural cavity type 2 innate lymphoid cells precede Th2 expansion in murine Litomosoides sigmodontis infection.

    PubMed

    Boyd, Alexis; Killoran, Kristin; Mitre, Edward; Nutman, Thomas B

    2015-12-01

    Recently, a family of innate cells has been identified that respond to IL-25 and IL-33 in murine intestinal helminths. Termed Type 2 innate lymphoid cells (ILC2s) they facilitate the development of Th2 responses responsible for helminth clearance. We evaluated these cells in a tissue-invasive helminth model. Using Litomosides sigmodontis (a strong Th2 polarizing filarial infection) we observed a robust Th2 response in the pleural cavity, where adult worms reside, marked by increased levels of IL-5 and IL-13 in infected mice. In parallel, ILC2s were expanded in the pleural cavity early in the infection, peaking during the pre-patent period. L. sigmodontis also elicits a strong systemic Th2 response, which includes significantly increased levels of IgG1, IgE and IL-5 in the plasma of infected mice. Although ILC2s were expanded locally, they were not expanded in the spleen, blood, or mediastinal lymph nodes in response to L. sigmodontis infection, suggesting that ILC2s function primarily at the site of infection. The increase in ILC2s in the pleural cavity and the expansion in Th2 responses indicates a probable role for these cells in initiating and maintaining the Th2 response and highlights the importance of these cells in helminth infections and their role in Th2 immunity. PMID:26394284

  2. [Characterization of endothelial progenitor cells and putative strategies to improve their expansion].

    PubMed

    Smadja, David M; Gaussem, Pascale

    2009-01-01

    Injection of endothelial progenitor cells (EPC) expanded ex vivo has been shown to increase neovascularization in preclinical models of ischemia and in adult patients, but the precise origin and identity of the cell population responsible for these clinical benefits are controversial. Given the potential usefulness of EPC as a cell therapy product, their thorough characterization is of major importance. This review describes the two cell populations currently called EPC and the means to find differential phenotypic markers. We have shown that BMP2/4 are specific markers of late EPC and play a key role in EPC commitment and outgrowth during neovascularization. Several authors have attempted to expand EPC ex vivo in order to obtain a homogeneous cell therapy product. One possible mean of expanding EPC ex vivo is to activate the thrombin receptor PAR-1 with the specific peptide SFLLRN. Indeed, PAR-1 activation increases angiogenic properties of EPC through activation of SDF-1, angiopoietin and IL-8 pathways. This review summarizes the characterization of EPC and different methods of ex vivo expansion. PMID:19527634

  3. Red Blood Cells Initiate Leukocyte Rolling in Postcapillary Expansions: A Lattice Boltzmann Analysis

    PubMed Central

    Sun, Chenghai; Migliorini, Cristiano; Munn, Lance L.

    2003-01-01

    Leukocyte rolling on the vascular endothelium requires initial contact between leukocytes circulating in the blood and the vessel wall. Although specific adhesion mechanisms are involved in leukocyte-endothelium interactions, adhesion patterns in vivo suggest other rheological mechanisms also play a role. Previous studies have proposed that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells (RBCs) and leukocytes as they enter postcapillary expansions, but the details of the fluid dynamics have not been elucidated. We have analyzed the interactions of red and white blood cells as they flow from a capillary into a postcapillary venule using a lattice Boltzmann approach. This technique provides the complete solution of the flow field and quantification of the particle-particle forces in a relevant geometry. Our results show that capillary-postcapillary venule diameter ratio, RBC configuration, and RBC shape are critical determinants of the initiation of cell rolling in postcapillary venules. The model predicts that an optimal configuration of the trailing red blood cells is required to drive the white blood cell to the wall. PMID:12829477

  4. Todralazine protects zebrafish from lethal effects of ionizing radiation: role of hematopoietic cell expansion.

    PubMed

    Dimri, Manali; Joshi, Jayadev; Chakrabarti, Rina; Sehgal, Neeta; Sureshbabu, Angara; Kumar, Indracanti Prem

    2015-02-01

    The Johns Hopkins Clinical Compound Library (JHCCL), a collection of Food and Drug Administration (FDA)-approved small molecules (1400), was screened in silico for identification of novel β2AR blockers and tested for hematopoietic stem cell (HSC) expansion and radioprotection in zebrafish embryos. Docking studies, followed by the capacity to hasten erythropoiesis, identified todralazine (Binding energy, -8.4 kcal/mol) as a potential HSC-modulating agent. Todralazine (5 μM) significantly increased erythropoiesis in caudal hematopoietic tissue (CHT) in wild-type and anemic zebrafish embryos (2.33- and 1.44-folds, respectively) when compared with untreated and anemic control groups. Todralazine (5 μM) treatment also led to an increased number of erythroid progenitors, as revealed from the increased expression of erythroid progenitor-specific genes in the CHT region. Consistent with these effects, zebrafish embryos, Tg(cmyb:gfp), treated with 5 μM todralazine from 24 to 36 hours post fertilization (hpf) showed increased (approximately two-folds) number of HSCs at the aorta-gonad-mesonephros region (AGM). Similarly, expression of HSC marker genes, runx1 (3.3-folds), and cMyb (1.41-folds) also increased in case of todralazine-treated embryos, further supporting its HSC expansion potential. Metoprolol, a known beta blocker, also induced HSC expansion (1.36- and 1.48-fold increase in runx1 and cMyb, respectively). Todralazine (5 μM) when added 30 min before 20 Gy gamma radiation, protected zebrafish from radiation-induced organ toxicity, apoptosis, and improved survival (80% survival advantage over 6 days). The 2-deoxyribose degradation test further suggested hydroxyl (OH) radical scavenging potential of todralazine, and the same is recapitulated in vivo. These results suggest that todralazine is a potential HSC expanding agent, which might be acting along with important functions, such as antioxidant and free radical scavenging, in manifesting radioprotection.

  5. Establishment and characterization of a cell based artificial antigen-presenting cell for expansion and activation of CD8+ T cells ex vivo.

    PubMed

    Gong, Weijuan; Ji, Mingchun; Cao, Zhengfeng; Wang, Liheng; Qian, Yayun; Hu, Maozhi; Qian, Li; Pan, Xingyuan

    2008-02-01

    Artificial antigen-presenting cells are expected to stimulate the expansion and acquisition of optimal therapeutic features of T cells before infusion. Here CD32 that binds to a crystallizable fragment of IgG monoclonal antibody was genetically expressed on human K562 leukemia cells to provide a ligand for T-cell receptor. CD86 and 4-1BBL, which are ligands of co-stimulating receptors of CD28 and 4-1BB, respectively, were also expressed on K562 cells. Then we accomplished the artificial antigen-presenting cells by coupling K32/CD86/4-1BBL cell with OKT3 monoclonal antibody against CD3, named K32/CD86/4-1BBL/OKT3 cells. These artificial modified cells had the abilities of inducing CD8+ T cell activation, promoting CD8+ T cell proliferation, division, and long-term growth, inhibiting CD8+ T cell apoptosis, and enhancing CD8+ T cell secretion of IFN-gamma and perforin. Furthermore, antigen-specific cytotoxic T lymphocytes could be retained in the culture stimulated with K32/CD86/4-1BBL/OKT3 cells at least within 28 days. This approach was robust, simple, reproducible and economical for expansion and activation of CD8+ T cells and may have important therapeutic implications for adoptive immunotherapy. PMID:18318994

  6. Metformin selectively affects human glioblastoma tumor-initiating cell viability

    PubMed Central

    Würth, Roberto; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adirana; Corsaro, Alessandro; Parodi, Alessia; Sirito, Rodolfo; Massollo, Michela; Marini, Cecilia; Zona, Gianluigi; Fenoglio, Daniela; Sambuceti, Gianmario; Filaci, Gilberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

    2013-01-01

    Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect. PMID:23255107

  7. Low- and high-LET radiation drives clonal expansion of lung progenitor cells in vivo

    PubMed Central

    Farin, Alicia M.; Manzo, Nicholas D.; Kirsch, David G.; Stripp, Barry R.

    2015-01-01

    Abundant populations of epithelial progenitor cells maintain the epithelium along the proximal-to-distal axis of the airway. Exposure of lung tissue to ionizing radiation leads to tissue remodeling and potential cancer initiation or progression. However, little is known about the effects of ionizing radiation on airway epithelial progenitor cells. We hypothesized that ionizing radiation exposure will alter the behavior of airway epithelial progenitor cells in a radiation dose- and quality-dependent manner. To address this hypothesis, we cultured primary airway epithelial cells isolated from mice exposed to various doses of 320 kVp X-ray or 600 MeV/nucleon 56Fe ions in a 3D epithelial-fibroblast co-culture system. Colony-forming efficiency of the airway epithelial progenitor cells was assessed at culture day 14. In vivo clonogenic and proliferative potentials of airway epithelial progenitor cells were measured after exposure to ionizing radiation by lineage tracing and IdU incorporation. Exposure to both X-rays and 56Fe resulted in a dose dependent decrease in the ability of epithelial progenitors to form colonies in vitro. In vivo evidence for increased clonogenic expansion of epithelial progenitors was observed after exposure to both X-rays and 56Fe. Interestingly, we found no significant increase in the epithelial proliferative index, indicating that ionizing radiation does not promote increased turnover of the airway epithelium. Therefore, we propose a model in which radiation induces a dose-dependent decrease in the pool of available progenitor cells, leaving fewer progenitors able to maintain the airway long-term. This work provides novel insights into the effects of ionizing radiation exposure on airway epithelial progenitor cell behavior. PMID:25564721

  8. Influence of IL-3 functional fragment on cord blood stem cell ex vivo expansion and differentiation

    PubMed Central

    Ren, Zhihua; Zhang, Yu; Zhang, Yanxi; Jiang, Wenhong; Dai, Wei; Ding, Xinxin

    2016-01-01

    Background Recombinant human interleukin-3 (rhIL-3) is a multiple hematopoietic growth factor, which enhances stem cell expansion and hematopoiesis regeneration in vitro and in vivo, when administrated in combination with other cytokines. However, the structure-function study of rhIL-3 remains rarely studied, so far. The purpose of this study was to recognize the short peptide with similar function as rhIL-3, and assess the hematopoietic efficacy in umbilical cord blood (UCB) stem cell culture as well. Methods Two novel monoclonal antibodies (mAb) (C1 and E1) were generated against rhIL-3 using hybridoma technique. Eleven short peptides were depicted and synthesized to overlap covering the full length sequence of rhIL-3. ELISA was employed to distinguish the antibody-binding peptide from the negative peptides. In addition, the multi-potential hematopoiesis capabilities of the positive peptides were evaluated by adding 25 ng/mL of each peptide to the culture medium of hematopoietic stem cells (HSCs) derived from UCB. Total nucleated cell number and the CD34+ cell number from each individual treatment group were calculated on day 7. Correlated antibodies at 0.5 or 2 molar fold to each peptide were also tested in the stem cell expansion experiment, to further confirm the bioactivity of the peptides. Results Two peptides were recognized by the novel generated antibodies, using ELISA. Peptide 3 and 8 exhibited comparable hematopoiesis potentials, with 25.01±0.14 fold, and 19.89±0.12 fold increase of total nucleated cell number on day 7, respectively, compared with the basal medium control (4.93±0.55 fold). These biological effects were neutralized by adding the corresponding mAb at a dose dependent manner. Conclusions Our results identified two specific regions of rhIL-3 responsible for HSC proliferation and differentiation, which were located from 28 to 49 amino acids (P3), and 107 to 127 amino acids (P8), respectively. The short peptide 3 and 8 might act

  9. Application of porous glycosaminoglycan-based scaffolds for expansion of human cord blood stem cells in perfusion culture.

    PubMed

    Cho, Cheul H; Eliason, James F; Matthew, Howard W T

    2008-07-01

    In vitro expansion of hematopoietic stem cells (HSCs) has been employed to obtain sufficient numbers of stem cells for successful engraftment after HSC transplantation. A three-dimensional perfusion bioreactor system with a heparin-chitosan scaffold was designed and evaluated for its capability to support maintenance and expansion of HSCs. Porous chitosan scaffolds were fabricated by a freeze-drying technique and N-desulfated heparin was covalently immobilized within the scaffolds using carbodiimide chemistry. CD34+ HSCs isolated from umbilical cord blood by immunomagnetic separation were cultured within the porous scaffold in a perfusion bioreactor system. Control cultures were maintained on dishes coated with similar heparin-chitosan films. Oxygen uptake was measured during the culture period. After 7 days of culture, scaffolds were harvested for analysis. Cellular phenotype and HSC characteristics were evaluated via flow cytometry and colony forming unit assays. The results indicate good cell retention and proliferation within the perfused scaffolds. Oxygen consumption in the perfusion bioreactor system increased continuously during the culture, indicating steady cell growth. Cells from the perfused scaffold cultures showed higher percentages of primitive progenitors and exhibited superior colony forming unit performance as compared to cells from static cultures. In addition, perfusion culture at low oxygen (5%) enhanced the expansion of CD34+ cells and colony-forming activity compared to high oxygen (19%) cultures. The results suggest that perfusion culture of cord blood CD34+ cells under bone marrow-like conditions enhances HSC expansion compared to static cultures.

  10. Angiotensin II type-1 receptor (AT1R) regulates expansion, differentiation, and functional capacity of antigen-specific CD8+ T cells

    PubMed Central

    Silva-Filho, João Luiz; Caruso-Neves, Celso; Pinheiro, Ana Acacia Sá

    2016-01-01

    Angiotensin II (Ang II) and its receptor AT1 (AT1R), an important effector axis of renin-angiotensin system (RAS), have been demonstrated to regulate T-cell responses. However, these studies characterized Ang II and AT1R effects using pharmacological tools, which do not target only Ang II/AT1R axis. The specific role of AT1R expressed by antigen-specific CD8+ T cells is unknown. Then we immunized transgenic mice expressing a T-cell receptor specific for SIINFEKL epitope (OT-I mice) with sporozoites of the rodent malaria parasite Plasmodium berghei expressing the cytotoxic epitope SIINFEKL. Early priming events after immunization were not affected but the expansion and contraction of AT1R-deficient (AT1R−/−) OT-I cells was decreased. Moreover, they seemed more activated, express higher levels of CTLA-4, PD-1, LAG-3, and have decreased functional capacity during the effector phase. Memory AT1R−/− OT-I cells exhibited higher IL-7Rα expression, activation, and exhaustion phenotypes but less cytotoxic capacity. Importantly, AT1R−/− OT-I cells show better control of blood parasitemia burden and ameliorate mice survival during lethal disease induced by blood-stage malaria. Our study reveals that AT1R in antigen-specific CD8+ T cells regulates expansion, differentiation, and function during effector and memory phases of the response against Plasmodium, which could apply to different infectious agents. PMID:27782175

  11. Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

    PubMed Central

    Osiecki, Michael J.; Michl, Thomas D.; Kul Babur, Betul; Kabiri, Mahboubeh; Atkinson, Kerry; Lott, William B.; Griesser, Hans J.; Doran, Michael R.

    2015-01-01

    Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs. PMID:26660475

  12. Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells.

    PubMed

    Osiecki, Michael J; Michl, Thomas D; Kul Babur, Betul; Kabiri, Mahboubeh; Atkinson, Kerry; Lott, William B; Griesser, Hans J; Doran, Michael R

    2015-01-01

    Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.

  13. Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells.

    PubMed

    Osiecki, Michael J; Michl, Thomas D; Kul Babur, Betul; Kabiri, Mahboubeh; Atkinson, Kerry; Lott, William B; Griesser, Hans J; Doran, Michael R

    2015-01-01

    Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs. PMID:26660475

  14. Brain and muscle Arnt-like 1 promotes skeletal muscle regeneration through satellite cell expansion.

    PubMed

    Chatterjee, Somik; Yin, Hongshan; Nam, Deokhwa; Li, Yong; Ma, Ke

    2015-02-01

    Circadian clock is an evolutionarily conserved timing mechanism governing diverse biological processes and the skeletal muscle possesses intrinsic functional clocks. Interestingly, although the essential clock transcription activator, Brain and muscle Arnt-like 1 (Bmal1), participates in maintenance of muscle mass, little is known regarding its role in muscle growth and repair. In this report, we investigate the in vivo function of Bmal1 in skeletal muscle regeneration using two muscle injury models. Bmal1 is highly up-regulated by cardiotoxin injury, and its genetic ablation significantly impairs regeneration with markedly suppressed new myofiber formation and attenuated myogenic induction. A similarly defective regenerative response is observed in Bmal1-null mice as compared to wild-type controls upon freeze injury. Lack of satellite cell expansion accounts for the regeneration defect, as Bmal1(-/-) mice display significantly lower satellite cell number with nearly abolished induction of the satellite cell marker, Pax7. Furthermore, satellite cell-derived primary myoblasts devoid of Bmal1 display reduced growth and proliferation ex vivo. Collectively, our results demonstrate, for the first time, that Bmal1 is an integral component of the pro-myogenic response that is required for muscle repair. This mechanism may underlie its role in preserving adult muscle mass and could be targeted therapeutically to prevent muscle-wasting diseases.

  15. Expansion of chromosome territories with chromatin decompaction in BAF53-depleted interphase cells.

    PubMed

    Lee, Kiwon; Kang, Mi Jin; Kwon, Su Jin; Kwon, Yunhee Kim; Kim, Ki Woo; Lim, Jae-Hwan; Kwon, Hyockman

    2007-10-01

    Chromosomes are compartmentalized into discrete chromosome territories during interphase in mammalian cells. A chromosome territory is generated by the tendency of chromatin to occupy the smallest shell volume, which is determined by the polymeric properties and interactions of the internal meshwork of the chromatin fiber. Here, we show that BAF53 knockdown by small interfering RNA interference led to the expansion of chromosome territories. This was accompanied by a reduction in chromatin compaction, an increase in the micrococcal nuclease sensitivity of the chromatin, and an alteration in H3-K9 and H3-K79 dimethylation. Interestingly, the BAF53 knockdown cells suffer a cell cycle defect. Despite the significant irregularity and decompaction of the polynucleosomes isolated from the BAF53 knockdown cells, the chromatin loading of H1 and core histones remained unaltered, as did the nucleosome spacing. The histone hyperacetylation and down-regulation of BRG-1, mBrm, and Tip49, the catalytic components of the SWI/SNF complex and the TIP60 complex, respectively, did not expand chromosome territories. These results indicate that BAF53 contributes to the polymeric properties and/or the internal meshwork interactions of the chromatin fiber probably via a novel mechanism. PMID:17652455

  16. Dnmt3a Regulates Myeloproliferation and Liver-Specific Expansion of Hematopoietic Stem and Progenitor Cells

    PubMed Central

    Guryanova, Olga A.; Lieu, Yen K.; Garrett-Bakelman, Francine E.; Spitzer, Barbara; Glass, Jacob L.; Shank, Kaitlyn; Valencia Martinez, Ana Belen; Rivera, Sharon A.; Durham, Benjamin H.; Rapaport, Franck; Keller, Matthew D.; Pandey, Suveg; Bastian, Lennart; Tovbin, Daniel; Weinstein, Abby R.; Teruya-Feldstein, Julie; Abdel-Wahab, Omar; Santini, Valeria; Mason, Christopher E.; Melnick, Ari M.; Mukherjee, Siddhartha; Levine, Ross L.

    2015-01-01

    DNMT3A mutations are observed in myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). Transplantation studies have elucidated an important role for Dnmt3a in stem cell self-renewal and in myeloid differentiation. Here we investigated the impact of conditional hematopoietic Dnmt3a loss on disease phenotype in primary mice. Mx1-Cre-mediated Dnmt3a ablation led to the development of a lethal, fully penetrant myeloproliferative neoplasm with myelodysplasia (MDS/MPN) characterized by peripheral cytopenias and by marked, progressive hepatomegaly. We detected expanded stem/progenitor populations in the liver of Dnmt3a-ablated mice. The MDS/MPN induced by Dnmt3a ablation was transplantable, including the marked hepatomegaly. Homing studies showed that Dnmt3a-deleted bone marrow cells preferentially migrated to the liver. Gene expression and DNA methylation analyses of progenitor cell populations identified differential regulation of hematopoietic regulatory pathways, including fetal liver hematopoiesis transcriptional programs. These data demonstrate that Dnmt3a ablation in the hematopoietic system leads to myeloid transformation in vivo, with cell autonomous aberrant tissue tropism and marked extramedullary hematopoiesis (EMH) with liver involvement. Hence, in addition to the established role of Dnmt3a in regulating self-renewal, Dnmt3a regulates tissue tropism and limits myeloid progenitor expansion in vivo. PMID:26710888

  17. The clock protein period 2 synchronizes mitotic expansion and decidual transformation of human endometrial stromal cells.

    PubMed

    Muter, Joanne; Lucas, Emma S; Chan, Yi-Wah; Brighton, Paul J; Moore, Jonathan D; Lacey, Lauren; Quenby, Siobhan; Lam, Eric W-F; Brosens, Jan J

    2015-04-01

    Implantation requires coordinated interactions between the conceptus and surrounding decidual cells, but the involvement of clock genes in this process is incompletely understood. Circadian oscillations are predicated on transcriptional-translational feedback loops, which balance the activities of the transcriptional activators CLOCK (circadian locomotor output cycles kaput) and brain muscle arnt-like 1 and repressors encoded by PER (Period) and Cryptochrome genes. We show that loss of PER2 expression silences circadian oscillations in decidualizing human endometrial stromal cells (HESCs). Down-regulation occurred between 12 and 24 hours following differentiation and coincided with reduced CLOCK binding to a noncanonical E-box enhancer in the PER2 promoter. RNA sequencing revealed that premature inhibition of PER2 by small interfering RNA knockdown leads to a grossly disorganized decidual response. Gene ontology analysis highlighted a preponderance of cell cycle regulators among the 1121 genes perturbed upon PER2 knockdown. Congruently, PER2 inhibition abrogated mitotic expansion of differentiating HESCs by inducing cell cycle block at G2/M. Analysis of 70 midluteal endometrial biopsies revealed an inverse correlation between PER2 transcript levels and the number of miscarriages in women suffering reproductive failure (Spearman rank test, ρ = -0.3260; P = 0.0046). Thus, PER2 synchronizes endometrial proliferation with initiation of aperiodic decidual gene expression; uncoupling of these events may cause recurrent pregnancy loss. PMID:25573754

  18. Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation

    PubMed Central

    2014-01-01

    Background Human adipose tissue is an ideal autologous source of mesenchymal stem cells (MSCs) for various regenerative medicine and tissue engineering strategies. Aged patients are one of the primary target populations for many promising applications. It has long been known that advanced age is negatively correlated with an organism’s reparative and regenerative potential, but little and conflicting information is available about the effects of age on the quality of human adipose tissue derived MSCs (hAT-MSCs). Methods To study the influence of age, the expansion and in vitro differentiation potential of hAT-MSCs from young (<30 years), adult (35-50 years) and aged (>60 years) individuals were investigated. MSCs were characterized for expression of the genes p16INK4a and p21 along with measurements of population doublings (PD), superoxide dismutase (SOD) activity, cellular senescence and differentiation potential. Results Aged MSCs displayed senescent features when compared with cells isolated from young donors, concomitant with reduced viability and proliferation. These features were also associated with significantly reduced differentiation potential in aged MSCs compared to young MSCs. Conclusions In conclusion, advancing age negatively impacts stem cell function and such age related alterations may be detrimental for successful stem cell therapies. PMID:24397850

  19. IDO expressing fibroblasts promote the expansion of antigen specific regulatory T cells.

    PubMed

    Curran, Terry-Ann; Jalili, Reza Baradar; Farrokhi, Ali; Ghahary, Aziz

    2014-01-01

    Regulatory CD4(+)CD25(+)Foxp3(+) T cells (Tregs) can be induced and expanded by dendritic cells (DCs) in the presence of the enzyme indoleamine 2,3-dioxygenase (IDO). Here we report that a possible alternative to DCs are IDO expressing dermal fibroblasts (DFs), which are easier to isolate and sustain in culture compared to DCs. When mouse splenocytes were co-cultured with IDO expressing DFs, a significant increase in frequency and the number of Tregs was found compared to those of control group (13.16%±1.8 vs. 5.53%±1.2, p<0.05). Despite observing a higher total number of dead CD4(+) cells in the IDO group, there was a more abundant live CD4(+)CD25(+) subpopulation in this group. Further analysis reveales that these CD4(+) CD25(+) cells have the capacity to expand in the presence of IDO expressing DFs. Greater number of CTLA-4(+) cells and high expression of TGF-β and IL-10 were found in CD4(+) cells of the IDO group compared to those of the controls. This finding confirmed a suppressive functionality of the expanded Tregs. Furthermore, CD4(+) CD25(+) cells isolated from the IDO group showed an alloantigen specific suppressive effect in a mixed lymphocyte reaction assay. These results confirm that IDO expressing dermal fibroblasts can expand a population of suppressive antigen specific Tregs. In conclusion, IDO expressing dermal fibroblasts have the capacity to stimulate the expansion of a subset of Tregs which can be used to generate antigen-specific immune tolerance.

  20. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

    NASA Astrophysics Data System (ADS)

    Lei, Yuguo; Schaffer, David V.

    2013-12-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 1072-fold expansion over 280 d), yield (∼2.0 × 107 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.

  1. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation.

    PubMed

    Lei, Yuguo; Schaffer, David V

    2013-12-24

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (~20-fold per 5-d passage, for a 10(72)-fold expansion over 280 d), yield (~2.0 × 10(7) cells per mL of hydrogel), and purity (~95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ~8 × 10(7) dopaminergic progenitors per mL of hydrogel and ~80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development. PMID:24248365

  2. Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

    SciTech Connect

    Kim, Rae-Kwon; Yoon, Chang-Hwan; Hyun, Kyung-Hwan; Lee, Hyejin; An, Sungkwan; Park, Myung-Jin; Kim, Min-Jung; Lee, Su-Jae

    2010-11-26

    Research highlights: {yields} Activation of Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the fractionated radiation-induced expansion of glioma stem-like cells. {yields} Inhibition of LCK prevents acquisition of fractionated radiation-induced resistance to chemotherapeutic treatment. {yields} LCK activity is critical for the maintenance of self-renewal in glioma stem-like cells. -- Abstract: Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In this study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133{sup +} cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and

  3. Number, frequency, self-renewal, and expansion of osteoprogenitor cells (CFU-O) in subcultured female rat vertebral cell populations.

    PubMed

    Bellows, Carlton G; Pei, Weidong; Heersche, Johan N M

    2004-01-01

    The present study was undertaken to determine whether the frequency and/or number of dexamethasone- and progesterone-responsive osteoprogenitors in cell populations derived from vertebrae of 6-week-old female rats could be increased relative to that of other progenitors. Frequencies and numbers of both progenitor types were determined for up to six subcultures using continuous subculturing, limiting dilution analysis, and colony assays. In dexamethasone-containing medium, subculturing resulted in an eightfold increase in the total number of dexamethasone-responsive osteoprogenitors and a 14-fold increase in progesterone-responsive osteoprogenitors in second subculture cells over first subculture cells without a significant increase in the frequency of these progenitors. From the third subculture onward, the frequency of both classes of osteoprogenitors decreased in a linear manner and none were observed after six subcultures. Similar results were obtained in progesterone-containing medium. Limiting dilution analysis in the presence of dexamethasone indicated that 2.61 % of cells represented a colony forming unit-fibroblast and 0.28 % represented an osteoprogenitor in first subculture cells, while in second subculture cells, these frequencies increased to 5.56 % and 0.40 %, respectively. Results show that while the frequency of colony forming unit-osteoprogenitor is not increased in the second subculture over the first, the total number of osteoprogenitors is greatly increased because of expansion of the total progenitor cell pool.

  4. Approximate smooth solutions of a mathematical model for the activation and clonal expansion of T cells.

    PubMed

    Criaco, D; Dolfin, M; Restuccia, L

    2013-02-01

    In a previous paper a mathematical model was developed for the dynamics of activation and clonal expansion of T cells during the immune response to a single type of antigen challenge, constructed phenomenologically in the macroscopic framework of a thermodynamic theory of continuum mechanics for reacting and proliferating fluid mixtures. The present contribution deals with approximate smooth solutions, called asymptotic waves, of the system of PDEs describing the introduced model, obtained using a suitable perturbative method. In particular, in the one-dimensional case, after deriving the expression of the velocity along the characteristic rays and the equation of the wave front, the transport equation for the first perturbation term of the asymptotic solution is obtained. Finally, it is shown that this transport equation can be reduced to an equation similar to Burgers equation.

  5. A Robust Single Primate Neuroepithelial Cell Clonal Expansion System for Neural Tube Development and Disease Studies

    PubMed Central

    Zhu, Xiaoqing; Li, Bo; Ai, Zongyong; Xiang, Zheng; Zhang, Kunshang; Qiu, Xiaoyan; Chen, Yongchang; Li, Yuemin; Rizak, Joshua D.; Niu, Yuyu; Hu, Xintian; Sun, Yi Eve; Ji, Weizhi; Li, Tianqing

    2015-01-01

    Summary Developing a model of primate neural tube (NT) development is important to promote many NT disorder studies in model organisms. Here, we report a robust and stable system to allow for clonal expansion of single monkey neuroepithelial stem cells (NESCs) to develop into miniature NT-like structures. Single NESCs can produce functional neurons in vitro, survive, and extensively regenerate neuron axons in monkey brain. NT formation and NESC maintenance depend on high metabolism activity and Wnt signaling. NESCs are regionally restricted to a telencephalic fate. Moreover, single NESCs can turn into radial glial progenitors (RGPCs). The transition is accurately regulated by Wnt signaling through regulation of Notch signaling and adhesion molecules. Finally, using the “NESC-TO-NTs” system, we model the functions of folic acid (FA) on NT closure and demonstrate that FA can regulate multiple mechanisms to prevent NT defects. Our system is ideal for studying NT development and diseases. PMID:26584544

  6. Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration

    PubMed Central

    Rost, Fabian; Nowoshilow, Sergej; Chara, Osvaldo; Tanaka, Elly M

    2015-01-01

    Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue. DOI: http://dx.doi.org/10.7554/eLife.10230.001 PMID:26568310

  7. Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins[W

    PubMed Central

    McMichael, Colleen M.; Reynolds, Gregory D.; Koch, Lisa M.; Wang, Chao; Jiang, Nan; Nadeau, Jeanette; Sack, Fred D.; Gelderman, Max B.; Pan, Jianwei; Bednarek, Sebastian Y.

    2013-01-01

    STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion. PMID:24179130

  8. Comparative Analysis of Media and Supplements on Initiation and Expansion of Adipose-Derived Stem Cells.

    PubMed

    Riis, Simone; Nielsen, Frederik Mølgaard; Pennisi, Cristian Pablo; Zachar, Vladimir; Fink, Trine

    2016-03-01

    Adipose-derived stem cells (ASCs) are being tested in clinical trials related to cell-based regenerative therapies. Although most of the current expansion protocols for ASCs use fetal calf serum (FCS), xenogeneic-free medium supplements are greatly desired. This study aims to compare the effect of FCS, human platelet lysate (hPL), and a fully defined medium on the initiation and maintenance of ASC cultures. ASCs obtained from five donors were cultured in five different media: StemPro, Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% hPL, or α-minimum essential medium (A-MEM) supplemented with 5% hPL, 10% hPL, or 10% FCS. The effect of media on proliferation, colony-forming units (CFUs), attachment, and morphology was assessed along with cell size, granularity, and immunophenotype. StemPro greatly compromised the initiation of ASC cultures, which could not survive more than a few passages. Cells cultured in A-MEM proliferated at a faster rate than in DMEM, and hPL significantly enhanced cell size, granularity, and proliferation compared with FCS. All media except StemPro supported CFUs equally well. Analysis of surface markers revealed higher levels of CD73 and CD105 in FCS-cultured ASCs, whereas increased levels of CD146 were found in hPL-cultured cells. Multiparametric flow cytometric analysis performed after seven passages revealed the existence of four distinct ASC subpopulations, all positive for CD73, CD90, and CD105, which mainly differed by their expression of CD146 and CD271. Analysis of the different subpopulations might represent an important biological measure when assessing different medium formulations for a particular clinical application. PMID:26838270

  9. Comparative Analysis of Media and Supplements on Initiation and Expansion of Adipose-Derived Stem Cells.

    PubMed

    Riis, Simone; Nielsen, Frederik Mølgaard; Pennisi, Cristian Pablo; Zachar, Vladimir; Fink, Trine

    2016-03-01

    Adipose-derived stem cells (ASCs) are being tested in clinical trials related to cell-based regenerative therapies. Although most of the current expansion protocols for ASCs use fetal calf serum (FCS), xenogeneic-free medium supplements are greatly desired. This study aims to compare the effect of FCS, human platelet lysate (hPL), and a fully defined medium on the initiation and maintenance of ASC cultures. ASCs obtained from five donors were cultured in five different media: StemPro, Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% hPL, or α-minimum essential medium (A-MEM) supplemented with 5% hPL, 10% hPL, or 10% FCS. The effect of media on proliferation, colony-forming units (CFUs), attachment, and morphology was assessed along with cell size, granularity, and immunophenotype. StemPro greatly compromised the initiation of ASC cultures, which could not survive more than a few passages. Cells cultured in A-MEM proliferated at a faster rate than in DMEM, and hPL significantly enhanced cell size, granularity, and proliferation compared with FCS. All media except StemPro supported CFUs equally well. Analysis of surface markers revealed higher levels of CD73 and CD105 in FCS-cultured ASCs, whereas increased levels of CD146 were found in hPL-cultured cells. Multiparametric flow cytometric analysis performed after seven passages revealed the existence of four distinct ASC subpopulations, all positive for CD73, CD90, and CD105, which mainly differed by their expression of CD146 and CD271. Analysis of the different subpopulations might represent an important biological measure when assessing different medium formulations for a particular clinical application.

  10. C9ORF72 intermediate repeat expansion in patients affected by atypical parkinsonian syndromes or Parkinson's disease complicated by psychosis or dementia in a Sardinian population.

    PubMed

    Cannas, Antonino; Solla, Paolo; Borghero, Giuseppe; Floris, Gian Luca; Chio, Adriano; Mascia, Marcello Mario; Modugno, Nicola; Muroni, Antonella; Orofino, Gianni; Di Stefano, Francesca; Calvo, Andrea; Moglia, Cristina; Restagno, Gabriella; Meloni, Mario; Farris, Rita; Ciaccio, Daniela; Puddu, Roberta; Vacca, Melisa Iris; Melis, Rosanna; Murru, Maria Rita; Tranquilli, Stefania; Corongiu, Daniela; Rolesu, Marcella; Cuccu, Stefania; Marrosu, Maria Giovanna; Marrosu, Francesco

    2015-11-01

    The hexanucleotide repeat expansion GGGGCC in the C9ORF72 gene larger than 30 repeats has been identified as a major genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent papers investigated the possible pathogenic role and associated clinical phenotypes of intermediate C9ORF72 repeat expansion ranging between 20 and 30 repeats. Some studies suggested its pathogenicity for typical Parkinson's disease (PD), atypical parkinsonian syndromes, FTD with/without parkinsonism, and ALS with/without parkinsonism or with/without dementia. In our study, we aimed to screen patients affected by atypical parkinsonian syndromes or PD complicated by psychosis or dementia for the presence of C9ORF72 repeat expansions, and in unrelated age- and sex-matched healthy controls. Consecutive unrelated patients with atypical parkinsonian syndromes and patients with PD complicated by psychosis or dementia were included in this study. Atypical parkinsonian syndromes were further divided into two groups: one with patients who met the criteria for the classic forms of atypical parkinsonism [multiple system atrophy (MSA), Lewy body disease (LBD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD)] ;and patients who did not meet the above criteria, named non-classical atypical parkinsonism with or without dementia. Ninety-two unrelated patients (48 men, 44 women) were enrolled. None of the patients was found to be carriers of C9ORF72 repeat expansions with more than 30 repeats. Intermediate 20-30 repeat expansions were detected in four female patients (4.3 %). Three of them presented clinical features of atypical parkinsonian syndromes, two with non-classical atypical parkinsonism and dementia FTD-like, and one with non-classical atypical parkinsonism without dementia. The other patient presented clinical features of typical PD complicated by psychosis. Among 121 control subjects, none presented long or short expansion for the C9ORF

  11. Influenza infection results in local expansion of memory CD8+ T cells with antigen non-specific phenotype and function

    PubMed Central

    Sckisel, Gail D; Tietze, Julia K; Zamora, Anthony E; Hsiao, Hua-Hui; Priest, Stephen O; Wilkins, Danice E C; Lanier, Louis L; Blazar, Bruce R; Baumgarth, Nicole; Murphy, William J

    2014-01-01

    Primary viral infections induce activation of CD8+ T cells responsible for effective resistance. We sought to characterize the nature of the CD8+ T cell expansion observed after primary viral infection with influenza. Infection of naive mice with different strains of influenza resulted in the rapid expansion of memory CD8+ T cells exhibiting a unique bystander phenotype with significant up-regulation of natural killer group 2D (NKG2D), but not CD25, on the CD44highCD8+ T cells, suggesting an antigen non-specific phenotype. We further confirmed the non-specificity of this phenotype on ovalbumin-specific (OT-I) CD8+ T cells, which are not specific to influenza. These non-specific CD8+ T cells also displayed increased lytic capabilities and were observed primarily in the lung. Thus, influenza infection was shown to induce a rapid, antigen non-specific memory T cell expansion which is restricted to the specific site of inflammation. In contrast, CD8+ T cells of a similar phenotype could be observed in other organs following administration of systemic agonistic anti-CD40 and interleukin-2 immunotherapy, demonstrating that bystander expansion in multiple sites is possible depending on whether the nature of activation is either acute or systemic. Finally, intranasal blockade of NKG2D resulted in a significant increase in viral replication early during the course of infection, suggesting that NKG2D is a critical mediator of anti-influenza responses prior to the initiation of adaptive immunity. These results characterize further the local bystander expansion of tissue-resident, memory CD8+ T cells which, due to their early induction, may play an important NKG2D-mediated, antigen non-specific role during the early stages of viral infection. PMID:23937663

  12. The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells.

    PubMed

    Shortt, Alex J; Secker, Genevieve A; Lomas, Richard J; Wilshaw, Stacy P; Kearney, John N; Tuft, Stephen J; Daniels, Julie T

    2009-02-01

    Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (< or =4% in both intact and acellular groups) and ABCG2 (< or =3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (< or =6%) and ABCG2 (< or =8%). HAM frozen at -80 degrees C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (< or =68%) and ABCG2 (< or =62%) expression (p<0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs

  13. Chronic endotoxin exposure produces airflow obstruction and lung dendritic cell expansion.

    PubMed

    Lai, Peggy S; Fresco, Jennifer M; Pinilla, Miguel A; Macias, Alvaro A; Brown, Ronald D; Englert, Joshua A; Hofmann, Oliver; Lederer, James A; Hide, Winston; Christiani, David C; Cernadas, Manuela; Baron, Rebecca M

    2012-08-01

    Little is known about the mechanisms of persistent airflow obstruction that result from chronic occupational endotoxin exposure. We sought to analyze the inflammatory response underlying persistent airflow obstruction as a result of chronic occupational endotoxin exposure. We developed a murine model of daily inhaled endotoxin for periods of 5 days to 8 weeks. We analyzed physiologic lung dysfunction, lung histology, bronchoalveolar lavage fluid and total lung homogenate inflammatory cell and cytokine profiles, and pulmonary gene expression profiles. We observed an increase in airway hyperresponsiveness as a result of chronic endotoxin exposure. After 8 weeks, the mice exhibited an increase in bronchoalveolar lavage and lung neutrophils that correlated with an increase in proinflammatory cytokines. Detailed analyses of inflammatory cell subsets revealed an expansion of dendritic cells (DCs), and in particular, proinflammatory DCs, with a reduced percentage of macrophages. Gene expression profiling revealed the up-regulation of a panel of genes that was consistent with DC recruitment, and lung histology revealed an accumulation of DCs in inflammatory aggregates around the airways in 8-week-exposed animals. Repeated, low-dose LPS inhalation, which mirrors occupational exposure, resulted in airway hyperresponsiveness, associated with a failure to resolve the proinflammatory response, an inverted macrophage to DC ratio, and a significant rise in the inflammatory DC population. These findings point to a novel underlying mechanism of airflow obstruction as a result of occupational LPS exposure, and suggest molecular and cellular targets for therapeutic development.

  14. Estrogen deficiency heterogeneously affects tissue specific stem cells in mice.

    PubMed

    Kitajima, Yuriko; Doi, Hanako; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Kitajima, Michio; Miura, Kiyonori; Li, Tao-Sheng; Masuzaki, Hideaki

    2015-01-01

    Postmenopausal disorders are frequently observed in various organs, but their relationship with estrogen deficiency and mechanisms remain unclear. As tissue-specific stem cells have been found to express estrogen receptors, we examined the hypothesis that estrogen deficiency impairs stem cells, which consequently contributes to postmenopausal disorders. Six-week-old C57BL/6 female mice were ovariectomized, following which they received 17β-estradiol replacement or vehicle (control). Sham-operated mice were used as healthy controls. All mice were killed for evaluation 2 months after treatments. Compared with the healthy control, ovariectomy significantly decreased uterine weight, which was partially recovered by 17β-estradiol replacement. Ovariectomy significantly increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, but impaired their capacity to grow mixed cell-type colonies in vitro. Estrogen replacement further increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, without significantly affecting colony growth in vitro. The number of CD105-positive mesenchymal stem cells in bone marrow also significantly decreased after ovariectomy, but completely recovered following estrogen replacement. Otherwise, neither ovariectomy nor estrogen replacement changed the number of Pax7-positive satellite cells, which are a skeletal muscle-type stem cell. Estrogen deficiency heterogeneously affected tissue-specific stem cells, suggesting a likely and direct relationship with postmenopausal disorders. PMID:26245252

  15. Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells

    PubMed Central

    O'Leary, Claire E.; Riling, Christopher R.; Spruce, Lynn A.; Ding, Hua; Kumar, Suresh; Deng, Guoping; Liu, Yuhong; Seeholzer, Steven H.; Oliver, Paula M.

    2016-01-01

    Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation. We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells. Using a three-part proteomics approach in primary T cells, we identify stabilization of Jak1 in Ndfip1/2-deficient T cells stimulated through the TCR. Jak1 degradation is aborted in activated T cells that lack Ndfips. In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells. Thus, Ndfip1/Ndfip2 regulate the cross talk between the T-cell receptor and cytokine signalling pathways to limit inappropriate T-cell responses. PMID:27088444

  16. Expansion of antigen-specific regulatory T cells with the topical vitamin d analog calcipotriol.

    PubMed

    Ghoreishi, Mehran; Bach, Paxton; Obst, Jennifer; Komba, Mitsuhiro; Fleet, James C; Dutz, Jan P

    2009-05-15

    1,25-Dihydroxyvitamin D(3) is immunosuppressive both in vivo and in vitro. Topical vitamin D analogs such as calcipotriol alter keratinocyte function, but their effects on cutaneous immune responses are less well understood. We demonstrate that exposure of the skin to calcipotriol before transcutaneous immunization with OVA protein and CpG adjuvant prevents Ag-specific CD8(+) T cell priming coincident with Langerhans cell depletion in the skin. Immunization through calcipotriol-treated skin induces CD4(+)CD25(+) regulatory T cells (Treg) that prevent subsequent Ag-specific CD8(+) T cell proliferation and IFN-gamma production. Treg induced by calcipotriol are able to inhibit the induction and the elicitation of protein contact hypersensitivity. Topical calcipotriol treatment also induces RANKL (receptor activator of NF-kappaB ligand) expression by keratinocytes, a TNF family member involved in modulation of skin dendritic cells. UV light B induces Ag-specific tolerance when it is applied before transcutaneous immunization. We suggest that UV light B-induced tolerance is induced via a vitamin D receptor-dependent mechanism as vitamin D receptor (VDR) knockout mice fail to increase FoxP3(+) Treg in their peripheral draining lymph node following irradiation. Additionally, keratinocytes of VDR(-/-) mice fail to induce RANKL upon UV irradiation or calcipotriol treatment. The in vivo expansion of Ag-specific Treg with the topical application of the vitamin D analog calcipotriol followed by transcutaneous immunization is a simple method to augment functional Ag-specific CD4(+)CD25(+)Foxp3(+) Treg populations and mimics Ag-specific UV-induced tolerance.

  17. Preparation, quality criteria, and properties of human blood platelet lysate supplements for ex vivo stem cell expansion.

    PubMed

    Shih, Daniel Tzu-Bi; Burnouf, Thierry

    2015-01-25

    Most clinical applications of human multipotent mesenchymal stromal cells (MSCs) for cell therapy, tissue engineering, regenerative medicine, and treatment of immune and inflammatory diseases require a phase of isolation and ex vivo expansion allowing a clinically meaningful cell number to be reached. Conditions used for cell isolation and expansion should meet strict quality and safety requirements. This is particularly true for the growth medium used for MSC isolation and expansion. Basal growth media used for MSC expansion are supplemented with multiple nutrients and growth factors. Fetal bovine serum (FBS) has long been the gold standard medium supplement for laboratory-scale MSC culture. However, FBS has a poorly characterized composition and poses risk factors, as it may be a source of xenogenic antigens and zoonotic infections. FBS has therefore become undesirable as a growth medium supplement for isolating and expanding MSCs for human therapy protocols. In recent years, human blood materials, and most particularly lysates and releasates of platelet concentrates have emerged as efficient medium supplements for isolating and expanding MSCs from various origins. This review analyzes the advantages and limits of using human platelet materials as medium supplements for MSC isolation and expansion. We present the modes of production of allogeneic and autologous platelet concentrates, measures taken to ensure optimal pathogen safety profiles, and methods of preparing PLs for MSC expansion. We also discuss the supply of such blood preparations. Produced under optimal conditions of standardization and safety, human platelet materials can become the future 'gold standard' supplement for ex vivo production of MSCs for translational medicine and cell therapy applications.

  18. High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

    PubMed Central

    Spanholtz, Jan; Preijers, Frank; van der Meer, Arnold; Joosten, Irma; Schaap, Nicolaas; de Witte, Theo M.; Dolstra, Harry

    2010-01-01

    Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy. PMID:20169160

  19. Low oxygen tension favored expansion and hematopoietic reconstitution of CD34(+) CD38(-) cells expanded from human cord blood-derived CD34(+) Cells.

    PubMed

    Wang, Ziyan; Du, Zheng; Cai, Haibo; Ye, Zhaoyang; Fan, Jinli; Tan, Wen-Song

    2016-07-01

    Oxygen tension is an important factor that regulates hematopoietic stem cells (HSCs) in both in vivo hematopoietic microenvironment and ex vivo culture system. Although the effect of oxygen tension on ex vivo expansion of HSCs was extensively studied, there were no clear descriptions on physiological function and gene expression analysis of HSCs under different oxygen tensions. In this study, the effects of oxygen tension on ex vivo expansion characteristics of human umbilical cord blood (UCB)-derived CD34(+) cells are evaluated. Moreover, the physiological function of expanded CD34(+) cells was assessed by secondary expansion ability ex vivo and hematopoietic reconstitution ability in vivo. Also, genetic profiling was applied to analyze the expression of genes related to cell function. It was found that low oxygen tension favored expansion of CD34(+) CD38(-) cells. Additionally, CD34(+) cells expanded under low oxygen tension showed better secondary expansion ability and reconstitution ability than those under atmospheric oxygen concentration. Finally, the genetic profiling of CD34(+) CD38(-) cells cultured under low oxygen tension was more akin to freshly isolated cells. These results collectively demonstrate that low oxygen tension was able to better maintain both self-renewal and hematopoietic reconstitution potential and may lay an experimental basis for clinical transplantation of HSCs.

  20. Todralazine Protects Zebrafish from Lethal Effects of Ionizing Radiation: Role of Hematopoietic Cell Expansion

    PubMed Central

    Dimri, Manali; Joshi, Jayadev; Chakrabarti, Rina; Sehgal, Neeta; Sureshbabu, Angara

    2015-01-01

    Abstract The Johns Hopkins Clinical Compound Library (JHCCL), a collection of Food and Drug Administration (FDA)-approved small molecules (1400), was screened in silico for identification of novel β2AR blockers and tested for hematopoietic stem cell (HSC) expansion and radioprotection in zebrafish embryos. Docking studies, followed by the capacity to hasten erythropoiesis, identified todralazine (Binding energy, −8.4 kcal/mol) as a potential HSC-modulating agent. Todralazine (5 μM) significantly increased erythropoiesis in caudal hematopoietic tissue (CHT) in wild-type and anemic zebrafish embryos (2.33- and 1.44-folds, respectively) when compared with untreated and anemic control groups. Todralazine (5 μM) treatment also led to an increased number of erythroid progenitors, as revealed from the increased expression of erythroid progenitor-specific genes in the CHT region. Consistent with these effects, zebrafish embryos, Tg(cmyb:gfp), treated with 5 μM todralazine from 24 to 36 hours post fertilization (hpf) showed increased (approximately two-folds) number of HSCs at the aorta-gonad-mesonephros region (AGM). Similarly, expression of HSC marker genes, runx1 (3.3-folds), and cMyb (1.41-folds) also increased in case of todralazine-treated embryos, further supporting its HSC expansion potential. Metoprolol, a known beta blocker, also induced HSC expansion (1.36- and 1.48-fold increase in runx1 and cMyb, respectively). Todralazine (5 μM) when added 30 min before 20 Gy gamma radiation, protected zebrafish from radiation-induced organ toxicity, apoptosis, and improved survival (80% survival advantage over 6 days). The 2-deoxyribose degradation test further suggested hydroxyl (OH) radical scavenging potential of todralazine, and the same is recapitulated in vivo. These results suggest that todralazine is a potential HSC expanding agent, which might be acting along with important functions, such as antioxidant and free radical scavenging, in

  1. C3 Promotes Expansion of CD8+ and CD4+ T cells in a Listeria monocytogenes infection

    PubMed Central

    Nakayama, Yumi; Kim, Shin-Il; Kim, Eui Ho; Lambris, John D.; Sandor, Matyas; Suresh, M.

    2009-01-01

    It is known that C3 is required for optimal expansion of T cells during acute viral infections. However, it is not yet determined whether T cell responses to intracellular bacterial infections require C3. Therefore, we have investigated the requirement for C3 to elicit potent T cell responses to Listeria monocytogenes (LM). We show that expansion of antigen-specific CD8 and CD4 T cells during a primary response to LM was markedly reduced in the absence of C3 activity. Further studies indicated that, unlike in an influenza virus infection, the regulation of LM-specific T cell responses by C3 might not involve the downstream effector C5a. Moreover, reduced T cell responses to LM was not linked to defective maturation of DCs or developmental anomalies in the peripheral T cell compartment of C3-deficient mice. Experiments involving adoptive transfer of C3-deficient CD8 T cells into the C3-sufficient environment of wild type mice showed that these T cells do not have intrinsic proliferative defects and paracrine source of C3 will suffice for clonal expansion of CD8 T cells in vivo. However, stimulation of purified C3-deficient CD8 T cells by plastic-immobilized anti-CD3 showed that C3 promotes T cell proliferation directly, independent of its effects on antigen-presenting cells. Based on these findings, we propose that diminished T cell responses to LM in C3-deficient mice might be at least in part due to lack of direct effects of C3 on T cells. These studies have furthered our understanding of C3-mediated regulation of T cell immunity to intracellular pathogens. PMID:19648268

  2. ABSENCE OF SCLEROSTIN ADVERSELY AFFECTS B CELL SURVIVAL

    PubMed Central

    Cain, Corey J.; Rueda, Randell; McLelland, Bryce; Collette, Nicole M.; Loots, Gabriela G.; Manilay, Jennifer O.

    2012-01-01

    Increased osteoblast activity in sclerostin-knockout (Sost−/−) mice results in generalized hyperostosis and bones with small bone marrow cavities due to hyperactive mineralizing osteoblast populations. Hematopoietic cell fate decisions are dependent on their local microenvironment, which contains osteoblast and stromal cell populations that support both hematopoietic stem cell quiescence and facilitate B cell development. In this study, we investigated whether high bone mass environments affect B cell development via the utilization of Sost−/− mice, a model of sclerosteosis. We found the bone marrow of Sost−/− mice to be specifically depleted of B cells, due to elevated apoptosis at all B cell developmental stages. In contrast, B cell function in the spleen was normal. Sost expression analysis confirmed that Sost is primarily expressed in osteocytes and is not expressed in any hematopoietic lineage, which indicated that the B cell defects in Sost−/− mice are non-cell autonomous and this was confirmed by transplantation of wildtype (WT) bone marrow into lethally irradiated Sost−/− recipients. WT→Sost−/− chimeras displayed a reduction in B cells, whereas reciprocal Sost−/−→WT chimeras did not, supporting the idea that the Sost−/− bone environment cannot fully support normal B cell development. Expression of the pre-B cell growth stimulating factor, Cxcl12, was significantly lower in bone marrow stromal cells of Sost−/− mice while the Wnt target genes Lef-1 and Ccnd1 remained unchanged in B cells. Taken together, these results demonstrate a novel role for Sost in the regulation of bone marrow environments that support B cells. PMID:22434688

  3. Multiple types of data are required to identify the mechanisms influencing the spatial expansion of melanoma cell colonies

    PubMed Central

    2013-01-01

    Background The expansion of cell colonies is driven by a delicate balance of several mechanisms including cell motility, cell–to–cell adhesion and cell proliferation. New approaches that can be used to independently identify and quantify the role of each mechanism will help us understand how each mechanism contributes to the expansion process. Standard mathematical modelling approaches to describe such cell colony expansion typically neglect cell–to–cell adhesion, despite the fact that cell–to-cell adhesion is thought to play an important role. Results We use a combined experimental and mathematical modelling approach to determine the cell diffusivity, D, cell–to–cell adhesion strength, q, and cell proliferation rate, λ, in an expanding colony of MM127 melanoma cells. Using a circular barrier assay, we extract several types of experimental data and use a mathematical model to independently estimate D, q and λ. In our first set of experiments, we suppress cell proliferation and analyse three different types of data to estimate D and q. We find that standard types of data, such as the area enclosed by the leading edge of the expanding colony and more detailed cell density profiles throughout the expanding colony, does not provide sufficient information to uniquely identify D and q. We find that additional data relating to the degree of cell–to–cell clustering is required to provide independent estimates of q, and in turn D. In our second set of experiments, where proliferation is not suppressed, we use data describing temporal changes in cell density to determine the cell proliferation rate. In summary, we find that our experiments are best described using the range D=161−243μm2hour−1, q=0.3−0.5 (low to moderate strength) and λ=0.0305−0.0398hour−1, and with these parameters we can accurately predict the temporal variations in the spatial extent and cell density profile throughout the expanding melanoma cell colony. Conclusions Our

  4. IL-2-independent and TNFα-dependent Expansion of Vβ5+ Natural Regulatory T Cells During Retrovirus Infection

    PubMed Central

    Myers, Lara; Joedicke, Jara J.; Carmody, Aaron B.; Messer, Ronald J.; Kassiotis, George; Dudley, Jaquelin P.; Dittmer, Ulf; Hasenkrug, Kim J.

    2013-01-01

    Friend virus (FV) infection of mice induces the expansion and activation of regulatory T cells (Tregs) that dampen acute immune responses and promote the establishment and maintenance of chronic infection. Adoptive transfer experiments and the expression of Neuropilin 1 indicate that these cells are predominantly natural Tregs rather than virus-specific conventional CD4+ T cells that converted into induced Tregs. Analysis of Treg TCR Vβ chain usage revealed a broadly distributed polyclonal response with a high proportionate expansion of the Vβ5+ Treg subset, which are known to be responsive to endogenous retrovirus-encoded superantigens. In contrast to the major population of Tregs, the Vβ5+ subset expressed markers of terminally differentiated effector cells, and their expansion was associated with the level of the antiviral CD8+ T cell response rather than the level of FV infection. Surprisingly, the expansion and accumulation of the Vβ5+ Tregs was IL-2 independent but dependent upon TNFα. These experiments reveal a subset-specific Treg induction by a new pathway. PMID:23645880

  5. Genes Involved in Osmoregulation during Turgor-Driven Cell Expansion of Developing Cotton Fibers Are Differentially Regulated1

    PubMed Central

    Smart, Lawrence B.; Vojdani, Fakrieh; Maeshima, Masayoshi; Wilkins, Thea A.

    1998-01-01

    Cotton (Gossypium hirsutum L.) fibers are single-celled trichomes that synchronously undergo a phase of rapid cell expansion, then a phase including secondary cell wall deposition, and finally maturation. To determine if there is coordinated regulation of gene expression during fiber expansion, we analyzed the expression of components involved in turgor regulation and a cytoskeletal protein by measuring levels of mRNA and protein accumulation and enzyme activity. Fragments of the genes for the plasma membrane proton-translocating ATPase, vacuole-ATPase, proton-translocating pyrophosphatase (PPase), phosphoenolpyruvate carboxylase, major intrinsic protein, and α-tubulin were amplified by polymerase chain reaction and used as probes in ribonuclease protection assays of RNA from a fiber developmental series, revealing two discrete patterns of mRNA accumulation. Transcripts of all but the PPase accumulated to highest levels during the period of peak expansion (+12–15 d postanthesis [dpa]), then declined with the onset of secondary cell wall synthesis. The PPase was constitutively expressed through fiber development. Activity of the two proton-translocating-ATPases peaked at +15 dpa, whereas PPase activity peaked at +20 dpa, suggesting that all are involved in the process of cell expansion but with varying roles. Patterns of protein accumulation and enzyme activity for some of the proteins examined suggest posttranslational regulation through fiber development. PMID:9536073

  6. Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

    PubMed Central

    Heathman, Thomas R. J.; Glyn, Veronica A. M.; Picken, Andrew; Rafiq, Qasim A.; Nienow, Alvin W.; Kara, Bo; Hewitt, Christopher J.

    2015-01-01

    ABSTRACT Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot‐sizes required for commercial production. The use of animal‐derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot‐to‐lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large‐scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum‐free hMSC manufacturing process. Human bone‐marrow derived hMSCs were expanded on fibronectin‐coated, non‐porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 105 cells.mL−1 in serum‐free medium. The hMSCs were successfully harvested by our recently‐developed technique using animal‐free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post‐harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony‐forming potential. The hMSCs were held in suspension post‐harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum‐free vehicle solution using a controlled‐rate freezing process. Post‐thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component‐free hMSC production process from expansion through to cryopreservation

  7. Calnexin induces expansion of antigen-specific CD4+ T cells that confer immunity to fungal ascomycetes via conserved epitopes

    PubMed Central

    Wüthrich, Marcel; Brandhorst, Tristan T.; Sullivan, Thomas D.; Filutowicz, Hanna; Sterkel, Alana; Stewart, Douglas; Li, Mengyi; Lerksuthirat, Tassanee; LeBert, Vanessa; Shen, Zu Ting; Ostroff, Gary; Deepe, George S.; Hung, Chiung Yu; Cole, Garry; Walter, Jennifer A.; Jenkins, Marc K.; Klein, Bruce

    2015-01-01

    Fungal infections remain a threat due to the lack of broad spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown, but conserved antigen. Using transgenic CD4+ T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats induces expansion of calnexin-specific CD4+ T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4+ T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogeneticity and conservation of calnexin make this fungal protein a promising vaccine target. PMID:25800545

  8. Calnexin induces expansion of antigen-specific CD4(+) T cells that confer immunity to fungal ascomycetes via conserved epitopes.

    PubMed

    Wüthrich, Marcel; Brandhorst, Tristan T; Sullivan, Thomas D; Filutowicz, Hanna; Sterkel, Alana; Stewart, Douglas; Li, Mengyi; Lerksuthirat, Tassanee; LeBert, Vanessa; Shen, Zu Ting; Ostroff, Gary; Deepe, George S; Hung, Chiung Yu; Cole, Garry; Walter, Jennifer A; Jenkins, Marc K; Klein, Bruce

    2015-04-01

    Fungal infections remain a threat due to the lack of broad-spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown but conserved antigen. Using transgenic CD4(+) T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae, and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes, including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats, induces expansion of calnexin-specific CD4(+) T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4(+) T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogenicity and conservation of calnexin make this fungal protein a promising vaccine target. PMID:25800545

  9. Scalable ex vivo expansion of human mesenchymal stem/stromal cells in microcarrier-based stirred culture systems.

    PubMed

    Carmelo, Joana G; Fernandes-Platzgummer, Ana; Cabral, Joaquim M S; da Silva, Cláudia Lobato

    2015-01-01

    The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different microcarriers and xeno-free medium formulations.

  10. Scalable ex vivo expansion of human mesenchymal stem/stromal cells in microcarrier-based stirred culture systems.

    PubMed

    Carmelo, Joana G; Fernandes-Platzgummer, Ana; Cabral, Joaquim M S; da Silva, Cláudia Lobato

    2015-01-01

    The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different microcarriers and xeno-free medium formulations. PMID:25063496

  11. Dominant negative effect of polyglutamine expansion perturbs normal function of ataxin-3 in neuronal cells

    PubMed Central

    Neves-Carvalho, Andreia; Logarinho, Elsa; Freitas, Ana; Duarte-Silva, Sara; Costa, Maria do Carmo; Silva-Fernandes, Anabela; Martins, Margarida; Serra, Sofia Cravino; Lopes, André T.; Paulson, Henry L.; Heutink, Peter; Relvas, João B.; Maciel, Patrícia

    2015-01-01

    The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado–Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of α5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration. PMID:25143392

  12. Pharmacological preconditioning for short-term ex vivo expansion of human umbilical cord blood hematopoietic stem cells by filgrastim

    PubMed Central

    Grigoriadis, Nikolaos G; Grigoriadis, Ioannis G; Markoula, Sofia; Paschopoulos, Minas; Zikopoulos, Konstantinos; Apostolakopoulos, Panagiotis Gr; Vizirianakis, Ioannis S; Georgiou, Ioannis

    2016-01-01

    Although umbilical cord blood (UCB) hematopoietic stem cell transplantation (UCBT) has emerged as a promising haematological reconstitution therapy for leukemias and other related disorders, the insufficient UCB stem cell dosage still hinders better clinical outcomes. Previous research efforts, by focusing on ex vivo UCB expansion capabilities have sought to benefit from well-known mechanisms of self-renewal characteristics of UCB stem cells. However, the long-term (> 21 days) in vitro culture period and the low neutrophil recovery significantly reduce the transplantability of such ex vivo expanded UCB stem cells. To overcome the latter hurdles in this study, a post-thaw, short-term ex vivo expansion methodology of UCB mononuclear (UCB-MN) and CD34+ cells has been established. Notably, such effort was achieved through pharmacological preconditioned of UCB cultures by filgrastim agent already used in the clinical setting. In crucial cell populations implicated in the promotion of functional engraftment, the progression of free survival rates (PFS), a marked increase of 6.65 to 9.34 fold for UCB-MN and 35 to 49 fold for CD34+ cells has been noticed. Overall, these results indicate that transplantation of pharmacologically-preconditioned ex vivo expansion of UCB stem and progenitor cells keep high promise upon transplantation to enhance therapeutic potential in everyday clinical practice. PMID:27335700

  13. Scalable Expansion of Human Pluripotent Stem Cell-Derived Neural Progenitors in Stirred Suspension Bioreactor Under Xeno-free Condition.

    PubMed

    Nemati, Shiva; Abbasalizadeh, Saeed; Baharvand, Hossein

    2016-01-01

    Recent advances in neural differentiation technology have paved the way to generate clinical grade neural progenitor populations from human pluripotent stem cells. These cells are an excellent source for the production of neural cell-based therapeutic products to treat incurable central nervous system disorders such as Parkinson's disease and spinal cord injuries. This progress can be complemented by the development of robust bioprocessing technologies for large scale expansion of clinical grade neural progenitors under GMP conditions for promising clinical use and drug discovery applications. Here, we describe a protocol for a robust, scalable expansion of human neural progenitor cells from pluripotent stem cells as 3D aggregates in a stirred suspension bioreactor. The use of this platform has resulted in easily expansion of neural progenitor cells for several passages with a fold increase of up to 4.2 over a period of 5 days compared to a maximum 1.5-2-fold increase in the adherent static culture over a 1 week period. In the bioreactor culture, these cells maintained self-renewal, karyotype stability, and cloning efficiency capabilities. This approach can be also used for human neural progenitor cells derived from other sources such as the human fetal brain. PMID:26867543

  14. OX40 controls effector CD4+ T-cell expansion, not follicular T helper cell generation in acute Listeria infection

    PubMed Central

    Marriott, Clare L; Mackley, Emma C; Ferreira, Cristina; Veldhoen, Marc; Yagita, Hideo; Withers, David R

    2014-01-01

    To investigate the importance of OX40 signals for physiological CD4+ T-cell responses, an endogenous antigen-specific population of CD4+ T cells that recognise the 2W1S peptide was assessed and temporal control of OX40 signals was achieved using blocking or agonistic antibodies (Abs) in vivo. Following infection with Listeria monocytogenes expressing 2W1S peptide, OX40 was briefly expressed by the responding 2W1S-specific CD4+ T cells, but only on a subset that co-expressed effector cell markers. This population was specifically expanded by Ab-ligation of OX40 during priming, which also caused skewing of the memory response towards effector memory cells. Strikingly, this greatly enhanced effector response was accompanied by the loss of T follicular helper (TFH) cells and germinal centres. Mice deficient in OX40 and CD30 showed normal generation of TFH cells but impaired numbers of 2W1S-specific effector cells. OX40 was not expressed by 2W1S-specific memory cells, although it was rapidly up-regulated upon challenge whereupon Ab-ligation of OX40 specifically affected the effector subset. In summary, these data indicate that for CD4+ T cells, OX40 signals are important for generation of effector T cells rather than TFH cells in this response to acute bacterial infection. PMID:24771127

  15. CD3(+) and/or CD14(+) depletion from cord blood mononuclear cells before ex vivo expansion culture improves total nucleated cell and CD34(+) cell yields.

    PubMed

    Yang, H; Robinson, S N; Lu, J; Decker, W K; Xing, D; Steiner, D; Parmar, S; Shah, N; Champlin, R E; Munsell, M; Leen, A; Bollard, C; Simmons, P J; Shpall, E J

    2010-06-01

    Cord blood (CB) is used increasingly in transplant patients lacking sibling or unrelated donors. A major hurdle in the use of CB is its low cell dose, which is largely responsible for an elevated risk of graft failure and a significantly delayed neutrophil and platelet engraftment. As a positive correlation has been shown between the total nucleated cell (TNC) and CD34(+) cell dose transplanted and time to neutrophil and platelet engraftment, strategies to increase these measures are under development. One strategy includes the ex vivo expansion of CB mononuclear cells (MNC) with MSC in a cytokine cocktail. We show that this strategy can be further improved if CD3(+) and/or CD14(+) cells are first depleted from the CB MNC before ex vivo expansion. Ready translation of this depletion strategy to improve ex vivo CB expansion in the clinic is feasible as clinical-grade devices and reagents are available. Ultimately, the aim of improving TNC and CD34(+) transplant doses is to further improve the rate of neutrophil and platelet engraftment in CB recipients.

  16. Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy

    PubMed Central

    Chen, Can; Tan, Wee-Kiat; Chi, Zhixia; Xu, Xue-Hu; Wang, Shu

    2016-01-01

    Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer. PMID:27598655

  17. Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy.

    PubMed

    Du, Shou-Hui; Li, Zhendong; Chen, Can; Tan, Wee-Kiat; Chi, Zhixia; Kwang, Timothy Weixin; Xu, Xue-Hu; Wang, Shu

    2016-01-01

    Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer. PMID:27598655

  18. A Golgi and tonoplast localized S-acyl transferase is involved in cell expansion, cell division, vascular patterning and fertility in Arabidopsis

    PubMed Central

    Qi, Baoxiu; Doughty, James; Hooley, Richard

    2013-01-01

    S-acylation of eukaryotic proteins is the reversible attachment of palmitic or stearic acid to cysteine residues, catalysed by protein S-acyl transferases that share an Asp-His-His-Cys (DHHC) motif. Previous evidence suggests that in Arabidopsis S-acylation is involved in the control of cell size, polarity and the growth of pollen tubes and root hairs. Using a combination of yeast genetics, biochemistry, cell biology and loss of function genetics the roles of a member of the protein S-acyl transferase PAT family, AtPAT10 (At3g51390), have been explored. In keeping with its role as a PAT, AtPAT10 auto-S-acylates, and partially complements the yeast akr1 PAT mutant, and this requires Cys192 of the DHHC motif. In Arabidopsis AtPAT10 is localized in the Golgi stack, trans-Golgi network/early endosome and tonoplast. Loss-of-function mutants have a pleiotropic phenotype involving cell expansion and division, vascular patterning, and fertility that is rescued by wild-type AtPAT10 but not by catalytically inactive AtPAT10C192A. This supports the hypothesis that AtPAT10 is functionally independent of the other Arabidopsis PATs. Our findings demonstrate a growing importance of protein S-acylation in plants, and reveal a Golgi and tonoplast located S-acylation mechanism that affects a range of events during growth and development in Arabidopsis. PMID:23795888

  19. A Golgi and tonoplast localized S-acyl transferase is involved in cell expansion, cell division, vascular patterning and fertility in Arabidopsis.

    PubMed

    Qi, Baoxiu; Doughty, James; Hooley, Richard

    2013-10-01

    S-acylation of eukaryotic proteins is the reversible attachment of palmitic or stearic acid to cysteine residues, catalysed by protein S-acyl transferases that share an Asp-His-His-Cys (DHHC) motif. Previous evidence suggests that in Arabidopsis S-acylation is involved in the control of cell size, polarity and the growth of pollen tubes and root hairs. Using a combination of yeast genetics, biochemistry, cell biology and loss of function genetics the roles of a member of the protein S-acyl transferase PAT family, AtPAT10 (At3g51390), have been explored. In keeping with its role as a PAT, AtPAT10 auto-S-acylates, and partially complements the yeast akr1 PAT mutant, and this requires Cys(192) of the DHHC motif. In Arabidopsis AtPAT10 is localized in the Golgi stack, trans-Golgi network/early endosome and tonoplast. Loss-of-function mutants have a pleiotropic phenotype involving cell expansion and division, vascular patterning, and fertility that is rescued by wild-type AtPAT10 but not by catalytically inactive AtPAT10C(192) A. This supports the hypothesis that AtPAT10 is functionally independent of the other Arabidopsis PATs. Our findings demonstrate a growing importance of protein S-acylation in plants, and reveal a Golgi and tonoplast located S-acylation mechanism that affects a range of events during growth and development in Arabidopsis. PMID:23795888

  20. Residues 39-56 of Stem Cell Factor Protein Sequence Are Capable of Stimulating the Expansion of Cord Blood CD34+ Cells

    PubMed Central

    Shen, Bin; Jiang, Wenhong; Fan, Jie; Dai, Wei; Ding, Xinxin; Jiang, Yongping

    2015-01-01

    Background Stem cell factor (SCF) can stimulate hematopoietic stem cell (HSC) expansion; however, the specific structural region(s) of SCF protein that are critical for this function are still unknown. A novel monoclonal antibody (named 23C8) against recombinant human SCF (rhSCF) was previously found to inhibit the ability of rhSCF to enhance HSC expansion, making it possible to identify the relevant active region to HSC. Methods Eleven polypeptides were synthesized, which were designed to cover the full-length of rhSCF, with overlaps that are at least 3 amino acids long. ELISA was used to identify the polypeptide(s) that specifically react with the anti-SCF. The effects of the synthetic polypeptides on human HSC expansion, or on the ability of the full-length rhSCF to stimulate cell proliferation, were evaluated ex vivo. Total cell number was monitored using hemocytometer whereas CD34+ cell number was calculated based on the proportion determined via flow cytometry on day 6 of culture. Results Of all polypeptides analyzed, only one, named P0, corresponding to the SCF protein sequence at residues 39–56, was recognized by 23C8 mAb during ELISA. P0 stimulated the expansion of CD34+ cells derived from human umbilical cord blood (UCB). Addition of P0 increased the numbers of total mononucleated cells and CD34+ cells, by ~2 fold on day 6. P0 also showed partial competition against full-length rhSCF in the ex vivo cell expansion assay. Conclusion Residues 39–56 of rhSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34+ cells. The peptide P0 is a potential candidate for further development as a synthetic substitute for rhSCF in laboratory and clinical applications. PMID:26505626

  1. An Improved Harvest and in Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Xu, Song; De Becker, Ann; Van Camp, Ben; Vanderkerken, Karin; Van Riet, Ivan

    2010-01-01

    Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time. PMID:21197440

  2. Human amniotic epithelial cells as novel feeder layers for promoting ex vivo expansion of limbal epithelial progenitor cells.

    PubMed

    Chen, Ying-Ting; Li, Wei; Hayashida, Yasutaka; He, Hua; Chen, Szu-Yu; Tseng, David Y; Kheirkhah, Ahmad; Tseng, Scheffer C G

    2007-08-01

    Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan-cytokeratins (pan-CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan-CK(+)/Vim(+) or pan-CK(+)/Vim(-) were maintained in serum-free media with high calcium. In contrast, all HAECs became pan-CK(+)/Vim(+) in serum-containing media, which also promoted HAEC proliferation for at least eight passages, especially supplemented with epidermal growth factor and insulin. Mitomycin C-arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC-supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43 but higher levels of stem cell-associated markers such as p63, Musashi-1, and ATP-binding cassette subfamily G2 than those of 3T3-supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63-positive epithelial progenitor cells as well as nestin-positive neuronal-like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus. Disclosure of potential conflicts of interest is found at the end of this article.

  3. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    PubMed Central

    2011-01-01

    Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation. PMID:22208362

  4. Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells.

    PubMed

    Hoffmann, Petra; Eder, Ruediger; Kunz-Schughart, Leoni A; Andreesen, Reinhard; Edinger, Matthias

    2004-08-01

    CD4(+)CD25+ regulatory T (Treg) cells are pivotal for the maintenance of self-tolerance, and their adoptive transfer gives protection from autoimmune diseases and pathogenic alloresponses after solid organ or bone marrow transplantation in murine model systems. In vitro, human CD4(+)CD25+ Treg cells display phenotypic and functional characteristics similar to those of murine CD4(+)CD25+ Treg cells: namely, hyporesponsiveness to T-cell receptor (TCR) stimulation and suppression of CD25- T cells. Thus far, the detailed characterization and potential clinical application of human CD4(+)CD25+ Treg cells have been hampered by their paucity in peripheral blood and the lack of appropriate expansion protocols. Here we describe the up to 40 000-fold expansion of highly purified human CD4(+)CD25high T cells in vitro through the use of artificial antigen-presenting cells for repeated stimulation via CD3 and CD28 in the presence of high-dose interleukin 2 (IL-2). Expanded CD4(+)CD25high T cells were polyclonal, maintained their phenotype, exceeded the suppressive activity of freshly isolated CD4(+)CD25high T cells, and maintained expression of the lymph node homing receptors L-selectin (CD62L) and CCR7. The ability to rapidly expand human CD4(+)CD25high Treg cells on a large scale will not only facilitate their further exploration but also accelerate their potential clinical application in T cell-mediated diseases and transplantation medicine. PMID:15090447

  5. Modulators affecting the immune dialogue between human immune and colon cancer cells

    PubMed Central

    Djaldetti, Meir; Bessler, Hanna

    2014-01-01

    cells leading to a balance in cytokine production. It is conceivable that the prevalence of anti-inflammatory cytokine production by PBMC in the affected colonic mucosa will contribute to the delay, or even to halt down malignant expansion. Targeting the interplay between immune and cancer cells by mediators capable to alter cytokine secretion toward increased anti-inflammatory cytokine release by PBMC and tumor associated macrophages, may serve as an additional strategy for treatment of malignant diseases. This review will focus on the inflammatory events preceding tumorigenesis in general, and on a number of modulators capable to affect colon cancer cell-induced production of inflammatory cytokines by PBMC through alteration of the immune cross-talk between PBMC and cancer cells. PMID:24834143

  6. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    PubMed

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  7. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    PubMed

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  8. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development

    PubMed Central

    Peremyslov, Valera V.; Cole, Rex A.; Fowler, John E.; Dolja, Valerian V.

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  9. Polyclonal expansion of cervical cytobrush-derived T cells to investigate HIV-specific responses in the female genital tract

    PubMed Central

    Bere, Alfred; Denny, Lynette; Burgers, Wendy A; Passmore, Jo-Ann S

    2010-01-01

    Human immunodeficiency virus (HIV) -specific T-cell responses are detectable in the female genital tract of HIV-infected women but little is known about their frequency or the factors that influence their detection. We investigated the feasibility of polyclonal in vitro expansion of cervical cytobrush-derived T cells to investigate HIV-specific responses in the female genital tract in HIV-infected women. Cytobrush-derived cervical cells were isolated from 22 HIV-infected women and expanded with anti-CD3 and recombinant interleukin-2. Cervical T-cell lines were investigated for Gag-specific responses by interferon-γ ELISPOT and compared with those detected in matched blood samples. Cervical T-cell lines were established from 16/22 (72·7%) participants. Although the absolute number of CD3± cells recovered after expansion was positively associated with the number of cells isolated ex vivo (P = 0·01; R = 0·62), we observed a significant negative correlation between fold expansion and ex vivo cell number (P = 0·004; R = −0·68). We show that both the magnitude (P = 0·002; R = 0·7) and specific Gag regions targeted by cervical T-cell lines (P < 0·0001; R = 0·5) correlated significantly with those detected in blood. With one exception, cervical interferon-γ T-cell responses to Gag were detected only in HIV-infected women with blood Gag-specific response > 1000 spot-forming units/106 cells. We conclude that cervical Gag-specific T-cell responses in expanded lines are most easily detectable in women who have corresponding high-magnitude Gag-specific T-cell responses in blood. PMID:20201983

  10. Human iPS cell-derived fibroblast-like cells as feeder layers for iPS cell derivation and expansion.

    PubMed

    Du, Shou-Hui; Tay, Johan Chin-Kang; Chen, Can; Tay, Felix-Chang; Tan, Wee-Kiat; Li, Zhen-Dong; Wang, Shu

    2015-08-01

    Mouse embryonic fibroblasts (MEFs) are commonly used as feeder cells for the generation of human induced pluripotent stem cells (hiPSCs). However, medical applications of cell derivatives of hiPSCs generated with a MEF feeder system run the risk of having xeno-factor contamination due to long-term cell culturing under an animal factor-containing environment. We developed a new method for the derivation of human fibroblast-like cells (FLCs) from a previously established hiPSC line in an FLC differentiation medium. The method was based on direct differentiation of hiPSCs seeded on Matrigel followed by expansion of differentiating cells on gelatin. Using inactivated FLCs as feeder layers, primary human foreskin fibroblasts were successfully reprogrammed into a state of pluripotency by Oct4, Sox2 Klf4, and c-Myc (OSKM) transcription factor genes, with a reprogramming efficiency under an optimized condition superior to that obtained on MEF feeder layers. Furthermore, the FLCs were more effective in supporting the growth of human pluripotent stem cells. The pluripotency and differentiation capability of the cells cultured on FLC feeder layers were well retained. Our results suggest that FLCs are a safe alternative to MEFs for hiPSC generation and expansion, especially in the clinical settings wherein hiPSC derivatives will be used for medical treatment.

  11. NKG2C+CD57+ Natural Killer Cell Expansion Parallels Cytomegalovirus-Specific CD8+ T Cell Evolution towards Senescence

    PubMed Central

    Heath, John; Newhook, Nicholas; Comeau, Emilie; Gallant, Maureen; Fudge, Neva

    2016-01-01

    Objective. Measuring NKG2C+CD57+ natural killer (NK) cell expansion to investigate NK responses against human cytomegalovirus (HCMV) and assessing relationships with adaptive immunity against HCMV. Methods. Expansion of NKG2C+CD57+ NK was measured in peripheral blood mononuclear cells (PBMC) from groups distinguished by HCMV and human immunodeficiency virus (HIV) infection status. Anti-HCMV antibody levels against HCMV-infected MRC-5 cell lysate were assessed by ELISA and HCMV-specific CD8+ T cell responses characterized by intracellular flow cytometry following PBMC stimulation with immunodominant HCMV peptides. Results. Median NK, antibody, and CD8+ T cell responses against HCMV were significantly greater in the HCMV/HIV coinfected group than the group infected with CMV alone. The fraction of CMV-specific CD8+ T cells expressing CD28 correlated inversely with NKG2C+CD57+ NK expansion in HIV infection. Conclusion. Our data reveal no significant direct relationships between NK and adaptive immunity against HCMV. However, stronger NK and adaptive immune responses against HCMV and an inverse correlation between NKG2C+CD57+ NK expansion and proliferative reserve of HCMV-specific CD8+ T cells, as signified by CD28 expression, indicate parallel evolution of NK and T cell responses against HCMV in HIV infection. Similar aspects of chronic HCMV infection may drive both NK and CD8+ T cell memory inflation. PMID:27314055

  12. In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage

    PubMed Central

    Hare, Ian; Gencheva, Marieta; Evans, Rebecca; Fortney, James; Piktel, Debbie; Vos, Jeffrey A.; Howell, David; Gibson, Laura F.

    2016-01-01

    Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation. PMID:26880992

  13. Osteoprogenitor cells from bone marrow and cortical bone: understanding how the environment affects their fate.

    PubMed

    Corradetti, Bruna; Taraballi, Francesca; Powell, Sebastian; Sung, David; Minardi, Silvia; Ferrari, Mauro; Weiner, Bradley K; Tasciotti, Ennio

    2015-05-01

    Bone is a dynamic organ where skeletal progenitors and hematopoietic cells share and compete for space. Presumptive mesenchymal stem cells (MSC) have been identified and harvested from the bone marrow (BM-MSC) and cortical bone fragments (CBF-MSC). In this study, we demonstrate that despite the cells sharing a common ancestor, the differences in the structural properties of the resident tissues affect cell behavior and prime them to react differently to stimuli. Similarly to the bone marrow, the cortical portion of the bone contains a unique subset of cells that stains positively for the common MSC-associated markers. These cells display different multipotent differentiation capability, clonogenic expansion, and immunosuppressive potential. In particular, when compared with BM-MSC, CBF-MSC are bigger in size, show a lower proliferation rate at early passages, have a greater commitment toward the osteogenic lineage, constitutively produce nitric oxide as a mediator for bone remodeling, and more readily respond to proinflammatory cytokines. Our data suggest that the effect of the tissue's microenvironment makes the CBF-MSC a superior candidate in the development of new strategies for bone repair.

  14. Constitutive Expressor of Pathogenesis-Related Genes5 affects cell wall biogenesis and trichome development

    PubMed Central

    Brininstool, Ginger; Kasili, Remmy; Simmons, L Alice; Kirik, Viktor; Hülskamp, Martin; Larkin, John C

    2008-01-01

    Background The Arabidopsis thaliana CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene has been previously implicated in disease resistance, cell proliferation, cell death, and sugar sensing, and encodes a putative membrane protein of unknown biochemical function. Trichome development is also affected in cpr5 plants, which have leaf trichomes that are reduced in size and branch number. Results In the work presented here, the role of CPR5 in trichome development was examined. Trichomes on cpr5 mutants had reduced birefringence, suggesting a difference in cell wall structure between cpr5 and wild-type trichomes. Consistent with this, leaf cell walls of cpr5 plants contained significantly less paracrystalline cellulose and had an altered wall carbohydrate composition. We also found that the effects of cpr5 on trichome size and endoreplication of trichome nuclear DNA were epistatic to the effects of mutations in triptychon (try) or overexpression of GLABRA3, indicating that these trichome developmental regulators are dependant on CPR5 function for their effects on trichome expansion and endoreplication. Conclusion Our results suggest that CPR5 is unlikely to be a specific regulator of pathogen response pathways or senescence, but rather functions either in cell wall biogenesis or in multiple cell signaling or transcription response pathways. PMID:18485217

  15. Clonal expansion of CD8 T cells in the systemic circulation precedes development of ipilimumab-induced toxicities

    PubMed Central

    Subudhi, Sumit K.; Aparicio, Ana; Gao, Jianjun; Zurita, Amado J.; Araujo, John C.; Logothetis, Christopher J.; Tahir, Salahaldin A.; Korivi, Brinda R.; Slack, Rebecca S.; Vence, Luis; Emerson, Ryan O.; Yusko, Erik; Vignali, Marissa; Robins, Harlan S.; Sun, Jingjing; Allison, James P.; Sharma, Padmanee

    2016-01-01

    Immune checkpoint therapies, such as ipilimumab, induce dramatic antitumor responses in a subset of patients with advanced malignancies, but they may also induce inflammatory responses and toxicities termed immune-related adverse events (irAEs). These irAEs are often low grade and manageable, but severe irAEs may lead to prolonged hospitalizations or fatalities. Early intervention is necessary to minimize morbidities that occur with severe irAEs. However, correlative biomarkers are currently lacking. In a phase II clinical trial that treated 27 patients with metastatic prostate cancer, we aimed to test the safety and efficacy of androgen deprivation therapy plus ipilimumab. In this study, we observed grade 3 toxicities in >40% of treated patients, which led to early closure of the study. Because ipilimumab enhances T-cell responses, we hypothesized that increased clonal T-cell responses in the systemic circulation may contribute to irAEs. Sequencing of the T-cell receptor β-chains in purified T cells revealed clonal expansion of CD8 T cells, which occurred in blood samples collected before the onset of grade 2–3 irAEs. These initial results suggested that expansion of ≥55 CD8 T-cell clones preceded the development of severe irAEs. We further evaluated available blood samples from a second trial and determined that patients who experienced grade 2–3 irAEs also had expansion of ≥55 CD8 T-cell clones in blood samples collected before the onset of irAEs. We propose that CD8 T-cell clonal expansion may be a correlative biomarker to enable close monitoring and early intervention for patients receiving ipilimumab. PMID:27698113

  16. Feline left ventricular oxygen consumption is not affected by volume expansion, ejection or redevelopment of pressure during relaxation.

    PubMed

    Duwel, C M; Westerhof, N

    1988-09-01

    We studied the dependency of myocardial oxygen consumption on the mechanical events during left ventricular relaxation in isolated supported cat hearts. The volume of the left ventricle was controlled by means of a balloon connected to a membrane pump. Oxygen consumption (MVO2 in cm3.min-1.100 g-1) for three protocols (PROT) performed at peak isovolumic pressure, was studied: (1) rapid ejection to zero pressure, (2) partial rapid ejection followed by redevelopment of pressure, (3) volume expansion during relaxation, and compared with oxygen consumption of isovolumic (ISOV) beats. We found (mean +/- SD): (table; see text) In the protocols 1 and 3 the differences were not significant (paired Student's t-test, p greater than 0.05). In protocols 1 and 2 left ventricular volume was decreased up to 2.15 cm3 (i.e. stroke volume, SV) during the pressure release. We studied the specific effect of ejection (i.e., wall muscle shortening) in a fourth protocol in which the ventricle ejected up to 2.7 cm3 under nearly zero pressure load (isobaric contraction). There was a small amount of oxygen consumption associable with this unloaded ejection i.e. MVO2 = 3.38 (+/- 0.47) + 0.30 (+/- 0.16) SV, but it was too small to compensate for a decrease in MVO2 expected from the pressure release according to the tension time index. These findings suggest that oxygen consumption does not depend on the mechanical events during ventricular relaxation. PMID:3174398

  17. Gender and cell-type specific effects of the transcription coupled repair protein, ERCC6/CSB, on repeat expansion in a mouse model of the Fragile X-related disorders

    PubMed Central

    Zhao, Xiao-Nan; Usdin, Karen

    2014-01-01

    The Repeat Expansion Diseases (REDs) are human genetic disorders that arise from expansion of a tandem repeat tract. The Fragile X-related disorders are members of this disease group in which the repeat unit is CGG/CCG and is located in the 5′ untranslated region of the FMR1 gene. Affected individuals often show mosaicism with respect to repeat number resulting from both expansion and contraction of the repeat tract, however, the mechanism responsible for these changes in repeat number are unknown. Work from a variety of model systems suggests that Transcription Coupled Repair (TCR) may contribute to repeat instability in diseases resulting from CAG/CTG-repeat expansion. To test whether TCR could contribute to repeat instability in the Fragile X-related disorders, we tested the effect of mutations in Csb (Cockayne Syndrome group B), a gene essential for TCR, in a knock-in mouse model of these disorders. We found that the loss of CSB affects expansions in a gender and cell type-specific manner. Our data also show an unanticipated gender difference in instability even in Csb+/+ animals that may have implications for our understanding of the mechanism of repeat expansion in the FX mouse model and perhaps for humans as well. PMID:24352881

  18. Rigorous modelling of light scattering in solar cells based on finite element method and Huygens' expansion.

    PubMed

    Sever, Martin; Krč, Janez; Čampa, Andrej; Topič, Marko

    2015-11-30

    Finite element method is coupled with Huygens' expansion to determine light intensity distribution of scattered light in solar cells and other optoelectronic devices. The rigorous foundation of the modelling enables calculation of the light intensity distribution at a chosen distance and surface of observation in chosen material in reflection or in transmission for given wavelength of the incident light. The calculation of scattering or anti-reflection properties is not limited to a single textured interface, but can be done above more complex structures with several scattering interfaces or even with particles involved. Both scattering at periodic and at random textures can be efficiently handled with the modelling approach. A procedure for minimisation of the effect of small-area sample, which is considered in the finite element method calculation, is proposed and implemented into the modelling. Angular distribution function, total transmission and total reflection of the scattering interface or structure can be determined using the model. Furthermore, a method for calculation of the haze parameter of reflected or transmitted light is proposed. The modelling approach is applied to periodic and random nano-textured samples for photovoltaic applications, showing good agreement with measured data. PMID:26698803

  19. Action of Obestatin in Skeletal Muscle Repair: Stem Cell Expansion, Muscle Growth, and Microenvironment Remodeling

    PubMed Central

    Gurriarán-Rodríguez, Uxía; Santos-Zas, Icía; González-Sánchez, Jessica; Beiroa, Daniel; Moresi, Viviana; Mosteiro, Carlos S; Lin, Wei; Viñuela, Juan E; Señarís, José; García-Caballero, Tomás; Casanueva, Felipe F; Nogueiras, Rubén; Gallego, Rosalía; Renaud, Jean-Marc; Adamo, Sergio; Pazos, Yolanda; Camiña, Jesús P

    2015-01-01

    The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration. PMID:25762009

  20. [Feedback control mechanisms of plant cell expansion]. Progress report, [June 1989--June 1992

    SciTech Connect

    Cosgrove, D.J.

    1992-12-31

    We have generated considerable evidence for the significance of wall stress relaxation in the control of plant growth and found that several agents (gibberellin, light, genetic loci for dwarf stature) influence growth rate via alteration of wall relaxation. We have refined our methods for measuring wall relaxation and, moreover, have found that wall relaxation properties bear only a distance relationship to wall mechanical properties. We have garnered novel insights into the nature of cell expansion mechanisms by analyzing spontaneous fluctuations of plant growth rate in seedlings. These experiments involved the application of mathematical techniques for analyzing growth rate fluctuations and the development of new instrumentation for measuring and forcing plant growth in a controlled fashion. These studies conclude that growth rate fluctuations generated by the plant as consequence of a feedback control system. This conclusion has important implications for the nature of wall loosening processes and demands a different framework for thinking about growth control. It also implies the existence of a growth rate sensor.

  1. Rigorous modelling of light scattering in solar cells based on finite element method and Huygens' expansion.

    PubMed

    Sever, Martin; Krč, Janez; Čampa, Andrej; Topič, Marko

    2015-11-30

    Finite element method is coupled with Huygens' expansion to determine light intensity distribution of scattered light in solar cells and other optoelectronic devices. The rigorous foundation of the modelling enables calculation of the light intensity distribution at a chosen distance and surface of observation in chosen material in reflection or in transmission for given wavelength of the incident light. The calculation of scattering or anti-reflection properties is not limited to a single textured interface, but can be done above more complex structures with several scattering interfaces or even with particles involved. Both scattering at periodic and at random textures can be efficiently handled with the modelling approach. A procedure for minimisation of the effect of small-area sample, which is considered in the finite element method calculation, is proposed and implemented into the modelling. Angular distribution function, total transmission and total reflection of the scattering interface or structure can be determined using the model. Furthermore, a method for calculation of the haze parameter of reflected or transmitted light is proposed. The modelling approach is applied to periodic and random nano-textured samples for photovoltaic applications, showing good agreement with measured data.

  2. Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling.

    PubMed

    Gurriarán-Rodríguez, Uxía; Santos-Zas, Icía; González-Sánchez, Jessica; Beiroa, Daniel; Moresi, Viviana; Mosteiro, Carlos S; Lin, Wei; Viñuela, Juan E; Señarís, José; García-Caballero, Tomás; Casanueva, Felipe F; Nogueiras, Rubén; Gallego, Rosalía; Renaud, Jean-Marc; Adamo, Sergio; Pazos, Yolanda; Camiña, Jesús P

    2015-06-01

    The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration.

  3. Critical steps in the isolation and expansion of adipose-derived stem cells for translational therapy.

    PubMed

    Riis, S; Zachar, V; Boucher, S; Vemuri, M C; Pennisi, C P; Fink, T

    2015-06-08

    Since the discovery of adipose-derived stem cells (ASCs), there have been high expectations of their putative clinical use. Recent advances support these expectations, and it is expected that the transition from pre-clinical and clinical studies to implementation as a standard treatment modality is imminent. However ASCs must be isolated and expanded according to good manufacturing practice guidelines and a basic assurance of quality, safety, and medical effectiveness is needed for authorisation by regulatory agencies, such as European Medicines Agency and US Food and Drug Administration. In this review, a collection of studies investigating the influence of different steps of the isolation and expansion protocol on the yield and functionality of ASCs has been presented in an attempt to come up with best recommendations that ensure potential beneficial clinical outcome of using ASCs in any therapeutic setting. If the findings confirm the initial observations of beneficial effects of ASCs, the path is paved for implementing these ASC-based therapies as standard treatment options.

  4. Thermal expansion coefficient prediction of fuel-cell seal materials from silica sand

    NASA Astrophysics Data System (ADS)

    Hidayat, Nurul; Triwikantoro, Baqiya, Malik A.; Pratapa, Suminar

    2013-09-01

    This study is focused on the prediction of coefficient of thermal expansion (CTE) of silica-sand-based fuel-cell seal materials (FcSMs) which in principle require a CTE value in the range of 9.5-12 ppm/°C. A semi-quantitative theoretical method to predict the CTE value is proposed by applying the analyzed phase compositions from XRD data and characterized density-porosity behavior. A typical silica sand was milled at 150 rpm for 1 hour followed by heating at 1000 °C for another hour. The sand and heated samples were characterized by means of XRD to perceive the phase composition correlation between them. Rietveld refinement was executed to investigate the weight fraction of the phase contained in the samples, and then converted to volume fraction for composite CTE calculations. The result was applied to predict their potential physical properties for FcSM. Porosity was taken into account in the calculation after which it was directly measured by the Archimedes method.

  5. Rosmarinic Acid and Melissa officinalis Extracts Differently Affect Glioblastoma Cells

    PubMed Central

    Ramanauskiene, Kristina; Raudonis, Raimondas

    2016-01-01

    Lemon balm (Melissa officinalis L.) has many biological effects but especially important is its neuroprotective activity. The aim of the study is to produce different extracts of Melissa officinalis and analyse their chemical composition and biological properties on rat glioblastoma C6 cells. Results revealed that rosmarinic acid (RA) is the predominant compound of lemon balm extracts. RA has cytotoxic effect on glioblastoma cells (LC50 290.5 μM after the incubation of 24 h and LC50 171.3 μM after 48 h). RA at concentration 80–130 μM suppresses the cell proliferation and has an antioxidant effect. 200 μM and higher concentrations of RA have a prooxidant effect and initiate cell death through necrosis. The aqueous extract of lemon balm is also enriched in phenolic compounds: protocatechuic, caftaric, caffeic, ferulic, and cichoric acids and flavonoid luteolin-7-glucoside. This extract at concentrations 50 μM–200 μM RA has cytotoxic activity and initiates cell death through apoptosis. Extracts prepared with 70% ethanol contain the biggest amount of active compounds. These extracts have the highest cytotoxic activity on glioblastoma cells. They initiate generation of intracellular ROS and cell death through apoptosis and necrosis. Our data suggest that differently prepared lemon balm extracts differently affect glioblastoma cells and can be used as neuroprotective agents in several therapeutic strategies.

  6. Rosmarinic Acid and Melissa officinalis Extracts Differently Affect Glioblastoma Cells

    PubMed Central

    Ramanauskiene, Kristina; Raudonis, Raimondas

    2016-01-01

    Lemon balm (Melissa officinalis L.) has many biological effects but especially important is its neuroprotective activity. The aim of the study is to produce different extracts of Melissa officinalis and analyse their chemical composition and biological properties on rat glioblastoma C6 cells. Results revealed that rosmarinic acid (RA) is the predominant compound of lemon balm extracts. RA has cytotoxic effect on glioblastoma cells (LC50 290.5 μM after the incubation of 24 h and LC50 171.3 μM after 48 h). RA at concentration 80–130 μM suppresses the cell proliferation and has an antioxidant effect. 200 μM and higher concentrations of RA have a prooxidant effect and initiate cell death through necrosis. The aqueous extract of lemon balm is also enriched in phenolic compounds: protocatechuic, caftaric, caffeic, ferulic, and cichoric acids and flavonoid luteolin-7-glucoside. This extract at concentrations 50 μM–200 μM RA has cytotoxic activity and initiates cell death through apoptosis. Extracts prepared with 70% ethanol contain the biggest amount of active compounds. These extracts have the highest cytotoxic activity on glioblastoma cells. They initiate generation of intracellular ROS and cell death through apoptosis and necrosis. Our data suggest that differently prepared lemon balm extracts differently affect glioblastoma cells and can be used as neuroprotective agents in several therapeutic strategies. PMID:27688825

  7. Expansion of B-1a Cells with Germline Heavy Chain Sequence in Lupus Mice

    PubMed Central

    Holodick, Nichol E.; Zeumer, Leilani; Rothstein, Thomas L.; Morel, Laurence

    2016-01-01

    B6.Sle1.Sle2.Sle3 (B6.TC) lupus-prone mice carrying the NZB allele of Cdkn2c, encoding for the cyclin-dependent kinase inhibitor P18INK4, accumulate B-1a cells due to a higher rate of proliferative self-renewal. However, it is unclear whether this affects primarily early-appearing B-1a cells of fetal origin or later-appearing B-1a cells that emerge from bone marrow. B-1a cells are the major source of natural autoantibodies, and it has been shown that their protective nature is associated with a germline-like sequence, which is characterized by few N-nucleotide insertions and a repertoire skewed toward rearrangements predominated during fetal life, VH11 and VH12. To determine the nature of B-1a cells expanded in B6.TC mice, we amplified immunoglobulin genes by PCR from single cells in mice. Sequencing showed a significantly higher proportion of B-1a cell antibodies that display fewer N-additions in B6.TC mice than in B6 control mice. Following this lower number of N-insertions within the CDR-H3 region, the B6.TC B-1a cells display shorter CDR-H3 length than B6 B-1a cells. The absence of N-additions is a surrogate for fetal origin, as TdT expression starts after birth in mice. Therefore, our results suggest that the B-1a cell population is not only expanded in autoimmune B6.TC mice but also qualitatively different with the majority of cells from fetal origin. Accordingly, our sequencing results also demonstrated the overuse of VH11 and VH12 in autoimmune B6.TC mice as compared to B6 controls. These results suggest that the development of lupus autoantibodies in these mice is coupled with skewing of the B-1a cell repertoire and possible retention of protective natural antibodies. PMID:27047495

  8. Angiopoietin-Like Protein 3 Promotes Preservation of Stemness during Ex Vivo Expansion of Murine Hematopoietic Stem Cells

    PubMed Central

    Farahbakhshian, Elnaz; Verstegen, Monique M.; Visser, Trudi P.; Kheradmandkia, Sima; Geerts, Dirk; Arshad, Shazia; Riaz, Noveen; Grosveld, Frank; van Til, Niek P.; Meijerink, Jules P. P.

    2014-01-01

    Allogeneic hematopoietic stem cell (HSC) transplantations from umbilical cord blood or autologous HSCs for gene therapy purposes are hampered by limited number of stem cells. To test the ability to expand HSCs in vitro prior to transplantation, two growth factor cocktails containing stem cell factor, thrombopoietin, fms-related tyrosine kinase-3 ligand (STF) or stem cell factor, thrombopoietin, insulin-like growth factor-2, fibroblast growth factor-1 (STIF) either with or without the addition of angiopoietin-like protein-3 (Angptl3) were used. Culturing HSCs in STF and STIF media for 7 days expanded long-term repopulating stem cells content in vivo by ∼6-fold and ∼10-fold compared to freshly isolated stem cells. Addition of Angptl3 resulted in increased expansion of these populations by ∼17-fold and ∼32-fold, respectively, and was further supported by enforced expression of Angptl3 in HSCs through lentiviral transduction that also promoted HSC expansion. As expansion of highly purified lineage-negative, Sca-1+, c-Kit+ HSCs was less efficient than less pure lineage-negative HSCs, Angptl3 may have a direct effect on HCS but also an indirect effect on accessory cells that support HSC expansion. No evidence for leukemia or toxicity was found during long-term follow up of mice transplanted with ex vivo expanded HSCs or manipulated HSC populations that expressed Angptl3. We conclude that the cytokine combinations used in this study to expand HSCs ex vivo enhances the engraftment in vivo. This has important implications for allogeneic umbilical cord-blood derived HSC transplantations and autologous HSC applications including gene therapy. PMID:25170927

  9. Substrate elasticity affects bovine satellite cell activation kinetics in vitro.

    PubMed

    Lapin, M R; Gonzalez, J M; Johnson, S E

    2013-05-01

    Satellite cells support efficient postnatal skeletal muscle hypertrophy through fusion into the adjacent muscle fiber. Nuclear contribution allows for maintenance of the fiber myonuclear domain and proficient transcription of myogenic genes. Niche growth factors affect satellite cell biology; however, the interplay between fiber elasticity and microenvironment proteins remains largely unknown. The objective of the experiment was to examine the effects of hepatocyte growth factor (HGF) and surface elasticity on bovine satellite cell (BSC) activation kinetics in vitro. Young's elastic modulus was calculated for the semimembranosus (SM) and LM muscles of young bulls (5 d; n = 8) and adult cows (27 mo; n = 4) cattle. Results indicate that LM elasticity decreased (P < 0.05) with age; no difference in Young's modulus for the SM was noted. Bovine satellite cells were seeded atop polyacrylamide bioscaffolds with surface elasticities that mimic young bull and adult cow LM or traditional cultureware. Cells were maintained in low-serum media supplemented with 5 ng/mL HGF or vehicle only for 24 or 48 h. Activation was evaluated by proliferating cell nuclear antigen (PCNA) immunocytochemistry. Results indicate that BSC maintained on rigid surfaces were activated at 24 h and refractive to HGF supplementation. By contrast, fewer (P < 0.05) BSC had exited quiescence after 24 h of culture on surfaces reflective of either young bull (8.1 ± 1.7 kPa) or adult cow (14.6 ± 1.6 kPa) LM. Supplementation with HGF promoted activation of BSC cultured on bioscaffolds as measured by an increase (P < 0.05) in PCNA immunopositive cells. Culture on pliant surfaces affected neither activation kinetics nor numbers of Paired box 7 (Pax7) immunopositive muscle stem cells (P > 0.05). However, with increasing surface elasticity, an increase (P < 0.05) in the numbers of muscle progenitors was observed. These results confirm that biophysical and biochemical signals regulate BSC activation.

  10. Brief Report: The Deletion of the Phosphatase Regulator NIPP1 Causes Progenitor Cell Expansion in the Adult Liver.

    PubMed

    Boens, Shannah; Verbinnen, Iris; Verhulst, Stefaan; Szekér, Kathelijne; Ferreira, Monica; Gevaert, Thomas; Baes, Myriam; Roskams, Tania; van Grunsven, Leo A; Van Eynde, Aleyde; Bollen, Mathieu

    2016-08-01

    The Ppp1r8 gene encodes NIPP1, a nuclear interactor of protein phosphatase PP1. The deletion of NIPP1 is embryonic lethal at the gastrulation stage, which has hampered its functional characterization in adult tissues. Here, we describe the effects of a conditional deletion of NIPP1 in mouse liver epithelial cells. Ppp1r8(-/-) livers developed a ductular reaction, that is, bile-duct hyperplasia with associated fibrosis. The increased proliferation of biliary epithelial cells was at least partially due to an expansion of the progenitor cell compartment that was independent of liver injury. Gene-expression analysis confirmed an upregulation of progenitor cell markers in the liver knockout livers but showed no effect on the expression of liver-injury associated regulators of cholangiocyte differentiation markers. Consistent with an inhibitory effect of NIPP1 on progenitor cell proliferation, Ppp1r8(-/-) livers displayed an increased sensitivity to diet-supplemented 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which also causes bile-duct hyperplasia through progenitor cell expansion. In contrast, the liver knockouts responded normally to injuries (partial hepatectomy, single CCl4 administration) that are restored through proliferation of differentiated parenchymal cells. Our data indicate that NIPP1 does not regulate the proliferation of hepatocytes but is a suppressor of biliary epithelial cell proliferation, including progenitor cells, in the adult liver. Stem Cells 2016;34:2256-2262. PMID:27068806

  11. Expression of MHC class I receptors confers functional intraclonal heterogeneity to a reactive expansion of gammadelta T cells.

    PubMed

    Lafarge, Xavier; Pitard, Vincent; Ravet, Sophie; Roumanes, David; Halary, Franck; Dromer, Claire; Vivier, Eric; Paul, Pascale; Moreau, Jean-François; Déchanet-Merville, Julie

    2005-06-01

    NK cell receptors for MHC class I molecules (MHC-NKR) can be expressed by T cell subsets. The restricted repertoire and phenotypic characteristics of MHC-NKR(+) T cells indicate that expression of MHC-NKR is acquired upon antigenic challenge and might promote expansion of T cells. Previous studies performed on in vitro generated alphabeta T cell clones concluded that MHC-NKR expression was not a clonal attribute. Here, we examined a massive monoclonal expansion of a non-leukemic gammadelta T cell population found in the peripheral blood of a lung-transplanted patient who suffered from a cytomegalovirus infection. Despite their monoclonality, these T cells displayed a heterogeneous and stable in vivo Ig- and lectin-like MHC-NKR phenotype. Twenty percent of the cells displayed a CD94(+)NKG2A(+) phenotype, and 10% were labeled with an anti-CD158b1/b2/j monoclonal antibody. A CD158b/j(+) gammadelta T cell clone derived in vitro from patient's peripheral blood lymphocytes was shown to express the activating form CD158j (KIR2DS2), which once cross-linked stimulated the clone cytolytic function and costimulated the TCR-induced production of cytokines, independently of the killer-activating receptor-associated protein (KARAP). In conclusion, heterogeneity of MHC-NKR expression confers a functional intraclonal diversity that may participate to induction of specific gammadelta T cell effector functions or proliferation upon pathogen challenge.

  12. Direct visualization of endogenous Salmonella-specific B cells reveals a marked delay in clonal expansion and germinal center development

    PubMed Central

    Nanton, Minelva R.; Lee, Seung-Joo; Atif, Shaikh M.; Nuccio, Sean-Paul; Taylor, Justin J.; Bäumler, Andreas J.; Way, Sing Sing; McSorley, Stephen J.

    2014-01-01

    CD4+ T cells and B cells are both essential for acquired immunity to Salmonella infection. It is well established that Salmonella inhibit host CD4+ T-cell responses, but a corresponding inhibitory effect on B cells is less well defined. Here, we utilize an antigen tetramer and pull-down enrichment strategy to directly visualize OVA-specific B cells in mice, as they respond to infection with Salmonella-OVA. Surprisingly, OVA-specific B-cell expansion and germinal center formation was not detected until bacteria were cleared from the host. Furthermore, Salmonella infection also actively inhibited both B- and T-cell responses to the same coinjected antigen but this did not require the presence of iNOS. The SPI2 locus has been shown to be responsible for inhibition of Salmonella-specific CD4+ T-cell responses, and an examination of SPI2-deficient bacteria demonstrated a recovery in B-cell expansion in infected mice. Together, these data suggest that Salmonella can simultaneously inhibit host B- and T-cell responses using SPI2-dependent mechanisms. PMID:25346524

  13. Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer.

    PubMed

    Meng, Qingda; Liu, Zhenjiang; Rangelova, Elena; Poiret, Thomas; Ambati, Aditya; Rane, Lalit; Xie, Shanshan; Verbeke, Caroline; Dodoo, Ernest; Del Chiaro, Marco; Löhr, Matthias; Segersvärd, Ralf; Maeurer, Markus J

    2016-01-01

    Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-[gamma] production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR V[beta] was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8(+) T cells. CD3(+)CD8(+), CD3(+)CD4(+), and CD3(+)CD4(-)CD8(-)[double-negative (DN) T cells] resided predominantly in central (CD45RA(-)CCR7(+)) and effector (CD45RA-CCR7-) memory subsets. CD8(+) TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4(+) TILs showed only up to 12% LAG-3(+) staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8(+)) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-[alpha] and IFN-[gamma] production. Twelve of 17 of CD8(+) TILs showed preferential expansion of certain TCR V[beta] families (eg, 99.2% V[beta]13.2 in CD8(+) TILs, 77% in the V[beta]1, 65.9% in the V[beta]22, and 63.3% in the V[beta]14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs

  14. AtDOF5.4/OBP4, a DOF Transcription Factor Gene that Negatively Regulates Cell Cycle Progression and Cell Expansion in Arabidopsis thaliana

    PubMed Central

    Xu, Peipei; Chen, Haiying; Ying, Lu; Cai, Weiming

    2016-01-01

    In contrast to animals, plant development involves continuous organ formation, which requires strict regulation of cell proliferation. The core cell cycle machinery is conserved across plants and animals, but plants have developed new mechanisms that precisely regulate cell proliferation in response to internal and external stimuli. Here, we report that the DOF transcription factor OBP4 negatively regulates cell proliferation and expansion. OBP4 is a nuclear protein. Constitutive and inducible overexpression of OBP4 reduced the cell size and number, resulting in dwarf plants. Inducible overexpression of OBP4 in Arabidopsis also promoted early endocycle onset and inhibited cell expansion, while inducible overexpression of OBP4 fused to the VP16 activation domain in Arabidopsis delayed endocycle onset and promoted plant growth. Furthermore, gene expression analysis showed that cell cycle regulators and cell wall expansion factors were largely down-regulated in the OBP4 overexpression lines. Short-term inducible analysis coupled with in vivo ChIP assays indicated that OBP4 targets the CyclinB1;1, CDKB1;1 and XTH genes. These results strongly suggest that OBP4 is a negative regulator of cell cycle progression and cell growth. These findings increase our understanding of the transcriptional regulation of the cell cycle in plants. PMID:27297966

  15. High antigen levels induce an exhausted phenotype in a chronic infection without impairing T cell expansion and survival.

    PubMed

    Utzschneider, Daniel T; Alfei, Francesca; Roelli, Patrick; Barras, David; Chennupati, Vijaykumar; Darbre, Stephanie; Delorenzi, Mauro; Pinschewer, Daniel D; Zehn, Dietmar

    2016-08-22

    Chronic infections induce T cells showing impaired cytokine secretion and up-regulated expression of inhibitory receptors such as PD-1. What determines the acquisition of this chronic phenotype and how it impacts T cell function remain vaguely understood. Using newly generated recombinant antigen variant-expressing chronic lymphocytic choriomeningitis virus (LCMV) strains, we uncovered that T cell differentiation and acquisition of a chronic or exhausted phenotype depend critically on the frequency of T cell receptor (TCR) engagement and less significantly on the strength of TCR stimulation. In fact, we noted that low-level antigen exposure promotes the formation of T cells with an acute phenotype in chronic infections. Unexpectedly, we found that T cell populations with an acute or chronic phenotype are maintained equally well in chronic infections and undergo comparable primary and secondary expansion. Thus, our observations contrast with the view that T cells with a typical chronic infection phenotype are severely functionally impaired and rapidly transition into a terminal stage of differentiation. Instead, our data unravel that T cells primarily undergo a form of phenotypic and functional differentiation in the early phase of a chronic LCMV infection without inheriting a net survival or expansion deficit, and we demonstrate that the acquired chronic phenotype transitions into the memory T cell compartment. PMID:27455951

  16. High-Dose Nicotinamide Suppresses ROS Generation and Augments Population Expansion during CD8+ T Cell Activation

    PubMed Central

    Choi, Ho Jin; Jang, So-Young; Hwang, Eun Seong

    2015-01-01

    During T cell activation, mitochondrial content increases to meet the high energy demand of rapid cell proliferation. With this increase, the level of reactive oxygen species (ROS) also increases and causes the rapid apoptotic death of activated cells, thereby facilitating T cell homeostasis. Nicotinamide (NAM) has previously been shown to enhance mitochondria quality and extend the replicative life span of human fibroblasts. In this study, we examined the effect of NAM on CD8+ T cell activation. NAM treatment attenuated the increase of mitochondrial content and ROS in T cells activated by CD3/CD28 antibodies. This was accompanied by an accelerated and higher-level clonal expansion resulting from attenuated apoptotic death but not increased division of the activated cells. Attenuation of ROS-triggered pro-apoptotic events and upregulation of Bcl-2 expression appeared to be involved. Although cells activated in the presence of NAM exhibited compromised cytokine gene expression, our results suggest a means to augment the size of T cell expansion during activation without consuming their limited replicative potential. PMID:26442863

  17. Long-Term In Vitro Expansion of Salivary Gland Stem Cells Driven by Wnt Signals

    PubMed Central

    Maimets, Martti; Rocchi, Cecilia; Bron, Reinier; Pringle, Sarah; Kuipers, Jeroen; Giepmans, Ben N.G.; Vries, Robert G.J.; Clevers, Hans; de Haan, Gerald; van Os, Ronald; Coppes, Robert P.

    2015-01-01

    Summary Adult stem cells are the ultimate source for replenishment of salivary gland (SG) tissue. Self-renewal ability of stem cells is dependent on extrinsic niche signals that have not been unraveled for the SG. The ductal compartment in SG has been identified as the location harboring stem cells. Here, we report that rare SG ductal EpCAM+ cells express nuclear β-catenin, indicating active Wnt signaling. In cell culture experiments, EpCAMhigh cells respond potently to Wnt signals stimulating self-renewal and long-term expansion of SG organoids, containing all differentiated SG cell types. Conversely, Wnt inhibition ablated long-term organoid cultures. Finally, transplantation of cells pre-treated with Wnt agonists into submandibular glands of irradiated mice successfully and robustly restored saliva secretion and increased the number of functional acini in vivo. Collectively, these results identify Wnt signaling as a key driver of adult SG stem cells, allowing extensive in vitro expansion and enabling restoration of SG function upon transplantation. PMID:26724906

  18. High-Dose Nicotinamide Suppresses ROS Generation and Augments Population Expansion during CD8(+) T Cell Activation.

    PubMed

    Choi, Ho Jin; Jang, So-Young; Hwang, Eun Seong

    2015-10-01

    During T cell activation, mitochondrial content increases to meet the high energy demand of rapid cell proliferation. With this increase, the level of reactive oxygen species (ROS) also increases and causes the rapid apoptotic death of activated cells, thereby facilitating T cell homeostasis. Nicotinamide (NAM) has previously been shown to enhance mitochondria quality and extend the replicative life span of human fibroblasts. In this study, we examined the effect of NAM on CD8(+) T cell activation. NAM treatment attenuated the increase of mitochondrial content and ROS in T cells activated by CD3/CD28 antibodies. This was accompanied by an accelerated and higher-level clonal expansion resulting from attenuated apoptotic death but not increased division of the activated cells. Attenuation of ROS-triggered pro-apoptotic events and upregulation of Bcl-2 expression appeared to be involved. Although cells activated in the presence of NAM exhibited compromised cytokine gene expression, our results suggest a means to augment the size of T cell expansion during activation without consuming their limited replicative potential. PMID:26442863

  19. Bmal1 is required for beta cell compensatory expansion, survival and metabolic adaptation to diet-induced obesity in mice

    PubMed Central

    Rakshit, Kuntol; Hsu, Tu Wen

    2016-01-01

    Aims/hypothesis Obesity and consequent insulin resistance are known risk factors for type 2 diabetes. A compensatory increase in beta cell function and mass in response to insulin resistance permits maintenance of normal glucose homeostasis, whereas failure to do so results in beta cell failure and type 2 diabetes. Recent evidence suggests that the circadian system is essential for proper metabolic control and regulation of beta cell function. We set out to address the hypothesis that the beta cell circadian clock is essential for the appropriate functional and morphological beta cell response to insulin resistance. Methods We employed conditional deletion of the Bmal1 (also known as Arntl) gene (encoding a key circadian clock transcription factor) in beta cells using the tamoxifen-inducible CreERT recombination system. Upon adulthood, Bmal1 deletion in beta cells was achieved and mice were exposed to either chow or high fat diet (HFD). Changes in diurnal glycaemia, glucose tolerance and insulin secretion were longitudinally monitored in vivo and islet morphology and turnover assessed by immunofluorescence. Isolated islet experiments in vitro were performed to delineate changes in beta cell function and transcriptional regulation of cell proliferation. Results Adult Bmal1 deletion in beta cells resulted in failed metabolic adaptation to HFD characterised by fasting and diurnal hyperglycaemia, glucose intolerance and loss of glucose-stimulated insulin secretion. Importantly, HFD-induced beta cell expansion was absent following beta cell Bmal1 deletion indicating impaired beta cell proliferative and regenerative potential, which was confirmed by assessment of transcriptional profiles in isolated islets. Conclusion/interpretation Results of the study suggest that the beta cell circadian clock is a novel regulator of compensatory beta cell expansion and function in response to increased insulin demand associated with diet-induced obesity. PMID:26762333

  20. Factors affecting white cell content in platelet concentrates.

    PubMed

    Champion, A B; Carmen, R A

    1985-01-01

    In this study, we investigated the factors affecting white cell content in platelet concentrates. White cell yields can be reduced 50 percent by stopping platelet-rich plasma expression when the interface is 1 cm from the top of the blood bag as compared to stopping expression when the interface reaches the top of the bag. Further reductions can be achieved by careful handling during transfer of units from the centrifuge cups to expressors (after the first spin) and by carefully balancing units against each other to ensure proper rotor balance during the first spin. Following these suggestions, blood banks should be able to produce platelet concentrates with white cell yields between 2 and 6 X 10(7) and with platelet yields between 7.5 and 8 X 10(10). Transfusion of this product may reduce febrile reactions and lower the incidence of alloimmunizations. PMID:4024231

  1. Noggin 1 overexpression in retinal progenitors affects bipolar cell generation.

    PubMed

    Messina, Andrea; Bridi, Simone; Bozza, Angela; Bozzi, Yuri; Baudet, Marie-Laure; Casarosa, Simona

    2016-01-01

    Waves of Bone Morphogenetic Proteins (BMPs) and their antagonists are present during initial eye development, but their possible roles in retinogenesis are still unknown. We have recently shown that noggin 1, a BMP antagonist, renders pluripotent cells able to differentiate into retinal precursors, and might be involved in the maintenance of retinal structures in the adult vertebrate eye. Here, we report that noggin 1, differently from noggin 2 and noggin 4, is expressed during all phases of Xenopus laevis retinal development. Gain-of-function experiments by electroporation in the optic vesicle show that overexpression of noggin 1 significantly decreases the number of bipolar cells in the inner nuclear layer of the retina, without significantly affecting the generation of the other retinal cell types. Our data suggest that BMP signaling could be involved in the differentiation of retinal progenitors into specific retinal subtypes during late phases of vertebrate retinal development. PMID:27389985

  2. ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells

    PubMed Central

    Moreb, Jan S; Baker, Henry V; Chang, Lung-Ji; Amaya, Maria; Lopez, M Cecilia; Ostmark, Blanca; Chou, Wayne

    2008-01-01

    Background Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over expression have been studied. Methods We have employed oligonucleotide microarrays to analyze changes in gene profiles in A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were used to identify gene expression patterns that are specific to the knock down of ALDH activity and significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with green fluorescent protein (GFP) siRNA. Results We confirmed significant and specific down regulation of ALDH1A1 and ALDH3A1 in Lenti 1+3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray analysis were validated by real time RT-PCR on RNA obtained from Lenti 1+3 or WT cells treated with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were significantly different (P < 0.001) and their expression changed by ≥ 2 folds in the Lenti 1+3 group versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration and adhesion were among the most affected pathways. Conclusion These molecular effects of the ALDH knock-down are associated with in vitro functional changes in the proliferation and motility of these cells and demonstrate the significance of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung cancer. PMID:19025616

  3. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. II. Quantity and quality of light required

    NASA Technical Reports Server (NTRS)

    Van Volkenburgh, E.; Cleland, R. E.; Watanabe, M.

    1990-01-01

    The quantity and quality of light required for light-stimulated cell expansion in leaves of Phaseolus vulgaris L. have been determined. Seedlings were grown in dim red light (RL; 4 micromoles photons m-2 s-1) until cell division in the primary leaves was completed, then excised discs were incubated in 10 mM sucrose plus 10 mM KCl in a variety of light treatments. The growth response of discs exposed to continuous white light (WL) for 16 h was saturated at 100 micromoles m-2 s-1, and did not show reciprocity. Extensive, but not continuous, illumination was needed for maximal growth. The wavelength dependence of disc expansion was determined from fluence-response curves obtained from 380 to 730 nm provided by the Okazaki Large Spectrograph. Blue (BL; 460 nm) and red light (RL; 660 nm) were most effective in promoting leaf cell growth, both in photosynthetically active and inhibited leaf discs. Far-red light (FR; 730 nm) reduced the effectiveness of RL, but not BL, indicating that phytochrome and a separate blue-light receptor mediate expansion of leaf cells.

  4. POLYGALACTURONASE INVOLVED IN EXPANSION1 Functions in Cell Elongation and Flower Development in Arabidopsis[C][W

    PubMed Central

    Xiao, Chaowen; Somerville, Chris; Anderson, Charles T.

    2014-01-01

    Pectins are acidic carbohydrates that comprise a significant fraction of the primary walls of eudicotyledonous plant cells. They influence wall porosity and extensibility, thus controlling cell and organ growth during plant development. The regulated degradation of pectins is required for many cell separation events in plants, but the role of pectin degradation in cell expansion is poorly defined. Using an activation tag screen designed to isolate genes involved in wall expansion, we identified a gene encoding a putative polygalacturonase that, when overexpressed, resulted in enhanced hypocotyl elongation in etiolated Arabidopsis thaliana seedlings. We named this gene POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1). Plants lacking PGX1 display reduced hypocotyl elongation that is complemented by transgenic PGX1 expression. PGX1 is expressed in expanding tissues throughout development, including seedlings, roots, leaves, and flowers. PGX1-GFP (green fluorescent protein) localizes to the apoplast, and heterologously expressed PGX1 displays in vitro polygalacturonase activity, supporting a function for this protein in apoplastic pectin degradation. Plants either overexpressing or lacking PGX1 display alterations in total polygalacturonase activity, pectin molecular mass, and wall composition and also display higher proportions of flowers with extra petals, suggesting PGX1’s involvement in floral organ patterning. These results reveal new roles for polygalacturonases in plant development. PMID:24681615

  5. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion.

    PubMed

    Miller, Michelle Erin; Rosten, Patty; Lemieux, Madeleine E; Lai, Courteney; Humphries, R Keith

    2016-01-01

    Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1's importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1's functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sor(tm1(Cre/ERT)Nat)/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1's role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation. PMID:26986211

  6. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion

    PubMed Central

    Miller, Michelle Erin; Rosten, Patty; Lemieux, Madeleine E.; Lai, Courteney; Humphries, R. Keith

    2016-01-01

    Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1’s importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1’s functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sortm1(Cre/ERT)Nat/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1’s role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation. PMID:26986211

  7. The Important Role of FLT3-L in Ex Vivo Expansion of Hematopoietic Stem Cells following Co-Culture with Mesenchymal Stem Cells

    PubMed Central

    Oubari, Farhad; Amirizade, Naser; Mohammadpour, Hemn; Nakhlestani, Mozhdeh; Zarif, Mahin Nikougoftar

    2015-01-01

    Objective Hematopoietic stem cells (HSCs) transplantation using umbilical cord blood (UCB) has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand (FLT3-L) as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell (MSC) feeder. Materials and Methods In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count (TNC), cluster of differentiation 34+(CD34+) cell count, colony forming unit assay (CFU), long-term culture initiating cell (LTC-IC), homeobox protein B4 (HoxB4) mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test. Results HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines (P<0.05). FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor (SCF), thrombopoietin (TPO) and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased (P<0.05). Conclusion FLT3-L co-culture with MSCs can induce high yield expansion of HSCs and be a substitute for the universal cocktail of SCF, TPO and FLT3-L in feeder-free culture. PMID:26199899

  8. Expansion of recipient-derived antiviral T cells may influence donor chimerism after allogeneic stem cell transplantation.

    PubMed

    Borchers, S; Weissinger, E M; Pabst, B; Ganzenmueller, T; Dammann, E; Luther, S; Diedrich, H; Ganser, A; Stadler, M

    2013-12-01

    Donor chimerism (DC) analysis is an important marker in the hematopoietic stem cell transplant follow-up. Here, we present evidence for a possible relationship of infectious complications and declines in DC. We analyzed the DC in patients experiencing cytomegalovirus (CMV) reactivation. In addition, in some patients chimerism analyses of T-cell subsets were performed. CMV-specific cytotoxic T-lymphocytes (CMV-CTL) were monitored using human leukocyte antigen-restricted multimer staining. Interestingly, CMV reactivation was accompanied by changes in DC in 11 of 67 patients transplanted. For example, DC declined in a cord blood recipient, in both total leukocytes and CD4 and CD8 T-cell subsets upon CMV reactivation. The latter was controlled after only 5 days through expanding CMV-CTL of 96% recipient origin, according to chimerism analysis of CMV-CTL (enriched beyond 50%). In another patient, transplanted after reduced-intensity conditioning from a DQB1 mismatched, CMV seronegative donor, incipient CMV reactivation was completely aborted by CMV-CTL of recipient origin. However, at the same time, mixed chimerism dropped from 51% to 0% donor type, resulting in late graft rejection. Our data indicate that chimerism analyses in subset populations lead to a better understanding of declining total leukocyte chimerism. Furthermore, recipient-derived CMV-CTL may be able to control CMV reactivation after reduced-intensity conditioning. We speculate that autologous CMV-CTL may be instrumental to overcome recurrent CMV reactivations, especially in patients transplanted from CMV-seronegative donors. In addition, the expansion of recipient-derived CMV-CTL may contribute to both, graft failure or to conversion to full DC.

  9. Vitamin D Attenuates Kidney Fibrosis via Reducing Fibroblast Expansion, Inflammation, and Epithelial Cell Apoptosis.

    PubMed

    Arfian, Nur; Muflikhah, Khusnul; Soeyono, Sri Kadarsih; Sari, Dwi Cahyani Ratna; Tranggono, Untung; Anggorowati, Nungki; Romi, Muhammad Mansyur

    2016-01-01

    Kidney fibrosis is the common final pathway of chronic kidney diseases (CKD). It is characterized by myofibroblast formation, inflammation, and epithelial architecture damage. Vitamin D is known as a renoprotective agent, although the precise mechanism is not well understood. This study aimed to elucidate the effect of vitamin D in fibroblast expansion, inflammation, and apoptosis in kidney fibrosis. We performed unilateral ureteral obstruction (UUO) in male Swiss-Webster background mice (3 months, 30-40 grams) to induce kidney fibrosis. The mice (n=25) were divided into five groups: UUO, 3 groups treated with different oral vitamin D doses (0.125 µg/kg (UUO+VD1), 0.25 µg/kg (UUO+VD2), and 0.5 µg/kg (UUO+VD3), and a Sham operation (SO) group with ethanol 0.2% supplementation. We sacrificed the mice on day14 after the operation and harvested the kidney. We made paraffin sections for histological analysis. Tubular injury and fibrosis were quantified based on periodic acid-Schiff (PAS) and Sirius Red (SR) staining. Immunostaining was done for examination of myofibroblasts (αSMA), fibroblasts (PDGFRβ), TLR4, and apoptosis (TUNEL). We did RNA extraction and cDNA for Reverse transcriptase PCR (RT-PCR) experiment for measuring MCP-1, ICAM-1, TLR4, and collagen 1 expression. TGFβ1 level was quantified using ELISA. We observed a significantly lower levels of fibrosis (p<0.001), tubular injury scores (p<0.001), and myofibroblast areas (p<0.001) in the groups treated with vitamin D compared with the UUO group. The TGFβ1 levels and the fibroblast quantifications were also significantly lower in the former group. However, we did not find any significant difference among the various vitamin D-treated groups. Concerning the dose-independent effect, we only compared the UUO+VD-1 group with SO group and found by TUNEL assay that UUO+VD-1 had a significantly lower epithelial cell apoptosis. RT-PCR analysis showed lower expression of collagen1, as well as inflammation

  10. Thermal Expansion of Three Closed Cell Polymeric Foams at Cryogenic Temperatures

    NASA Technical Reports Server (NTRS)

    Stokes, Eric

    2006-01-01

    The Space Shuttle External Tank (ET) contains the liquid H2 fuel and liquid oxygen oxidizer and supplies them under pressure to the three space shuttle main engines (SSME) in the orbiter during lift-off and ascent. The ET thermal protection system consists of sprayed-on foam insulation and pre-molded ablator materials. The closed-cell foams are the external coating on the ET and are responsible for minimizing the amount of moisture that condenses out and freezes on the tank from the humid air in Florida while it is on the pad with cryogenic propellant awaiting launch. This effort was part of the overall drive to understand the behavior of these materials under use-conditions. There are four specially-engineered closed-cell foams used on the tank. The thermal expansion (contraction) of three of the polyurethane and polyisocyanurate foams were measured from -423 F (the temperature of liquid hydrogen) to 125 F under atmospheric conditions and under vacuum. One of them, NCFI 24-124, is a mechanically-applied material and covers the main acreage of the tank, accounting for 77 percent of the total foam used. Another, BX-265, is also a mechanically-applied and hand-sprayed material used on the tank's "closeout" areas. PDL 1034 is a hand-poured foam used for filling odd-shaped cavities in the tank, Measurements were made in triplicate in the three primary material directions in the case of the first two materials and the two primary material directions in the case of the last. Task 1 was developing the techniques for getting a uniform heating rate and minimizing axial and radial thermal gradients in the specimens. Temperature measurements were made at four locations in the specimens during this initial development phase of testing. Major challenges that were overcome include developing techniques for transferring the coolant, liquid helium (-452 F), from its storage container to the test facility with a minimal transfer of heat to the coolant and control of the heating

  11. Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro

    SciTech Connect

    Wright, Lynda S.; Prowse, Karen R.; Wallace, Kyle; Linskens, Maarten H.K.; Svendsen, Clive N. . E-mail: svendsen@waisman.wisc.edu

    2006-07-01

    Isolation of a true self-renewing stem cell from the human brain would be of great interest as a reliable source of neural tissue. Here, we report that human fetal cortical cells grown in epidermal growth factor expressed low levels of telomerase and telomeres in these cultures shortened over time leading to growth arrest after 30 weeks. Following leukemia inhibitory factor (LIF) supplementation, growth rates and telomerase expression increased. This was best demonstrated following cell cycle synchronization and staining for telomerase using immunocytochemistry. This increase in activity resulted in the maintenance of telomeres at approximately 7 kb for more than 60 weeks in vitro. However, all cultures displayed a lack of oligodendrotye production, decreases in neurogenesis over time and underwent replicative senescence associated with increased expression of p21 before 70 weeks in vitro. Thus, under our culture conditions, these cells are not stable, multipotent, telomerase expressing self-renewing stem cells. They may be more accurately described as human neural progenitor cells (hNPC) with limited lifespan and bi-potent potential (neurons/astrocytes). Interestingly, hNPC follow a course of proliferation, neuronal production and growth arrest similar to that seen during expansion and development of the human cortex, thus providing a possible model neural system. Furthermore, due to their high expansion potential and lack of tumorogenicity, these cells remain a unique and safe source of tissue for clinical transplantation.

  12. Inhibitors of apoptosis proteins (IAPs) are required for effective T-cell expansion/survival during antiviral immunity in mice.

    PubMed

    Gentle, Ian E; Moelter, Isabel; Lechler, Nadja; Bambach, Sarah; Vucikuja, Smiljka; Häcker, Georg; Aichele, Peter

    2014-01-30

    Inhibitors of apoptosis proteins (IAPs) were originally described as regulating apoptosis by direct binding to caspases. More recently, IAPs have been identified as important modulators of canonical and noncanonical nuclear factor κB signaling via their ubiquitin-E3 ligase activity. IAPs are therefore, not only gatekeepers of cell death, but are probably also involved in the regulation of inflammation, as well as innate and adaptive immunity. In this study, we analyzed the role of IAPs in T-cell immunity during lymphocytic choriomeningitis virus (LCMV) infection by pharmacological targeting with an IAP antagonist/second mitochondria-derived activator of caspase-mimetic. Expansion of virus-specific CD8 T cells was drastically reduced in LCMV-infected mice exposed to IAP antagonists. Accordingly, virus control was substantially impaired, indicated by high virus titres in the spleen and the spread of LCMV to peripheral organs. The profound negative effect of IAP antagonists on T-cell immunity was partially linked to tumor necrosis factor-mediated cell death of activated T cells and required inhibition of X-linked inhibitor of apoptosis, as well as cellular IAP-1. Thus, IAPs play an important role in T-cell expansion and survival in the context of a highly inflammatory environment such as a virus infection, indicating that IAP antagonists may interfere with immune responses.

  13. Melanopsin, photosensitive ganglion cells, and seasonal affective disorder.

    PubMed

    Roecklein, Kathryn A; Wong, Patricia M; Miller, Megan A; Donofry, Shannon D; Kamarck, Marissa L; Brainard, George C

    2013-03-01

    In two recent reports, melanopsin gene variations were associated with seasonal affective disorder (SAD), and in changes in the timing of sleep and activity in healthy individuals. New studies have deepened our understanding of the retinohypothalamic tract, which translates environmental light received by the retina into neural signals sent to a set of nonvisual nuclei in the brain that are responsible for functions other than sight including circadian, neuroendocrine and neurobehavioral regulation. Because this pathway mediates seasonal changes in physiology, behavior, and mood, individual variations in the pathway may explain why approximately 1-2% of the North American population develops mood disorders with a seasonal pattern (i.e., Major Depressive and Bipolar Disorders with a seasonal pattern, also known as seasonal affective disorder/SAD). Components of depression including mood changes, sleep patterns, appetite, and cognitive performance can be affected by the biological and behavioral responses to light. Specifically, variations in the gene sequence for the retinal photopigment, melanopsin, may be responsible for significant increased risk for mood disorders with a seasonal pattern, and may do so by leading to changes in activity and sleep timing in winter. The retinal sensitivity of SAD is hypothesized to be decreased compared to controls, and that further decrements in winter light levels may combine to trigger depression in winter. Here we outline steps for new research to address the possible role of melanopsin in seasonal affective disorder including chromatic pupillometry designed to measure the sensitivity of melanopsin containing retinal ganglion cells.

  14. Large-scale in vitro expansion of human regulatory T cells with potent xenoantigen-specific suppression.

    PubMed

    Jin, Xi; Lu, Yanrong; Zhao, Ye; Yi, Shounan

    2016-08-01

    Xenotransplantation is a potential solution to the organ donor shortage. Immunosuppression is required for successful application of xenotransplantation but may lead to infection and cancer. Thus, strategies for immune tolerance induction need to be developed. Polyclonal regulatory T cells (Treg) play a central role in the induction and maintenance of immune tolerance and have been shown to protect against islet xenograft rejection in vivo. However, global immune suppression may be mediated by polyclonal Treg immunotherapy and a simple method for in vitro expansion of xenoantigen-specific Treg for efficient Treg application becomes necessary. Human Treg isolated from peripheral blood mononuclear cells (PBMCs) were initially cultured with anti-CD3/CD28 beads, rapamycin and IL-2 for 7 days as polyclonal expansion. Expanded Treg were then cocultured with irradiated porcine PBMC as xenoantigen stimulation for three subsequent cycles with 7 days for each cycle in the presence of IL-2 and anti-CD3/CD28 beads. Treg phenotype and suppressive capacity were assessed after each cycle of xenoantigen stimulation. Treg expanded with one cycle of xenoantigen stimulation retained Treg suppressive phenotype but acquired no xenoantigen specificity along with poor expansion efficiency, whereas expansion with two-cycle xenoantigen stimulation resulted in not only more than 800-fold Treg expansion but highly suppressive xenoantigen-specific Treg with effector Treg phenotype. However further increase of stimulation cycles resulted in reduced Treg suppressive potency. This study provides a simple approach to obtain high numbers of xenoantigen-specific Treg for immune tolerance induction in xenotransplantation.

  15. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    PubMed

    Chou, Ming-Li; Burnouf, Thierry; Wang, Tsung-Jen

    2014-01-01

    Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory

  16. STAT5-regulated microRNA-193b controls haematopoietic stem and progenitor cell expansion by modulating cytokine receptor signalling

    PubMed Central

    Haetscher, Nadine; Feuermann, Yonatan; Wingert, Susanne; Rehage, Maike; Thalheimer, Frederic B.; Weiser, Christian; Bohnenberger, Hanibal; Jung, Klaus; Schroeder, Timm; Serve, Hubert; Oellerich, Thomas; Hennighausen, Lothar; Rieger, Michael A.

    2015-01-01

    Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b−/− mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion. PMID:26603207

  17. Trichostatin A Stabilizes the Expression of Pluripotent Genes in Human Mesenchymal Stem Cells during Ex Vivo Expansion

    PubMed Central

    Han, Bing; Li, Jing; Li, Zhilong; Guo, Ling; Wang, Shan; Liu, Peishu; Wu, Yaojiong

    2013-01-01

    Background Mesenchymal stem cells (MSCs) have been considered as ideal cells for the treatment of a variety of diseases. However, aging and spontaneous differentiation of MSCs during culture expansion dampen their effectiveness. Previous studies suggest that ex vivo aging of MSCs is largely caused by epigenetic changes particularly a decline of histone H3 acetylation levels in promoter regions of pluripotent genes due to inappropriate growth environment. Methodology/Principal Findings In this study, we examined whether histone deacetylase inhibitor trichostatin A (TSA) could suppress the histone H3 deacetylation thus maintaining the primitive property of MSCs. We found that in regular adherent culture, human MSCs became flatter and larger upon successive passaging, while the expression of pluripotent genes such as Oct4, Sox2, Nanog, Rex-1, CD133 and TERT decreased markedly. Administration of low concentrations of TSA in culture significantly suppressed the morphological changes in MSCs otherwise occurred during culture expansion, increased their proliferation while retaining their cell contact growth inhibition property and multipotent differentiation ability. Moreover, TSA stabilized the expression of the above pluripotent genes and histone H3 acetylation levels in K9 and K14 in promoter regions of Oct4, Sox2 and TERT. Conclusions/Significance Our results suggest that TSA may serve as an effective culture additive to maintain the primitive feature of MSCs during culture expansion. PMID:24312356

  18. A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

    PubMed Central

    Shelley, Brandon C.; Gowing, Geneviève; Svendsen, Clive N.

    2014-01-01

    A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products. PMID:24962813

  19. Would cancer stem cells affect the future investment in stem cell therapy.

    PubMed

    Rameshwar, Pranela

    2012-04-20

    The common goal within the overwhelming interests in stem cell research is to safely translate the science to patients. Although there are various methods by which this goal can be reached, this editorial emphasizes the safety of mesenchymal stem cell (MSC) transplant and possible confounds by the growing information on cancer stem cells (CSCs). There are several ongoing clinical trials with MSCs and their interactions with CSCs need to be examined. The rapid knowledge on MSCs and CSCs has now collided with regards to the safe treatment of MSCs. The information discussed on MSCs can be extrapolated to other stem cells with similar phenotype and functions such as placenta stem cells. MSCs are attractive for cell therapy, mainly due to reduced ethical concerns, ease in expansion and reduced ability to be transformed. Also, MSCs can exert both immune suppressor and tissue regeneration simultaneously. It is expected that any clinical trial with MSCs will take precaution to ensure that the cells are not transformed. However, going forward, the different centers should be aware that MSCs might undergo oncogenic events, especially as undifferentiated cells or early differentiated cells. Another major concern for MSC therapy is their ability to promote tumor growth and perhaps, to protect CSCs by altered immune responses. These issues are discussed in light of a large number of undiagnosed cancers.

  20. Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells

    PubMed Central

    Yanovsky-Dagan, Shira; Avitzour, Michal; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Mitrani-Rosenbaum, Stella; Levy-Lahad, Ephrat; Birnbaum, Ramon Y.; Gepstein, Lior; Epsztejn-Litman, Silvina; Eiges, Rachel

    2015-01-01

    Summary CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation. PMID:26190529

  1. T Helper Cell Activation and Expansion Is Sensitive to Glutaminase Inhibition under Both Hypoxic and Normoxic Conditions.

    PubMed

    Sener, Zeynep; Cederkvist, Fritjof H; Volchenkov, Roman; Holen, Halvor L; Skålhegg, Bjørn S

    2016-01-01

    Immune responses often take place where nutrients and O2 availability are limited. This has an impact on T cell metabolism and influences activation and effector functions. T cell proliferation and expansion are associated with increased consumption of glutamine which is needed in a number of metabolic pathways and regulate various physiological processes. The first step in endogenous glutamine metabolism is reversible and is regulated by glutaminase (GLS1 and GLS2) and glutamine synthase (GLUL). There are two isoforms of GLS1, Kidney type glutaminase (KGA) and Glutaminase C (GAC). The aim of this study is to investigate the expression, localization and role of GLS1 and GLUL in naïve and activated human CD4+ T cells stimulated through the CD3 and CD28 receptors under normoxia and hypoxia. In proliferating cells, GAC was upregulated and KGA was downregulated, and both enzymes were located to the mitochondria irrespective of O2 levels. By contrast GLUL is localized to the cytoplasm and was upregulated under hypoxia. Proliferation was dependent on glutamine consumption, as glutamine deprivation and GLS1 inhibition decreased proliferation and expression of CD25 and CD226, regardless of O2 availability. Again irrespective of O2, GLS1 inhibition decreased the proportion of CCR6 and CXCR3 expressing CD4+ T cells as well as cytokine production. We propose that systemic Th cell activation and expansion might be dependent on glutamine but not O2 availability. PMID:27467144

  2. Human Adipose Stromal Cells (ASC) for the Regeneration of Injured Cartilage Display Genetic Stability after In Vitro Culture Expansion

    PubMed Central

    Neri, Simona; Bourin, Philippe; Peyrafitte, Julie-Anne; Cattini, Luca; Facchini, Andrea; Mariani, Erminia

    2013-01-01

    Mesenchymal stromal cells are emerging as an extremely promising therapeutic agent for tissue regeneration due to their multi-potency, immune-modulation and secretome activities, but safety remains one of the main concerns, particularly when in vitro manipulation, such as cell expansion, is performed before clinical application. Indeed, it is well documented that in vitro expansion reduces replicative potential and some multi-potency and promotes cell senescence. Furthermore, during in vitro aging there is a decrease in DNA synthesis and repair efficiency thus leading to DNA damage accumulation and possibly inducing genomic instability. The European Research Project ADIPOA aims at validating an innovative cell-based therapy where autologous adipose stromal cells (ASCs) are injected in the diseased articulation to activate regeneration of the cartilage. The primary objective of this paper was to assess the safety of cultured ASCs. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype and microsatellite instability analysis. In addition, RT-PCR array-based evaluation of the expression of genes related to DNA damage signaling pathways was performed. Finally, the senescence and replicative potential of cultured cells was evaluated by telomere length and telomerase activity assessment, whereas anchorage-independent clone development was tested in vitro by soft agar growth. We found that cultured ASCs do not show genetic alterations and replicative senescence during the period of observation, nor anchorage-independent growth, supporting an argument for the safety of ASCs for clinical use. PMID:24205017

  3. Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

    SciTech Connect

    Baconnais, S.; Delavoie, F. |; Zahm, J.M.; Milliot, M.; Castillon, N.; Terryn, C.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E. , E-Mail: edith.puchelle@univ-reims.fr; Balossier, G.

    2005-10-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

  4. Human adipose stromal cells (ASC) for the regeneration of injured cartilage display genetic stability after in vitro culture expansion.

    PubMed

    Neri, Simona; Bourin, Philippe; Peyrafitte, Julie-Anne; Cattini, Luca; Facchini, Andrea; Mariani, Erminia

    2013-01-01

    Mesenchymal stromal cells are emerging as an extremely promising therapeutic agent for tissue regeneration due to their multi-potency, immune-modulation and secretome activities, but safety remains one of the main concerns, particularly when in vitro manipulation, such as cell expansion, is performed before clinical application. Indeed, it is well documented that in vitro expansion reduces replicative potential and some multi-potency and promotes cell senescence. Furthermore, during in vitro aging there is a decrease in DNA synthesis and repair efficiency thus leading to DNA damage accumulation and possibly inducing genomic instability. The European Research Project ADIPOA aims at validating an innovative cell-based therapy where autologous adipose stromal cells (ASCs) are injected in the diseased articulation to activate regeneration of the cartilage. The primary objective of this paper was to assess the safety of cultured ASCs. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype and microsatellite instability analysis. In addition, RT-PCR array-based evaluation of the expression of genes related to DNA damage signaling pathways was performed. Finally, the senescence and replicative potential of cultured cells was evaluated by telomere length and telomerase activity assessment, whereas anchorage-independent clone development was tested in vitro by soft agar growth. We found that cultured ASCs do not show genetic alterations and replicative senescence during the period of observation, nor anchorage-independent growth, supporting an argument for the safety of ASCs for clinical use.

  5. T Helper Cell Activation and Expansion Is Sensitive to Glutaminase Inhibition under Both Hypoxic and Normoxic Conditions

    PubMed Central

    Sener, Zeynep; Cederkvist, Fritjof H.; Volchenkov, Roman; Holen, Halvor L.; Skålhegg, Bjørn S.

    2016-01-01

    Immune responses often take place where nutrients and O2 availability are limited. This has an impact on T cell metabolism and influences activation and effector functions. T cell proliferation and expansion are associated with increased consumption of glutamine which is needed in a number of metabolic pathways and regulate various physiological processes. The first step in endogenous glutamine metabolism is reversible and is regulated by glutaminase (GLS1 and GLS2) and glutamine synthase (GLUL). There are two isoforms of GLS1, Kidney type glutaminase (KGA) and Glutaminase C (GAC). The aim of this study is to investigate the expression, localization and role of GLS1 and GLUL in naïve and activated human CD4+ T cells stimulated through the CD3 and CD28 receptors under normoxia and hypoxia. In proliferating cells, GAC was upregulated and KGA was downregulated, and both enzymes were located to the mitochondria irrespective of O2 levels. By contrast GLUL is localized to the cytoplasm and was upregulated under hypoxia. Proliferation was dependent on glutamine consumption, as glutamine deprivation and GLS1 inhibition decreased proliferation and expression of CD25 and CD226, regardless of O2 availability. Again irrespective of O2, GLS1 inhibition decreased the proportion of CCR6 and CXCR3 expressing CD4+ T cells as well as cytokine production. We propose that systemic Th cell activation and expansion might be dependent on glutamine but not O2 availability. PMID:27467144

  6. T Helper Cell Activation and Expansion Is Sensitive to Glutaminase Inhibition under Both Hypoxic and Normoxic Conditions.

    PubMed

    Sener, Zeynep; Cederkvist, Fritjof H; Volchenkov, Roman; Holen, Halvor L; Skålhegg, Bjørn S

    2016-01-01

    Immune responses often take place where nutrients and O2 availability are limited. This has an impact on T cell metabolism and influences activation and effector functions. T cell proliferation and expansion are associated with increased consumption of glutamine which is needed in a number of metabolic pathways and regulate various physiological processes. The first step in endogenous glutamine metabolism is reversible and is regulated by glutaminase (GLS1 and GLS2) and glutamine synthase (GLUL). There are two isoforms of GLS1, Kidney type glutaminase (KGA) and Glutaminase C (GAC). The aim of this study is to investigate the expression, localization and role of GLS1 and GLUL in naïve and activated human CD4+ T cells stimulated through the CD3 and CD28 receptors under normoxia and hypoxia. In proliferating cells, GAC was upregulated and KGA was downregulated, and both enzymes were located to the mitochondria irrespective of O2 levels. By contrast GLUL is localized to the cytoplasm and was upregulated under hypoxia. Proliferation was dependent on glutamine consumption, as glutamine deprivation and GLS1 inhibition decreased proliferation and expression of CD25 and CD226, regardless of O2 availability. Again irrespective of O2, GLS1 inhibition decreased the proportion of CCR6 and CXCR3 expressing CD4+ T cells as well as cytokine production. We propose that systemic Th cell activation and expansion might be dependent on glutamine but not O2 availability.

  7. R-spondin 1 and noggin facilitate expansion of resident stem cells from non-damaged gallbladders.

    PubMed

    Lugli, Natalia; Kamileri, Irene; Keogh, Adrian; Malinka, Thomas; Sarris, Michalis E; Talianidis, Iannis; Schaad, Olivier; Candinas, Daniel; Stroka, Deborah; Halazonetis, Thanos D

    2016-05-01

    Pioneering studies within the last few years have allowed the in vitro expansion of tissue-specific adult stem cells from a variety of endoderm-derived organs, including the stomach, small intestine, and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R-spondin 1 and is likely facilitated by the fact that in healthy adults the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential that is accompanied by the emergence of Lgr5-positive stem cells; these cells can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non-regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem/progenitor cells that can be propagated in culture as organoids for more than a year. Growth of these organoids was stimulated by R-spondin 1 and noggin, whereas in the absence of these growth factors, the organoids differentiated partially toward the hepatocyte fate. When transplanted under the liver capsule, gallbladder-derived organoids maintained their architecture for 2 weeks. Furthermore, single cells prepared from dissociated organoids and injected into the mesenteric vein populated the liver parenchyma of carbon tetrachloride-treated mice. Human gallbladders were also a source of organoid-forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro, and induced to differentiate toward the hepatocyte lineage. PMID:26993089

  8. Genome rearrangement affects RNA virus adaptability on prostate cancer cells

    PubMed Central

    Pesko, Kendra; Voigt, Emily A.; Swick, Adam; Morley, Valerie J.; Timm, Collin; Yin, John; Turner, Paul E.

    2015-01-01

    Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3′ UTR – core protein genes – envelope protein genes – RNA-dependent RNA-polymerase gene – 5′ UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5′ end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a

  9. Isolation, expansion and neural differentiation of stem cells from human plucked hair: a further step towards autologous nerve recovery.

    PubMed

    Gho, Coen G; Schomann, Timo; de Groot, Simon C; Frijns, Johan H M; Rivolta, Marcelo N; Neumann, Martino H A; Huisman, Margriet A

    2016-10-01

    Stem cells from the adult hair follicle bulge can differentiate into neurons and glia, which is advantageous for the development of an autologous cell-based therapy for neurological diseases. Consequently, bulge stem cells from plucked hair may increase opportunities for personalized neuroregenerative therapy. Hairs were plucked from the scalps of healthy donors, and the bulges were cultured without prior tissue treatment. Shortly after outgrowth from the bulge, cellular protein expression was established immunohistochemically. The doubling time was calculated upon expansion, and the viability of expanded, cryopreserved cells was assessed after shear stress. The neuroglial differentiation potential was assessed from cryopreserved cells. Shortly after outgrowth, the cells were immunopositive for nestin, SLUG, AP-2α and SOX9, and negative for SOX10. Each bulge yielded approximately 1 × 10(4) cells after three passages. Doubling time was 3.3 (±1.5) days. Cellular viability did not differ significantly from control cells after shear stress. The cells expressed class III β-tubulin (TUBB3) and synapsin-1 after 3 weeks of neuronal differentiation. Glial differentiation yielded KROX20- and MPZ-immunopositive cells after 2 weeks. We demonstrated that human hair follicle bulge-derived stem cells can be cultivated easily, expanded efficiently and kept frozen until needed. After cryopreservation, the cells were viable and displayed both neuronal and glial differentiation potential.

  10. Spatial and Temporal Effects of Free-Air CO2 Enrichment (POPFACE) on Leaf Growth, Cell Expansion, and Cell Production in a Closed Canopy of Poplar1

    PubMed Central

    Taylor, Gail; Tricker, Penny J.; Zhang, Fang Z.; Alston, Victoria J.; Miglietta, Franco; Kuzminsky, Elena

    2003-01-01

    Leaf expansion in the fast-growing tree, Populus × euramericana was stimulated by elevated [CO2] in a closed-canopy forest plantation, exposed using a free air CO2 enrichment technique enabling long-term experimentation in field conditions. The effects of elevated [CO2] over time were characterized and related to the leaf plastochron index (LPI), and showed that leaf expansion was stimulated at very early (LPI, 0–3) and late (LPI, 6–8) stages in development. Early and late effects of elevated [CO2] were largely the result of increased cell expansion and increased cell production, respectively. Spatial effects of elevated [CO2] were also marked and increased final leaf size resulted from an effect on leaf area, but not leaf length, demonstrating changed leaf shape in response to [CO2]. Leaves exhibited a basipetal gradient of leaf development, investigated by defining seven interveinal areas, with growth ceasing first at the leaf tip. Interestingly, and in contrast to other reports, no spatial differences in epidermal cell size were apparent across the lamina, whereas a clear basipetal gradient in cell production rate was found. These data suggest that the rate and timing of cell production was more important in determining leaf shape, given the constant cell size across the leaf lamina. The effect of elevated [CO2] imposed on this developmental gradient suggested that leaf cell production continued longer in elevated [CO2] and that basal increases in cell production rate were also more important than altered cell expansion for increased final leaf size and altered leaf shape in elevated [CO2]. PMID:12529526

  11. Iron affects the structure of cell membrane molecular models.

    PubMed

    Suwalsky, M; Martínez, F; Cárdenas, H; Grzyb, J; Strzałka, K

    2005-03-01

    The effects of Fe(3+) and Fe(2+) on molecular models of biomembranes were investigated. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids located in the outer and inner moieties of cell membranes, respectively. X-ray studies showed that very low concentrations of Fe(3+) affected DMPC organization and 10(-3)M induced a total loss of its multilamellar periodic stacking. Experiments carried out with Fe(2+) on DMPC showed weaker effects than those induced by Fe(3+) ions. Similar experiments were performed on DMPE bilayers. Fe(3+) from 10(-7)M up to 10(-4)M had practically no effect on DMPE structure. However, 10(-3)M Fe(3+) induced a deep perturbation of the multilamellar structure of DMPE. However, 10(-3)M Fe(2+) had no effect on DMPE organization practically. Differential scanning calorimetry measurements also revealed different effects of Fe(3+) and Fe(2+) on the phase transition and other thermal properties of the examined lipids. In conclusion, the results obtained indicate that iron ions interact with phospholipid bilayers perturbing their structures. These findings are consistent with the observation that iron ions change cell membrane fluidity and, therefore, affect its functions. PMID:15752465

  12. In Vivo Expansion of Endogenous Regulatory T Cell Populations Induces Long-Term Suppression of Contact Hypersensitivity.

    PubMed

    El Beidaq, Asmaa; Link, Christopher W M; Hofmann, Katharina; Frehse, Britta; Hartmann, Karin; Bieber, Katja; Martin, Stefan F; Ludwig, Ralf J; Manz, Rudolf A

    2016-09-01

    Contact hypersensitivity (CHS) of murine skin serves as a model of allergic contact dermatitis. Hapten-specific CD8 T cells and neutrophils represent the major effector cells driving this inflammatory reaction whereas Foxp3(+) regulatory T cells (Tregs) control the severity of inflammation. However, whether in vivo expansion of endogenous Tregs can downregulate CHS-mediated inflammation remains to be elucidated. In this study, we addressed this issue by using injection of an IL-2/anti-IL-2 mAb JES6-1 complex (IL-2/JES6-1) as a means of Treg induction in 2,4,6-trinitrochlorobenzene-induced CHS. IL-2/JES6-1 injection before or after hapten sensitization led to a considerable reduction of skin inflammation, even when rechallenged up to 3 wk after the last treatment. Conversely, Treg depletion re-established the CHS response in IL-2/JES6-1-treated mice. IL-2/JES6-1 injection resulted in increased frequencies of natural and peripheral Tregs in spleen and draining lymph nodes (LNs), elevated IL-10 and TGF-β production by CD4 T cells, reduced CD86 expression by dendritic cells, and led to lower numbers of hapten-specific IFN-γ-producing CD8 T effector cells in LNs. Neutrophil and CD8 T cell infiltration was reduced in inflamed ear tissue, whereas CTLA-4(+)Foxp3(+) Treg frequencies were augmented. Adoptive transfer of LN cells of sensitized mice into recipients treated with IL-2/JES6-1 showed impaired CHS. Our results show that in vivo Treg expansion results in a prolonged CHS suppression, a sustained reduction of hapten-specific CD8 T cells, and a decrease in effector cell influx in inflamed tissue. PMID:27439515

  13. IGF-1-mediated osteoblastic niche expansion enhances long-term hematopoietic stem cell engraftment after murine bone marrow transplantation.

    PubMed

    Caselli, Anna; Olson, Timothy S; Otsuru, Satoru; Chen, Xiaohua; Hofmann, Ted J; Nah, Hyun-Duck; Grisendi, Giulia; Paolucci, Paolo; Dominici, Massimo; Horwitz, Edwin M

    2013-10-01

    The efficiency of hematopoietic stem cell (HSC) engraftment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation. Here, we show that this reorganization represents proliferation of mature endosteal osteoblasts which seem to arise from a small subset of high-proliferative, relatively radio-resistant endosteal cells. Multiple layers of osteoblasts form along the endosteal surface within 48 hours after total body irradiation, concomitant with a peak in marrow cytokine expression. This niche reorganization fosters homing of the transplanted hematopoietic cells to the host marrow space and engraftment of long-term-HSC. Inhibition of insulin-like growth factor (IGF)-1-receptor tyrosine kinase signaling abrogates endosteal osteoblast proliferation and donor HSC engraftment, suggesting that the cytokine IGF-1 is a crucial mediator of endosteal niche reorganization and consequently donor HSC engraftment. Further understanding of this novel mechanism of IGF-1-dependent osteoblastic niche expansion and HSC engraftment may yield clinical applications for improving engraftment efficiency after clinical HSC transplantation.

  14. Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells

    PubMed Central

    Morhayim, Jess; van de Peppel, Jeroen; Braakman, Eric; Rombouts, Elwin W. J. C.; ter Borg, Mariette N. D.; Dudakovic, Amel; Chiba, Hideki; van der Eerden, Bram C. J.; Raaijmakers, Marc H.; van Wijnen, Andre J.; Cornelissen, Jan J.; van Leeuwen, Johannes P.

    2016-01-01

    Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders. PMID:27585950

  15. The Arabidopsis CLASP Gene Encodes a Microtubule-Associated Protein Involved in Cell Expansion and Division[W

    PubMed Central

    Ambrose, J. Christian; Shoji, Tsubasa; Kotzer, Amanda M.; Pighin, Jamie A.; Wasteneys, Geoffrey O.

    2007-01-01

    Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein–CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants. PMID:17873093

  16. Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells

    NASA Astrophysics Data System (ADS)

    Morhayim, Jess; van de Peppel, Jeroen; Braakman, Eric; Rombouts, Elwin W. J. C.; Ter Borg, Mariette N. D.; Dudakovic, Amel; Chiba, Hideki; van der Eerden, Bram C. J.; Raaijmakers, Marc H.; van Wijnen, Andre J.; Cornelissen, Jan J.; van Leeuwen, Johannes P.

    2016-09-01

    Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.

  17. Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells.

    PubMed

    Morhayim, Jess; van de Peppel, Jeroen; Braakman, Eric; Rombouts, Elwin W J C; Ter Borg, Mariette N D; Dudakovic, Amel; Chiba, Hideki; van der Eerden, Bram C J; Raaijmakers, Marc H; van Wijnen, Andre J; Cornelissen, Jan J; van Leeuwen, Johannes P

    2016-01-01

    Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34(+) HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34(+) cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders. PMID:27585950

  18. Efficient and scalable expansion of human pluripotent stem cells under clinically compliant settings: a view in 2013

    PubMed Central

    Wang, Ying; Cheng, Linzhao; Gerecht, Sharon

    2015-01-01

    Human pluripotent stem cells (hPSCs) hold great promise for revolutionizing regenerative medicine for their potential applications in disease modeling, drug discovery, and cellular therapy. Many their applications require robust and scalable expansion of hPSCs, even under settings compliant to good clinical practices. Rapid evolution of media and substrates provided safer and more defined culture conditions for long-term expansion of undifferentiated hPSCs in either adhesion or suspension. With well-designed automatic systems or fully controlled bioreactors, production of a clinically relevant quantity of hPSCs could be achieved in the near future. The goal is to find a scalable, xeno-free, chemically defined, and economic culture system for clinical-grade expansion of hPSCs that complies the requirements of current Good Manufacturing Practices (cGMP). This review provides an updated overview of the current development and challenges on the way to accomplish this goal, including discussions on basic principles for bioprocess design, serum-free media, extracellular matric or synthesized substrate, microcarrier- or cell aggregate-based suspension culture, and scalability and practicality of equipment. PMID:24132657

  19. Efficient and scalable expansion of human pluripotent stem cells under clinically compliant settings: a view in 2013.

    PubMed

    Wang, Ying; Cheng, Linzhao; Gerecht, Sharon

    2014-07-01

    Human pluripotent stem cells (hPSCs) hold great promise for revolutionizing regenerative medicine for their potential applications in disease modeling, drug discovery, and cellular therapy. Many their applications require robust and scalable expansion of hPSCs, even under settings compliant to good clinical practices. Rapid evolution of media and substrates provided safer and more defined culture conditions for long-term expansion of undifferentiated hPSCs in either adhesion or suspension. With well-designed automatic systems or fully controlled bioreactors, production of a clinically relevant quantity of hPSCs could be achieved in the near future. The goal is to find a scalable, xeno-free, chemically defined, and economic culture system for clinical-grade expansion of hPSCs that complies the requirements of current good manufacturing practices. This review provides an updated overview of the current development and challenges on the way to accomplish this goal, including discussions on basic principles for bioprocess design, serum-free media, extracellular matric or synthesized substrate, microcarrier- or cell aggregate-based suspension culture, and scalability and practicality of equipment. PMID:24132657

  20. Bursicon functions within the Drosophila CNS to modulate wing expansion behavior, hormone secretion, and cell death.

    PubMed

    Peabody, Nathan C; Diao, Fengqiu; Luan, Haojiang; Wang, Howard; Dewey, Elizabeth M; Honegger, Hans-Willi; White, Benjamin H

    2008-12-31

    Hormones are often responsible for synchronizing somatic physiological changes with changes in behavior. Ecdysis (i.e., the shedding of the exoskeleton) in insects has served as a useful model for elucidating the molecular and cellular mechanisms of this synchronization, and has provided numerous insights into the hormonal coordination of body and behavior. An example in which the mechanisms have remained enigmatic is the neurohormone bursicon, which, after the final molt, coordinates the plasticization and tanning of the initially folded wings with behaviors that drive wing expansion. The somatic effects of the hormone are governed by bursicon that is released into the blood from neurons in the abdominal ganglion (the B(AG)), which die after wing expansion. How bursicon induces the behavioral programs required for wing expansion, however, has remained unknown. Here we show by targeted suppression of excitability that a pair of bursicon-immunoreactive neurons distinct from the B(AG) and located within the subesophageal ganglion in Drosophila (the B(SEG)) is involved in controlling wing expansion behaviors. Unlike the B(AG), the B(SEG) arborize widely in the nervous system, including within the abdominal neuromeres, suggesting that, in addition to governing behavior, they also may modulate the B(AG.) Indeed, we show that animals lacking bursicon receptor function have deficits both in the humoral release of bursicon and in posteclosion apoptosis of the B(AG). Our results reveal novel neuromodulatory functions for bursicon and support the hypothesis that the B(SEG) are essential for orchestrating both the behavioral and somatic processes underlying wing expansion.

  1. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

    SciTech Connect

    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul; Rohaya, M. A. W.

    2013-11-27

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however

  2. Suspension-Expansion of Bone Marrow Results in Small Mesenchymal Stem Cells Exhibiting Increased Transpulmonary Passage Following Intravenous Administration.

    PubMed

    Zanetti, Andrea; Grata, Michelle; Etling, Emily B; Panday, Regeant; Villanueva, Flordeliza S; Toma, Catalin

    2015-07-01

    Mesenchymal stem cells (MSCs) have been extensively explored in a variety of regenerative medicine applications. The relatively large size of MSCs expanded in tissue culture flasks leads to retention in the microcirculation of the lungs following intravenous delivery, reducing their capacity to reach target sites. We explored whether the expansion of whole marrow in suspension cultures would yield smaller MSCs with increased capacity to traverse the pulmonary microcirculation compared with traditional monolayer cultures. We tested this hypothesis using rat marrow in a suspension bioreactor culture with fibronectin-coated microcarriers, leading to sustained expansion of both the microbead-adherent cells, as well as of a nonadherent cell fraction. Magnetic depletion of CD45(+) cells from the bioreactor cultures after 5 weeks led to a highly enriched CD73(+)/CD90(+)/CD105(+) MSC population. The bioreactor-grown MSCs were significantly smaller than parallel monolayer MSCs (15.1 ± 0.9 μm vs. 18.5 ± 2.3 μm diameter, p<0.05). When fluorescently labeled bioreactor-grown MSCs were intravenously injected into rats, the peak cell concentration in the arterial circulation was an order of magnitude higher than similarly delivered monolayer-grown MSCs (94.8 ± 29.6 vs. 8.2 ± 5.6/10(6) nucleated blood cells, respectively, p<0.05). At 24 h after intravenous injection of the LacZ-labeled bioreactor-grown MSCs, there was a significant threefold decrease in the LacZ-labeled MSCs trapped in the lungs, with a significant increase in the cells reaching the spleen and liver in comparison to their monolayer MSC counterparts. Bioreactor-grown whole marrow cell cultures yielded smaller MSCs with increased capacity to traverse the pulmonary microcirculation compared with traditionally expanded monolayer MSCs. This may significantly improve the capacity and efficiency of these cells to home to injury sites downstream of the lungs. PMID:25567723

  3. Human periosteal-derived cell expansion in a perfusion bioreactor system: proliferation, differentiation and extracellular matrix formation.

    PubMed

    Sonnaert, M; Papantoniou, I; Bloemen, V; Kerckhofs, G; Luyten, F P; Schrooten, J

    2014-09-01

    Perfusion bioreactor systems have shown to be a valuable tool for the in vitro development of three-dimensional (3D) cell-carrier constructs. Their use for cell expansion, however, has been much less explored. Since maintenance of the initial cell phenotype is essential in this process, it is imperative to obtain insight into the bioreactor-related variables determining cell fate. Therefore, this study investigated the influence of fluid flow-induced shear stress on the proliferation, differentiation and matrix deposition of human periosteal-derived cells in the absence of additional differentiation-inducing stimuli; 120 000 cells were seeded on additive manufactured 3D Ti6Al4V scaffolds and cultured for up to 28 days at different flow rates in the range 0.04-6 ml/min. DNA measurements showed, on average, a three-fold increase in cell content for all perfused conditions in comparison to static controls, whereas the magnitude of the flow rate did not have an influence. Contrast-enhanced nanofocus X-ray computed tomography showed substantial formation of an engineered neotissue in all perfused conditions, resulting in a filling (up to 70%) of the total internal void volume, and no flow rate-dependent differences were observed. The expression of key osteogenic markers, such as RunX2, OCN, OPN and Col1, did not show any significant changes in comparison to static controls after 28 days of culture, with the exception of OSX at high flow rates. We therefore concluded that, in the absence of additional osteogenic stimuli, the investigated perfusion conditions increased cell proliferation but did not significantly enhance osteogenic differentiation, thus allowing for this process to be used for cell expansion. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25186024

  4. Tricellulin deficiency affects tight junction architecture and cochlear hair cells

    PubMed Central

    Nayak, Gowri; Lee, Sue I.; Yousaf, Rizwan; Edelmann, Stephanie E.; Trincot, Claire; Van Itallie, Christina M.; Sinha, Ghanshyam P.; Rafeeq, Maria; Jones, Sherri M.; Belyantseva, Inna A.; Anderson, James M.; Forge, Andrew; Frolenkov, Gregory I.; Riazuddin, Saima

    2013-01-01

    The two compositionally distinct extracellular cochlear fluids, endolymph and perilymph, are separated by tight junctions that outline the scala media and reticular lamina. Mutations in TRIC (also known as MARVELD2), which encodes a tricellular tight junction protein known as tricellulin, lead to nonsyndromic hearing loss (DFNB49). We generated a knockin mouse that carries a mutation orthologous to the TRIC coding mutation linked to DFNB49 hearing loss in humans. Tricellulin was absent from the tricellular junctions in the inner ear epithelia of the mutant animals, which developed rapidly progressing hearing loss accompanied by loss of mechanosensory cochlear hair cells, while the endocochlear potential and paracellular permeability of a biotin-based tracer in the stria vascularis were unaltered. Freeze-fracture electron microscopy revealed disruption of the strands of intramembrane particles connecting bicellular and tricellular junctions in the inner ear epithelia of tricellulin-deficient mice. These ultrastructural changes may selectively affect the paracellular permeability of ions or small molecules, resulting in a toxic microenvironment for cochlear hair cells. Consistent with this hypothesis, hair cell loss was rescued in tricellulin-deficient mice when generation of normal endolymph was inhibited by a concomitant deletion of the transcription factor, Pou3f4. Finally, comprehensive phenotypic screening showed a broader pathological phenotype in the mutant mice, which highlights the non-redundant roles played by tricellulin. PMID:23979167

  5. T-cell defect in diffuse large B-cell lymphomas involves expansion of myeloid-derived suppressor cells.

    PubMed

    Azzaoui, Imane; Uhel, Fabrice; Rossille, Delphine; Pangault, Celine; Dulong, Joelle; Le Priol, Jerome; Lamy, Thierry; Houot, Roch; Le Gouill, Steven; Cartron, Guillaume; Godmer, Pascal; Bouabdallah, Krimo; Milpied, Noel; Damaj, Gandhi; Tarte, Karin; Fest, Thierry; Roussel, Mikael

    2016-08-25

    In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils represents an independent prognostic factor. These cell subsets include monocytic and granulocytic myeloid-derived suppressor cells (M- and G-MDSCs) defined by their ability to suppress T-cell responses. MDSCs are a heterogeneous population described in inflammatory and infectious diseases and in numerous tumors including multiple myeloma, chronic lymphocytic leukemia, and DLBCL. However, their mechanisms of action remain unclear. We broadly assessed the presence and mechanisms of suppression of MDSC subsets in DLBCL. First, a myeloid suppressive signature was identified by gene expression profiling in DLBCL peripheral blood. Accordingly, we identified, in a cohort of 66 DLBCL patients, an increase in circulating G-MDSC (Lin(neg)HLA-DR(neg)CD33(pos)CD11b(pos)) and M-MDSC (CD14(pos)HLA-DR(low)) counts. Interestingly, only M-MDSC number was correlated with the International Prognostic Index, event-free survival, and number of circulating Tregs. Furthermore, T-cell proliferation was restored after monocyte depletion. Myeloid-dependent T-cell suppression was attributed to a release of interleukin-10 and S100A12 and increased PD-L1 expression. In summary, we identified expanded MDSC subsets in DLBCL, as well as new mechanisms of immunosuppression in DLBCL. PMID:27338100

  6. CD27 costimulation contributes substantially to the expansion of functional memory CD8(+) T cells after peptide immunization.

    PubMed

    Taraban, Vadim Y; Rowley, Tania F; Kerr, Jonathan P; Willoughby, Jane E; Johnson, Peter M W; Al-Shamkhani, Aymen; Buchan, Sarah L

    2013-12-01

    Naive T cells require signals from multiple costimulatory receptors to acquire full effector function and differentiate to long-lived memory cells. The costimulatory receptor, CD27, is essential for optimal T-cell priming and memory differentiation in a variety of settings, although whether CD27 is similarly required during memory CD8(+) T-cell reactivation remains controversial. We have used OVA and anti-CD40 to establish a memory CD8(+) T-cell population and report here that their secondary expansion, driven by peptide and anti-CD40, polyI:C, or LPS, requires CD27. Furthermore, antigenic peptide and a soluble form of the CD27 ligand, CD70 (soluble recombinant CD70 (sCD70)), is sufficient for secondary memory CD8(+) T-cell accumulation at multiple anatomical sites, dependent on CD80/86. Prior to boost, resting effector- and central-memory CD8(+) T cells both expressed CD27 with greater expression on central memory cells. Nonetheless, both populations upregulated CD27 after TCR engagement and accumulated in proportion after boosting with Ag and sCD70. Mechanistically, sCD70 increased the frequency of divided and cytolytic memory T cells, conferred resistance to apoptosis and enabled retardation of tumor growth in vivo. These data demonstrate the central role played by CD27/70 during secondary CD8(+) T-cell activation to a peptide Ag, and identify sCD70 as an immunotherapeutic adjuvant for antitumor immunity.

  7. Granulocytic Myeloid Derived Suppressor Cells Expansion during Active Pulmonary Tuberculosis Is Associated with High Nitric Oxide Plasma Level

    PubMed Central

    El Daker, Sary; Sacchi, Alessandra; Tempestilli, Massimo; Carducci, Claudia; Goletti, Delia; Vanini, Valentina; Colizzi, Vittorio; Lauria, Francesco Nicola; Martini, Federico; Martino, Angelo

    2015-01-01

    Tuberculosis (TB) is still the principal cause of death caused by a single infectious agent, and the balance between the bacillus and host defense mechanisms reflects the different manifestations of the pathology. The aim of this work was to study the role of myeloid-derived suppressor cells (MDSCs) during active pulmonary tuberculosis at the site of infection. We observed an expansion of MDSCs in the lung and blood of patients with active TB, which are correlated with an enhanced amount of nitric oxide in the plasma. We also found that these cells have the remarkable ability to suppress T-cell response, suggesting an important role in the modulation of the immune response against TB. Interestingly, a trend in the diminution of MDSCs was found after an efficacious anti-TB therapy, suggesting that these cells may be used as a potential biomarker for monitoring anti-TB therapy efficacy. PMID:25879532

  8. NEVERSHED and INFLORESCENCE DEFICIENT IN ABSCISSION are differentially required for cell expansion and cell separation during floral organ abscission in Arabidopsis thaliana.

    PubMed

    Liu, Bin; Butenko, Melinka A; Shi, Chun-Lin; Bolivar, Jenny L; Winge, Per; Stenvik, Grethe-Elisabeth; Vie, Ane Kjersti; Leslie, Michelle E; Brembu, Tore; Kristiansen, Wenche; Bones, Atle M; Patterson, Sara E; Liljegren, Sarah J; Aalen, Reidunn B

    2013-12-01

    Floral organ shedding is a cell separation event preceded by cell-wall loosening and generally accompanied by cell expansion. Mutations in NEVERSHED (NEV) or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) block floral organ abscission in Arabidopsis thaliana. NEV encodes an ADP-ribosylation factor GTPase-activating protein, and cells of nev mutant flowers display membrane-trafficking defects. IDA encodes a secreted peptide that signals through the receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2). Analyses of single and double mutants revealed unique features of the nev and ida phenotypes. Cell-wall loosening was delayed in ida flowers. In contrast, nev and nev ida mutants displayed ectopic enlargement of abscission zone (AZ) cells, indicating that cell expansion alone is not sufficient to trigger organ loss. These results suggest that NEV initially prevents precocious cell expansion but is later integral for cell separation. IDA is involved primarily in the final cell separation step. A mutation in KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1), a suppressor of the ida mutant, could not rescue the abscission defects of nev mutant flowers, indicating that NEV-dependent activity downstream of KNAT1 is required. Transcriptional profiling of mutant AZs identified gene clusters regulated by IDA-HAE/HSL2. Several genes were more strongly downregulated in nev-7 compared with ida and hae hsl2 mutants, consistent with the rapid inhibition of organ loosening in nev mutants, and the overlapping roles of NEV and IDA in cell separation. A model of the crosstalk between the IDA signalling pathway and NEV-mediated membrane traffic during floral organ abscission is presented.

  9. αB-Crystallin regulates expansion of CD11b+Gr-1+ immature myeloid cells during tumor progression

    PubMed Central

    Dieterich, Lothar C.; Schiller, Petter; Huang, Hua; Wawrousek, Eric F.; Loskog, Angelica; Wanders, Alkwin; Moons, Lieve; Dimberg, Anna

    2013-01-01

    The molecular chaperone αB-crystallin has emerged as a target for cancer therapy due to its expression in human tumors and its role in regulating tumor angiogenesis. αB-crystallin also reduces neuroinflammation, but its role in other inflammatory conditions has not been investigated. Here, we examined whether αB-crystallin regulates inflammation associated with tumors and ischemia. We found that CD45+ leukocyte infiltration is 3-fold increased in tumors and ischemic myocardium in αB-crystallin-deficient mice. Notably, αB-crystallin is prominently expressed in CD11b+ Gr-1+ immature myeloid cells (IMCs), known as regulators of angiogenesis and immune responses, while lymphocytes and mature granulocytes show low αB-crystallin expression. αB-Crystallin deficiency results in a 3-fold higher accumulation of CD11b+ Gr-1+ IMCs in tumors and a significant rise in CD11b+ Gr-1+ IMCs in spleen and bone marrow. Similarly, we noted a 2-fold increase in CD11b+ Gr-1+ IMCs in chronically inflamed livers in αB-crystallin-deficient mice. The effect of αB-crystallin on IMC accumulation is limited to pathological conditions, as CD11b+ Gr-1+ IMCs are not elevated in naive mice. Through ex vivo differentiation of CD11b+ Gr-1+ cells, we provide evidence that αB-crystallin regulates systemic expansion of IMCs through a cell-intrinsic mechanism. Our study suggests a key role of αB-crystallin in limiting expansion of CD11b+ Gr-1+ IMCs in diverse pathological conditions.—Dieterich, L. C., Schiller, P., Huang, H., Wawrousek, E. F., Loskog, A., Wanders, A., Moons, L., Dimberg, A. αB-Crystallin regulates expansion of CD11b+Gr-1+ immature myeloid cells during tumor progression. PMID:23033322

  10. Interleukin-15 enhances the expansion and function of natural killer T cells from adult peripheral and umbilical cord blood.

    PubMed

    Lin, Syh-Jae; Huang, Ying-Cheng; Cheng, Po-Jen; Lee, Pei-Tzu; Hsiao, Hsiu-Shan; Kuo, Ming-Ling

    2015-12-01

    Invariant natural killer T cells (iNKT cells) are innate-like non-conventional T cells restricted by the CD1d molecule that are unique in their ability to play a pivotal role in immune regulation. Deficient iNKT function has been reported in patients receiving umbilical cord blood (UCB) transplantation. We sought to determine the effect of interleukin (IL)-15 on α-galactosylceramide (α-GalCer)-expanded iNKT cell function from UCB and adult peripheral blood (APB) mononuclear cells (MNCs). Fresh APB and UCB MNCs were cultured with IL-15 (50 ng/ml) in the presence or absence of α-GalCer (100 ng/ml) for 10 days. Cells were harvested for examination of cell yield, apoptosis, cytokine production and cytotoxic function of Vα24(+)/Vβ11(+) iNKT cells. We observed that α-GalCer-expanded APB and UCB iNKT cells and such expansion was further enhanced with IL-15. The percentage of CD3(+)CD56(+) NKT-like cells in both APB and UCB MNCs was increased with IL-15 but not with α-GalCer. Apoptosis of UCB iNKT cells was ameliorated by IL-15. Although APB and UCB iNKT cells secreted lower IFN-γ, it could be enhanced with IL-15. The expression of perforin in APB iNKT cells can also be enhanced with IL-15. UCB Vα24(+)Vβ11(+) iNKT cells further augmented K562 cytotoxicity mediated by IL-15. Taken together, these results demonstrated the relative functional deficiencies of α-GalCer induced UCB iNKT cells, which can be ameliorated by IL-15. Our findings suggest a therapeutic benefit of IL-15 immunotherapy during the post-UCB transplant period when iNKT function remains poor.

  11. CD4+ invariant natural killer T cells protect from murine GVHD lethality through expansion of donor CD4+CD25+FoxP3+ regulatory T cells.

    PubMed

    Schneidawind, Dominik; Pierini, Antonio; Alvarez, Maite; Pan, Yuqiong; Baker, Jeanette; Buechele, Corina; Luong, Richard H; Meyer, Everett H; Negrin, Robert S

    2014-11-20

    Dysregulated donor T cells lead to destruction of host tissues resulting in graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). We investigated the impact of highly purified (>95%) donor CD4(+) invariant natural killer T (iNKT) cells on GVHD in a murine model of allogeneic HCT. We found that low doses of adoptively transferred donor CD4(+) iNKT cells protect from GVHD morbidity and mortality through an expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). These Tregs express high levels of the Ikaros transcription factor Helios and expand from the Treg pool of the donor graft. Furthermore, CD4(+) iNKT cells preserve T-cell-mediated graft-versus-tumor effects. Our studies reveal new aspects of the cellular interplay between iNKT cells and Tregs in the context of tolerance induction after allogeneic HCT and set the stage for clinical translation. PMID:25293774

  12. The tarani mutation alters surface curvature in Arabidopsis leaves by perturbing the patterns of surface expansion and cell division

    PubMed Central

    Karidas, Premananda; Challa, Krishna Reddy; Nath, Utpal

    2015-01-01

    The leaf surface usually stays flat, maintained by coordinated growth. Growth perturbation can introduce overall surface curvature, which can be negative, giving a saddle-shaped leaf, or positive, giving a cup-like leaf. Little is known about the molecular mechanisms that underlie leaf flatness, primarily because only a few mutants with altered surface curvature have been isolated and studied. Characterization of mutants of the CINCINNATA-like TCP genes in Antirrhinum and Arabidopsis have revealed that their products help maintain flatness by balancing the pattern of cell proliferation and surface expansion between the margin and the central zone during leaf morphogenesis. On the other hand, deletion of two homologous PEAPOD genes causes cup-shaped leaves in Arabidopsis due to excess division of dispersed meristemoid cells. Here, we report the isolation and characterization of an Arabidopsis mutant, tarani (tni), with enlarged, cup-shaped leaves. Morphometric analyses showed that the positive curvature of the tni leaf is linked to excess growth at the centre compared to the margin. By monitoring the dynamic pattern of CYCLIN D3;2 expression, we show that the shape of the primary arrest front is strongly convex in growing tni leaves, leading to excess mitotic expansion synchronized with excess cell proliferation at the centre. Reduction of cell proliferation and of endogenous gibberellic acid levels rescued the tni phenotype. Genetic interactions demonstrated that TNI maintains leaf flatness independent of TCPs and PEAPODs. PMID:25711708

  13. EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression.

    PubMed

    Moschou, Panagiotis N; Savenkov, Eugene I; Minina, Elena A; Fukada, Kazutake; Reza, Salim Hossain; Gutierrez-Beltran, Emilio; Sanchez-Vera, Victoria; Suarez, Maria F; Hussey, Patrick J; Smertenko, Andrei P; Bozhkov, Peter V

    2016-10-01

    The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific.

  14. EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression.

    PubMed

    Moschou, Panagiotis N; Savenkov, Eugene I; Minina, Elena A; Fukada, Kazutake; Reza, Salim Hossain; Gutierrez-Beltran, Emilio; Sanchez-Vera, Victoria; Suarez, Maria F; Hussey, Patrick J; Smertenko, Andrei P; Bozhkov, Peter V

    2016-10-01

    The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific. PMID:27229374

  15. Recent Hadley cell expansion: The role of internal atmospheric variability in reconciling modeled and observed trends

    NASA Astrophysics Data System (ADS)

    Garfinkel, Chaim I.; Waugh, Darryn W.; Polvani, Lorenzo M.

    2015-12-01

    Several studies have reported that global climate models underestimate the observed trend in tropical expansion, with the implication that such models are missing key processes of the climate system. We show here that integrations of a chemistry-climate model forced with observed sea surface temperatures (SSTs), greenhouse gases, and ozone-depleting substances can produce 1980 to 2009 expansion trends comparable to those found in most reanalyses data products. Correct representation of the SSTs changes is important for the Northern Hemisphere, while correct representation of stratospheric ozone changes is important for the Southern Hemisphere. The ensemble mean trend (which captures only the forced response) is nearly always much weaker than trends in reanalyses. This suggests that a large fraction of the recently observed changes may, in fact, be a consequence of internal atmospheric variability and not a response of the climate system to anthropogenic forcings.

  16. Loss of B7-H1 expression by recipient parenchymal cells leads to expansion of infiltrating donor CD8+ T cells and persistence of GVHD

    PubMed Central

    Li, Xiaofan; Deng, Ruishu; He, Wei; Liu, Can; Wang, Miao; Young, James; Meng, Zhipeng; Du, Chantal; Huang, Wendong; Chen, Lieping; Chen, Yuan-Zhong; Martin, Paul; Forman, Stephen; Zeng, Defu

    2013-01-01

    Previous experimental studies have shown that acute graft-versus-host disease (GVHD) is associated with two waves of donor CD8+ T cell expansion. In the current studies, we used in vivo bioluminescent imaging (BLI), in vivo BrdU-labeling, and three different experimental GVHD systems to show that B7-H1 expression by recipient parenchymal cells controls the second wave of alloreactive donor CD8+ T cell expansion and the associated second phase of GVHD. Loss of B7-H1 expression by parenchymal cells during the course of GVHD was associated with persistent proliferation of donor CD8+ T cells in GVHD target tissues and continued tissue injury, whereas persistent expression of B7-H1 expression by parenchymal cells led to reduced proliferation of donor CD8+ T cells in GVHD target tissues and resolution of GVHD. These studies demonstrate that parenchymal cell expression of B7-H1 is required for tolerizing infiltrating T cells and preventing the persistence of GVHD. Our results suggest that therapies designed to preserve or restore expression of B7-H1 expression by parenchymal tissues in the recipient could prevent or ameliorate GVHD in humans. PMID:22156590

  17. Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures

    PubMed Central

    Duinhouwer, Lucia E.; Tüysüz, Nesrin; Rombouts, Elwin W. J. C.; ter Borg, Mariette N. D.; Mastrobattista, Enrico; Spanholtz, Jan; Cornelissen, Jan J.; ten Berge, Derk; Braakman, Eric

    2015-01-01

    Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC. PMID:25807521

  18. Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures.

    PubMed

    Duinhouwer, Lucia E; Tüysüz, Nesrin; Rombouts, Elwin W J C; Ter Borg, Mariette N D; Mastrobattista, Enrico; Spanholtz, Jan; Cornelissen, Jan J; Ten Berge, Derk; Braakman, Eric

    2015-01-01

    Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC.

  19. New common variants affecting susceptibility to basal cell carcinoma.

    PubMed

    Stacey, Simon N; Sulem, Patrick; Masson, Gisli; Gudjonsson, Sigurjon A; Thorleifsson, Gudmar; Jakobsdottir, Margret; Sigurdsson, Asgeir; Gudbjartsson, Daniel F; Sigurgeirsson, Bardur; Benediktsdottir, Kristrun R; Thorisdottir, Kristin; Ragnarsson, Rafn; Scherer, Dominique; Hemminki, Kari; Rudnai, Peter; Gurzau, Eugene; Koppova, Kvetoslava; Botella-Estrada, Rafael; Soriano, Virtudes; Juberias, Pablo; Saez, Berta; Gilaberte, Yolanda; Fuentelsaz, Victoria; Corredera, Cristina; Grasa, Matilde; Höiom, Veronica; Lindblom, Annika; Bonenkamp, Johannes J; van Rossum, Michelle M; Aben, Katja K H; de Vries, Esther; Santinami, Mario; Di Mauro, Maria G; Maurichi, Andrea; Wendt, Judith; Hochleitner, Pia; Pehamberger, Hubert; Gudmundsson, Julius; Magnusdottir, Droplaug N; Gretarsdottir, Solveig; Holm, Hilma; Steinthorsdottir, Valgerdur; Frigge, Michael L; Blondal, Thorarinn; Saemundsdottir, Jona; Bjarnason, Hjördis; Kristjansson, Kristleifur; Bjornsdottir, Gyda; Okamoto, Ichiro; Rivoltini, Licia; Rodolfo, Monica; Kiemeney, Lambertus A; Hansson, Johan; Nagore, Eduardo; Mayordomo, José I; Kumar, Rajiv; Karagas, Margaret R; Nelson, Heather H; Gulcher, Jeffrey R; Rafnar, Thorunn; Thorsteinsdottir, Unnur; Olafsson, Jon H; Kong, Augustine; Stefansson, Kari

    2009-08-01

    In a follow-up to our previously reported genome-wide association study of cutaneous basal cell carcinoma (BCC), we describe here several new susceptibility variants. SNP rs11170164, encoding a G138E substitution in the keratin 5 (KRT5) gene, affects risk of BCC (OR = 1.35, P = 2.1 x 10(-9)). A variant at 9p21 near CDKN2A and CDKN2B also confers susceptibility to BCC (rs2151280[C]; OR = 1.19, P = 6.9 x 10(-9)), as does rs157935[T] at 7q32 near the imprinted gene KLF14 (OR = 1.23, P = 5.7 x 10(-10)). The effect of rs157935[T] is dependent on the parental origin of the risk allele. None of these variants were found to be associated with melanoma or fair-pigmentation traits. A melanoma- and pigmentation-associated variant in the SLC45A2 gene, L374F, is associated with risk of both BCC and squamous cell carcinoma. Finally, we report conclusive evidence that rs401681[C] in the TERT-CLPTM1L locus confers susceptibility to BCC but protects against melanoma. PMID:19578363

  20. Sall1 in renal stromal progenitors non-cell autonomously restricts the excessive expansion of nephron progenitors.

    PubMed

    Ohmori, Tomoko; Tanigawa, Shunsuke; Kaku, Yusuke; Fujimura, Sayoko; Nishinakamura, Ryuichi

    2015-10-29

    The mammalian kidney develops from reciprocal interactions between the metanephric mesenchyme and ureteric bud, the former of which contains nephron progenitors. The third lineage, the stroma, fills up the interstitial space and is derived from distinct progenitors that express the transcription factor Foxd1. We showed previously that deletion of the nuclear factor Sall1 in nephron progenitors leads to their depletion in mice. However, Sall1 is expressed not only in nephron progenitors but also in stromal progenitors. Here we report that specific Sall1 deletion in stromal progenitors leads to aberrant expansion of nephron progenitors, which is in sharp contrast with a nephron progenitor-specific deletion. The mutant mice also exhibited cystic kidneys after birth and died before adulthood. We found that Decorin, which inhibits Bmp-mediated nephron differentiation, was upregulated in the mutant stroma. In contrast, the expression of Fat4, which restricts nephron progenitor expansion, was reduced mildly. Furthermore, the Sall1 protein binds to many stroma-related gene loci, including Decorin and Fat4. Thus, the expression of Sall1 in stromal progenitors restricts the excessive expansion of nephron progenitors in a non-cell autonomous manner, and Sall1-mediated regulation of Decorin and Fat4 might at least partially underlie the pathogenesis.

  1. Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice.

    PubMed

    Yang, Cai-Rong; Lowther, Katie M; Lalioti, Maria D; Seli, Emre

    2016-01-01

    Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab(-/-) female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab(-/-) mice. Using a coculture system of oocytectomized cumulus oophorus complexes (OOXs) with denuded oocytes, we found that when wild-type OOXs were cocultured with Epab(-/-) oocytes, or when Epab(-/-) OOXs were cocultured with WT oocytes, cumulus expansion failed to occur in response to epidermal growth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab(-/-) mice fail to send and receive the necessary signals required for cumulus expansion. The abnormalities in Epab(-/-) CCs are not due to lower expression of the oocyte-derived factors growth differentiation factor 9 or bone morphogenetic protein 15, because Epab(-/-) oocytes express these proteins at comparable levels with WT. Epab(-/-) granulosa cells (GCs) exhibit decreased levels of phosphorylated MEK1/2, ERK1/2, and p90 ribosomal S6 kinase in response to lutenizing hormone and EGF treatment, as well as decreased phosphorylation of the EGF receptor. In conclusion, EPAB, which is oocyte specific, is required for the ability of CCs and GCs to become responsive to LH and EGF signaling. These results emphasize the importance of oocyte-somatic communication for GC and CC function.

  2. Expansion of polyclonal B-cell precursors in bone marrow from children treated for acute lymphoblastic leukemia.

    PubMed

    Duval, M; Fenneteau, O; Cave, H; Gobillot, C; Rohrlich, P; Guidal, C; Lescoeur, B; Legac, S; Schlegel, N; Sterkers, G; Vilmer, E

    1997-06-01

    In a series of 12 patients (mean age: 3 years at diagnosis) receiving chemotherapy for acute lymphoblastic leukemia, bone marrow examinations performed during hematopoietic recovery following treatment-induced agranulocytosis or completion of maintenance treatment showed at least 15% of non malignant immature cells which were sometimes hardly distinguishable from leukemic cells. No comparable data was observed in patients treated with G-CSF. The cytological features of these cells as well as their immunophenotyping were defined. Results showed that the majority of cells expressed HLA-DR, CD19, CD10 and cytoplasmic IgM but not the CD34 markers. This predominant and homogeneous pre-B cell population which likely represents the expansion of a minor population detectable in normal bone marrow is phenotypically indistinguishable from leukemic cells. The pattern of IgH gene rearrangements studied by PCR amplification of the CDRIII region showed that these cells were polyclonal. Except in one patient, minimal residual disease was not detected using probes specific for IgH or TCR gene rearrangement of the malignant clone. In children during the hematopoietic recovery after chemotherapy, immature marrow cells in great numbers, even with an highly homogeneous immunophenotype identical to the malignant clone's, are not sufficient for the diagnosis of relapse.

  3. Oriented cell division affects the global stress and cell packing geometry of a monolayer under stretch.

    PubMed

    Xu, Guang-Kui; Liu, Yang; Zheng, Zhaoliang

    2016-02-01

    Cell division plays a vital role in tissue morphogenesis and homeostasis, and the division plane is crucial for cell fate. For isolated cells, extensive studies show that the orientation of divisions is sensitive to cell shape and the direction of extrinsic mechanical forces. However, it is poorly understood that how the cell divides within a cell monolayer and how the local stress change, due to the division, affects the global stress of epithelial monolayers. Here, we use the vertex dynamics models to investigate the effects of division orientation on the configurations and mechanics of a cell monolayer under stretch. We examine three scenarios of the divisions: dividing along the stretch axis, dividing along the geometric long axis of cells, and dividing at a random angle. It is found that the division along the long cell axis can induce the minimal energy difference, and the global stress of the monolayer after stretch releases more rapidly in this case. Moreover, the long-axis division can result in more random cell orientations and more isotropic cell shapes within the monolayer, comparing with other two cases. This study helps understand the division orientation of cells within a monolayer under mechanical stimuli, and may shed light on linking individual cell's behaviors to the global mechanics and patterns of tissues. PMID:26774292

  4. Oriented cell division affects the global stress and cell packing geometry of a monolayer under stretch.

    PubMed

    Xu, Guang-Kui; Liu, Yang; Zheng, Zhaoliang

    2016-02-01

    Cell division plays a vital role in tissue morphogenesis and homeostasis, and the division plane is crucial for cell fate. For isolated cells, extensive studies show that the orientation of divisions is sensitive to cell shape and the direction of extrinsic mechanical forces. However, it is poorly understood that how the cell divides within a cell monolayer and how the local stress change, due to the division, affects the global stress of epithelial monolayers. Here, we use the vertex dynamics models to investigate the effects of division orientation on the configurations and mechanics of a cell monolayer under stretch. We examine three scenarios of the divisions: dividing along the stretch axis, dividing along the geometric long axis of cells, and dividing at a random angle. It is found that the division along the long cell axis can induce the minimal energy difference, and the global stress of the monolayer after stretch releases more rapidly in this case. Moreover, the long-axis division can result in more random cell orientations and more isotropic cell shapes within the monolayer, comparing with other two cases. This study helps understand the division orientation of cells within a monolayer under mechanical stimuli, and may shed light on linking individual cell's behaviors to the global mechanics and patterns of tissues.

  5. Anabolic androgens affect the competitive interactions in cell migration and adhesion between normal mouse urothelial cells and urothelial carcinoma cells.

    PubMed

    Huang, Chi-Ping; Hsieh, Teng-Fu; Chen, Chi-Cheng; Hung, Xiao-Fan; Yu, Ai-Lin; Chang, Chawnshang; Shyr, Chih-Rong

    2014-09-26

    The urothelium is constantly rebuilt by normal urothelial cells to regenerate damaged tissues caused by stimuli in urine. However, the urothelial carcinoma cells expand the territory by aberrant growth of tumor cells, which migrate and occupy the damaged tissues to spread outside and disrupt the normal cells and organized tissues and form a tumor. Therefore, the interaction between normal urothelial cells and urothelial carcinoma cells affect the initiation and progression of urothelial tumors if normal urothelial cells fail to migrate and adhere to the damages sites to regenerate the tissues. Here, comparing normal murine urothelial cells with murine urothelial carcinoma cells (MBT-2), we found that normal cells had less migration ability than carcinoma cells. And in our co-culture system we found that carcinoma cells had propensity migrating toward normal urothelial cells and carcinoma cells had more advantages to adhere than normal cells. To reverse this condition, we used anabolic androgen, dihyrotestosterone (DHT) to treat normal cells and found that DHT treatment increased the migration ability of normal urothelial cells toward carcinoma cells and the adhesion capacity in competition with carcinoma cells. This study provides the base of a novel therapeutic approach by using anabolic hormone-enforced normal urothelial cells to regenerate the damage urothelium and defend against the occupancy of carcinoma cells to thwart cancer development and recurrence.

  6. [Effect of different cytokine combinations on the expression of CD49d and CXCR4 and ex vivo expansion of umbilical cord blood mononuclear cells].

    PubMed

    Mao, Ping; Xu, Li; Mo, Wen-Jian; Yin, Yi; Xu, Yan-Li; Lin, Xiu-Mei

    2006-04-01

    This study was purposed to explore the effect of different cytokine combinations on the expansion of the mononuclear cells drived from umbilical cord blood (CB) ex vivo and expression of CXCR4 and CD49d on CD34+ cells after expansion. Human fresh CB mononuclear cells were cultured in serum-free and stroma-free medium containing different combinations of cytokine for 7 days. At day o and 7, the total cells were counted, CD34+ cells and CD34+CXCR4+, CD34+CD49d+ cells were assayed by flow cytometry, and CFU were determined. According to the different combinations of cytokine, experiments were divided into four groups: control, SF group (SCF + FL), SFT group (SCF + FL + TPO) and SFT6 group (SCF + FL + TPO + IL-6). The results showed that the SF (SF group) combination supported only low expansion of total cells, CD34+ cells and CFU. The addition of TPO in SF group restored UCB stem/progenitors expansion to a higher level than that in SF group, while there was no difference between groups SFT and SFT6 (P > 0.05). The cytokine combinations in groups SF, SFT and SFT6 all could upregulate the expression levels of CD49d and CXCR4 on expanded cord blood CD34+ cells, but there were no significant differences between groups SF, SFT and SFT6 (P > 0.05). It is concluded that SCF + FL has no strong synergistic effects on primitive hematopoietic cells. TPO plays an important role in enhancing expansion of umbilical cord blood hematopoietic cells, while IL-6 only shows a neutral effect on it. SCF + FL + TPO combination not only promotes progenitor cells expansion but also upregulates the expression of CD49d and CXCR4 on CD34+ cells from cord blood.

  7. Growth Factors Cross-Linked to Collagen Microcarriers Promote Expansion and Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

    PubMed

    Bertolo, Alessandro; Arcolino, Fanny; Capossela, Simona; Taddei, Anna Rita; Baur, Martin; Pötzel, Tobias; Stoyanov, Jivko

    2015-10-01

    Tissue engineering is a field in progressive expansion and requires constant updates in methods and devices. One of the central fields is the development of biocompatible, biodegradable, and injectable scaffolds, such as collagen microcarriers. To enhance cell attachment and produce a cost-effective cell culture solution with local stimulation of cells, basic fibroblast growth factor (bFGF) or transforming growth factor-β1 (TGF-β1) was covalently immobilized on microcarriers either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) or riboflavin/UV (RB/UV) light-mediated cross-linking. Collagen microcarriers cross-linked with bFGF or TGF-β1 were used for expansion and chondrogenic differentiation of human mesenchymal stem cells (MSCs). Evaluation methods included cell viability test, chondrogenic marker expression (aggrecan and collagen type I and type II), histological detection of proteoglycans, and immunohistochemical analysis. Cross-linking strengthened the collagen structure of the microcarriers and reduced collagenase-mediated degradation. MSCs effectively proliferated on microcarriers cross-linked with bFGF, especially by EDC/NHS cross-linking. Chondrogenic differentiation of MSCs was induced by TGF-β1 cross-linked on microcarriers, promoting gene expression and protein accumulation of aggrecan and collagen type I and type II, as well as proteoglycans. Cross-linking by RB/UV enhanced chondrogenesis more than any other group. In addition, cross-linking reduced scaffold shrinkage exerted by MSCs during chondrogenesis, a desirable feature for microcarriers if used as tissue defect filler. In conclusion, cross-linking of bFGF or TGF-β1 to collagen microcarriers supported in vitro proliferation and chondrogenesis, respectively. If translated in vivo and in clinical practice, such approach might lead a step closer to development of a cost-effective and locally acting device for cell-based therapy. PMID:26222829

  8. Targeting STAT3 in adoptively transferred T cells promotes their in vivo expansion and antitumor effects

    PubMed Central

    Kujawski, Maciej; Zhang, Chunyan; Herrmann, Andreas; Reckamp, Karen; Scuto, Anna; Jensen, Michael; Deng, Jiehui; Forman, Stephen; Figlin, Robert; Yu, Hua

    2010-01-01

    Adoptive cell therapy with engineered T cells to improve natural immune response and antitumor functions has shown promise for treating cancer. However, the requirement for extensive ex vivo manipulation of T cells and the immunosuppressive effects of the tumor microenvironment limit this therapeutic modality. In the present study, we investigated the possibility to circumvent these limitations by engineering Stat3-deficient CD8+ T cells or by targeting Stat3 in the tumor microenvironment. We show that ablating Stat3 in CD8+ T cells prior to their transfer allows their efficient tumor infiltration and robust proliferation, resulting in increased tumor antigen-specific T cell activity and tumor growth inhibition. For potential clinical translation, we combined adoptive T cell therapy with an FDA-approved tyrosine kinase inhibitor, sunitinib, in renal cell carcinoma and melanoma tumor models. Sunitinib inhibited Stat3 in dendritic cells and T cells, reduced conversion of transferred Foxp3− T cells to tumor-associated T regulatory cells while increasing transferred CD8+ T cell infiltration and activation at the tumor site, leading to inhibition of primary tumor growth. These data demonstrate that adoptively transferred T cells can be expanded and activated in vivo either by engineering Stat3 silenced T cells or by targeting Stat3 systemically with small-molecule inhibitors. PMID:21118964

  9. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. I. Growth can occur without photosynthesis.

    PubMed

    Van Volkenburgh, E; Cleland, R E

    1990-08-01

    Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.

  10. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. I. Growth can occur without photosynthesis

    NASA Technical Reports Server (NTRS)

    Van Volkenburgh, E.; Cleland, R. E.

    1990-01-01

    Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.

  11. Ex-vivo expansion of red blood cells: How real for transfusion in humans?

    PubMed Central

    Migliaccio, Anna Rita; Masselli, Elena; Varricchio, Lilian; Whitsett, Carolyn

    2013-01-01

    Blood transfusion is indispensable for modern medicine. In developed countries, the blood supply is adequate and safe but blood for alloimmunized patients is often unavailable. Concerns are increasing that donations may become inadequate in the future as the population ages prompting a search for alternative transfusion products. Improvements in culture conditions and proof-of-principle studies in animal models have suggested that ex-vivo expanded red cells may represent such a product. Compared to other cell therapies transfusion poses the unique challenge of requiring great cell doses (2.5 × 1012 cells vs 107 cells). Although production of such cell numbers is theoretically possible, current technologies generate red cells in numbers sufficient only for safety studies. It is conceived that by the time these studies will be completed, technical barriers to mass cell production will have been eliminated making transfusion with ex-vivo generated red cells a reality. PMID:22177597

  12. Concise Review: Ex Vivo Expansion of Cord Blood-Derived Hematopoietic Stem and Progenitor Cells: Basic Principles, Experimental Approaches, and Impact in Regenerative Medicine

    PubMed Central

    Flores-Guzmán, Patricia; Fernández-Sánchez, Verónica

    2013-01-01

    Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play key roles in the production of mature blood cells and in the biology and clinical outcomes of hematopoietic transplants. The numbers of these cells, however, are extremely low, particularly in umbilical cord blood (UCB); thus, ex vivo expansion of human UCB-derived HSCs and HPCs has become a priority in the biomedical field. Expansion of progenitor cells can be achieved by culturing such cells in the presence of different combinations of recombinant stimulatory cytokines; in contrast, expansion of actual HSCs has proved to be more difficult because, in addition to needing recombinant cytokines, HSCs seem to deeply depend on the presence of stromal cells and/or elements that promote the activation of particular self-renewal signaling pathways. Hence, there is still controversy regarding the optimal culture conditions that should be used to achieve this. To date, UCB transplants using ex vivo-expanded cells have already been performed for the treatment of different hematological disorders, and although results are still far from being optimal, the advances are encouraging. Recent studies suggest that HSCs may also give rise to nonhematopoietic cells, such as neural, cardiac, mesenchymal, and muscle cells. Such plasticity and the possibility of producing nonhematopoietic cells at the clinical scale could bring new alternatives for the treatment of neural, metabolic, orthopedic, cardiac, and neoplastic disorders. Once standardized, ex vivo expansion of human HSCs/HPCs will surely have a positive impact in regenerative medicine. PMID:24101670

  13. Matrix-mediated retention of osteogenic differentiation potential by human adult bone marrow stromal cells during ex vivo expansion.

    PubMed

    Mauney, Joshua R; Kaplan, David L; Volloch, Vladimir

    2004-07-01

    During prolonged cultivation ex vivo, adult bone marrow stromal stem cells (BMSCs) undergo two probably interdependent processes, replicative aging and a decline in differentiation potential. Recently, our results with primary human fibroblasts indicated that growth on denatured collagen (DC) matrix results in the reduction of the rate of cellular aging. The present study has been undertaken to test whether the growth of human BMSCs under the same conditions would translate into preservation of cellular aging-attenuated functions, such as the ability to express HSP70 in response to stress as well as of osteogenic differentiation potential. We report here that growth of BMSCs on a DC matrix versus tissue culture polystyrene significantly reduced one of the main manifestations of cellular aging, the attenuation of the ability to express a major protective stress response component, HSP70, increased the proliferation capacity of ex vivo expanded BMSCs, reduced the rate of morphological changes, and resulted in a dramatic increase in the retention of the potential to express osteogenic-specific functions and markers upon treatment with osteogenic stimulants. BMSCs are a promising and increasingly important cell source for tissue engineering as well as cell and gene therapeutic strategies. For use of BMSCs in these applications, ex vivo expansion is necessary to obtain a sufficient, therapeutically useful, number of cells; however, this results in the loss of differentiation potential. This problem is especially acute in older patients where more extensive in vitro expansion of smaller number of stem/progenitor cells is needed. The finding that growth on certain biomaterials preserves aging-attenuated functions, enhances proliferation capacity, and maintains differentiation potential of BMSCs indicates a promising approach to address this problem.

  14. Factors affecting the cryosurvival of mouse two-cell embryos.

    PubMed

    Critser, J K; Arneson, B W; Aaker, D V; Huse-Benda, A R; Ball, G D

    1988-01-01

    A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Interleukin-5 Supports the Expansion of Fas Ligand-Expressing Killer B Cells that Induce Antigen-Specific Apoptosis of CD4+ T Cells and Secrete Interleukin-10

    PubMed Central

    Klinker, Matthew W.; Reed, Tamra J.; Fox, David A.; Lundy, Steven K.

    2013-01-01

    Beyond their critical role in humoral immunity, B lymphocytes can employ a variety of immunomodulatory mechanisms including expression of the apoptosis-inducing molecule Fas ligand (FasL; CD178). Here, we extensively characterized the surface phenotype of FasL+ killer B cells, showing they are enriched in the IgMhighCD5+CD1dhigh B cell subset previously reported to contain a higher frequency of B cells producing interleukin-10 (IL-10). A rare population of B cells expressing IL-10 was present among FasL+ B cells, but most FasL+ B cells did not produce IL-10. We also identify interleukin-5 (IL-5) as a novel inducer of killer B cell function. Constitutively FasL+ B cells expressed higher levels of the IL-5 receptor, and treating B cells with IL-5 and CD40L resulted in the expansion of a B cell population enriched for FasL+ cells. B cells stimulated with IL-5 and CD40L were potent inducers of apoptosis in activated primary CD4+ T cells, and this killing function was antigen-specific and dependent upon FasL. IL-5 also enhanced IL-10 secretion in B cells stimulated with CD40L. Taken together these findings elucidate the relationship of FasL+ B cells and IL-10-producing B cells and demonstrate that IL-5 can induce or enhance both killer B cell activity and IL-10 secretion in B cells. Finally, we found that the killer B cell activity induced by IL-5 was completely blocked by IL-4, suggesting the existence of a previously unknown antagonistic relationship between these type-2 cytokines in modulating the activity of killer B cells. Targeting this IL-5/IL-4 signaling axis may therefore represent a novel area of drug discovery in inflammatory disorders. PMID:23940537

  16. A model of cell wall expansion based on thermodynamics of polymer networks

    NASA Technical Reports Server (NTRS)

    Veytsman, B. A.; Cosgrove, D. J.

    1998-01-01

    A theory of cell wall extension is proposed. It is shown that macroscopic properties of cell walls can be explained through the microscopic properties of interpenetrating networks of cellulose and hemicellulose. The qualitative conclusions of the theory agree with the existing experimental data. The dependence of the cell wall yield threshold on the secretion of the wall components is discussed.

  17. Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture.

    PubMed

    Kempf, Henning; Olmer, Ruth; Kropp, Christina; Rückert, Michael; Jara-Avaca, Monica; Robles-Diaz, Diana; Franke, Annika; Elliott, David A; Wojciechowski, Daniel; Fischer, Martin; Roa Lara, Angelica; Kensah, George; Gruh, Ina; Haverich, Axel; Martin, Ulrich; Zweigerdt, Robert

    2014-12-01

    To harness the potential of human pluripotent stem cells (hPSCs), an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion). Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity) was enabled that were directly applicable to bioartificial cardiac tissue formation. PMID:25454631

  18. The WNT-controlled transcriptional regulator LBH is required for mammary stem cell expansion and maintenance of the basal lineage

    PubMed Central

    Lindley, Linsey E.; Curtis, Kevin M.; Sanchez-Mejias, Avencia; Rieger, Megan E.; Robbins, David J.; Briegel, Karoline J.

    2015-01-01

    The identification of multipotent mammary stem cells (MaSCs) has provided an explanation for the unique regenerative capacity of the mammary gland throughout adult life. However, it remains unclear what genes maintain MaSCs and control their specification into the two epithelial lineages: luminal and basal. LBH is a novel transcription co-factor in the WNT pathway with hitherto unknown physiological function. LBH is expressed during mammary gland development and aberrantly overexpressed in aggressive ‘basal’ subtype breast cancers. Here, we have explored the in vivo role of LBH in mammopoiesis. We show that in postnatal mammary epithelia, LBH is predominantly expressed in the Lin−CD29highCD24+ basal MaSC population. Upon conditional inactivation of LBH, mice exhibit pronounced delays in mammary tissue expansion during puberty and pregnancy, accompanied by increased luminal differentiation at the expense of basal lineage specification. These defects could be traced to a severe reduction in the frequency and self-renewal/differentiation potential of basal MaSCs. Mechanistically, LBH induces expression of key epithelial stem cell transcription factor ΔNp63 to promote a basal MaSC state and repress luminal differentiation genes, mainly that encoding estrogen receptor α (Esr1/ERα). Collectively, these studies identify LBH as an essential regulator of basal MaSC expansion/maintenance, raising important implications for its potential role in breast cancer pathogenesis. PMID:25655704

  19. Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi.

    PubMed

    Bacher, P; Kniemeyer, O; Schönbrunn, A; Sawitzki, B; Assenmacher, M; Rietschel, E; Steinbach, A; Cornely, O A; Brakhage, A A; Thiel, A; Scheffold, A

    2014-07-01

    Foxp3(+) regulatory T cells (Treg) have a central role for keeping the balance between pro- and anti-inflammatory immune responses against chronically encountered antigens at mucosal sites. However, their antigen specificity especially in humans is largely unknown. Here we used a sensitive enrichment technology for antigen-reactive T cells to directly compare the conventional vs. regulatory CD4(+) T-cell response directed against two ubiquitous mucosal fungi, Aspergillus fumigatus and Candida albicans. In healthy humans, fungus-specific CD4(+)CD25(+)CD127(-)Foxp3(+) Treg are strongly expanded in peripheral blood and possess phenotypic, epigenetic and functional features of thymus-derived Treg. Intriguingly, for A. fumigatus, the strong Treg response contrasts with minimal conventional T-cell memory, indicating selective Treg expansion as an effective mechanism to prevent inappropriate immune activation in healthy individuals. By contrast, in subjects with A. fumigatus allergies, specific Th2 cells were strongly expanded despite the presence of specific Treg. Taken together, we demonstrate a largely expanded Treg population specific for mucosal fungi as part of the physiological human T-cell repertoire and identify a unique capacity of A. fumigatus to selectively generate Treg responses as a potentially important mechanism for the prevention of allergic reactions.

  20. Microbial colonization drives expansion of IL-1 receptor 1 expressing, IL-17 producing γ/δ T cells

    PubMed Central

    Duan, Jinyou; Chung, Hachung; Troy, Erin; Kasper, Dennis L.

    2014-01-01

    SUMMARY IL-17 cytokine production by the Th17 T-cell subset is regulated by intestinal commmensals. We show microbial colonization also regulates innate IL-17 production. A population of CD62L− γ/δ T cells, in particular a lineage expressing the IL-1 receptor 1 (IL-1R1), can be quickly activated by microbes to produce IL-17. Antibiotic-treatment and monocolonization of mice suggest specific commensals—but not metronidazole-sensitive anaerobes like Bacteroides species—are required for maintaining IL-1R1+ γ/δ T cells. Signaling through the guanine nucleotide exchange factor VAV1 but not through Toll-like receptors or antigen presentation pathways is essential for inducing IL-1R1+ γ/δ T cells. Furthermore, IL-1R1+ γ/δ T cells are a potential source of IL-17 that can be activated by IL-23 and IL-1 in both infectious and noninfectious settings in vitro and in vivo. Thus, commensals orchestrate the expansion of phenotypically distinct γδ T cells and innate immunity is a three-way interaction between host, pathogens and microbiota. PMID:20159619

  1. The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion.

    PubMed

    Su, Liyan; Bassa, Carole; Audran, Corinne; Mila, Isabelle; Cheniclet, Catherine; Chevalier, Christian; Bouzayen, Mondher; Roustan, Jean-Paul; Chervin, Christian

    2014-11-01

    Auxin is known to regulate cell division and cell elongation, thus controlling plant growth and development. Part of the auxin signaling pathway depends on the fine-tuned degradation of the auxin/indole acetic acid (Aux/IAA) transcriptional repressors. Recent evidence indicates that Aux/IAA proteins play a role in fruit development in tomato (Solanum lycopersicum Mill.), a model species for fleshy fruit development. We report here on the functional characterization of Sl-IAA17 during tomato fruit development. Silencing of Sl-IAA17 by an RNA interference (RNAi) strategy resulted in the production of larger fruit than the wild type. Histological analyses of the fruit organ and tissues demonstrated that this phenotype was associated with a thicker pericarp, rather than larger locules and/or a larger number of seeds. Microscopic analysis demonstrated that the higher pericarp thickness in Sl-IAA17 RNAi fruits was not due to a larger number of cells, but to the increase in cell size. Finally, we observed that the cell expansion in the transgenic fruits is tightly coupled with higher ploidy levels than in the wild type, suggesting a stimulation of the endoreduplication process. In conclusion, this work provides new insights into the function of the Aux/IAA pathway in fleshy fruit development, especially fruit size and cell size determination in tomato.

  2. A comparative analysis of in vitro expansion of natural killer cells of a patient with autoimmune haemolytic anaemia and ovarian cancer with patients with other solid tumours

    PubMed Central

    DEDEEPIYA, VIDYASAGAR DEVAPRASAD; TERUNUMA, HIROSHI; DENG, XUEWEN; BASKAR, SUBRAMANI; MANJUNATH, SADANANDA RAO; SENTHILKUMAR, RAJAPPA; MURUGAN, PALANISAMY; THAMARAIKANNAN, PARAMASIVAM; SRINIVASAN, THANGAVELU; PREETHY, SENTHILKUMAR; ABRAHAM, SAMUEL J.K.

    2012-01-01

    The functional profile of natural killer (NK) cells has been reported to be lower in auto-immune haemolytic anaemia (AIHA). In this study, we report a comparative analysis of peripheral blood mononuclear cells (PBMNCs) and the in vitro expansion of NK cells in a patient with AIHA and cancer, with that of other cancer patients without AIHA. PBMNCs and in vitro NK-cell expansion of a 64-year old female patient with ovarian cancer and AIHA was compared with that of four other patients with cancer without AIHA who underwent autologous immune enhancement therapy (AIET). The NK cells were cultured using autologous plasma without feeder layers. The quantities of PBMNCs, NK cells and CD3−CD56+ cells were compared. The average quantity of PBMNCs per ml in Cases I to V were 10.71, 39.2, 49.26, 65.16 and 49.33×104, respectively, and the average maximum count of NK cells was 3.9, 1730.03, 1824.16, 1058.61 and 761×106, respectively. The average percentage of CD3−CD56+ cells in Cases I to V following in vitro expansion was 1.2, 65.7, 28.63, 65.9 and 40%, respectively. In the present study, probably the first in the literature, the in vitro expansion of NK cells was found to be significantly lower in the AIHA patient. Previously, only a lower NK-cell functional profile was reported. Further studies are required to establish the association between AIHA and NK-cell profile and in vitro expansion, and to find common antibodies between red blood cells (RBCs) and NK cells. PMID:22740927

  3. Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis.

    PubMed

    Wang, Qishan; Bag, Jnanankur

    2006-02-17

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including alpha-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.

  4. Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis

    SciTech Connect

    Wang Qishan; Bag, Jnanankur . E-mail: jbag@uoguelph.ca

    2006-02-17

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including {alpha}-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.

  5. Expansion of quiescent lung adenocarcinoma CD8+ T cells by MUC1-8-mer peptide-T2 cell-β2 microglobulin complexes.

    PubMed

    Atzin-Méndez, J A; López-González, J S; Báez, R; Arenas-Del Angel, M C; Montaño, L F; Silva-Adaya, D; Lascurain, R; Gorocica, P

    2016-01-01

    Adoptive immunotherapy requires the isolation of CD8+ T cells specific for tumor-associated antigens, their expansion in vitro and their transfusion to the patient to mediate a therapeutic effect. MUC1 is an important adenocarcinoma antigen immunogenic for T cells. The MUC1-derived SAPDTRPA (MUC1-8-mer) peptide is a potent epitope recognized by CD8+ T cells in murine models. Likewise, the T2 cell line has been used as an antigen-presenting cell to activate CD8+ T cells, but so far MUC1 has not been assessed in this context. We evaluated whether the MUC1-8-mer peptide can be presented by T2 cells to expand CD25+CD8+ T cells isolated from HLA-A2+ lung adenocarcinoma patients with stage III or IV tumors. The results showed that MUC1-8-mer peptide-loaded T2 cells activated CD8+ T cells from cancer HLA-A2+ patients when anti-CD2, anti-CD28 antibodies and IL-2 were added. The percentage of CD25+CD8+ T cells was 3-fold higher than those in the non-stimulated cells (P=0.018). HLA-A2+ patient cells showed a significant difference (2.3-fold higher) in activation status than HLA-A2+ healthy control cells (P=0.04). Moreover, 77.6% of MUC1-8-mer peptide-specific CD8+ T cells proliferated following a second stimulation with MUC1-8-mer peptide-loaded T2 cells after 10 days of cell culture. There were significant differences in the percentage of basal CD25+CD8+ T cells in relation to the cancer stage; this difference disappeared after MUC1-8-mer peptide stimulation. In conclusion, expansion of CD25+CD8+ T cells by MUC1-8 peptide-loaded T2 cells plus costimulatory signals via CD2, CD28 and IL-2 can be useful in adoptive immunotherapy.

  6. Expansion of quiescent lung adenocarcinoma CD8+ T cells by MUC1-8-mer peptide-T2 cell-β2 microglobulin complexes

    PubMed Central

    ATZIN-MÉNDEZ, J.A.; LÓPEZ-GONZÁLEZ, J.S.; BÁEZ, R.; ARENAS-DEL ANGEL, M.C.; MONTAÑO, L.F.; SILVA-ADAYA, D.; LASCURAIN, R.; GOROCICA, P.

    2016-01-01

    Adoptive immunotherapy requires the isolation of CD8+ T cells specific for tumor-associated antigens, their expansion in vitro and their transfusion to the patient to mediate a therapeutic effect. MUC1 is an important adenocarcinoma antigen immunogenic for T cells. The MUC1-derived SAPDTRPA (MUC1-8-mer) peptide is a potent epitope recognized by CD8+ T cells in murine models. Likewise, the T2 cell line has been used as an antigen-presenting cell to activate CD8+ T cells, but so far MUC1 has not been assessed in this context. We evaluated whether the MUC1-8-mer peptide can be presented by T2 cells to expand CD25+CD8+ T cells isolated from HLA-A2+ lung adenocarcinoma patients with stage III or IV tumors. The results showed that MUC1-8-mer peptide-loaded T2 cells activated CD8+ T cells from cancer HLA-A2+ patients when anti-CD2, anti-CD28 antibodies and IL-2 were added. The percentage of CD25+CD8+ T cells was 3-fold higher than those in the non-stimulated cells (P=0.018). HLA-A2+ patient cells showed a significant difference (2.3-fold higher) in activation status than HLA-A2+ healthy control cells (P=0.04). Moreover, 77.6% of MUC1-8-mer peptide-specific CD8+ T cells proliferated following a second stimulation with MUC1-8-mer peptide-loaded T2 cells after 10 days of cell culture. There were significant differences in the percentage of basal CD25+CD8+ T cells in relation to the cancer stage; this difference disappeared after MUC1-8-mer peptide stimulation. In conclusion, expansion of CD25+CD8+ T cells by MUC1-8 peptide-loaded T2 cells plus costimulatory signals via CD2, CD28 and IL-2 can be useful in adoptive immunotherapy. PMID:26498650

  7. Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies

    PubMed Central

    Davis, Darryl R; Kizana, Eddy; Terrovitis, John; Barth, Andreas S.; Zhang, Yiqiang; Smith, Rachel Ruckdeschel; Miake, Junichiro; Marbán, Eduardo

    2010-01-01

    The adult heart contains reservoirs of progenitor cells that express embryonic and stem cell-related antigens. While these antigenically-purified cells are promising candidates for autologous cell therapy, clinical application is hampered by their limited abundance and tedious isolation methods. Methods that involve an intermediate cardiosphere-forming step have proven successful and are being tested clinically, but it is unclear whether the cardiosphere step is necessary. Accordingly, we investigated the molecular profile and functional benefit of cells that spontaneously emigrate from cardiac tissue in primary culture. Adult Wistar-Kyoto rat hearts were minced, digested and cultured as separate anatomical regions. Loosely-adherent cells that surround the plated tissue were harvested weekly for a total of five harvests. Genetic lineage tracing demonstrated that a small proportion of the direct outgrowth from cardiac samples originates from myocardial cells. This outgrowth contains sub-populations of cells expressing embryonic (SSEA-1) and stem cell-related antigens (c-Kit, abcg2) that varied with time in culture but not with the cardiac chamber of origin. This direct outgrowth, and its expanded progeny, underwent marked in vitro angiogenic/cardiogenic differentiation and cytokine secretion (IGF-1, VGEF). In vivo effects included long-term functional benefits as gauged by MRI following cell injection in a rat model of myocardial infarction. Outgrowth cells afforded equivalent functional benefits to cardiosphere-derived cells, which require more processing steps to manufacture. These results provide the basis for a simplified and efficient process to generate autologous cardiac progenitor cells (and mesenchymal supporting cells) to augment clinically-relevant approaches for myocardial repair. PMID:20211627

  8. Selective deletion of Smad4 in postnatal germ cells does not affect spermatogenesis or fertility in mice.

    PubMed

    Hao, Xiao-Xia; Chen, Su-Ren; Tang, Ji-Xin; Li, Jian; Cheng, Jin-Mei; Jin, Cheng; Wang, Xiu-Xia; Liu, Yi-Xun

    2016-07-01

    SMAD4 is the central component of canonical signaling in the transforming growth factor beta (TGFβ) superfamily. Loss of Smad4 in Sertoli cells affects the expansion of the fetal testis cords, whereas selective deletion of Smad4 in Leydig cells alone does not appreciably alter fetal or adult testis development. Loss of Smad4 in Sertoli and Leydig cells, on the other hand, leads to testicular dysgenesis, and tumor formation in mice. Within the murine testes, Smad4 is also expressed in germ cells of the seminiferous tubules. We therefore, crossed Ngn3-Cre or Stra8-Cre transgenic mice with Smad4-flox mice to generate conditional knockout animals in which Smad4 was specifically deleted in postnatal germ cells to further uncover cell type-specific requirement of Smad4. Unexpectedly, these germ-cell-knockout mice were fertile and did not exhibit any detectable abnormalities in spermatogenesis, indicating that Smad4 is not required for the production of sperm; instead, these data indicate a cell type-specific requirement of Smad4 primarily during testis development. Mol. Reprod. Dev. 83: 615-623, 2016. © 2016 Wiley Periodicals, Inc. PMID:27265621

  9. Culturing and expansion of "clinical grade" precursors cells from the fetal human central nervous system.

    PubMed

    Gelati, Maurizio; Profico, Daniela; Projetti-Pensi, Massimo; Muzi, Gianmarco; Sgaravizzi, Giada; Vescovi, Angelo Luigi

    2013-01-01

    NSCs have been demonstrated to be very useful in grafts into the mammalian central nervous system to investigate the exploitation of NSC for the therapy of neurodegenerative disorders in animal models of neurodegenerative diseases. To push cell therapy in CNS on stage of clinical application, it is necessary to establish a continuous and standardized, clinical grade (i.e., produced following the good manufacturin