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Sample records for drosophila tendon cells

  1. Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila

    PubMed Central

    Cattenoz, Pierre B.; Popkova, Anna; Southall, Tony D.; Aiello, Giuseppe; Brand, Andrea H.; Giangrande, Angela

    2016-01-01

    High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades. PMID:26567182

  2. Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila.

    PubMed

    Cattenoz, Pierre B; Popkova, Anna; Southall, Tony D; Aiello, Giuseppe; Brand, Andrea H; Giangrande, Angela

    2016-01-01

    High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain-containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades.

  3. Establishment of the Muscle-Tendon Junction During Thorax Morphogenesis in Drosophila Requires the Rho-Kinase.

    PubMed

    Vega-Macaya, Franco; Manieu, Catalina; Valdivia, Mauricio; Mlodzik, Marek; Olguín, Patricio

    2016-11-01

    The assembly of the musculoskeletal system in Drosophila relies on the integration of chemical and mechanical signaling between the developing muscles with ectodermal cells specialized as "tendon cells." Mechanical tension generated at the junction of flight muscles and tendon cells of the notum epithelium is required for muscle morphogenesis, and is balanced by the epithelium in order to not deform. We report that Drosophila Rho kinase (DRok) is necessary in tendon cells to assemble stable myotendinous junctions (MTJ), which are required for muscle morphogenesis and survival. In addition, DRok is required in tendon cells to maintain epithelial shape and cell orientation in the notum, independently of chascon (chas). Loss of DRok function in tendon cells results in mis-orientation of tendon cell extensions and abnormal accumulation of Thrombospondin and βPS-integrin, which may cause abnormal myotendinous junction formation and muscle morphogenesis. This role does not depend exclusively on nonmuscular Myosin-II activation (Myo-II), indicating that other DRok targets are key in this process. We propose that DRok function in tendon cells is key to promote the establishment of MTJ attachment and to balance mechanical tension generated at the MTJ by muscle compaction. Copyright © 2016 by the Genetics Society of America.

  4. A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction

    PubMed Central

    Tiwari, Prabhat; Malhotra, Vivek; VijayRaghavan, K.

    2015-01-01

    Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV) and BM-40 from hemocytes and fat cells. PMID:26488612

  5. "Importin" signaling roles for import proteins: the function of Drosophila importin-7 (DIM-7) in muscle-tendon signaling.

    PubMed

    Liu, Ze Cindy; Geisbrecht, Erika R

    2012-01-01

    The formation of a mature myotendinous junction (MTJ) between a muscle and its site of attachment is a highly regulated process that involves myofiber migration, cell-cell signaling, and culminates with the stable adhesion between the adjacent muscle-tendon cells. Improper establishment or maintenance of muscle-tendon attachment sites results in a decrease in force generation during muscle contraction and progressive muscular dystrophies in vertebrate models. Many studies have demonstrated the important role of the integrins and integrin-associated proteins in the formation and maintenance of the MTJ. We recently demonstrated that moleskin (msk), the gene that encodes for Drosophila importin-7 (DIM-7), is required for the proper formation of muscle-tendon adhesion sites in the developing embryo. Further studies demonstrated an enrichment of DIM-7 to the ends of muscles where the muscles attach to their target tendon cells. Genetic analysis supports a model whereby msk is required in the muscle and signals via the secreted epidermal growth factor receptor (Egfr) ligand Vein to regulate tendon cell maturation. These data demonstrate a novel role for the canonical nuclear import protein DIM-7 in establishment of the MTJ.

  6. Mesenchymal stem cell applications to tendon healing

    PubMed Central

    Chaudhury, Salma

    2012-01-01

    Summary Tendons are often subject to age related degenerative changes that coincide with a diminished regenerative capacity. Torn tendons often heal by forming scar tissue that is structurally weaker than healthy native tendon tissue, predisposing to mechanical failure. There is increasing interest in providing biological stimuli to increase the tendon reparative response. Stem cells in particular are an exciting and promising prospect as they have the potential to provide appropriate cellular signals to encourage neotendon formation during repair rather than scar tissue. Currently, a number of issues need to be investigated further before it can be determined whether stem cells are an effective and safe therapeutic option for encouraging tendon repair. This review explores the in-vitro and invivo evidence assessing the effect of stem cells on tendon healing, as well as the potential clinical applications. PMID:23738300

  7. Amontillado is required for Drosophila Slit processing and for tendon-mediated muscle patterning

    PubMed Central

    Ordan, Elly

    2016-01-01

    ABSTRACT Slit cleavage into N-terminal and C-terminal polypeptides is essential for restricting the range of Slit activity. Although the Slit cleavage site has been characterized previously and is evolutionally conserved, the identity of the protease that cleaves Slit remains elusive. Our previous analysis indicated that Slit cleavage is essential to immobilize the active Slit-N at the tendon cell surfaces, mediating the arrest of muscle elongation. In an attempt to identify the protease required for Slit cleavage we performed an RNAi-based assay in the ectoderm and followed the process of elongation of the lateral transverse muscles toward tendon cells. The screen led to the identification of the Drosophila homolog of pheromone convertase 2 (PC2), Amontillado (Amon), as an essential protease for Slit cleavage. Further analysis indicated that Slit mobility on SDS polyacrylamide gel electrophoresis (SDS-PAGE) is slightly up-shifted in amon mutants, and its conventional cleavage into the Slit-N and Slit-C polypeptides is attenuated. Consistent with the requirement for amon to promote Slit cleavage and membrane immobilization of Slit-N, the muscle phenotype of amon mutant embryos was rescued by co-expressing a membrane-bound form of full-length Slit lacking the cleavage site and knocked into the slit locus. The identification of a novel protease component essential for Slit processing may represent an additional regulatory step in the Slit signaling pathway. PMID:27628033

  8. Tendon-derived stem cells as a new cell source for tendon tissue engineering.

    PubMed

    Zhang, Qiang; Cheng, Biao

    2013-01-01

    Tendon injuries are very common in occupational and athletic settings, and the elderly population. Tendons repair and regenerate slowly and inefficiently in vivo after injury. The limited ability of tendons to self-repair and the general inefficiency of current treatment strategies have intensified the need for an effective therapeutic approach. Tendon-derived stem cells (TDSCs) have recently been identified within tendon tissues. TDSCs exhibit universal stem cell characteristics, such as clonogenicity, a high proliferative capacity, multi-differentiation potential, non-immunogenicity, and immunosuppression. As a result, implanting TDSCs at damaged sites within tendons may be an effective way for tendon regeneration. This review summarizes the properties of TDSCs and discusses the advantages of its use in tendon tissue engineering.

  9. Stem Cells for Augmenting Tendon Repair

    PubMed Central

    Gulotta, Lawrence V.; Chaudhury, Salma; Wiznia, Daniel

    2012-01-01

    Tendon healing is fraught with complications such as reruptures and adhesion formation due to the formation of scar tissue at the injury site as opposed to the regeneration of native tissue. Stem cells are an attractive option in developing cell-based therapies to improve tendon healing. However, several questions remain to be answered before stem cells can be used clinically. Specifically, the type of stem cell, the amount of cells, and the proper combination of growth factors or mechanical stimuli to induce differentiation all remain to be seen. This paper outlines the current literature on the use of stem cells for tendon augmentation. PMID:22190960

  10. What understanding tendon cell differentiation can teach us about pathological tendon ossification.

    PubMed

    Magne, D; Bougault, C

    2015-08-01

    Tendons are the structures that attach muscles to bones and transmit mechanical forces. Tendon cells are composed of mature tenocytes and a rare population of tendon stem cells. Both cell types ensure homeostasis and repair of tendon extracellular matrix to guarantee its specific mechanical properties. Moreover, tendon cells seem to present a marked potential for trans-differentiation, predominantly into the chondrocyte and osteoblast lineages. In this review article, we first present chronic tendon pathologies associated with abnormal ossification, such as spondyloarthritis and calcifying tendinopathy, and discuss how tendon cell differentiation and trans-differentiation may participate in these diseases. We moreover present the factors known to influence tendon cell differentiation and trans-differentiation, with a particular emphasis on extracellular environment, mechanical stimulation and several soluble factors that can tip the balance toward one or another lineage. A better understanding of the neglected tendon cell biology may be extremely useful to understand the pathological mechanisms of spondyloarthritis and calcifying tendinopathy.

  11. Loss of Drosophila A-type lamin C initially causes tendon abnormality including disintegration of cytoskeleton and nuclear lamina in muscular defects.

    PubMed

    Uchino, Ryo; Nonaka, Yu-Ki; Horigome, Tuneyoshi; Sugiyama, Shin; Furukawa, Kazuhiro

    2013-01-01

    Lamins are the major components of nuclear envelope architecture, being required for both the structural and informational roles of the nuclei. Mutations of lamins cause a spectrum of diseases in humans, including muscular dystrophy. We report here that the loss of the A-type lamin gene, lamin C in Drosophila resulted in pupal metamorphic lethality caused by tendon defects, matching the characteristics of human A-type lamin revealed by Emery-Dreifuss muscular dystrophy (EDMD). In tendon cells lacking lamin C activity, overall cell morphology was affected and organization of the spectraplakin family cytoskeletal protein Shortstop which is prominently expressed in tendon cells gradually disintegrated, notably around the nucleus and in a manner correlating well with the degradation of musculature. Furthermore, lamin C null mutants were efficiently rescued by restoring lamin C expression to shortstop-expressing cells, which include tendon cells but exclude skeletal muscle cells. Thus the critical function of A-type lamin C proteins in Drosophila musculature is to maintain proper function and morphology of tendon cells.

  12. The cell biology of suturing tendons

    PubMed Central

    Wong, J.K.F.; Alyouha, S.; Kadler, K.E.; Ferguson, M.W.J.; McGrouther, D.A.

    2010-01-01

    Trauma by suturing tendon form areas devoid of cells termed “acellular zones” in the matrix. This study aimed to characterise the cellular insult of suturing and acellular zone formation in mouse tendon. Acellular zone formation was evaluated using single grasping sutures placed using flexor tendons with time lapse cell viability imaging for a period of 12 h. Both tension and injury were required to induce cell death and cell movement in the formation of the acellular zone. DNA fragmentation studies and transmission electron microscopy indicated that cells necrosed. Parallel in vivo studies showed that cell-to-cell contacts were disrupted following grasping by the suture in tensioned tendon. Without tension, cell death was lessened and cell-to-cell contacts remained intact. Quantitative immunohistochemistry and 3D cellular profile mapping of wound healing markers over a one year time course showed that acellular zones arise rapidly and showed no evidence of healing whilst the wound healing response occurred in the surrounding tissues. The acellular zones were also evident in a standard modified “Kessler” clinical repair. In conclusion, the suture repair of injured tendons produces acellular zones, which may potentially cause early tendon failure. PMID:20600895

  13. Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

    PubMed Central

    Clohisey, Sara M. R.; Dzhindzhev, Nikola S.; Ohkura, Hiroyuki

    2014-01-01

    Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells. PMID:25203404

  14. Informing Stem Cell-Based Tendon Tissue Engineering Approaches with Embryonic Tendon Development.

    PubMed

    Okech, William; Kuo, Catherine K

    Adult tendons fail to regenerate normal tissue after injury, and instead form dysfunctional scar tissue with abnormal mechanical properties. Surgical repair with grafts is the current standard to treat injuries, but faces significant limitations including pain and high rates of re-injury. To address this, we aim to regenerate new, normal tendons to replace dysfunctional tendons. A common approach to tendon tissue engineering is to design scaffolds and bioreactors based on adult tendon properties that can direct adult stem cell tenogenesis. Despite significant progress, advances have been limited due, in part, to a need for markers and potent induction cues. Our goal is to develop novel tendon tissue engineering approaches informed by embryonic tendon development. We are characterizing structure-property relationships of embryonic tendon to identify design parameters for three-dimensional scaffolds and bioreactor mechanical loading systems to direct adult stem cell tenogenesis. We will review studies in which we quantified changes in the mechanical and biochemical properties of tendon during embryonic development and elucidated specific mechanisms of functional property elaboration. We then examined the effects of these mechanical and biochemical factors on embryonic tendon cell behavior. Using custom-designed bioreactors, we also examined the effects of dynamic mechanical loading and growth factor treatment on embryonic tendon cells. Our findings have established cues to induce tenogenesis as well as metrics to evaluate differentiation. We finish by discussing how we have evaluated the tenogenic differentiation potential of adult stem cells by comparing their responses to that of embryonic tendon cells in these culture systems.

  15. Efficacy of tendon stem cells in fibroblast-derived matrix for tendon tissue engineering.

    PubMed

    Jiang, Dapeng; Xu, Bo; Yang, Mowen; Zhao, Zheng; Zhang, Yubo; Li, Zhaozhu

    2014-05-01

    After injury, tendons often heal with poor tissue quality and inferior mechanical properties. Tissue engineering using tendon stem cells (TSCs) is a promising approach in the repair of injured tendon. Tenogenic differentiation of TSCs needs an appropriate environment. More recently, the acellular extracellular matrix (ECM) generated from fibroblasts has been used to construct various engineering tissues. In this study, we successfully developed an engineered tendon tissue formed by seeding TSCs in de-cellularized fibroblast-derived matrix (dFM). Patellar TSCs and dermal fibroblast were isolated and cultured. Using the method of osmotic shock, dFM was obtained from dermal fibroblast. ECM proteins in dFM were examined. TSCs at passage 3 were seeded in dFM for 1 week. Proliferative capacity and characterization of TSCs cultured in dFM were determined by population doubling time, immunofluorescence staining and quantitative reverse transcriptase polymerase chain reaction. Engineered tendon tissue was prepared with dFM and TSCs. Its potentials for neo-tendon formation and promoting tendon healing were investigated. dFM is suitable for growth and tenogenic differentiation of TSCs in vitro. Neo-tendon tissue was formed with tendon-specific protein expression when TSCs were implanted together with dFM. In a patellar tendon injury model, implantation of engineered tendon tissue significantly improved the histologic and mechanical properties of injured tendon. The findings obtained from our study provide a basis for potential use of engineered tendon tissue containing dFM and TSCs in tendon repair and regeneration. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  16. Famotidine suppresses osteogenic differentiation of tendon cells in vitro and pathological calcification of tendon in vivo.

    PubMed

    Yamamoto, Kenichi; Hojo, Hironori; Koshima, Isao; Chung, Ung-il; Ohba, Shinsuke

    2012-12-01

    Heterotopic ossification or calcification follows any type of musculoskeletal trauma and is known to occur after arthroplasties of hip, knee, shoulder, or elbow; fractures; joint dislocations; or tendon ruptures. Histamine receptor H2 (Hrh2) has been shown to be effective for reducing pain and decreasing calcification in patients with calcifying tendinitis, which suggested that H2 blockers were effective for the treatment of tendon ossification or calcification. However, the detailed mechanisms of its action on tendon remain to be clarified. We investigated the mechanisms underlying H2 blocker-mediated suppression of tendon calcification, with a focus on the direct action of the drug on tendon cells. Famotidine treatment suppressed the mRNA expressions of Col10a1 and osteocalcin, ossification markers, in a tendon-derived cell line TT-D6, as well as a preosteoblastic one MC3T3-E1. Both of the cell lines expressed Hrh2; histamine treatment induced osteocalcin expression in these cells. Famotidine administration suppressed calcification in the Achilles tendon of ttw mice, a mouse model of ectopic ossification. These data suggest that famotidine inhibits osteogenic differentiation of tendon cells in vitro, and this inhibition may underlie the anti-calcification effects of the drug in vivo. This study points to the use of H2 blockers as a promising strategy for treating heterotopic ossification or calcification in tendon, and provides evidence in support of the clinical use of famotidine.

  17. Novel Model of Tendon Regeneration Reveals Distinct Cell Mechanisms Underlying Regenerative and Fibrotic Tendon Healing

    PubMed Central

    Howell, Kristen; Chien, Chun; Bell, Rebecca; Laudier, Damien; Tufa, Sara F.; Keene, Douglas R.; Andarawis-Puri, Nelly; Huang, Alice H.

    2017-01-01

    To date, the cell and molecular mechanisms regulating tendon healing are poorly understood. Here, we establish a novel model of tendon regeneration using neonatal mice and show that neonates heal via formation of a ‘neo-tendon’ that differentiates along the tendon specific lineage with functional restoration of gait and mechanical properties. In contrast, adults heal via fibrovascular scar, aberrant differentiation toward cartilage and bone, with persistently impaired function. Lineage tracing identified intrinsic recruitment of Scx-lineage cells as a key cellular mechanism of neonatal healing that is absent in adults. Instead, adult Scx-lineage tenocytes are not recruited into the defect but transdifferentiate into ectopic cartilage; in the absence of tenogenic cells, extrinsic αSMA-expressing cells persist to form a permanent scar. Collectively, these results establish an exciting model of tendon regeneration and uncover a novel cellular mechanism underlying regenerative vs non-regenerative tendon healing. PMID:28332620

  18. Characterization of age-related changes of tendon stem cells from adult human tendons.

    PubMed

    Ruzzini, Laura; Abbruzzese, Franca; Rainer, Alberto; Longo, Umile Giuseppe; Trombetta, Marcella; Maffulli, Nicola; Denaro, Vincenzo

    2014-11-01

    The present study evaluated the presence of stem cells in hamstring tendons from adult subjects of different ages. The aim was to isolate, characterize and expand these cells in vitro, and to evaluate whether cell activities are influenced by age. Tendon stem cells (TSCs) were isolated through magnetic sorting from the hamstring tendons of six patients. TSC percentage, morphology and clonogenic potential were evaluated, as well as the expression of specific surface markers. TSC multi-potency was also investigated as a function of age, and quantitative polimerase chain reaction was used to evaluate gene expression of TSCs cultured in suitable differentiating media. The presence of easily harvestable stem cell population within adult human hamstring tendons was demonstrated. These cells exhibit features such as clonogenicity, multi-potency and mesenchymal stem cells markers expression. The age-related variations in human TSCs affect the number of isolated cells and their self-renewal potential, while multi-potency assays are not influenced by tendon ageing, even though cells from younger individuals expressed higher levels of osteogenic and adipogenic genes, while chondrogenic genes were highly expressed in cells from older individuals. These results may open new opportunities to study TSCs to better understand tendon physiology, healing and pathological processes such as tendinopathy and degenerative age-related changes opening new frontiers in the management of tendinopathy and tendon ruptures.

  19. Multilayer tendon slices seeded with bone marrow stromal cells: a novel composite for tendon engineering.

    PubMed

    Omae, Hiromichi; Zhao, Chunfeng; Sun, Yu Long; An, Kai-Nan; Amadio, Peter C

    2009-07-01

    The ideal scaffold for tendon engineering would possess the basic structure of the tendon, native extracellular matrix, and capability of cell seeding. The purpose of this study was to assess the tissue engineering potential of a novel composite consisting of a decellularized multilayer sliced tendon (MST) scaffold seeded with bone marrow stromal cells (BMSC). BMSC and infraspinatus tendons were harvested from 20 dogs. The tendons were sectioned in longitudinal slices with a thickness of 50 microm. The slices were decellularized, seeded with BMSC, and then bundled into one composite. The composite was incubated in culture media for 14 days. The resulting BMSC-seeded MST was evaluated by qRT-PCR and histology. The BMSC viability was assessed by a fluorescent tracking marker. Histology showed that the seeded cells aligned between the collagen fibers of the tendon slices. Analysis by qRT-PCR showed higher tenomodulin and MMP13 expression and lower collagen type I expression in the composite than in the BMSC before seeding. BMSC labeled with fluorescent tracking marker were observed in the composite after culture. Mechanical testing showed no differences between scaffolds with or without BMSC. BMSC can survive in a MST scaffold. The increased tenomodulin expression suggests that BMSC might express a tendon phenotype in this environment. This new composite might be useful as a model of tendon tissue engineering.

  20. The effect of decellularized matrices on human tendon stem/progenitor cell differentiation and tendon repair.

    PubMed

    Yin, Zi; Chen, Xiao; Zhu, Ting; Hu, Jia-jie; Song, Hai-xin; Shen, Wei-liang; Jiang, Liu-yun; Heng, Boon Chin; Ji, Jun-feng; Ouyang, Hong-Wei

    2013-12-01

    It is reported that decellularized collagen matrices derived from dermal skin and bone have been clinically used for tendon repair. However, the varying biological and physical properties of matrices originating from different tissues may influence the differentiation of tendon stem cells, which has not been systematically evaluated. In this study, the effects of collagenous matrices derived from different tissues (tendon, bone and dermis) on the cell differentiation of human tendon stem/progenitor cells (hTSPCs) were investigated, in the context of tendon repair. It was found that all three matrices supported the adhesion and proliferation of hTSPCs despite differences in topography. Interestingly, tendon-derived decellularized matrix promoted the tendinous phenotype in hTSPCs and inhibited their osteogenesis, even under osteogenic induction conditions, through modulation of the teno- and osteolineage-specific transcription factors Scleraxis and Runx2. Bone-derived decellularized matrix robustly induced osteogenic differentiation of hTSPCs, whereas dermal skin-derived collagen matrix had no apparent effect on hTSPC differentiation. Based on the specific biological function of the tendon-derived decellularized matrix, a tissue-engineered tendon comprising TSPCs and tendon-derived matrix was successfully fabricated for Achilles tendon reconstruction. Implantation of this cell-scaffold construct led to a more mature structure (histology score: 4.08 ± 0.61 vs. 8.51 ± 1.66), larger collagen fibrils (52.2 ± 1.6 nm vs. 47.5 ± 2.8 nm) and stronger mechanical properties (stiffness: 21.68 ± 7.1 Nm m(-1) vs.13.2 ± 5.9 Nm m(-1)) of repaired tendons compared to the control group. The results suggest that stem cells promote the rate of repair of Achilles tendon in the presence of a tendinous matrix. This study thus highlights the potential of decellularized matrix for future tissue engineering applications, as well as developing a practical strategy for functional tendon

  1. Tendon synovial cells secrete fibronectin in vivo and in vitro

    SciTech Connect

    Banes, A.J.; Link, G.W.; Bevin, A.G.; Peterson, H.D.; Gillespie, Y.; Bynum, D.; Watts, S.; Dahners, L.

    1988-01-01

    The chemistry and cell biology of the tendon have been largely overlooked due to the emphasis on collagen, the principle structural component of the tendon. The tendon must not only transmit the force of muscle contraction to bone to effect movement, but it must also glide simultaneously over extratendonous tissues. Fibronectin is classified as a cell attachment molecule that induces cell spreading and adhesion to substratum. The external surface of intact avian flexor tendon stained positively with antibody to cellular fibronectin. However, if the surface synovial cells were first removed with collagenase, no positive reaction with antifibronectin antibody was detected. Analysis of immunologically stained frozen sections of tendon also revealed fibronectin at the tendon synovium, but little was associated with cells internal in tendon. The staining pattern with isolated, cultured synovial cells and fibroblasts from the tendon interior substantiated the histological observations. Analysis of polyacrylamide gel profiles of /sup 35/S-methionine-labeled proteins synthesized by synovial cells and internal fibroblasts indicated that fibronectin was synthesized principally by synovial cells. Fibronectin at the tendon surface may play a role in cell attachment to prevent cell removal by the friction of gliding. Alternatively, fibronectin, with its binding sites for hyaluronic acid and collagen, may act as a complex for boundary lubrication.

  2. The effect of tendon surface treatment on cell attachment for potential enhancement of tendon graft healing: an ex vivo model.

    PubMed

    Hashimoto, Takahiro; Sun, Yu-Long; An, Kai-Nan; Amadio, Peter C; Zhao, Chunfeng

    2012-12-01

    For both tendon allografts and autografts, the surface, initially optimized for gliding, may not be ideal to facilitate tissue integration for graft healing to host tendon or bone. As a prelude to studying tendon-bone integration, we investigated the effect of surface treatments with trypsin or mechanical abrasion on cell attachment to the tendon surface in a canine ex vivo intrasynovial tendon tissue culture model. Intrasynovial tendon allograft surfaces were seeded with cells after the following treatments: (1) no treatment, (2) mechanical abrasion, (3) trypsin, and (4) abrasion and trypsin. The area covered by cells was determined using confocal laser microscopy at one and two weeks. Results were compared to untreated extrasynovial tendon. Additional tendons were characterized with scanning electron microscopy. Tendons with trypsin treatment had significantly more surface coverage with cells than the other groups, after both one and two weeks of culture. In terms of the cellular shape and size, cells on tendons with trypsin treatment spread more and were more polygonal in shape, whereas tendons with mechanical abrasion with/without trypsin treatment contained smaller, more spindle-like cells. Surface roughening can affect cell behavior with topographical stimulation. Trypsin surface digestion exposes a mesh-like structure on the tendon surface, which could enhance cell adherence and, possibly, tendon/bone healing.

  3. Harnessing endogenous stem/progenitor cells for tendon regeneration.

    PubMed

    Lee, Chang H; Lee, Francis Y; Tarafder, Solaiman; Kao, Kristy; Jun, Yena; Yang, Guodong; Mao, Jeremy J

    2015-07-01

    Current stem cell-based strategies for tissue regeneration involve ex vivo manipulation of these cells to confer features of the desired progenitor population. Recently, the concept that endogenous stem/progenitor cells could be used for regenerating tissues has emerged as a promising approach that potentially overcomes the obstacles related to cell transplantation. Here we applied this strategy for the regeneration of injured tendons in a rat model. First, we identified a rare fraction of tendon cells that was positive for the known tendon stem cell marker CD146 and exhibited clonogenic capacity, as well as multilineage differentiation ability. These tendon-resident CD146+ stem/progenitor cells were selectively enriched by connective tissue growth factor delivery (CTGF delivery) in the early phase of tendon healing, followed by tenogenic differentiation in the later phase. The time-controlled proliferation and differentiation of CD146+ stem/progenitor cells by CTGF delivery successfully led to tendon regeneration with densely aligned collagen fibers, normal level of cellularity, and functional restoration. Using siRNA knockdown to evaluate factors involved in tendon generation, we demonstrated that the FAK/ERK1/2 signaling pathway regulates CTGF-induced proliferation and differentiation of CD146+ stem/progenitor cells. Together, our findings support the use of endogenous stem/progenitor cells as a strategy for tendon regeneration without cell transplantation and suggest this approach warrants exploration in other tissues.

  4. Cystic fibrosis transmembrane conductance regulator mediates tenogenic differentiation of tendon-derived stem cells and tendon repair: accelerating tendon injury healing by intervening in its downstream signaling.

    PubMed

    Liu, Yang; Xu, Jia; Xu, Liangliang; Wu, Tianyi; Sun, Yuxin; Lee, Yuk-Wai; Wang, Bin; Chan, Hsiao-Chang; Jiang, Xiaohua; Zhang, Jinfang; Li, Gang

    2017-09-01

    Tendons are a mechanosensitive tissue, which enables them to transmit to bone forces that are derived from muscle. Patients with tendon injuries, such as tendinopathy or tendon rupture, were often observed with matrix degeneration, and the healing of tendon injuries remains a challenge as a result of the limited understanding of tendon biology. Our study demonstrates that the stretch-mediated activation channel, cystic fibrosis transmembrane conductance regulator (CFTR), was up-regulated in tendon-derived stem cells (TDSCs) during tenogenic differentiation under mechanical stretching. Tendon tissues in CFTR-dysfunctional DF508 mice exhibited irregular cell arrangement, uneven fibril diameter distribution, weak mechanical properties, and less matrix formation in a tendon defect model. Moreover, both tendon tissues and TDSCs isolated from DF508 mice showed significantly decreased levels of tendon markers, such as scleraxis, tenomodulin, Col1A1 (collagen type I α 1 chain), and decorin Furthermore, by RNA sequencing analysis, we demonstrated that Wnt/β-catenin signaling was abnormally activated in TDSCs from DF508 mice, thereby further activating the pERK1/2 signaling pathway. Of most importance, we found that intervention in pERK1/2 signaling could promote tenogenic differentiation and tendon regeneration both in vitro and in vivo Taken together, our study demonstrates that CFTR plays an important role in tenogenic differentiation and tendon regeneration by inhibiting the β-catinin/pERK1/2 signaling pathway. The therapeutic strategy of intervening in the CFTR/β-catenin/pERK1/2 regulatory axis may be helpful for accelerating tendon injury healing, which has implications for tendon injury management.-Liu, Y., Xu, J., Xu, L., Wu, T., Sun, Y., Lee, Y.-W., Wang, B., Chan, H.-C., Jiang, X., Zhang, J., Li, G. Cystic fibrosis transmembrane conductance regulator mediates tenogenic differentiation of tendon-derived stem cells and tendon repair: accelerating tendon injury

  5. Harnessing endogenous stem/progenitor cells for tendon regeneration

    PubMed Central

    Lee, Chang H.; Lee, Francis Y.; Tarafder, Solaiman; Kao, Kristy; Jun, Yena; Yang, Guodong; Mao, Jeremy J.

    2015-01-01

    Current stem cell–based strategies for tissue regeneration involve ex vivo manipulation of these cells to confer features of the desired progenitor population. Recently, the concept that endogenous stem/progenitor cells could be used for regenerating tissues has emerged as a promising approach that potentially overcomes the obstacles related to cell transplantation. Here we applied this strategy for the regeneration of injured tendons in a rat model. First, we identified a rare fraction of tendon cells that was positive for the known tendon stem cell marker CD146 and exhibited clonogenic capacity, as well as multilineage differentiation ability. These tendon-resident CD146+ stem/progenitor cells were selectively enriched by connective tissue growth factor delivery (CTGF delivery) in the early phase of tendon healing, followed by tenogenic differentiation in the later phase. The time-controlled proliferation and differentiation of CD146+ stem/progenitor cells by CTGF delivery successfully led to tendon regeneration with densely aligned collagen fibers, normal level of cellularity, and functional restoration. Using siRNA knockdown to evaluate factors involved in tendon generation, we demonstrated that the FAK/ERK1/2 signaling pathway regulates CTGF-induced proliferation and differentiation of CD146+ stem/progenitor cells. Together, our findings support the use of endogenous stem/progenitor cells as a strategy for tendon regeneration without cell transplantation and suggest this approach warrants exploration in other tissues. PMID:26053662

  6. Stem cells for tendon tissue engineering and regeneration.

    PubMed

    Yin, Zi; Chen, Xiao; Chen, Jia-Lin; Ouyang, Hong-Wei

    2010-05-01

    Tendon injuries are common especially in sports activities, but tendon is a unique connective tissue with poor self-repair capability. With advances in stem cell biology, tissue engineering is becoming increasingly powerful for tissue regeneration. Stem cells with capacity of multipotency and self-renewal are an ideal cell source for tissue engineering. This review focus on discussing the potential strategies including inductive growth factors, bio-scaffolds, mechanical stimulation, genetic modification and co-culture techniques to direct tendon-lineage differentiation of stem cells for complete tendon regeneration. Attempting to use embryonic stem cells as seed cells for tendon tissue engineering have achieved encouraging results. The combination of chemical and physical signals in stem cell microenvironment could be regulated to induce differentiation of the embryonic stem cells into tendon. We summarize fundamental questions, as well as future directions in tendon biology and tissue engineering. Multifaceted technologies are increasingly required to control stem cell differentiation, to develop novel stem cell-based therapy, and, ultimately, to achieve more effective repair or regeneration of injured tendons.

  7. Stem Cell Applications in Tendon Disorders: A Clinical Perspective

    PubMed Central

    Young, Mark

    2012-01-01

    Tendon injuries are a common cause of morbidity and a significant health burden on society. Tendons are structural tissues connecting muscle to bone and are prone to tearing and tendinopathy, an overuse or degenerative condition that is characterized by failed healing and cellular depletion. Current treatments, for tendon tear are conservative, surgical repair or surgical scaffold reconstruction. Tendinopathy is treated by exercises, injection therapies, shock wave treatments or surgical tendon debridement. However, tendons usually heal with fibrosis and scar tissue, which has suboptimal tensile strength and is prone to reinjury, resulting in lifestyle changes with activity restriction. Preclinical studies show that cell therapies have the potential to regenerate rather than repair tendon tissue, a process termed tenogenesis. A number of different cell lines, with varying degrees of differentiation, have being evaluated including stem cells, tendon derived cells and dermal fibroblasts. Even though cellular therapies offer some potential in treating tendon disorders, there have been few published clinical trials to determine the ideal cell source, the number of cells to administer, or the optimal bioscaffold for clinical use. PMID:22448174

  8. The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration.

    PubMed

    Chang, Chung-Hsun; Tsai, Wen-Chung; Lin, Miao-Sui; Hsu, Ya-Hui; Pang, Jong-Hwei Su

    2011-03-01

    Pentadecapeptide BPC 157, composed of 15 amino acids, is a partial sequence of body protection compound (BPC) that is discovered in and isolated from human gastric juice. Experimentally it has been demonstrated to accelerate the healing of many different wounds, including transected rat Achilles tendon. This study was designed to investigate the potential mechanism of BPC 157 to enhance healing of injured tendon. The outgrowth of tendon fibroblasts from tendon explants cultured with or without BPC 157 was examined. Results showed that BPC 157 significantly accelerated the outgrowth of tendon explants. Cell proliferation of cultured tendon fibroblasts derived from rat Achilles tendon was not directly affected by BPC 157 as evaluated by MTT assay. However, the survival of BPC 157-treated cells was significantly increased under the H(2)O(2) stress. BPC 157 markedly increased the in vitro migration of tendon fibroblasts in a dose-dependent manner as revealed by transwell filter migration assay. BPC 157 also dose dependently accelerated the spreading of tendon fibroblasts on culture dishes. The F-actin formation as detected by FITC-phalloidin staining was induced in BPC 157-treated fibroblasts. The protein expression and activation of FAK and paxillin were determined by Western blot analysis, and the phosphorylation levels of both FAK and paxillin were dose dependently increased by BPC 157 while the total amounts of protein was unaltered. In conclusion, BPC 157 promotes the ex vivo outgrowth of tendon fibroblasts from tendon explants, cell survival under stress, and the in vitro migration of tendon fibroblasts, which is likely mediated by the activation of the FAK-paxillin pathway.

  9. Planar cell polarity in Drosophila

    PubMed Central

    Maung, Saw Myat Thanda W

    2011-01-01

    In all multicellular organisms, epithelial cells are not only polarized along the apical-basal axis, but also within the epithelial plane, giving cells a sense of direction. Planar cell polarity (PCP) signaling regulates establishment of polarity within the plane of an epithelium. The outcomes of PCP signaling are diverse and include the determination of cell fates, the generation of asymmetric but highly aligned structures, such as the stereocilia in the human inner ear or the hairs on a fly wing, or the directional migration of cells during convergence and extension during vertebrate gastrulation. In humans, aberrant PCP signaling can result in severe developmental defects, such as open neural tubes (spina bifida), and can cause cystic kidneys. In this review, we discuss the basic mechanism and more recent findings of PCP signaling focusing on Drosophila melanogaster, the model organism in which most key PCP components were initially identified. PMID:21983142

  10. The Drosophila cyst stem cell lineage

    PubMed Central

    Zoller, Richard; Schulz, Cordula

    2012-01-01

    In all animals, germline cells differentiate in intimate contact with somatic cells and interactions between germline and soma are particularly important for germline development and function. In the male gonad of Drosophila melanogaster, the developing germline cells are enclosed by somatic cyst cells. The cyst cells are derived from cyst stem cells (CySCs) of somatic origin and codifferentiate with the germline cells. The fast generation cycle and the genetic tractability of Drosophila has made the Drosophila testis an excellent model for studying both the roles of somatic cells in guiding germline development and the interdependence of two separate stem cell lineages. This review focuses on our current understanding of CySC specification, CySC self-renewing divisions, cyst cell differentiation, and soma-germline interactions. Many of the mechanisms guiding these processes in Drosophila testes are similarly essential for the development and function of tissues in other organisms, most importantly for gametogenesis in mammals. PMID:23087834

  11. Markers for the identification of tendon-derived stem cells in vitro and tendon stem cells in situ - update and future development.

    PubMed

    Lui, Pauline Po Yee

    2015-06-02

    The efficacy of tendon-derived stem cells (TDSCs) for the promotion of tendon and tendon-bone junction repair has been reported in animal studies. Modulation of the tendon stem cell niche in vivo has also been reported to influence tendon structure. There is a need to have specific and reliable markers that can define TDSCs in vitro and tendon stem cells in situ for several reasons: to understand the basic biology of TDSCs and their subpopulations in vitro; to understand the identity, niches and functions of tendon/progenitor stem cells in vivo; to meet the governmental regulatory requirements for quality of TDSCs when translating the exciting preclinical findings into clinical trial/practice; and to develop new treatment strategies for mobilizing endogenous stem/progenitor cells in tendon. TDSCs were reported to express the common mesenchymal stem cell (MSC) markers and some embryonic stem cell (ESC) markers, and there were attempts to use these markers to label tendon stem cells in situ. Are these stem cell markers useful for the identification of TDSCs in vitro and tracking of tendon stem cells in situ? This review aims to discuss the values of the panel of MSC, ESC and tendon-related markers for the identification of TDSCs in vitro. Important factors influencing marker expression by TDSCs are discussed. The usefulness and limitations of the panel of MSC, ESC and tendon-related markers for tracking stem cells in tendon, especially tendon stem cells, in situ are then reviewed. Future research directions are proposed.

  12. Combined effects of engineered tendon matrix and GDF-6 on bone marrow mesenchymal stem cell-based tendon regeneration.

    PubMed

    Jiang, Dapeng; Gao, Peng; Zhang, Yubo; Yang, Shulong

    2016-05-01

    To examine whether an engineered tendon matrix (ETM) environment and growth and differentiation factor-6 (GDF-6) have synergistic effects on the tenogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the quality of tendon repair. ETM and GDF-6 promote tenogenic differentiation of BMSCs in vitro. Implantation of GDF-6-incorporated ETM containing BMSCs into a tendon injury model significantly improved the histological and mechanical properties of the repaired tendon. GDF-6-incorporated ETM containing BMSCs represents a promising strategy for tendon injury repair.

  13. Tendon Progenitor Cells in Injured Tendons Have Strong Chondrogenic Potential: The CD105-Negative Subpopulation Induces Chondrogenic Degeneration

    PubMed Central

    Asai, Shuji; Otsuru, Satoru; Candela, Maria Elena; Cantley, Leslie; Uchibe, Kenta; Hofmann, Ted J.; Zhang, Kairui; Wapner, Keith L.; Soslowsky, Louis J; Horwitz, Edwin M.; Enomoto-Iwamoto, Motomi

    2014-01-01

    To study the cellular mechanism of the tendon repair process, we used a mouse Achilles tendon injury model to focus on the cells recruited to the injured site. The cells isolated from injured tendon 1 week after the surgery and uninjured tendons contained the connective tissue progenitor populations as determined by colony-forming capacity, cell surface markers and multipotency. When the injured tendon-derived progenitor cells (inTPCs) were transplanted into injured Achilles tendons, they were not only integrated in the regenerating area expressing tenogenic phenotype but also trans-differentiated into chondrogenic cells in the degenerative lesion that underwent ectopic endochondral ossification. Surprisingly, the micromass culture of the inTPCs rapidly underwent chondrogenic differentiation even in the absence of exogenous BMPs or TGFβs. The cells isolated from human ruptured tendon tissues also showed connective tissue progenitor properties and exhibited stronger chondrogenic ability than bone marrow stromal cells. The mouse inTPCs contained two subpopulations one positive and one negative for CD105, a co-receptor of the TGFβ superfamily. The CD105-negative cells showed superior chondrogenic potential in vitro and induced larger chondroid degenerative lesions in mice as compared to the CD105-positive cells. These findings indicate that tendon progenitor cells are recruited to the injured site of tendons and have a strong chondrogenic potential and that the CD105-negative population of these cells would be the cause for chondroid degeneration in injured tendons. The newly identified cells recruited to the injured tendon may provide novel targets to develop therapeutic strategies to facilitate tendon repair. PMID:25220576

  14. Tendon progenitor cells in injured tendons have strong chondrogenic potential: the CD105-negative subpopulation induces chondrogenic degeneration.

    PubMed

    Asai, Shuji; Otsuru, Satoru; Candela, Maria Elena; Cantley, Leslie; Uchibe, Kenta; Hofmann, Ted J; Zhang, Kairui; Wapner, Keith L; Soslowsky, Louis J; Horwitz, Edwin M; Enomoto-Iwamoto, Motomi

    2014-12-01

    To study the cellular mechanism of the tendon repair process, we used a mouse Achilles tendon injury model to focus on the cells recruited to the injured site. The cells isolated from injured tendon 1 week after the surgery and uninjured tendons contained the connective tissue progenitor populations as determined by colony-forming capacity, cell surface markers, and multipotency. When the injured tendon-derived progenitor cells (inTPCs) were transplanted into injured Achilles tendons, they were not only integrated in the regenerating area expressing tenogenic phenotype but also trans-differentiated into chondrogenic cells in the degenerative lesion that underwent ectopic endochondral ossification. Surprisingly, the micromass culture of the inTPCs rapidly underwent chondrogenic differentiation even in the absence of exogenous bone morphogenetic proteins or TGFβs. The cells isolated from human ruptured tendon tissues also showed connective tissue progenitor properties and exhibited stronger chondrogenic ability than bone marrow stromal cells. The mouse inTPCs contained two subpopulations one positive and one negative for CD105, a coreceptor of the TGFβ superfamily. The CD105-negative cells showed superior chondrogenic potential in vitro and induced larger chondroid degenerative lesions in mice as compared to the CD105-positive cells. These findings indicate that tendon progenitor cells are recruited to the injured site of tendons and have a strong chondrogenic potential and that the CD105-negative population of these cells would be the cause for chondroid degeneration in injured tendons. The newly identified cells recruited to the injured tendon may provide novel targets to develop therapeutic strategies to facilitate tendon repair.

  15. Tendon-derived stem cells (TDSCs): from basic science to potential roles in tendon pathology and tissue engineering applications.

    PubMed

    Lui, Pauline Po Yee; Chan, Kai Ming

    2011-11-01

    Traditionally, tendons are considered to only contain tenocytes that are responsible for the maintenance, repair and remodeling of tendons. Stem cells, which are termed tendon-derived stem cells (TDSCs), have recently been identified in tendons. This review aims to summarize the current information about the in vitro characteristics of TDSCs, including issues related to TDSC isolation and culture, their cell morphology, immunophenotypes, proliferation and differentiation characteristics and senescence during in vitro passaging. The challenges in studying the functions of these cells are also discussed. The niche where TDSCs resided essentially provides signals that are conducive to the maintenance of definitive stem cell properties of TDSCs. Yet the niche may also induce pathologies by imposing an aberrant function on TDSCs or other targets. The possible niche factors of TDSCs are herein discussed. We presented current evidences supporting the potential pathogenic role of TDSCs in the development of tendinopathy with reference to the recent findings on the altered biological responses of these cells in response to their potential niche factors. The use of resident stem cells may promote engraftment and differentiation of transplanted cells in tendon and tendon-bone junction repair because the tendon milieu is an ideal and familiar environment to the transplanted cells. Evidences are presented to show the potential advantages and results of using TDSCs as a new cell source for tendon and tendon-bone junction repair. Issues pertaining to the use of TDSCs for tissue repair are also discussed.

  16. Cell phenotypic variation in normal and damaged tendons

    PubMed Central

    Clegg, Peter D; Strassburg, Sandra; Smith, Roger K

    2007-01-01

    Injuries to tendons are common in both human athletes as well as in animals, such as the horse, which are used for competitive purposes. Furthermore, such injuries are also increasing in prevalence in the ageing, sedentary population. Tendon diseases often respond poorly to treatment and require lengthy periods of rehabilitation. The tendon has a unique extracellular matrix, which has developed to withstand the mechanical demands of such tensile-load bearing structures. Following injury, any repair process is inadequate and results in tissue that is distinct from original tendon tissue. There is growing evidence for the key role of the tendon cell (tenocyte) in both the normal physiological homeostasis and regulation of the tendon matrix and the pathological derangements that occur in disease. In particular, the tenocyte is considered to have a major role in effecting the subclinical matrix degeneration that is thought to occur prior to clinical disease, as well as in the severe degradative events that occur in the tendon at the onset of clinical disease. Furthermore, the tenocyte is likely to have a central role in the production of the biologically inadequate fibrocartilaginous repair tissue that develops subsequent to tendinopathy. Understanding the biology of the tenocyte is central to the development of appropriate interventions and drug therapies that will either prevent the onset of disease, or lead to more rapid and appropriate repair of injured tendon. Central to this is a full understanding of the proteolytic response in the tendon in disease by such enzymes as metalloproteinases, as well as the control of the inappropriate fibrocartilaginous differentiation. Finally, it is important that we understand the role of both intrinsic and extrinsic cellular elements in the repair process in the tendon subsequent to injury. PMID:17696903

  17. The role of engineered tendon matrix in the stemness of tendon stem cells in vitro and the promotion of tendon-like tissue formation in vivo

    PubMed Central

    Zhang, Jianying; Li, Bin; Wang, James H-C.

    2011-01-01

    When injured, tendons tend to heal but with poor structure and compromised function. Tissue engineering is a promising approach to enhancing the quality of healing tendons. Our group and others have identified tendon stem cells (TSCs), a type of tendon-specific stem cells which may be optimal for cellular interventions seeking to restore normal structure and function to injured tendons. However, in vitro expanding of TSCs on regular plastic cell culture dishes only yields a limited number of TSCs before they lose the stemness, i.e., the self-renewal capability and multipotency. In this study, we developed a substrate material for TSCs, engineered tendon matrix (ETM) from decellularized tendon tissues. We showed that ETM in vitro was able to stimulate TSC proliferation and better preserve the stemness of TSCs than plastic culture surfaces. In vivo, implantation of ETM-TSC composite promoted tendon-like tissue formation whereas implantation of TSCs alone led to little such tissue formation. Together, the findings of this study indicate that ETM may be used to effectively expand TSCs in vitro and with TSCs, to enhance repair of injured tendons in vivo. PMID:21703682

  18. Tendon Regeneration with Tendon Hydrogel-Based Cell Delivery: A Comparison of Fibroblasts and Adipose-Derived Stem Cells.

    PubMed

    Chattopadhyay, Arhana; Galvez, Michael G; Bachmann, Michael; Legrand, Anais; McGoldrick, Rory; Lovell, Alberto; Jacobs, Mollie; Crowe, Chris; Umansky, Elise; Chang, James

    2016-09-01

    Tendon hydrogel is a promising biomaterial for improving repair strength after tendon injury. This study compares the capacity of fibroblasts and adipose-derived stem cells to proliferate, survive, and acquire tenogenic properties when seeded into tendon hydrogel in vitro and in vivo. The effect of cell density on hydrogel contraction was measured macroscopically. To assess tenogenic properties, RNA was isolated from cells seeded in vitro in hydrogel, and tenocyte markers were quantified. To assess in vitro proliferation and survival, MTS and live-dead assays were performed. Finally, to assess the in vivo survival of cells in hydrogel, subcutaneous injections were performed on rats and in vivo imaging was performed. At 0.5 million cells/ml, both the fibroblasts and adipose-derived stem cells induced minimal hydrogel contraction compared with higher cellular concentrations. Fibroblasts and adipose-derived stem cells seeded at 0.5 million cells/ml in tendon hydrogel up-regulated several tenocyte markers after 1 week. On MTS assay, fibroblasts and adipose-derived stem cells proliferated in hydrogel at similar rates. On live-dead assay, fibroblasts survived longer than adipose-derived stem cells. With use of the in vivo imaging system and histologic evaluation, fibroblasts survived longer than adipose-derived stem cells in hydrogel in vivo. Tendon healing is mediated by the proliferation, survival, and tenogenic differentiation of cells at the site of injury. Tendon hydrogel delivering dermal fibroblasts may improve and stimulate this process compared with adipose-derived stem cells. Future studies will be needed to evaluate the effects of this hydrogel-based cell delivery on chronic tendon injuries.

  19. Asymmetric stem cell division: lessons from Drosophila.

    PubMed

    Wu, Pao-Shu; Egger, Boris; Brand, Andrea H

    2008-06-01

    Asymmetric cell division is an important and conserved strategy in the generation of cellular diversity during animal development. Many of our insights into the underlying mechanisms of asymmetric cell division have been gained from Drosophila, including the establishment of polarity, orientation of mitotic spindles and segregation of cell fate determinants. Recent studies are also beginning to reveal the connection between the misregulation of asymmetric cell division and cancer. What we are learning from Drosophila as a model system has implication both for stem cell biology and also cancer research.

  20. The long head of the biceps tendon is a suitable cell source for tendon tissue regeneration

    PubMed Central

    Pietschmann, Matthias F.; Gülecyüz, Mehmet F.; Ficklscherer, Andreas; Jansson, Volkmar; Müller, Peter E.

    2014-01-01

    Introduction Tendon tissue engineering (TTE) tries to produce tendinous tissue of high quality to replace dysfunctional tissue. One possible application of TTE might be the replacement of ruptured tissue of the rotator cuff. Autologous tenocytes seem to be most suitable as no differentiation in vitro is necessary. Today it is still uncertain if there is a difference between tendon-derived cells (TDC) of different native tissues. Moreover, the search for suitable scaffolds is another important issue in TTE. Material and methods This study compared TDC of the long head of the biceps tendon (LHB), the anterior cruciate ligament (ACL) and the tendon of the musculus semitendinosus (TMS). The TDC were isolated using the cell migration method. Cell morphology was assessed using light microscopy and gene expression was performed using polymerase chain reaction (PCR). Afterwards, cell seeding efficiency and proliferation were tested on a collagen I scaffold using the WST-1 assay. Results were confirmed using H + E staining. Results The TDC of the LHB showed higher expression levels of collagen type I and decorin (p < 0.01) compared to TDC of other origin. Results showed efficient cell seeding and proliferation within the scaffold. Proliferation within the scaffold was not as high as when cells were cultivated without a scaffold. Conclusions The TDC of the LHB seems to be the most suitable cell source. Further research is necessary to find out if the results can be transferred to an in vivo model. The new collagen I scaffold seems to offer an opportunity to combine good biocompatibility and mechanical strength. PMID:25097592

  1. Live cell imaging in Drosophila melanogaster.

    PubMed

    Parton, Richard M; Vallés, Ana Maria; Dobbie, Ian M; Davis, Ilan

    2010-04-01

    Although many of the techniques of live cell imaging in Drosophila melanogaster are also used by the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties with regard to keeping the cells alive, introducing fluorescent probes, and imaging through thick, hazy cytoplasm. This article outlines the major tissue types amenable to study by time-lapse cinematography and different methods for keeping the cells alive. It describes various imaging and associated techniques best suited to following changes in the distribution of fluorescently labeled molecules in real time in these tissues. Imaging, in general, is a rapidly developing discipline, and recent advances in imaging technology are able to greatly extend what can be achieved with live cell imaging of Drosophila tissues. As far as possible, this article includes the latest technical developments and discusses likely future developments in imaging methods that could have an impact on research using Drosophila.

  2. Tendon injuries

    PubMed Central

    Wu, Fan; Nerlich, Michael; Docheva, Denitsa

    2017-01-01

    Tendons connect muscles to bones, ensuring joint movement. With advanced age, tendons become more prone to degeneration followed by injuries. Tendon repair often requires lengthy periods of rehabilitation, especially in elderly patients. Existing medical and surgical treatments often fail to regain full tendon function. The development of novel treatment methods has been hampered due to limited understanding of basic tendon biology. Recently, it was discovered that tendons, similar to other mesenchymal tissues, contain tendon stem/progenitor cells (TSPCs) which possess the common stem cell properties. The current strategies for enhancing tendon repair consist mainly of applying stem cells, growth factors, natural and artificial biomaterials alone or in combination. In this review, we summarise the basic biology of tendon tissues and provide an update on the latest repair proposals for tendon tears. Cite this article: EFORT Open Rev 2017;2:332-342. DOI: 10.1302/2058-5241.2.160075 PMID:28828182

  3. The effects of high glucose on tendon-derived stem cells: implications of the pathogenesis of diabetic tendon disorders.

    PubMed

    Lin, Yu-Cheng; Li, Ying-Juan; Rui, Yun-Feng; Dai, Guang-Chun; Shi, Liu; Xu, Hong-Liang; Ni, Ming; Zhao, Song; Chen, Hui; Wang, Chen; Li, Gang; Teng, Gao-Jun

    2017-03-14

    Patients with diabetes are at great risk to suffer many musculoskeletal disorders, such as tendinopathy, tendon rupture and impaired tendon healing. However, the pathogenesis of these tendon disorders still remains unclear. In this study, we aimed to investigate the effects of high glucose on cell proliferation, cell apoptosis and tendon-related markers expression of tendon-derived stem cells (TDSCs) in vitro. These findings might provide new insights into the pathogenesis of diabetic tendon disorders. The cell proliferative ability and apoptosis rate of TDSCs in different groups were evaluated by MTT assay and Annexin V-FITC/PI staining assay. The mRNA expression of tendon-related markers (Scleraxis and Collagen I alpha 1 chain) were assessed by qRT-PCR. The protein expression of tendon-related markers (Tenomodulin and Collagen I) were measured by Western blotting. The proliferative ability of TDSCs treated with high glucose (15mM and 25mM) decreased significantly at day1, day3 and day5. The cell apoptosis of TDSCs increased significantly when they were cultured with high glucose for 48h in vitro. The gene expression of Scleraxis and Collagen I alpha 1 chain in TDSCs decreased significantly when they were treated with high glucose for 24h and 48h. The protein expression of Tenomodulin and Collagen I in TDSCs decreased significantly when they were treated with high glucose for 24h and 48h. High glucose could inhibit cell proliferation, induce cell apoptosis and suppress the tendon-related markers expression of TDSCs in vitro. These findings might account for some pathological mechanisms underlying the pathogenesis of diabetic tendon disorders.

  4. Human iPSC-Derived Neural Crest Stem Cells Promote Tendon Repair in a Rat Patellar Tendon Window Defect Model

    PubMed Central

    Xu, Wei; Wang, Yequan; Liu, Erfu; Sun, Yanjun; Luo, Ziwei; Xu, Zhiling; Liu, Wanqian; Zhong, Li; Lv, Yonggang; Wang, Aijun; Tang, Zhenyu; Li, Song

    2013-01-01

    Induced pluripotent stem cells (iPSCs) hold great potential for cell therapy and tissue engineering. Neural crest stem cells (NCSCs) are multipotent that are capable of differentiating into mesenchymal lineages. In this study, we investigated whether iPSC-derived NCSCs (iPSC-NCSCs) have potential for tendon repair. Human iPSC-NCSCs were suspended in fibrin gel and transplanted into a rat patellar tendon window defect. At 4 weeks post-transplantation, macroscopical observation showed that the repair of iPSC-NCSC-treated tendons was superior to that of non-iPSC-NCSC-treated tendons. Histological and mechanical examinations revealed that iPSC-NCSCs treatment significantly enhanced tendon healing as indicated by the improvement in matrix synthesis and mechanical properties. Furthermore, transplanted iPSC-NCSCs produced fetal tendon-related matrix proteins, stem cell recruitment factors, and tenogenic differentiation factors, and accelerated the host endogenous repair process. This study demonstrates a potential strategy of employing iPSC-derived NCSCs for tendon tissue engineering. PMID:23815150

  5. A comparison of tenocytes and mesenchymal stem cells for use in flexor tendon tissue engineering.

    PubMed

    Kryger, Gil S; Chong, Alphonsus K S; Costa, Melinda; Pham, Hung; Bates, Steven J; Chang, James

    2007-01-01

    Tissue-engineered tendon grafts will meet an important clinical need. To engineer tendons, we used acellularized allogeneic tendon as scaffold material. To determine the ideal cell type to seed the scaffolds, we studied in vitro characteristics of epitenon tenocytes, tendon sheath fibroblasts, bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived mesenchymal stem cells (ASCs). Subsequently, we implanted reseeded acellularized tendons in vivo as flexor tendon grafts. Tenocytes, sheath fibroblasts, BMSCs, and ASCs were obtained from adult rabbits. For all cell lines, collagen 1, 2, and 3 immunocytochemistry was performed, and proliferation was assessed by hemacytometry and senescence by beta-galactosidase staining. Flexor tendons were acellularized after harvest. Tendons were assessed by histology after in vitro reseeding with each of the cell types after 1, 4, and 8 weeks. Finally, reseeded tendons and controls were implanted in a flexor profundus tendon defect. After 6 weeks, the reseeded tendons were harvested and assessed by histology. Statistical analysis for cell proliferation was performed using analysis of variance and t-tests with Bonferroni correction. All cell types had similar collagen expression. Cell proliferation was higher in ASCs in late passage compared with early passage and in ASCs compared with epitenon tenocytes at late passage. The other cell types were similar in growth characteristics. No senescence was detected. In vitro assessment of reseeded constructs showed the presence of cells on the construct surface. In vivo assessment after implantation showed viable cells seen within the tendon architecture in all cell types. This study suggests that the four cell types may be successfully used to engineer tendons. Adipoderived mesenchymal stem cells proliferate faster in cell culture, but the cell types were similar in other respects. All could be used to successfully repopulate acellularized tendon in vivo as flexor tendon grafts.

  6. Tendon cell outgrowth rates and morphology associated with kevlar-49.

    PubMed

    Zimmerman, M; Gordon, K E

    1988-12-01

    A rat tendon cell model was used to evaluate the in vitro biocompatibility of kevlar-49. The cell response to kevlar was compared to carbon AS-4 and nylon sutures. Three trials were run and cell growth rates were statistically similar for all the materials tested. A separate experiment was conducted in which the same fiber materials were placed in the same Petri dish. Again, the rates were similar for each material. Finally, the cells were observed with a scanning electron microscope, and the three classic cell morphologies associated with this tendon cell model were observed. Also, cellular attachment to the fiber and cellular encapsulation of the fiber were identical for the three materials tested. Kevlar-49 proved to be comparable to carbon AS4 and nylon sutures in terms of cellular response and cell outgrowth rates.

  7. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  8. Stem cells in the Drosophila digestive system.

    PubMed

    Zeng, Xiankun; Chauhan, Chhavi; Hou, Steven X

    2013-01-01

    Adult stem cells maintain tissue homeostasis by continuously replenishing damaged, aged and dead cells in any organism. Five types of region and organ-specific multipotent adult stem cells have been identified in the Drosophila digestive system: intestinal stem cells (ISCs) in the posterior midgut; hindgut intestinal stem cells (HISCs) at the midgut/hindgut junction; renal and nephric stem cells (RNSCs) in the Malpighian Tubules; type I gastric stem cells (GaSCs) at foregut/midgut junction; and type II gastric stem cells (GSSCs) at the middle of the midgut. Despite the fact that each type of stem cell is unique to a particular organ, they share common molecular markers and some regulatory signaling pathways. Due to the simpler tissue structure, ease of performing genetic analysis, and availability of abundant mutants, Drosophila serves as an elegant and powerful model system to study complex stem cell biology. The recent discoveries, particularly in the Drosophila ISC system, have greatly advanced our understanding of stem cell self-renewal, differentiation, and the role of stem cells play in tissue homeostasis/regeneration and adaptive tissue growth.

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

    SciTech Connect

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

    2014-07-18

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

  10. Repopulation of Intrasynovial Flexor Tendon Allograft with Bone Marrow Stromal Cells: An Ex Vivo Model

    PubMed Central

    Amadio, Peter C.; Thoreson, Andrew R.; An, Kai-Nan

    2014-01-01

    Purpose: Delayed healing is a common problem whenever tendon allografts are used for tendon or ligament reconstruction. Repopulating the allograft with host cells may accelerate tendon regeneration, but cell penetration into the allograft tendon is limited. Processing the tendon surface with slits that guide cells into the allograft substrate may improve healing. The purpose of this study was to describe a surface modification of allograft tendon that includes slits to aid cell repopulation and lubrication to enhance tendon gliding. Methods: Canine flexor digitorum profundus tendons were used for this study. Cyclic gliding resistance was measured over 1000 cycles. Tensile stiffness was assessed for normal tendon, tendon decellularized with trypsin and Triton X-100 (decellularized group), tendon decellularized and perforated with multiple slits (MS group) and tendon decellularized, perforated with slits and treated with a carbodiimide-derivatized hyaluronic acid and gelatin (cd-HA-gelatin) surface modification (MS-SM group). To assess tendon repopulation, bone marrow stromal cells (BMSCs) were used in the decellularized and MS groups. DNA concentration and histology were evaluated and compared to normal tendons and nonseeded decellularized tendons. Results: The gliding resistance of the decellularized and MS groups was significantly higher compared with the normal group. There was no significant difference in gliding resistance between the decellularized and MS group. Gliding resistance of the normal group and MS-SM group was not significantly different. The Young's modulus was not significantly different among the four groups. The DNA concentration in the MS group was significantly lower than in normal tendons, but significantly higher than in decellularized tendons, with or without BMSCs. Viable BMSCs were found in the slits after 2 weeks in tissue culture. Conclusions: Tendon slits can successfully harbor BMSCs without compromising their survival and without

  11. Flexor tendon tissue engineering: temporal distribution of donor tenocytes versus recipient cells.

    PubMed

    Thorfinn, Johan; Saber, Sepideh; Angelidis, Ioannis K; Ki, Sae H; Zhang, Andrew Y; Chong, Alphonsus K; Pham, Hung M; Lee, Gordon K; Chang, James

    2009-12-01

    Tissue-engineered tendon material may address tendon shortages in mutilating hand injuries. Tenocytes from rabbit flexor tendon can be successfully seeded onto acellularized tendons that are used as tendon constructs. These constructs in vivo exhibit a population of tenocyte-like cells; however, it is not known to what extent these cells are of donor or recipient origin. Furthermore, the temporal distribution is also not known. Tenocytes from New Zealand male rabbits were cultured and seeded onto acellularized rabbit forepaw flexor tendons (n = 48). These tendon constructs were transplanted into female recipients. Tendons were examined after 3, 6, 12, and 30 weeks using fluorescent in situ hybridization to detect the Y chromosome in the male donor cells. One unseeded, acellularized allograft in each animal was used as a control. The donor male tenocytes populate the epitenon and endotenon of the grafts at greater numbers than the recipient female tenocytes at 3 and 6 weeks. The donor and recipient tenocytes are present jointly in the grafts until 12 weeks. At 30 weeks, nearly all cells are recipient tenocyte-like cells. Donor male cells survive in decreasing numbers over time until 30 weeks. The presence of cells in tissue-engineered tendon grafts has been shown in prior studies to add to the strength of the constructs in vitro. This study shows that recipient cells can migrate into and repopulate the tendon construct. Cell seeding onto tendon material may create stronger constructs that will allow the initiation of motion earlier.

  12. Determination of Blastoderm Cells in Drosophila melanogaster

    PubMed Central

    Chan, L.-N.; Gehring, W.

    1971-01-01

    A method for culturing blastoderm cells of Drosophila in vivo has been developed that allows these cells to differentiate into larval or adult structures. By intermixture of genetically marked cells from bisected and whole embryos, it was shown that blastoderm cells are restricted in their potential for forming adult epidermal structures. Cells isolated from anterior-half embryos are determined for forming head and thoracic structures, whereas cells from posterior-half embryos are determined for forming thoracic and abdominal structures. The specificity of determination and the localization of determinative factors is discussed. Images PMID:5002429

  13. Proliferative control in Drosophila stem cells.

    PubMed

    Kohlmaier, Alexander; Edgar, Bruce A

    2008-12-01

    The relationship between cell growth (cell mass increase over time) and cell division is poorly understood in animal stem cells. Recent studies in several Drosophila stem cell types have provided the tools to interrogate this relationship. In several cases (brat, mei-P26, pros, bam, lethal giant larvae, polo), mutations have been defined that trigger tumorous overproliferation of progenitor cells and reveal how unrestricted self-renewing capacity is controlled. Moreover, microRNAs have been discovered as essential regulators of stem cell division rate and identity, suggesting that stem cell self-renewal depends on protein translational control. Biosynthetic capacity has also been found to be limiting for stem cell division rates. Finally, asymmetric cell division can impose dominant differentiation signals in a stem cell's daughter, and this can inhibit the stem cell-specific proliferation signature and lock in cell cycle exit.

  14. Effects of Lubricant and Autologous Bone Marrow Stromal Cell Augmentation on Immobilized Flexor Tendon Repairs

    PubMed Central

    Zhao, Chunfeng; Ozasa, Yasuhiro; Shimura, Haruhiko; Reisdorf, Ramona L.; Thoreson, Andrew R.; Jay, Gregory; Moran, Steven L.; An, Kai-Nan; Amadio, Peter C.

    2016-01-01

    The purpose of the study was to test a novel treatment that carbodiimide-derivatized-hyaluronic acid-lubricin (cd-HA-lubricin) combined cell-based therapy in an immobilized flexor tendon repair in a canine model. Seventy-eight flexor tendons from 39 dogs were transected. One tendon was treated with cd-HA-lubricin plus an interpositional graft of 8 × 105 BMSCs and GDF-5. The other tendon was repaired without treatment. After 21 day of immobilization, 19 dogs were sacrificed; the remaining 20 dogs underwent a 21-day rehabilitation protocol before euthanasia. The work of flexion, tendon gliding resistance, and adhesion score in treated tendons were significantly less than the untreated tendons (p < 0.05). The failure strength of the untreated tendons was higher than the treated tendons at 21 and 42 days (p < 0.05). However, there is no significant difference in stiffness between two groups at day 42. Histologic analysis of treated tendons showed a smooth surface and viable transplanted cells 42 days after the repair, whereas untreated tendons showed severe adhesion formation around the repair site. The combination of lubricant and cell treatment resulted in significantly improved digit function, reduced adhesion formation. This novel treatment can address the unmet needs of patients who are unable to commence an early mobilization protocol after flexor tendon repair. PMID:26177854

  15. Therapeutic strategies for tendon healing based on novel biomaterials, factors and cells.

    PubMed

    Gross, Gerhard; Hoffmann, Andrea

    2013-01-01

    The repair of tendon injuries still presents a major clinical challenge to orthopedic medicine. Tendons, like some other tissues, are poorly vascularized and heal slowly. In addition, healing often leads to the formation of fibrous tissue and scar tissue which lack flexibility and biomechanical properties. So the treatment of tendon injuries is challenging. We give an overview of the structure and composition of tendons, pathological states of tendon and natural healing, as well as therapeutic options. We focus in particular on biomaterials that have been specifically developed or suggested for the successful repair of tendon injuries. In addition, we also review factor- and cell-dependent strategies to heal tendon and ligament disorders. Although brief, we hope that this review will be helpful, particularly for those readers who are new to the field of tendon tissue engineering. Copyright © 2013 S. Karger AG, Basel.

  16. Altered Fate of Tendon-Derived Stem Cells Isolated from a Failed Tendon-Healing Animal Model of Tendinopathy

    PubMed Central

    Rui, Yun Feng; Wong, Yin Mei; Tan, Qi; Chan, Kai Ming

    2013-01-01

    We hypothesized that altered fate of tendon-derived stem cells (TDSCs) might contribute to chondro-ossification and failed healing in the collagenase-induced (CI) tendon injury model. This study aimed to compare the yield, proliferative capacity, immunophenotypes, senescence, and differentiation potential of TDSCs isolated from healthy (HT) and CI tendons. TDSCs were isolated from CI and healthy Sprague-Dawley rat patellar tendons. The yield, proliferative capacity, immunophenotypes, and senescence of TDSCs (CI) and TDSCs (HT) were compared by colony-forming unit assay, BrdU assay, flow cytometry, and β-galactosidase activity assay, respectively. Their osteogenic and chondrogenic differentiation potentials and mRNA expression of tendon-related markers were compared using standard assays. More TDSCs, which showed a lower proliferative potential and a higher cellular senescence were present in the CI patellar tendons compared to HT tendons. There was a higher alkaline phosphatase activity and mineralization in TDSCs (CI) in both basal and osteogenic media. More chondrocyte-like cells and higher proteoglycan deposition, Sox9 and collagen type II expression were observed in TDSCs (CI) pellets upon chondrogenic induction. There was a higher protein expression of Sox9, but a lower mRNA expression of Col1a1, Scx, and Tnmd in TDSCs (CI) in a basal medium. In conclusion, TDSCs (CI) showed altered fate, a higher cellular senescence, but a lower proliferative capacity compared to TDSCs (HT), which might contribute to pathological chondro-ossification and failed tendon healing in this animal model. PMID:23106341

  17. Uncovering the cellular and molecular changes in tendon stem/progenitor cells attributed to tendon aging and degeneration.

    PubMed

    Kohler, Julia; Popov, Cvetan; Klotz, Barbara; Alberton, Paolo; Prall, Wolf Christian; Haasters, Florian; Müller-Deubert, Sigrid; Ebert, Regina; Klein-Hitpass, Ludger; Jakob, Franz; Schieker, Matthias; Docheva, Denitsa

    2013-12-01

    Although the link between altered stem cell properties and tissue aging has been recognized, the molecular and cellular processes of tendon aging have not been elucidated. As tendons contain stem/progenitor cells (TSPC), we investigated whether the molecular and cellular attributes of TSPC alter during tendon aging and degeneration. Comparing TSPC derived from young/healthy (Y-TSPC) and aged/degenerated human Achilles tendon biopsies (A-TSPC), we observed that A-TSPC exhibit a profound self-renewal and clonogenic deficits, while their multipotency was still retained. Senescence analysis showed a premature entry into senescence of the A-TSPC, a finding accompanied by an upregulation of p16(INK4A). To identify age-related molecular factors, we performed microarray and gene ontology analyses. These analyses revealed an intriguing transcriptomal shift in A-TSPC, where the most differentially expressed probesets encode for genes regulating cell adhesion, migration, and actin cytoskeleton. Time-lapse analysis showed that A-TSPC exhibit decelerated motion and delayed wound closure concomitant to a higher actin stress fiber content and a slower turnover of actin filaments. Lastly, based on the expression analyses of microarray candidates, we suggest that dysregulated cell-matrix interactions and the ROCK kinase pathway might be key players in TSPC aging. Taken together, we propose that during tendon aging and degeneration, the TSPC pool is becoming exhausted in terms of size and functional fitness. Thus, our study provides the first fundamental basis for further exploration into the molecular mechanisms behind tendon aging and degeneration as well as for the selection of novel tendon-specific therapeutical targets. © 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  18. LRT, a tendon-specific leucine-rich repeat protein, promotes muscle-tendon targeting through its interaction with Robo.

    PubMed

    Wayburn, Bess; Volk, Talila

    2009-11-01

    Correct muscle migration towards tendon cells, and the adhesion of these two cell types, form the basis for contractile tissue assembly in the Drosophila embryo. While molecules promoting the attraction of muscles towards tendon cells have been described, signals involved in the arrest of muscle migration following the arrival of myotubes at their corresponding tendon cells have yet to be elucidated. Here, we describe a novel tendon-specific transmembrane protein, which we named LRT due to the presence of a leucine-rich repeat domain (LRR) in its extracellular region. Our analysis suggests that LRT acts non-autonomously to better target the muscle and/or arrest its migration upon arrival at its corresponding tendon cell. Muscles in embryos lacking LRT exhibited continuous formation of membrane extensions despite arrival at their corresponding tendon cells, and a partial failure of muscles to target their correct tendon cells. In addition, overexpression of LRT in tendon cells often stalled muscles located close to the tendon cells. LRT formed a protein complex with Robo, and we detected a functional genetic interaction between Robo and LRT at the level of muscle migration behavior. Taken together, our data suggest a novel mechanism by which muscles are targeted towards tendon cells as a result of LRT-Robo interactions. This mechanism may apply to the Robo-dependent migration of a wide variety of cell types.

  19. Multipotent mesenchymal stem cells from human subacromial bursa: potential for cell based tendon tissue engineering.

    PubMed

    Song, Na; Armstrong, April D; Li, Feng; Ouyang, Hongsheng; Niyibizi, Christopher

    2014-01-01

    Rotator cuff injuries are a common clinical problem either as a result of overuse or aging. Biological approaches to tendon repair that involve use of scaffolding materials or cell-based approaches are currently being investigated. The cell-based approaches are focused on applying multipotent mesenchymal stem cells (MSCs) mostly harvested from bone marrow. In the present study, we focused on characterizing cells harvested from tissues associated with rotator cuff tendons based on an assumption that these cells would be more appropriate for tendon repair. We isolated MSCs from bursa tissue associated with rotator cuff tendons and characterized them for multilineage differentiation in vitro and in vivo. Human bursa was obtained from patients undergoing rotator cuff surgery and cells within were isolated using collagenase and dispase digestion. The cells isolated from the tissues were characterized for osteoblastic, adipogenic, chondrogenic, and tenogenic differentiation in vitro and in vivo. The results showed that the cells isolated from bursa tissue exhibited MSCs characteristics as evidenced by the expression of putative cell surface markers attributed to MSCs. The cells exhibited high proliferative capacity and differentiated toward cells of mesenchymal lineages with high efficiency. Bursa-derived cells expressed markers of tenocytes when treated with bone morphogenetic protein-12 (BMP-12) and assumed aligned morphology in culture. Bursa cells pretreated with BMP-12 and seeded in ceramic scaffolds formed extensive bone, as well as tendon-like tissue in vivo. Bone formation was demonstrated by histological analysis and immunofluorescence for DMP-1 in tissue sections made from the scaffolds seeded with the cells. Tendon-like tissue formed in vivo consisted of parallel collagen fibres typical of tendon tissues. Bursa-derived cells also formed a fibrocartilagenous tissue in the ceramic scaffolds. Taken together, the results demonstrate a new source of MSCs with a

  20. Therapeutic Roles of Tendon Stem/Progenitor Cells in Tendinopathy

    PubMed Central

    Zhang, Xin; Lin, Yu-cheng; Rui, Yun-feng; Xu, Hong-liang; Chen, Hui; Wang, Chen; Teng, Gao-jun

    2016-01-01

    Tendinopathy is a tendon disorder characterized by activity-related pain, local edema, focal tenderness to palpation, and decreased strength in the affected area. Tendinopathy is prevalent in both athletes and the general population, highlighting the need to elucidate the pathogenesis of this disorder. Current treatments of tendinopathy are both conservative and symptomatic. The discovery of tendon stem/progenitor cells (TSPCs) and erroneous differentiation of TSPCs have provided new insights into the pathogenesis of tendinopathy. In this review, we firstly present the histopathological characteristics of tendinopathy and explore the cellular and molecular cues in the pathogenesis of tendinopathy. Current evidence of the depletion of the stem cell pool and altered TSPCs fate in the pathogenesis of tendinopathy has been presented. The potential regulatory factors for either tenogenic or nontenogenic differentiation of TSPCs are also summarized. The regulation of endogenous TSPCs or supplementation with exogenous TSPCs as therapeutic targets for the treatment of tendinopathy is proposed. Therefore, inhibiting the erroneous differentiation of TSPCs and regulating the differentiation of TSPCs into tendon cells might be important areas of future research and could provide new clinical treatments for tendinopathy. The current evidence suggests that TSPCs are promising therapeutic targets for the management of tendinopathy. PMID:27195010

  1. Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue

    PubMed Central

    Chen, Yu-Ying; He, Sheng-Teng; Yan, Fu-Hua; Zhou, Peng-Fei; Luo, Kai; Zhang, Yan-Ding; Xiao, Yin; Lin, Min-Kui

    2016-01-01

    Postnatal mesenchymal stem cells have the capacity to differentiate into multiple cell lineages. This study explored the possibility of dental pulp stem cells (DPSCs) for potential application in tendon tissue engineering. The expression of tendon-related markers such as scleraxis, tenascin-C, tenomodulin, eye absent homologue 2, collagens I and VI was detected in dental pulp tissue. Interestingly, under mechanical stimulation, these tendon-related markers were significantly enhanced when DPSCs were seeded in aligned polyglycolic acid (PGA) fibre scaffolds. Furthermore, mature tendon-like tissue was formed after transplantation of DPSC-PGA constructs under mechanical loading conditions in a mouse model. This study demonstrates that DPSCs could be a potential stem cell source for tissue engineering of tendon-like tissue. PMID:27811845

  2. Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue.

    PubMed

    Chen, Yu-Ying; He, Sheng-Teng; Yan, Fu-Hua; Zhou, Peng-Fei; Luo, Kai; Zhang, Yan-Ding; Xiao, Yin; Lin, Min-Kui

    2016-12-16

    Postnatal mesenchymal stem cells have the capacity to differentiate into multiple cell lineages. This study explored the possibility of dental pulp stem cells (DPSCs) for potential application in tendon tissue engineering. The expression of tendon-related markers such as scleraxis, tenascin-C, tenomodulin, eye absent homologue 2, collagens I and VI was detected in dental pulp tissue. Interestingly, under mechanical stimulation, these tendon-related markers were significantly enhanced when DPSCs were seeded in aligned polyglycolic acid (PGA) fibre scaffolds. Furthermore, mature tendon-like tissue was formed after transplantation of DPSC-PGA constructs under mechanical loading conditions in a mouse model. This study demonstrates that DPSCs could be a potential stem cell source for tissue engineering of tendon-like tissue.

  3. Do Cells Contribute to Tendon and Ligament Biomechanics?

    PubMed Central

    Hammer, Niels; Huster, Daniel; Fritsch, Sebastian; Hädrich, Carsten; Koch, Holger; Schmidt, Peter; Sichting, Freddy; Wagner, Martin Franz-Xaver; Boldt, Andreas

    2014-01-01

    Introduction Acellular scaffolds are increasingly used for the surgical repair of tendon injury and ligament tears. Despite this increased use, very little data exist directly comparing acellular scaffolds and their native counterparts. Such a comparison would help establish the effectiveness of the acellularization procedure of human tissues. Furthermore, such a comparison would help estimate the influence of cells in ligament and tendon stability and give insight into the effects of acellularization on collagen. Material and Methods Eighteen human iliotibial tract samples were obtained from nine body donors. Nine samples were acellularized with sodium dodecyl sulphate (SDS), while nine counterparts from the same donors remained in the native condition. The ends of all samples were plastinated to minimize material slippage. Their water content was adjusted to 69%, using the osmotic stress technique to exclude water content-related alterations of the mechanical properties. Uniaxial tensile testing was performed to obtain the elastic modulus, ultimate stress and maximum strain. The effectiveness of the acellularization procedure was histologically verified by means of a DNA assay. Results The histology samples showed a complete removal of the cells, an extensive, yet incomplete removal of the DNA content and alterations to the extracellular collagen. Tensile properties of the tract samples such as elastic modulus and ultimate stress were unaffected by acellularization with the exception of maximum strain. Discussion The data indicate that cells influence the mechanical properties of ligaments and tendons in vitro to a negligible extent. Moreover, acellularization with SDS alters material properties to a minor extent, indicating that this method provides a biomechanical match in ligament and tendon reconstruction. However, the given protocol insufficiently removes DNA. This may increase the potential for transplant rejection when acellular tract scaffolds are used in

  4. Platelet-rich plasma activates tendon-derived stem cells to promote regeneration of Achilles tendon rupture in rats.

    PubMed

    Xu, Kang; Al-Ani, Mohanad Kh; Sun, Yanjun; Xu, Wei; Pan, Lianhong; Song, Yang; Xu, ZhiLing; Pan, Xin; Yang, Li

    2017-04-01

    This study investigates whether platelet-rich plasma (PRP) is an activator of tendon-derived stem cells (TDSCs) to promote regeneration of Achilles tendon post-rupture in rats. In the in vitro study, PRGF (activated PRP) significantly enhanced cell DNA synthesis, improved viability and promoted proliferation, while facilitating cell migration and the recruitment of TDSCs. In addition, TDSCs were mixed with collagen and PRP to form collagen-TDSC constructs (CTC) and PRP-collagen-TDSC constructs (PCTCs). After 3 weeks of culture in vitro, we found that most of the encapsulated TDSCs in the CTCs and PCTCs were still alive, while cells in the PCTCs showed a more aligned arrangement compared to the CTCs. In addition, the micro-structure of PCTC showed more obvious fibre-like tissues and formed a cyclic microvascular structure. The tenocyte-related genes types I and III collagen, Tenascin-C and Scleraxis of TDSCs in the PCTCs and CTCs were upregulated with time, and PCTCs showed more significance than CTCs (p < 0.05). After in vivo transplantation, the CTCs and PCTCs showed stimulatory effects on Achilles tendon healing. Moreover, the PCTCs improved the macroscopic appearance, histological morphology and biomechanical strength of ruptured Achilles tendon better than CTC. These results indicate that PRP can activate TDSCs to improve the quality of Achilles tendon rupture healing in the early stages. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  5. A practical guide for the isolation and maintenance of stem cells from tendon.

    PubMed

    Lui, Pauline Po Yee

    2015-01-01

    Stem cells are unspecialized cells that can self-renew and have the ability to develop into cells of highly specialized functions. The study of stem cells holds enormous promise in the medical field ranging from their uses in cell therapies to their uses for greater understanding of tissue development and disease pathologies. Stem cells have been isolated from tendon tissue recently. These tendon-derived stem cells (TDSCs) are particularly relevant for tendon repair and the study of the potential roles of stem cells in tendon pathology as they are isolated from tendon tissues. This paper aims to describe the step-by-step protocol and the practical tips for the isolation and verification of stem cell characteristics of TDSCs. The cell seeding density and hence cell-cell contact has a significant impact on the isolation and expansion of TDSCs. Hence, I also describe our established protocol for the determination of the optimal seeding density for TDSC isolation and culture.

  6. Production of PGE(2) increases in tendons subjected to repetitive mechanical loading and induces differentiation of tendon stem cells into non-tenocytes.

    PubMed

    Zhang, Jianying; Wang, James H-C

    2010-02-01

    Whether tendon inflammation is involved in the development of tendinopathy or degenerative changes of the tendon remains a matter of debate. We explored this question by performing animal and cell culture experiments to determine the production and effects of PGE(2), a major inflammatory mediator in tendons. Mouse tendons were subjected to repetitive mechanical loading via treadmill running, and the effect of PGE(2) on proliferation and differentiation of tendon stem cells (TSCs) was assessed in vitro. Compared to levels in cage control mice, PGE(2) levels in mouse patellar and Achilles tendons were markedly increased in response to a bout of rigorous treadmill running. PGE(2) treatment of TSCs in culture decreased cell proliferation and induced both adipogenesis and osteogenesis of TSCs, as evidenced by accumulation of lipid droplets and calcium deposits, respectively. Effects of PGE(2) on both TSC proliferation and differentiation were apparently PGE(2)-dose-dependent. These findings suggest that high levels of PGE(2), which are present in tendons subjected to repetitive mechanical loading conditions in vivo as shown in this study, may result in degenerative changes of the tendon by decreasing proliferation of TSCs in tendons and also inducing differentiation of TSCs into adipocytes and osteocytes. The consequences of this PGE(2) effect on TSCs is the reduction of the pool of tenocytes for repair of tendons injured by mechanical loading, and production of fatty and calcified tissues within the tendon, often seen at the later stages of tendinopathy.

  7. Endoscopic Resection of Giant Cell Tumor of the Extensor Tendon of the Foot.

    PubMed

    Lui, Tun Hing

    2017-04-01

    The localized form of giant cell tumor of the tendon sheath is one of the most common soft tissue tumors of the foot and ankle region. It is characteristically a benign, sharply localized peritendinous fibrous mass in the synovial or tendinous spaces. The purpose of this Technical Note is to present the technical details of endoscopic resection of giant cell tumor of the extensor tendon of the foot with preservation of the tendon. It is indicated in the localized form of giant cell tumor of the extensor tendon at the foot dorsum. It is contraindicated in cases with a diffuse giant cell tumor, a giant cell tumor of the extensor tendon at the phalangeal level, or involvement of the flexor tendon.

  8. Cell response to sterilized electrospun poly(ɛ-caprolactone) scaffolds to aid tendon regeneration in vivo.

    PubMed

    Bhaskar, Prajwal; Bosworth, Lucy A; Wong, Richard; O'brien, Marie A; Kriel, Haydn; Smit, Eugene; McGrouther, Duncan A; Wong, Jason K; Cartmell, Sarah H

    2017-02-01

    The functional replacement of tendon represents an unmet clinical need in situations of tendon rupture, tendon grafting, and complex tendon reconstruction, as usually there is a finite source of healthy tendon to use as donors. The microfibrous architecture of tendon is critical to the function of tendon. This study investigates the use of electrospun poly(ɛ-caprolactone) scaffolds as potential biomaterial substitutes for tendon grafts. We assessed the performance of two electrospinning manufacturers (small- and large-scale) and the effect of two sterilization techniques-gamma irradiation and ethanol submersion-on cell response to these electrospun scaffolds after their implantation into a murine tendon model. Cell infiltration and proliferation analyses were undertaken to determine the effect on cell response within the implant over a 6-week period. Immunohistochemical analysis was performed to characterize inflammatory response and healing characteristics (proliferation, collagen deposition, myofibroblast activity, and apoptosis). Neither the sterilization techniques nor the manufacturer was observed to significantly affect the cell response to the scaffold. At each time point, cell response was similar to the autograft control. This suggests that ethanol submersion can be used for research purposes and that the scaffold can be easily reproduced by a large-scale manufacturer. These results further imply that this electrospun scaffold may provide an alternative to autograft, thus eliminating the need for sourcing healthy tendon tissue from a secondary site. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 389-397, 2017.

  9. Lovastatin-Mediated Changes in Human Tendon Cells.

    PubMed

    Kuzma-Kuzniarska, Maria; Cornell, Hannah R; Moneke, Michael C; Carr, Andrew J; Hulley, Philippa A

    2015-10-01

    Statins are among the most widely prescribed drugs worldwide. Numerous studies have shown their beneficial effects in prevention of cardiovascular disease through cholesterol-lowering and anti-atherosclerotic properties. Although some statin patients may experience muscle-related symptoms, severe side effects of statin therapy are rare, primarily due to extensive first-pass metabolism in the liver. Skeletal muscles appear to be the main site of side effects; however, recently some statin-related adverse effects have been described in tendon. The mechanism behind these side effects remains unknown. This is the first study that explores tendon-specific effects of statins in human primary tenocytes. The cells were cultured with different concentrations of lovastatin for up to 1 week. No changes in cell viability or morphology were observed in tenocytes incubated with therapeutic doses. Short-term exposure to lovastatin concentrations outside the therapeutic range had no effect on tenocyte viability; however, cell migration was reduced. Simvastatin and atorvastatin, two other drug family members, also reduced the migratory properties of the cells. Prolonged exposure to high concentrations of lovastatin induced changes in cytoskeleton leading to cell rounding and decreased levels of mRNA for matrix proteins, but increased BMP-2 expression. Gap junctional communication was impaired but due to cell shape change and separation rather than direct gap junction inhibition. These effects were accompanied by inhibition of prenylation of Rap1a small GTPase. Collectively, we showed that statins in a dose-dependent manner decrease migration of human tendon cells, alter their expression profile and impair the functional network, but do not inhibit gap junction function.

  10. The revitalisation of flexor tendon allografts with bone marrow stromal cells and mechanical stimulation: An ex vivo model revitalising flexor tendon allografts.

    PubMed

    Wu, J H; Thoreson, A R; Gingery, A; An, K N; Moran, S L; Amadio, P C; Zhao, C

    2017-03-01

    The present study describes a novel technique for revitalising allogenic intrasynovial tendons by combining cell-based therapy and mechanical stimulation in an ex vivo canine model. Specifically, canine flexor digitorum profundus tendons were used for this study and were divided into the following groups: (1) untreated, unprocessed normal tendon; (2) decellularised tendon; (3) bone marrow stromal cell (BMSC)-seeded tendon; and (4) BMSC-seeded and cyclically stretched tendon. Lateral slits were introduced on the tendon to facilitate cell seeding. Tendons from all four study groups were distracted by a servohydraulic testing machine. Tensile force and displacement data were continuously recorded at a sample rate of 20 Hz until 200 Newton of force was reached. Before testing, the cross-sectional dimensions of each tendon were measured with a digital caliper. Young's modulus was calculated from the slope of the linear region of the stress-strain curve. The BMSCs were labeled for histological and cell viability evaluation on the decellularized tendon scaffold under a confocal microscope. Gene expression levels of selected extracellular matrix tendon growth factor genes were measured. Results were reported as mean ± SD and data was analyzed with one-way ANOVAs followed by Tukey's post hoc multiple-comparison test. We observed no significant difference in cross-sectional area or in Young's modulus among the four study groups. In addition, histological sections showed that the BMSCs were aligned well and viable on the tendon slices after two-week culture in groups three and four. Expression levels of several extracellular matrix tendon growth factors, including collagen type I, collagen type III, and matrix metalloproteinase were significantly higher in group four than in group three (p < 0.05). Lateral slits introduced into de-cellularised tendon is a promising method of delivery of BMSCs without compromising cell viability and tendon mechanical properties. In addition

  11. Giant cell tumour of tendon sheath with simultaneous two tendon involvement of the foot treated with excision of the tumour and reconstruction of the flexor retinaculum using tibialis posterior tendon in a paediatric patient: A rare case report.

    PubMed

    Tiwari, Vivek; Ansari, Tahir; Mittal, Samarth; Sharma, Pankaj; Nalwa, Aasma

    2015-12-01

    Giant cell tumour of tendon sheath is a benign soft tissue tumour arising from the tendon sheath. The involvement of foot and ankle by such tumours is relatively rare. Children are not commonly afflicted by this condition. All such tumours are reported to arise either from a single tendon sheath or one joint. We report a case of giant cell tumour of tendon sheath in a 12-year-old child, arising simultaneously from the tendon sheaths of tibialis posterior and flexor digitorum longus tendons, as well as extending into the ankle joint. It was treated by complete excision of the mass along with the tendon sheaths with reconstruction of the flexor retinaculum. The location of the tumour, age of the patient, diffuse nature of the tumour and novel technique of reconstruction of the flexor retinaculum make this case extremely rare and the first to be reported in literature.

  12. Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality

    PubMed Central

    Yin, Zi; Hu, Jia-jie; Yang, Long; Zheng, Ze-Feng; An, Cheng-rui; Wu, Bing-bing; Zhang, Can; Shen, Wei-Liang; Liu, Huan-huan; Chen, Jia-lin; Heng, Boon Chin; Guo, Guo-ji; Chen, Xiao; Ouyang, Hong-Wei

    2016-01-01

    The repair of injured tendons remains a formidable clinical challenge because of our limited understanding of tendon stem cells and the regulation of tenogenesis. With single-cell analysis to characterize the gene expression profiles of individual cells isolated from tendon tissue, a subpopulation of nestin+ tendon stem/progenitor cells (TSPCs) was identified within the tendon cell population. Using Gene Expression Omnibus datasets and immunofluorescence assays, we found that nestin expression was activated at specific stages of tendon development. Moreover, isolated nestin+ TSPCs exhibited superior tenogenic capacity compared to nestin− TSPCs. Knockdown of nestin expression in TSPCs suppressed their clonogenic capacity and reduced their tenogenic potential significantly both in vitro and in vivo. Hence, these findings provide new insights into the identification of subpopulations of TSPCs and illustrate the crucial roles of nestin in TSPC fate decisions and phenotype maintenance, which may assist in future therapeutic strategies to treat tendon disease. PMID:28138519

  13. Giant cell tumor of the flexor hallucis longus tendon sheath: a case study.

    PubMed

    Findling, Jeff; Lascola, Natalie K; Groner, Thomas W

    2011-01-01

    Giant cell tumor of tendon sheath is infrequently documented in the foot and even less near the ankle. This case report involves such a tumor of the flexor hallucis longus tendon presenting at the posterior ankle. Diagnosis was aided by magnetic resonance imaging, and treatment consisted of complete surgical excision. Pathologic examination verified the diagnosis of giant cell tumor of tendon sheath, and follow-up magnetic resonance imaging revealed no remnants or recurrence of tumor 1 year after surgery.

  14. Tendon injury: from biology to tendon repair.

    PubMed

    Nourissat, Geoffroy; Berenbaum, Francis; Duprez, Delphine

    2015-04-01

    Tendon is a crucial component of the musculoskeletal system. Tendons connect muscle to bone and transmit forces to produce motion. Chronic and acute tendon injuries are very common and result in considerable pain and disability. The management of tendon injuries remains a challenge for clinicians. Effective treatments for tendon injuries are lacking because the understanding of tendon biology lags behind that of the other components of the musculoskeletal system. Animal and cellular models have been developed to study tendon-cell differentiation and tendon repair following injury. These studies have highlighted specific growth factors and transcription factors involved in tenogenesis during developmental and repair processes. Mechanical factors also seem to be essential for tendon development, homeostasis and repair. Mechanical signals are transduced via molecular signalling pathways that trigger adaptive responses in the tendon. Understanding the links between the mechanical and biological parameters involved in tendon development, homeostasis and repair is prerequisite for the identification of effective treatments for chronic and acute tendon injuries.

  15. Tissue engineering of flexor tendons: the effect of a tissue bioreactor on adipoderived stem cell-seeded and fibroblast-seeded tendon constructs.

    PubMed

    Angelidis, Ioannis K; Thorfinn, Johan; Connolly, Ian D; Lindsey, Derek; Pham, Hung M; Chang, James

    2010-09-01

    Tissue-engineered flexor tendons could eventually be used for reconstruction of large tendon defects. The goal of this project was to examine the effect of a tissue bioreactor on the biomechanical properties of tendon constructs seeded with adipoderived stem cells (ASCs) and fibroblasts (Fs). Rabbit rear paw flexor tendons were acellularized and seeded with ASCs or Fs. A custom bioreactor applied a cyclic mechanical load of 1.25 N at 1 cycle/minute for 5 days onto the tendon constructs. Three additional groups were used as controls: fresh tendons and tendons reseeded with either ASCs or Fs that were not exposed to the bioreactor treatment and were left in stationary incubation for 5 days. We compared the ultimate tensile stress (UTS) and elastic modulus (EM) of bioreactor-treated tendons with the unloaded control tendons and fresh tendons. Comparison across groups was assessed using one-way analysis of variance with the significance level set at p<.05. Pairwise comparison between the samples was determined by using the Tukey test. The UTS and EM values of bioreactor-treated tendons that were exposed to cyclic load were significantly higher than those of unloaded control tendons. Acellularized tendon constructs that were reseeded with ASCs and exposed to a cyclic load had a UTS of 66.76 MPa and an EM of 906.68 MPa; their unloaded equivalents had a UTS of 47.90 MPa and an EM of 715.57 MPa. Similar trends were found in the fibroblast-seeded tendon constructs that were exposed to the bioreactor treatment. The bioreactor-treated tendons approached the UTS and EM values of fresh tendons. Histologically, we found that cells reoriented themselves parallel to the direction of strain in response to cyclic strain. The application of cyclic strain on seeded tendon constructs that were treated with the bioreactor helped achieve a UTS and an EM comparable with those of fresh tendons. Bioreactor pretreatment and alternative cell lines, such as ASCs and Fs, might therefore

  16. Lovastatin‐Mediated Changes in Human Tendon Cells

    PubMed Central

    Cornell, Hannah R.; Moneke, Michael C.; Carr, Andrew J.; Hulley, Philippa A.

    2015-01-01

    Statins are among the most widely prescribed drugs worldwide. Numerous studies have shown their beneficial effects in prevention of cardiovascular disease through cholesterol‐lowering and anti‐atherosclerotic properties. Although some statin patients may experience muscle‐related symptoms, severe side effects of statin therapy are rare, primarily due to extensive first‐pass metabolism in the liver. Skeletal muscles appear to be the main site of side effects; however, recently some statin‐related adverse effects have been described in tendon. The mechanism behind these side effects remains unknown. This is the first study that explores tendon‐specific effects of statins in human primary tenocytes. The cells were cultured with different concentrations of lovastatin for up to 1 week. No changes in cell viability or morphology were observed in tenocytes incubated with therapeutic doses. Short‐term exposure to lovastatin concentrations outside the therapeutic range had no effect on tenocyte viability; however, cell migration was reduced. Simvastatin and atorvastatin, two other drug family members, also reduced the migratory properties of the cells. Prolonged exposure to high concentrations of lovastatin induced changes in cytoskeleton leading to cell rounding and decreased levels of mRNA for matrix proteins, but increased BMP‐2 expression. Gap junctional communication was impaired but due to cell shape change and separation rather than direct gap junction inhibition. These effects were accompanied by inhibition of prenylation of Rap1a small GTPase. Collectively, we showed that statins in a dose‐dependent manner decrease migration of human tendon cells, alter their expression profile and impair the functional network, but do not inhibit gap junction function. J. Cell. Physiol. 230: 2543–2551, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:25846724

  17. Tendon Reattachment to Bone in an Ovine Tendon Defect Model of Retraction Using Allogenic and Xenogenic Demineralised Bone Matrix Incorporated with Mesenchymal Stem Cells.

    PubMed

    Thangarajah, Tanujan; Shahbazi, Shirin; Pendegrass, Catherine J; Lambert, Simon; Alexander, Susan; Blunn, Gordon W

    2016-01-01

    Tendon-bone healing following rotator cuff repairs is mainly impaired by poor tissue quality. Demineralised bone matrix promotes healing of the tendon-bone interface but its role in the treatment of tendon tears with retraction has not been investigated. We hypothesized that cortical demineralised bone matrix used with minimally manipulated mesenchymal stem cells will result in improved function and restoration of the tendon-bone interface with no difference between xenogenic and allogenic scaffolds. In an ovine model, the patellar tendon was detached from the tibial tuberosity and a complete distal tendon transverse defect measuring 1 cm was created. Suture anchors were used to reattach the tendon and xenogenic demineralised bone matrix + minimally manipulated mesenchymal stem cells (n = 5), or allogenic demineralised bone matrix + minimally manipulated mesenchymal stem cells (n = 5) were used to bridge the defect. Graft incorporation into the tendon and its effect on regeneration of the enthesis was assessed using histomorphometry. Force plate analysis was used to assess functional recovery. Compared to the xenograft, the allograft was associated with significantly higher functional weight bearing at 6 (P = 0.047), 9 (P = 0.028), and 12 weeks (P = 0.009). In the allogenic group this was accompanied by greater remodeling of the demineralised bone matrix into tendon-like tissue in the region of the defect (p = 0.015), and a more direct type of enthesis characterized by significantly more fibrocartilage (p = 0.039). No failures of tendon-bone healing were noted in either group. Demineralised bone matrix used with minimally manipulated mesenchymal stem cells promotes healing of the tendon-bone interface in an ovine model of acute tendon retraction, with superior mechanical and histological results associated with use of an allograft.

  18. Tendon Reattachment to Bone in an Ovine Tendon Defect Model of Retraction Using Allogenic and Xenogenic Demineralised Bone Matrix Incorporated with Mesenchymal Stem Cells

    PubMed Central

    2016-01-01

    Background Tendon-bone healing following rotator cuff repairs is mainly impaired by poor tissue quality. Demineralised bone matrix promotes healing of the tendon-bone interface but its role in the treatment of tendon tears with retraction has not been investigated. We hypothesized that cortical demineralised bone matrix used with minimally manipulated mesenchymal stem cells will result in improved function and restoration of the tendon-bone interface with no difference between xenogenic and allogenic scaffolds. Materials and Methods In an ovine model, the patellar tendon was detached from the tibial tuberosity and a complete distal tendon transverse defect measuring 1 cm was created. Suture anchors were used to reattach the tendon and xenogenic demineralised bone matrix + minimally manipulated mesenchymal stem cells (n = 5), or allogenic demineralised bone matrix + minimally manipulated mesenchymal stem cells (n = 5) were used to bridge the defect. Graft incorporation into the tendon and its effect on regeneration of the enthesis was assessed using histomorphometry. Force plate analysis was used to assess functional recovery. Results Compared to the xenograft, the allograft was associated with significantly higher functional weight bearing at 6 (P = 0.047), 9 (P = 0.028), and 12 weeks (P = 0.009). In the allogenic group this was accompanied by greater remodeling of the demineralised bone matrix into tendon-like tissue in the region of the defect (p = 0.015), and a more direct type of enthesis characterized by significantly more fibrocartilage (p = 0.039). No failures of tendon-bone healing were noted in either group. Conclusion Demineralised bone matrix used with minimally manipulated mesenchymal stem cells promotes healing of the tendon-bone interface in an ovine model of acute tendon retraction, with superior mechanical and histological results associated with use of an allograft. PMID:27606597

  19. Flexor tendon tissue engineering: acellularized and reseeded tendon constructs.

    PubMed

    Chong, Alphonsus K S; Riboh, Jonathan; Smith, R Lane; Lindsey, Derek P; Pham, Hung M; Chang, James

    2009-06-01

    Tissue engineering of flexor tendons requires scaffolds with adequate strength and biocompatibility. The biomechanical properties of acellularized and reseeded flexor tendon scaffolds are unknown. Acellularized tendons and reseeded constructs were tested to determine whether the treatment process had altered their biomechanical properties. Rabbit flexor tendons were acellularized using a freeze-thaw cycle followed by trypsin and Triton-X treatment. Complete acellularization of the tendon samples was confirmed by histology and by attempting to obtain viable cells by trypsin treatment of acellularized tendon. Reseeded constructs were obtained by incubating acellularized tendons in a tenocyte suspension. Tensile testing was performed to compare the ultimate tensile stress and elastic modulus of acellularized tendons and reseeded flexor tendon constructs to control flexor tendons. The treatment protocol successfully acellularized flexor tendons. No cells were seen within the tendon on histologic assessment, and no viable cells could be obtained from acellularized tendon. Acellularized tendon was successfully reseeded with tenocytes, although cell adhesion was limited to the surface of the tendon scaffold. Tensile testing showed that acellularized tendon had the same ultimate stress and elastic modulus as normal tendons. Reseeded tendons had the same elastic modulus as normal tendons, but hind-paw tendon constructs showed a decrease in ultimate stress compared with normal tendons (50.09 MPa versus 66.01 MPa, p = 0.026). Acellularized flexor tendons are a potential high-strength scaffold for flexor tendon tissue engineering. This approach of acellularization and reseeding of flexor tendons may provide additional intrasynovial graft material for hand reconstruction.

  20. Stem cell technology for tendon regeneration: current status, challenges, and future research directions.

    PubMed

    Lui, Pauline Po Yee

    2015-01-01

    Tendon injuries are a common cause of physical disability. They present a clinical challenge to orthopedic surgeons because injured tendons respond poorly to current treatments without tissue regeneration and the time required for rehabilitation is long. New treatment options are required. Stem cell-based therapies offer great potential to promote tendon regeneration due to their high proliferative, synthetic, and immunomodulatory activities as well as their potential to differentiate to the target cell types and undergo genetic modification. In this review, I first recapped the challenges of tendon repair by reviewing the anatomy of tendon. Next, I discussed the advantages and limitations of using different types of stem cells compared to terminally differentiated cells for tendon tissue engineering. The safety and efficacy of application of stem cells and their modified counterparts for tendon tissue engineering were then summarized after a systematic literature search in PubMed. The challenges and future research directions to enhance, optimize, and standardize stem cell-based therapies for augmenting tendon repair were then discussed.

  1. [Advance of adipose-derived stem cells in tendon tissue engineering].

    PubMed

    Yan, Mingming; Ni, Jiangdong

    2014-02-01

    Tendon tissue engineering is a novel therapeutic strategy for severe tendon injury and loss. Adipose derived stem cells (ASCs) have been studied extensively, due to their potency to differentiate into musculoskeletal tissue precursors such as osteoblasts, chondrocytes, adipocytes, and tendocytes under specific cues and high ability of proliferation. Resources of ASCs are ubiquitous and isolation of ASCs is secure, simple and minimally invasive. Mounting evidences demonstrate that ASCs may be involved in tendon tissue engineering and repair the severe injury of tendon under stimulation of various growth factors and other appropriate fittings.

  2. Drosophila neurotactin mediates heterophilic cell adhesion.

    PubMed Central

    Barthalay, Y; Hipeau-Jacquotte, R; de la Escalera, S; Jiménez, F; Piovant, M

    1990-01-01

    Neurotactin is a 135 kd membrane glycoprotein which consists of a core protein, with an apparent molecular weight of 120 kd, and of N-linked oligosaccharides. In vivo, the protein can be phosphorylated in presence of radioactive orthophosphate. Neurotactin expression in the larval CNS and in primary embryonic cell cultures suggests that it behaves as a contact molecule between neurons or epithelial cells. Electron microscopy studies reveal that neurotactin is uniformly expressed along the areas of contacts between cells, without, however, being restricted to a particular type of junction. It putative adhesive properties have been tested by transfecting non adhesive Drosophila S2 cells with neurotactin cDNA. Heat shocked transfected cells do not aggregate, suggesting that neurotactin does not mediate homophilic cell adhesion. However, these transfected cells bind to a subpopulation of embryonic cells which probably possess a related ligand. The location at cellular junctions between specific neurons or epithelial cells, the heterophilic binding to a putative ligand and the ability to be phosphorylated are consistent with the suggestion that neurotactin functions as an adhesion molecule. Images Fig.1 Fig.2 Fig.3 Fig.4 Fig.5 PMID:2120048

  3. Mechanical factors in embryonic tendon development: Potential cues for stem cell tenogenesis

    PubMed Central

    Schiele, Nathan R.; Marturano, Joseph E.; Kuo, Catherine K.

    2013-01-01

    Tendons are connective tissues required for motion and are frequently injured. Poor healing and inadequate return to normal tissue structure and mechanical function make tendon a prime candidate for tissue engineering, however functional tendons have yet to be engineered. The physical environment, from substrate stiffness to dynamic mechanical loading, may regulate tenogenic stem cell differentiation. Tissue stiffness and loading parameters derived from embryonic development may enhance tenogenic stem cell differentiation and tendon tissue formation. We highlight current understanding of the mechanical environment experienced by embryonic tendons and how progenitor cells may sense and respond to physical inputs. We further discuss how mechanical factors have only recently been used to induce tenogenic fate in stem cells. PMID:23916867

  4. Mechanical factors in embryonic tendon development: potential cues for stem cell tenogenesis.

    PubMed

    Schiele, Nathan R; Marturano, Joseph E; Kuo, Catherine K

    2013-10-01

    Tendons are connective tissues required for motion and are frequently injured. Poor healing and inadequate return to normal tissue structure and mechanical function make tendon a prime candidate for tissue engineering; however functional tendons have yet to be engineered. The physical environment, from substrate stiffness to dynamic mechanical loading, may regulate tenogenic stem cell differentiation. Tissue stiffness and loading parameters derived from embryonic development may enhance tenogenic stem cell differentiation and tendon tissue formation. We highlight the current understanding of the mechanical environment experienced by embryonic tendons and how progenitor cells may sense and respond to physical inputs. We further discuss how mechanical factors have only recently been used to induce tenogenic fate in stem cells.

  5. Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study.

    PubMed

    de Mos, Marieke; Koevoet, Wendy J L M; Jahr, Holger; Verstegen, Monique M A; Heijboer, Marinus P; Kops, Nicole; van Leeuwen, Johannes P T M; Weinans, Harrie; Verhaar, Jan A N; van Osch, Gerjo J V M

    2007-02-23

    Tendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential. Cells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs). Tendon-derived cells stained D7-FIB (fibroblast-marker) positive, but alpha-SMA (marker for smooth muscle cells and pericytes) negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker), and 73% positive for CD105 (mesenchymal progenitor-cell marker). In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4) and PPARG (peroxisome proliferative activated receptor gamma). In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations. This study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.

  6. Spatial control of the actin cytoskeleton in Drosophila epithelial cells.

    PubMed

    Baum, B; Perrimon, N

    2001-10-01

    The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.

  7. Enhanced tenogenic differentiation and tendon-like tissue formation by CHIP overexpression in tendon-derived stem cells.

    PubMed

    Han, Weifeng; Chen, Lei; Liu, Junpeng; Guo, Ai

    2017-04-01

    The carboxyl terminus of Hsc70-interacting protein (CHIP, also known as STUB1) plays critical roles in the proliferation and differentiation of many types of cells. The potential function of CHIP in tendon-derived stem cells (TDSCs) remains largely unknown at present. Here, we investigated the effects of CHIP on tenogenic differentiation of TDSCs via lentivirus-mediated overexpression. Forced expression of CHIP induced morphological changes and significantly enhanced cell proliferation, as well as tendon differentiation in vitro. Upon stimulation with differentiation induction medium, CHIP-overexpressing TDSCs displayed significant inhibition of differentiation into osteogenic and adipogenic lineages. Subsequent implantation of TDSCs overexpressing CHIP with collagen sponges into nude mice induced a marked increase in ectopic tendon formation in vivo, compared with the control group. Our findings collectively suggest that CHIP is an important contributory factor to tenogenic tissue formation. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Lineage tracing of resident tendon progenitor cells during growth and natural healing.

    PubMed

    Dyment, Nathaniel A; Hagiwara, Yusuke; Matthews, Brya G; Li, Yingcui; Kalajzic, Ivo; Rowe, David W

    2014-01-01

    Unlike during embryogenesis, the identity of tissue resident progenitor cells that contribute to postnatal tendon growth and natural healing is poorly characterized. Therefore, we utilized 1) an inducible Cre driven by alpha smooth muscle actin (SMACreERT2), that identifies mesenchymal progenitors, 2) a constitutively active Cre driven by growth and differentiation factor 5 (GDF5Cre), a critical regulator of joint condensation, in combination with 3) an Ai9 Cre reporter to permanently label SMA9 and GDF5-9 populations and their progeny. In growing mice, SMA9+ cells were found in peritendinous structures and scleraxis-positive (ScxGFP+) cells within the tendon midsubstance and myotendinous junction. The progenitors within the tendon midsubstance were transiently labeled as they displayed a 4-fold expansion from day 2 to day 21 but reduced to baseline levels by day 70. SMA9+ cells were not found within tendon entheses or ligaments in the knee, suggesting a different origin. In contrast to the SMA9 population, GDF5-9+ cells extended from the bone through the enthesis and into a portion of the tendon midsubstance. GDF5-9+ cells were also found throughout the length of the ligaments, indicating a significant variation in the progenitors that contribute to tendons and ligaments. Following tendon injury, SMA9+ paratenon cells were the main contributors to the healing response. SMA9+ cells extended over the defect space at 1 week and differentiated into ScxGFP+ cells at 2 weeks, which coincided with increased collagen signal in the paratenon bridge. Thus, SMA9-labeled cells represent a unique progenitor source that contributes to the tendon midsubstance, paratenon, and myotendinous junction during growth and natural healing, while GDF5 progenitors contribute to tendon enthesis and ligament development. Understanding the mechanisms that regulate the expansion and differentiation of these progenitors may prove crucial to improving future repair strategies.

  9. Passage and concentration-dependent effects of Indomethacin on tendon derived cells

    PubMed Central

    Mallick, Emad; Scutt, Nanette; Scutt, Andy; Rolf, Christer

    2009-01-01

    Background Non-steroidal anti-inflammatory drugs (NSAID) are commonly used in the treatment of tendinopathies such as tendonitis and tendinosis. Despite this, little is known of their direct actions on tendon-derived cells. As NSAIDs have been shown to delay healing in a number of mesenchymal tissues we have investigated the direct effects of indomethacin on the proliferation of tendon-derived cells. Results and Discussion The results obtained were dependent on both the type of cells used and the method of measurement. When measured using the Alamar blue assay, a common method for the measurement of cell proliferation and viability, no effect of indomethacin was seen regardless of cell source. It is likely that this lack of effect was due to a paucity of mitochondrial enzymes in tendon cells. However, when cell number was assessed using the methylene blue assay, which is a simple nuclear staining technique, an Indomethacin-induced inhibition of proliferation was seen in primary cells but not in secondary subcultures. Conclusion These results suggest that firstly, care must be taken when deciding on methodology used to investigate tendon-derived cells as these cells have a quite different metabolism to other mesenchymal derive cells. Secondly, Indomethacin can inhibit the proliferation of primary tendon derived cells and that secondary subculture selects for a population of cells that is unresponsive to this drug. PMID:19341464

  10. Mesenchymal stem cells for tendon healing: what is on the horizon?

    PubMed

    Veronesi, Francesca; Salamanna, Francesca; Tschon, Matilde; Maglio, Melania; Nicoli Aldini, Nicolo; Fini, Milena

    2016-09-06

    Tendon injuries are a noteworthy morbidity but at present there are few effective scientifically proven treatments. In recent decades, a number of new strategies including tissue engineering with mesenchymal stem cells (MSCs) have been proposed to enhance tendon healing. Although MSCs are an interesting and promising approach, many questions regarding their use in tendon repair remain unanswered. This descriptive overview of the literature of the last decade explores the in vivo studies on tendon healing, in small and large animal models, which used MSCs harvested from different tissues, and the state of the art in clinical applications. It was observed that there are still doubts about the optimum amount of MSCs to use and their source and the type of scaffolds to deliver the cells. Thus, further studies are needed to determine the best protocol for MSC use in tendon healing. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Biology and mechano-response of tendon cells: progress overview and perspectives†

    PubMed Central

    Sun, Hui B.; Schaniel, Christoph; Leong, Daniel J.; Wang, James H-C.

    2015-01-01

    In this review, we summarize the group discussions on Cell Biology & Mechanics from the 2014 ORS/ISMMS New Frontiers in Tendon Research Conference. The major discussion topics included: 1) the biology of tendon stem/progenitor cells (TSPCs) and the potential of stem cell-based tendon therapy using TSPCs and other types of stem cells, namely, embryonic and/or induced pluripotent stem cells (iPSCs), 2) the biological concept and potential impact of cellular senescence on tendon aging, tendon injury repair and the development of degenerative disease, and 3) the effects of tendon cells’ mechano-response on tendon cell fate and metabolism. For each topic, a brief overview is presented which summarizes the major points discussed by the group participants. The focus of the discussions ranged from current research progress, challenges and opportunities, to future directions on these topics. In the preparation of this manuscript, authors consulted relevant references as a part of their efforts to present an accurate view on the topics discussed. PMID:25728946

  12. Assessment of stem cell carriers for tendon tissue engineering in pre-clinical models

    PubMed Central

    2014-01-01

    Tendon injuries are prevalent and problematic, especially among young and otherwise healthy individuals. The inherently slow innate healing process combined with the inevitable scar tissue formation compromise functional recovery, imposing the need for the development of therapeutic strategies. The limited number of low activity/reparative capacity tendon-resident cells has directed substantial research efforts towards the exploration of the therapeutic potential of various stem cells in tendon injuries and pathophysiologies. Severe injuries require the use of a stem cell carrier to enable cell localisation at the defect site. The present study describes advancements that injectable carriers, tissue grafts, anisotropically orientated biomaterials, and cell-sheets have achieved in preclinical models as stem cell carriers for tendon repair. PMID:25157898

  13. Assessment of stem cell carriers for tendon tissue engineering in pre-clinical models.

    PubMed

    Abbah, Sunny Akogwu; Spanoudes, Kyriakos; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I

    2014-01-01

    Tendon injuries are prevalent and problematic, especially among young and otherwise healthy individuals. The inherently slow innate healing process combined with the inevitable scar tissue formation compromise functional recovery, imposing the need for the development of therapeutic strategies. The limited number of low activity/reparative capacity tendon-resident cells has directed substantial research efforts towards the exploration of the therapeutic potential of various stem cells in tendon injuries and pathophysiologies. Severe injuries require the use of a stem cell carrier to enable cell localisation at the defect site. The present study describes advancements that injectable carriers, tissue grafts, anisotropically orientated biomaterials, and cell-sheets have achieved in preclinical models as stem cell carriers for tendon repair.

  14. Nutritional regulation of stem and progenitor cells in Drosophila

    PubMed Central

    Shim, Jiwon; Gururaja-Rao, Shubha; Banerjee, Utpal

    2013-01-01

    Stem cells and their progenitors are maintained within a microenvironment, termed the niche, through local cell-cell communication. Systemic signals originating outside the niche also affect stem cell and progenitor behavior. This review summarizes studies that pertain to nutritional effects on stem and progenitor cell maintenance and proliferation in Drosophila. Multiple tissue types are discussed that utilize the insulin-related signaling pathway to convey nutritional information either directly to these progenitors or via other cell types within the niche. The concept of systemic control of these cell types is not limited to Drosophila and may be functional in vertebrate systems, including mammals. PMID:24255094

  15. Analysis of Cell Cycle Switches in Drosophila Oogenesis.

    PubMed

    Jia, Dongyu; Huang, Yi-Chun; Deng, Wu-Min

    2015-01-01

    The study of Drosophila oogenesis provides invaluable information about signaling pathway regulation and cell cycle programming. During Drosophila oogenesis, a string of egg chambers in each ovariole progressively develops toward maturity. Egg chamber development consists of 14 stages. From stage 1 to stage 6 (mitotic cycle), main-body follicle cells undergo mitotic divisions. From stage 7 to stage 10a (endocycle), follicle cells cease mitosis but continue three rounds of endoreduplication. From stage 10b to stage 13 (gene amplification), instead of whole genome duplication, follicle cells selectively amplify specific genomic regions, mostly for chorion production. So far, Drosophila oogenesis is one of the most well studied model systems used to understand cell cycle switches, which furthers our knowledge about cell cycle control machinery and sheds new light on potential cancer treatments. Here, we give a brief summary of cell cycle switches, the associated signaling pathways and factors, and the detailed experimental procedures used to study the cell cycle switches.

  16. Tendon regeneration with a novel tendon hydrogel: in vitro effects of platelet-rich plasma on rat adipose-derived stem cells.

    PubMed

    Crowe, Christopher S; Chiou, Grace; McGoldrick, Rory; Hui, Kenneth; Pham, Hung; Chang, James

    2015-06-01

    Tendon hydrogel is a promising new injectable substance that has been shown to improve repair strength after tendon injury. This study assesses the capacity of platelet-rich plasma to stimulate proliferation and migration of rat adipose-derived stem cells in tendon hydrogel in vitro. To assess proliferation, adipose-derived stem cells were exposed to plasma, plasma supplemented with growth factors, or platelet-rich plasma in culture medium and tendon hydrogel. To assess migration, adipose-derived stem cells were plated onto tendon hydrogel -coated wells and covered with medium containing plasma, plasma supplemented with growth factors, platelet-rich plasma, or bovine serum albumin. Migration from cell-seeded to cell-free zones was assessed at 12-hour intervals. Platelet-rich plasma augmented proliferation to a greater extent compared with plasma and plasma supplemented with growth factors (10%: optical density, 1.18 versus 0.75 versus 0.98, respectively). Platelet-rich plasma was superior to plasma in tendon hydrogel (10%: optical density, 1.19 versus 0.85) but did not augment proliferation to the extent that plasma supplemented with growth factors did (10%: optical density, 1.19 versus 1.56). Platelet-rich plasma enhanced the migration of adipose-derived stem cells compared with serum-free medium (bovine serum albumin) (36 hours: platelet-rich plasma, 1.88; plasma, 1.51; plasma plus growth factor, 1.80; bovine serum albumin, 1.43). Tendon healing is mediated by migration of cells to the injured area and cellular proliferation at that site. Tendon hydrogel supplemented with platelet-rich plasma stimulates these processes. Future studies will evaluate this combination's ability to stimulate healing in chronic tendon injuries in vivo.

  17. Differences between the Cell Populations from the Peritenon and the Tendon Core with Regard to Their Potential Implication in Tendon Repair

    PubMed Central

    Cadby, Jennifer A.; Buehler, Evelyne; Godbout, Charles; van Weeren, P. René; Snedeker, Jess G.

    2014-01-01

    The role of intrinsic and extrinsic healing in injured tendons is still debated. In this study, we characterized cell plasticity, proliferative capacity, and migration characteristics as proxy measures of healing potential in cells derived from the peritenon (extrinsic healing) and compared these to cells from the tendon core (intrinsic healing). Both cell populations were extracted from horse superficial digital flexor tendon and characterized for tenogenic and matrix remodeling markers as well as for rates of migration and replication. Furthermore, colony-forming unit assays, multipotency assays, and real-time quantitative polymerase chain reaction analyses of markers of osteogenic and adipogenic differentiation after culture in induction media were performed. Finally, cellular capacity for differentiation towards a myofibroblastic phenotype was assessed. Our results demonstrate that both tendon- and peritenon-derived cell populations are capable of adipogenic and osteogenic differentiation, with higher expression of progenitor cell markers in peritenon cells. Cells from the peritenon also migrated faster, replicate more quickly, and show higher differentiation potential toward a myofibroblastic phenotype when compared to cells from the tendon core. Based on these data, we suggest that cells from the peritenon have substantial potential to influence tendon-healing outcome, warranting further scrutiny of their role. PMID:24651449

  18. Blood cells of Drosophila: cell lineages and role in host defence.

    PubMed

    Meister, Marie

    2004-02-01

    Drosophila haemopoiesis gives rise to three independent cell lineages: plasmatocytes, crystal cells and lamellocytes. The regulation of Drosophila stem cell proliferation and lineage specification involves transactivators and signalling pathways, many of which have mammalian counterparts that control haemopoietic processes. Drosophila plasmatocytes are professional phagocytes that resemble the monocyte/macrophage lineage, crystal cells play a critical role in defence-related melanisation, and lamellocytes encapsulate large invaders. Crystal cells and lamellocytes have no clear mammalian homologues. Research into the molecular mechanisms that underlie the various immune functions of Drosophila blood cells, such as non-self recognition, is now taking wing.

  19. Use of stem cells and growth factors in rotator cuff tendon repair.

    PubMed

    Akyol, Engin; Hindocha, Sandip; Khan, Wasim S

    2015-01-01

    In this review, we analysed the role of stem cell and growth factor therapy on rotator cuff tendon repair. The injury to the rotator cuff tendons can be sustained in numerous ways and generally causes significant pain and disability to the affected individual. Following surgical repair of ruptured rotator cuff tendons re-rupture rates can be as high as 20-60%. In order to augment this repair process and to decrease the re-rupture rates tissue engineering methods can be used. These include the use of stem cells and growth factors. Mesenchymal stem cells are stem cells which can differentiate into a variety of connective tissue cell types and can therefore be utilised in repairing tendons. So far there has only been one human study using stem cells in rotator cuff tendon repair. This study has produced a positive result but consisted of only 14 patients and lacks a control group for comparison. Similar work has also been done using growth factors. Both individual and combination growth factor therapy have been used to improve rotator cuff tendon repair. However, the results so far have been disappointing with growth factors. For the purpose of future studies better techniques should be explored with regards to the delivery of stem cells and growth factors as well as the possibility of combining growth factor and stem cell therapy to improve repair rates.

  20. Acute Carpal Tunnel Syndrome Caused by Diffuse Giant Cell Tumor of Tendon Sheath: A Case Report

    PubMed Central

    Ward, Christina M; Lueck, Nathan E; Steyers, Curtis M

    2007-01-01

    A 46 year old male developed spontaneous acute carpal tunnel syndrome of the right wrist without any antecedent trauma. Surgical exploration revealed hemorrhage secondary to diffuse giant cell tumor of tendon sheath as the underlying cause. PMID:17907439

  1. Human flexor tendon tissue engineering: in vivo effects of stem cell reseeding.

    PubMed

    Schmitt, Taliah; Fox, Paige M; Woon, Colin Y; Farnebo, Simon J; Bronstein, Joel A; Behn, Anthony; Pham, Hung; Chang, James

    2013-10-01

    Tissue-engineered human flexor tendons may be an option to aid in reconstruction of complex upper extremity injuries with significant tendon loss. The authors hypothesize that human adipose-derived stem cells remain viable following reseeding on human tendon scaffolds in vivo and aid in graft integration. Decellularized human flexor tendons harvested from fresh-frozen cadavers and reseeded with green fluorescent protein-labeled pooled human adipose-derived stem cells were examined with bioluminescent imaging and immunohistochemistry. Reseeded repaired tendons were compared biomechanically with unseeded controls following implantation in athymic rats at 2 and 4 weeks. The ratio of collagen I to collagen III at the repair site was examined using Sirius red staining. To confirm cell migration, reseeded and unseeded tendons were placed either in contact or with a 1-mm gap for 12 days. Green fluorescent protein signal was then detected. Following reseeding, viable cells were visualized at 12 days in vitro and 4 weeks in vivo. Biomechanical testing revealed no significant difference in ultimate load to failure and 2-mm gap force. Histologic evaluation showed host cell invasion and proliferation of the repair sites. No increase in collagen III was noted in reseeded constructs. Cell migration was confirmed from reseeded constructs to unseeded tendon scaffolds with tendon contact. Human adipose-derived stem cells reseeded onto decellularized allograft scaffolds are viable over 4 weeks in vivo. The movement of host cells into the scaffold and movement of adipose-derived stem cells along and into the scaffold suggests biointegration of the allograft.

  2. Tenogenesis of bone marrow-, adipose-, and tendon-derived stem cells in a dynamic bioreactor.

    PubMed

    Youngstrom, Daniel W; LaDow, Jade E; Barrett, Jennifer G

    2016-11-01

    Tendons are frequently damaged and fail to regenerate, leading to pain, loss of function, and reduced quality of life. Mesenchymal stem cells (MSCs) possess clinically useful tissue-regenerative properties and have been exploited for use in tendon tissue engineering and cell therapy. However, MSCs exhibit phenotypic heterogeneity based on the donor tissue used, and the efficacy of cell-based treatment modalities may be improved by optimizing cell source based on relative differentiation capacity. Equine MSCs were isolated from bone marrow (BM), adipose (AD), and tendon (TN), expanded in monolayer prior to seeding on decellularized tendon scaffolds (DTS), and cell-laden constructs were placed in a bioreactor designed to mimic the biophysical environment of the tendon. It was hypothesized that TN MSCs would differentiate toward a tendon cell phenotype better than BM and AD MSCs in response to a conditioning period involving cyclic mechanical stimulation for 1 hour per day at 3% strain and 0.33 Hz. All cell types integrated into DTS adopted an elongated morphology similar to tenocytes, expressed tendon marker genes, and improved tissue mechanical properties after 11 days. TN MSCs expressed the greatest levels of scleraxis, collagen type-I, and cartilage oligomeric matrix protein. Major histocompatibility class-II protein mRNA expression was not detected in any of the MSC types, suggesting low immunogenicity for allogeneic transplantation. The results suggest that TN MSCs are the ideal cell type for regenerative medicine therapies for tendinopathies, exhibiting the most mature tendon-like phenotype in vitro. When TN MSCs are unavailable, BM or AD MSCs may serve as robust alternatives.

  3. Intestinal stem cells in the adult Drosophila midgut

    SciTech Connect

    Jiang, Huaqi; Edgar, Bruce A.

    2011-11-15

    Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury. -- Highlights: Black-Right-Pointing-Pointer The homeostasis and regeneration of adult fly midguts are mediated by ISCs. Black-Right-Pointing-Pointer Damaged enterocytes induce the proliferation of intestinal stem cells (ISC). Black-Right-Pointing-Pointer EGFR and Jak/Stat signalings mediate compensatory ISC proliferation. Black-Right-Pointing-Pointer Notch signaling regulates ISC self-renewal and differentiation.

  4. Isolation and biological characterization of tendon-derived stem cells from fetal bovine.

    PubMed

    Yang, Jinjuan; Zhao, Qianjun; Wang, Kunfu; Liu, Hao; Ma, Caiyun; Huang, Hongmei; Liu, Yingjie

    2016-09-01

    The lack of appropriate candidates of cell sources for cell transplantation has hampered efforts to develop therapies for tendon injuries, such as tendon rupture, tendonitis, and tendinopathy. Tendon-derived stem cells (TDSCs) are a type of stem cells which may be used in the treatment of tendon injuries. In this study, TDSCs were isolated from 5-mo-old Luxi Yellow fetal bovine and cultured in vitro and further analyzed for their biological characteristics using immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR) assays. It was found that primary TDSCs could be expanded for 42 passages in vitro maintaining proliferation. The expressions of stem cell marker nucleostemin and tenocyte-related markers, such as collagen I, collagen II, collagen III, and tenascin-C, were observed on different passage cells by immunofluorescence. The results from RT-PCR show that TDSCs were positive for collagen type I, CD44, tenascin-C, and collagen type III but negative for collagen type II. Meanwhile, TDSC passage 4 was successfully induced to differentiate into osteoblasts, adipocytes, and chondrocytes. Our results indicate that the fetal bovine TDSCs not only had strong self-renewal capacity but also possess the potential for multi-lineage differentiation. This study provides theoretical basis and experimental foundation for potential therapeutic application of the fetal bovine TDSCs in the treatment of tendon injuries.

  5. Gastrocnemius tendon strain in a dog treated with autologous mesenchymal stem cells and a custom orthosis.

    PubMed

    Case, J Brad; Palmer, Ross; Valdes-Martinez, Alex; Egger, Erick L; Haussler, Kevin K

    2013-05-01

    To report clinical findings and outcome in a dog with gastrocnemius tendon strain treated with autologous mesenchymal stem cells and a custom orthosis. Clinical report. A 4-year-old spayed female Border Collie. Bone-marrow derived, autologous mesenchymal stem cells were transplanted into the tendon core lesion. A custom, progressive, dynamic orthosis was fit to the tarsus. Serial orthopedic examinations and ultrasonography as well as long-term force-plate gait analysis were utilized for follow up. Lameness subjectively resolved and peak vertical force increased from 43% to 92% of the contralateral pelvic limb. Serial ultrasonographic examinations revealed improved but incomplete restoration of normal linear fiber pattern of the gastrocnemius tendon. Findings suggest that autologous mesenchymal stem cell transplantation with custom, progressive, dynamic orthosis may be a viable, minimally invasive technique for treatment of calcaneal tendon injuries in dogs. © Copyright 2013 by The American College of Veterinary Surgeons.

  6. Transformation of Drosophila cell lines: an alternative approach to exogenous protein expression.

    PubMed

    Cherbas, Lucy; Cherbas, Peter

    2007-01-01

    Techniques and experimental applications are described for exogenous protein expression in Drosophila cell lines. Ways in which the Drosophila cell lines and the baculovirus expression vector system differ in their applications are emphasized.

  7. [Research progress of cell-scaffold complex in tendon tissue engineering].

    PubMed

    Zhu, Ying; Li, Min

    2013-04-01

    To review the research progress of cell-scaffold complex in the tendon tissue engineering. Recent literature concerning cell-scaffold complex in the tendon tissue engineering was reviewed, the research situation of the cell-scaffold complex was elaborated in the aspects of seed cells, scaffolds, cell culture, and application. In tendon tissue engineering, a cell-scaffold complex is built by appropriate seed cells and engineered scaffolds. Experiments showed that modified seed cells had better therapeutic effects. Further, scaffold functionality could be improved through surface modification, growth factor cure, mechanical stimulation, and contact guidance. Among these methods, mechanical stimulation revealed the most significant results in promoting cell proliferation and function. Through a variety of defect models, it is demonstrated that the use of cell-scaffold complex could achieve satisfactory results for tendon regeneration. The cell-scaffold complex for tendon tissue engineering is a popular research topic. Although it has not yet met the requirement of clinical use, it has broad application prospects.

  8. Inter-tissue mechanical stress affects Frizzled-mediated planar cell polarity in the Drosophila notum epidermis

    PubMed Central

    Olguín, Patricio; Glavic, Alvaro; Mlodzik, Marek

    2011-01-01

    Summary Frizzled/Planar Cell Polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the antero-posterior axis of the Drosophila thorax (notum) [1–4]. A subset of the trichome-producing notum cells differentiate as “tendon cells”, serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton [5]. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin [6], we show that maintenance of antero-posterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells and its loss-of-function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat (ft)/dachsous (ds) systems [7]. The chas phenotype arises during normal shortening of the IFMs [8] and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechano-response of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by interaction(s) with other tissues during development/organogenesis influences the maintenance of its shape and PCP features. PMID:21276726

  9. Effect of mechanical stimulation on bone marrow stromal cell-seeded tendon slice constructs: a potential engineered tendon patch for rotator cuff repair.

    PubMed

    Qin, Ting-Wu; Sun, Yu-Long; Thoreson, Andrew R; Steinmann, Scott P; Amadio, Peter C; An, Kai-Nan; Zhao, Chunfeng

    2015-05-01

    Cell-based tissue engineered tendons have potential to improve clinical outcomes following rotator cuff repair, especially in large or massive rotator cuff tears, which pose a great clinical challenge. The aim of this study was to develop a method of constructing a functional engineered tendon patch for rotator cuff repair with cyclic mechanical stimulation. Decellularized tendon slices (DTSs) were seeded with BMSCs and subjected to cyclic stretching for 1, 3, or 7 days. The mechanical properties, morphologic characteristics and tendon-related gene expression of the constructs were investigated. Viable BMSCs were observed on the DTS after 7 days. BMSCs penetrated into the DTSs and formed dense cell sheets after 7 days of mechanical stretching. Gene expression of type I collagen, decorin, and tenomodulin significantly increased in cyclically stretched BMSC-DTS constructs compared with the unstrained control group (P < 0.05). The ultimate tensile strength and stiffness of the cyclically stretched tendon constructs were similar to the unstrained control group (P > 0.05). In conclusion, mechanical stimulation of BMSC-DTS constructs upregulated expression of tendon-related proteins, promoted cell tenogenic differentiation, facilitated cell infiltration and formation of cell sheets, and retained mechanical properties. The patch could be used as a graft to enhance the surgical repair of rotator cuff tears. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. A review on the use of cell therapy in the treatment of tendon disease and injuries

    PubMed Central

    Sawadkar, Prasad; Mudera, Vivek

    2014-01-01

    Tendon disease and injuries carry significant morbidity worldwide in both athletic and non-athletic populations. It is estimated that tendon injuries account for 30%−50% of all musculoskeletal injuries globally. Current treatments have been inadequate in providing an accelerated process of repair resulting in high relapse rates. Modern concepts in tissue engineering and regenerative medicine have led to increasing interest in the application of cell therapy for the treatment of tendon disease. This review will explore the use of cell therapy, by bringing together up-to-date evidence from in vivo human and animal studies, and discuss the issues surrounding the safety and efficacy of its use in the treatment of tendon disease. PMID:25383170

  11. EGR1 induces tenogenic differentiation of tendon stem cells and promotes rabbit rotator cuff repair.

    PubMed

    Tao, Xu; Liu, Junpeng; Chen, Lei; Zhou, You; Tang, Kanglai

    2015-01-01

    The rate of healing failure after surgical repair of chronic rotator cuff tears is considerably high. The aim of this study was to investigate the function of the zinc finger transcription factor early growth response 1 (EGR1) in the differentiation of tendon stem cells (TSCs) and in tendon formation, healing, and tendon tear repair using an animal model of rotator cuff repair. Tenocyte, adipocyte, osteocyte, and chondrocyte differentiation as well as the expression of related genes were determined in EGR1-overexpressing TSCs (EGR1-TSCs) using tissue-specific staining, immunofluorescence staining, quantitative PCR, and western blotting. A rabbit rotator cuff repair model was established, and TSCs and EGR1-TSCs in a fibrin glue carrier were applied onto repair sites. The rabbits were sacrificed 8 weeks after repair operation, and tissues were histologically evaluated and tenocyte-related gene expression was determined. EGR1 induced tenogenic differentiation of TSCs and inhibited non-tenocyte differentiation of TSCs. Furthermore, EGR1 promoted tendon repair in a rabbit model of rotator cuff injury. The BMP12/Smad1/5/8 signaling pathway was involved in EGR1-induced tenogenic differentiation and rotator cuff tendon repair. EGR1 plays a key role in tendon formation, healing, and repair through BMP12/Smad1/5/8 pathway. EGR1-TSCs is a promising treatment for rotator cuff tendon repair surgeries. © 2015 S. Karger AG, Basel.

  12. Ageing does not result in a decline in cell synthetic activity in an injury prone tendon.

    PubMed

    Thorpe, C T; McDermott, B T; Goodship, A E; Clegg, P D; Birch, H L

    2016-06-01

    Advancing age is a well-known risk factor for tendon disease. Energy-storing tendons [e.g., human Achilles, equine superficial digital flexor tendon (SDFT)] are particularly vulnerable and it is thought that injury occurs following an accumulation of micro-damage in the extracellular matrix (ECM). Several authors suggest that age-related micro-damage accumulates due to a failure of the aging cell population to maintain the ECM or an imbalance between anabolic and catabolic pathways. We hypothesized that ageing results in a decreased ability of tendon cells to synthesize matrix components and matrix-degrading enzymes, resulting in a reduced turnover of the ECM and a decreased ability to repair micro-damage. The SDFT was collected from horses aged 3-30 years with no signs of tendon injury. Cell synthetic and degradative ability was assessed at the mRNA and protein levels. Telomere length was measured as an additional marker of cell ageing. There was no decrease in cellularity or relative telomere length with increasing age, and no decline in mRNA or protein levels for matrix proteins or degradative enzymes. The results suggest that the mechanism for age-related tendon deterioration is not due to reduced cellularity or a loss of synthetic functionality and that alternative mechanisms should be considered. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Adult Cells Combined With Platelet-Rich Plasma for Tendon Healing

    PubMed Central

    Rubio-Azpeitia, Eva; Sánchez, Pello; Delgado, Diego; Andia, Isabel

    2017-01-01

    Background: The combination of cells with platelet-rich plasma (PRP) may fulfill tendon deficits and help overcome the limited ability of tendons to heal. Purpose: To examine the suitability of 3 human cell types in combination with PRP and the potential impact of the tenocyte-conditioned media (CM) to enhance tendon healing. Study Design: Controlled laboratory study. Methods: Tenocytes, bone marrow–derived mesenchymal stem cells, and skin fibroblasts were cultured in 3-dimensional PRP hydrogels supplemented or not with CM, and cell proliferation and migration were examined. The effect of tendon-derived CM on matrix-forming phenotype and secretion of inflammatory proteins was determined through their administration to mesenchymal stem cells, tendon, and skin fibroblasts by reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results: Differences were found in the matrix-forming phenotype between each of the cell types. The ratio of collagen I:collagen III was greater in bone marrow–derived mesenchymal stem cells than in skin fibroblasts and tenocytes. The bone marrow–derived mesenchymal stem cells expressed increased levels of cartilage-related genes than tenocytes or skin fibroblasts. The presence of the tenocyte-CM stimulated basic healing mechanisms including proliferation and chemotaxis in all cell types. In addition, the tenocyte-CM modified the matrix-forming phenotype of every cell type when cultured in PRP hydrogels. Each cell type secreted interleukin-6, interleukin-8, and monocyte chemotactic protein-1 in PRP hydrogels, but mesenchymal stem cells secreted less interleukin-8 and monocyte chemotactic protein-1 than tenocytes or skin fibroblasts. Conclusion: The tenocyte-CM combined with PRP stimulated tenogenesis in mesenchymal stem cells and in skin fibroblasts and reduced the secretion of inflammatory proteins. Clinical Relevance: Modifying the target tissue with PRP prior to cell

  14. Optimization of an injectable tendon hydrogel: the effects of platelet-rich plasma and adipose-derived stem cells on tendon healing in vivo.

    PubMed

    Chiou, Grace Jane; Crowe, Christopher; McGoldrick, Rory; Hui, Kenneth; Pham, Hung; Chang, James

    2015-05-01

    Acute and chronic tendon injuries would benefit from stronger and more expeditious healing. We hypothesize that supplementation of a biocompatible tendon hydrogel with platelet-rich plasma (PRP) and adipose-derived stem cells (ASCs) would augment the tendon healing process. Using 55 Wistar rats, a full-thickness defect was created within the midsubstance of each Achilles tendon with the addition of one of five experimental conditions: (i) saline control (50-μL), (ii) tendon hydrogel (50-μL), (iii) tendon hydrogel (45-μL)+PRP (5-μL), (iv) tendon hydrogel (45-μL)+2×10(6)-ASCs/mL in phosphate buffered saline (5-μL), and (v) tendon hydrogel (45-μL)+2×10(6)-ASCs/mL in PRP (5-μL). Hydrogel was developed from decellularized, human cadaveric tendons. Fresh rat PRP was obtained per Amable et al.'s technique, and green fluorescent protein/luciferase-positive rat ASCs were utilized. Rats were sacrificed at weeks 1, 2, 4, and 8 after injury. Real-time in vivo bioluminescence imaging of groups with ASCs was performed. Upon sacrifice, Achilles tendons underwent biomechanical and histological evaluation. Comparisons across groups were analyzed using the two-sample Z-test for proportions and the Student's t-test for independent samples. Significance was set at p<0.05. (i) Bioluminescence imaging demonstrated that total photon flux was significantly increased for hydrogel+PRP+ASCs, versus hydrogel+ASCs for each postoperative day imaged (p<0.03). (ii) Mean ultimate failure load (UFL) was increased for hydrogel augmented with PRP and/or ASCs versus hydrogel alone at week 2 (p<0.03). By week 4, hydrogel alone reached a similar mean UFL to hydrogel augmented with PRP and/or ASCs (p>0.3). However, at week 8, hydrogel with PRP and ASCs demonstrated increased strength over other groups (p<0.05), except for hydrogel with PRP (p=0.25). (iii) Upon histological analysis, Hematoxylin and Eosin staining showed increased extracellular matrix formation in groups containing PRP and

  15. Fetal derived embryonic-like stem cells improve healing in a large animal flexor tendonitis model

    PubMed Central

    2011-01-01

    Introduction Tendon injury is a common problem in athletes, with poor tissue regeneration and a high rate of re-injury. Stem cell therapy is an attractive treatment modality as it may induce tissue regeneration rather than tissue repair. Currently, there are no reports on the use of pluripotent cells in a large animal tendon model in vivo. We report the use of intra-lesional injection of male, fetal derived embryonic-like stem cells (fdESC) that express Oct-4, Nanog, SSEA4, Tra 1-60, Tra 1-81 and telomerase. Methods Tendon injury was induced using a collagenase gel-physical defect model in the mid-metacarpal region of the superficial digital flexor tendon (SDFT) of eight female adult Thoroughbred or Thoroughbred cross horses. Tendon lesions were treated one week later with intra-lesional injection of male derived fdESCs in media or media alone. Therapy was blinded and randomized. Serial ultrasound examinations were performed and final analysis at eight weeks included magnetic resonance imaging (MRI), biochemical assays (total DNA, glycosaminoglycan, collagen), gene expression (TNC, TNMD, SCX, COL1A1, COL3A1, COMP, DCN, MMP1, MMP3, MMP13, 18S) and histology. Differences between groups were assessed with Wilcoxon's rank sum test. Results Cell survival was demonstrated via the presence of the SRY gene in fdESC treated, but not control treated, female SDFT at the end of the trial. There were no differences in tendon matrix specific gene expression or total proteoglycan, collagen or DNA of tendon lesions between groups. Tissue architecture, tendon size, tendon lesion size, and tendon linear fiber pattern were significantly improved on histologic sections and ultrasound in the fdESC treated tendons. Conclusions Such profound structural effects lend further support to the notion that pluripotent stem cells can effect musculoskeletal regeneration, rather than repair, even without in vitro lineage specific differentiation. Further investigation into the safety of

  16. Capacity of muscle derived stem cells and pericytes to promote tendon graft integration and ligamentization following anterior cruciate ligament reconstruction.

    PubMed

    Ćuti, Tomislav; Antunović, Maja; Marijanović, Inga; Ivković, Alan; Vukasović, Andreja; Matić, Igor; Pećina, Marko; Hudetz, Damir

    2017-06-01

    The aim of this study is to examine the capacity of muscle tissue preserved on hamstring tendons forming candy-stripe grafts in order to improve tendon to bone ingrowth and ligamentization. We hypothesized that muscle tissue does possess a stem cell population that could enhance the healing process of the ACL graft when preserved on the tendons. Human samples from gracilis and semitendinosus muscles were collected during ACL surgery from ten patients and from these tissue samples human muscle-derived stem cells and tendon-derived stem cells were isolated and propagated. Both stem cell populations were in-vitro differentiated into osteogenic lineage. Alkaline phosphatase activity was determined at days zero and 14 of the osteogenic induction and von Kossa staining to assess mineralization of the cultures. Total RNA was collected from osteoblast cultures and real time quantitative PCR was performed. Western-blot for osteocalcin and collagen type I followed protein isolation. Immunofluorescence double labeling of pericytes in muscle and tendon tissue was performed. Mesenchymal stem cells from muscle and tendon tissue were isolated and expanded in cell culture. More time was needed to grow the tendon derived culture compared to muscle derived culture. Muscle derived stem cells exhibited more alkaline phosphatase actvity compared to tendon derived stem cells, whereas tendon derived stem cells formed more mineralized nodules after 14 days of osteoinduction. Muscle derived stem cells exhibited higher expression levels of bone sialoprotein, and tendon derived stem cells showed higher expression of dental-matrix-protein 1 and osteocalcin. Immunofluorescent staining against pericytes indicated that they are more abundant in muscle tissue. These results indicate that muscle tissue is a better source of stem cells than tendon tissue. Achievement of this study is proof that there is vast innate capacity of muscle tissue for enhancement of bone-tendon integration and

  17. Tendon-derived progenitor cells improve healing of collagenase-induced flexor tendinitis.

    PubMed

    Durgam, Sushmitha S; Stewart, Allison A; Sivaguru, Mayandi; Wagoner Johnson, Amy J; Stewart, Matthew C

    2016-12-01

    Tendinitis is a common and a performance-limiting injury in athletes. This study describes the value of intralesional tendon-derived progenitor cell (TDPC) injections in equine flexor tendinitis. Collagenase-induced tendinitis was created in both front superficial digital flexor (SDF) tendons. Four weeks later, the forelimb tendon lesions were treated with 1 × 10(7) autogenous TDPCs or saline. Tendinitis was also induced by collagenase in one hind SDF tendon, to study the survival and distribution of DiI-labeled TDPCs 1, 2, 4, and 6 weeks after injection. The remaining normal tendon was used as a "control." Twelve weeks after forelimb TDPC injections, tendons were harvested for assessment of matrix gene expression, biochemical, biomechanical, and histological characteristics. DiI-labeled TDPCs were abundant 1 week after injection but gradually declined over time and were undetectable after 6 weeks. Twelve weeks after TDPC injection, collagens I and III, COMP and tenomodulin mRNA levels were similar (p = 0.3) in both TDPC and saline groups and higher (p < 0.05) than normal tendon. Yield and maximal stresses of the TDPC group were significantly greater (p = 0.005) than the saline group's and similar (p = 0.6) to normal tendon. However, the elastic modulus of the TDPC and saline groups were not significantly different (p = 0.32). Histological assessment of the repair tissues with Fourier transform-second harmonic generation imaging demonstrated that collagen alignment was significantly better (p = 0.02) in TDPC group than in the saline controls. In summary, treating collagenase-induced flexor tendon lesions with TDPCs improved the tensile strength and collagen fiber alignment of the repair tissue. Study Design © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2162-2171, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  18. Cell response to sterilized electrospun poly(ɛ‐caprolactone) scaffolds to aid tendon regeneration in vivo

    PubMed Central

    Bhaskar, Prajwal; Bosworth, Lucy A.; Wong, Richard; O'brien, Marie A.; Kriel, Haydn; Smit, Eugene; McGrouther, Duncan A.; Wong, Jason K.

    2016-01-01

    Abstract The functional replacement of tendon represents an unmet clinical need in situations of tendon rupture, tendon grafting, and complex tendon reconstruction, as usually there is a finite source of healthy tendon to use as donors. The microfibrous architecture of tendon is critical to the function of tendon. This study investigates the use of electrospun poly(ɛ‐caprolactone) scaffolds as potential biomaterial substitutes for tendon grafts. We assessed the performance of two electrospinning manufacturers (small‐ and large‐scale) and the effect of two sterilization techniques—gamma irradiation and ethanol submersion—on cell response to these electrospun scaffolds after their implantation into a murine tendon model. Cell infiltration and proliferation analyses were undertaken to determine the effect on cell response within the implant over a 6‐week period. Immunohistochemical analysis was performed to characterize inflammatory response and healing characteristics (proliferation, collagen deposition, myofibroblast activity, and apoptosis). Neither the sterilization techniques nor the manufacturer was observed to significantly affect the cell response to the scaffold. At each time point, cell response was similar to the autograft control. This suggests that ethanol submersion can be used for research purposes and that the scaffold can be easily reproduced by a large‐scale manufacturer. These results further imply that this electrospun scaffold may provide an alternative to autograft, thus eliminating the need for sourcing healthy tendon tissue from a secondary site. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 389–397, 2017. PMID:27649836

  19. Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction.

    PubMed

    Subramanian, Arul; Prokop, Andreas; Yamamoto, Misato; Sugimura, Kaoru; Uemura, Tadashi; Betschinger, Joerg; Knoblich, Juergen A; Volk, Talila

    2003-07-01

    Shot (previously named Kakapo), is a Drosophila Plakin family member containing both Actin binding and microtubule binding domains. In Drosophila, it is required for a wide range of processes, including axon extension, dendrite formation, axonal terminal arborization at the neuromuscular junction, tendon cell development, and adhesion of wing epithelium. To address how Shot exerts its activity at the molecular level, we investigated the molecular interactions of Shot with candidate proteins in mature larval tendon cells. We show that Shot colocalizes with EB1/APC1 and with a compact microtubule array extending between the muscle-tendon junction and the cuticle. Shot forms a protein complex with EB1 via its C-terminal EF-hands and GAS2-containing domains. In tendon cells with reduced Shot activity, EB1/APC1 dissociate from the muscle-tendon junction, and the microtubule array elongates. The resulting tendon cell, although associated with the muscle and the cuticle ends, loses its stress resistance and elongates. Our results suggest that Shot mediates tendon stress resistance by the organization of a compact microtubule network at the muscle-tendon junction. This is achieved by Shot association with the cytoplasmic faces of the basal hemiadherens junction and with the EB1/APC1 complex.

  20. In vivo tendon engineering with skeletal muscle derived cells in a mouse model.

    PubMed

    Chen, Bo; Wang, Bin; Zhang, Wen Jie; Zhou, Guangdong; Cao, Yilin; Liu, Wei

    2012-09-01

    Engineering a functional tendon with strong mechanical property remains an aim to be achieved for its eventual application. Both skeletal muscle and tendon are closely associated during their development and both can bear strong mechanical loading dynamically. This study explored the possibility of engineering stronger tendons with mouse skeletal muscle derived cells (MDCs) and with mouse tenocytes as a control. The results demonstrated that both MDCs and tenocytes shared the gene expression of growth differentiation factor-8 (GDF-8), collagens I, III, VI, scleraxis and tenomodulin, but with MyoD gene expression only in MDCs. Quantitatively, MDCs expressed higher levels of GDF-8, collagens III and VI (p < 0.05), whereas tenocytes expressed higher levels of collagen I, scleraxis and tenomodulin (p < 0.05). Interestingly, MDCs proliferated faster with more cells in S + G2/M phases than tenocytes (p < 0.05). After been seeded on polyglycolic acid (PGA) fibers, MDCs formed better quality engineered tendons with more mature collagen structure and thicker collagen fibrils as opposed to tenocyte engineered tendons. Biochemically, more collagen VI and decorin were produced in the former than in the later. Functionally, MDC engineered tendons exhibited stronger mechanical properties than tenocyte engineered tendons, including maximal load, stiffness, tensile strength and Young's modulus (p < 0.05). Furthermore, with the increase of implantation time, MDCs gradually lost their expression of myogenic molecules of MyoD and desmin and gained the expression of tenomodulin, a marker for tenocytes. Collectively, these results indicate that MDCs may serve as a desirable alternative cell source for engineering functional tendon tissue. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Dynamic loading of electrospun yarns guides mesenchymal stem cells towards a tendon lineage

    PubMed Central

    Bosworth, L.A.; Rathbone, S.R.; Bradley, R.S.; Cartmell, S.H.

    2014-01-01

    Alternative strategies are required when autograft tissue is not sufficient or available to reconstruct damaged tendons. Electrospun fibre yarns could provide such an alternative. This study investigates the seeding of human mesenchymal stem cells (hMSC) on electrospun yarns and their response when subjected to dynamic tensile loading. Cell seeded yarns sustained 3600 cycles per day for 21 days. Loaded yarns demonstrated a thickened cell layer around the scaffold׳s exterior compared to statically cultured yarns, which would suggest an increased rate of cell proliferation and/or matrix deposition, whilst maintaining a predominant uniaxial cell orientation. Tensile properties of cell-seeded yarns increased with time compared to acellular yarns. Loaded scaffolds demonstrated an up-regulation in several key tendon genes, including collagen Type I. This study demonstrates the support of hMSCs on electrospun yarns and their differentiation towards a tendon lineage when mechanically stimulated. PMID:25129861

  2. Study of Bone Marrow Mesenchymal and Tendon-Derived Stem Cells Transplantation on the Regenerating Effect of Achilles Tendon Ruptures in Rats

    PubMed Central

    Al-ani, Mohanad Kh; Xu, Kang; Sun, Yanjun; Pan, Lianhong; Xu, ZhiLing; Yang, Li

    2015-01-01

    Comparative therapeutic significance of tendon-derived stem cells (TDSCs) and bone marrow mesenchymal stem cells (BMSCs) transplantation to treat ruptured Achilles tendon was studied. Three groups of SD rats comprising 24 rats each, designated as TDSCs and BMSCs, and nontreated were studied for regenerative effects through morpho-histological evaluations and ultimate failure load. For possible mechanism in tendon repair/regeneration through TDSCs and BMSCs, we measured Collagen-I (Col-I), Col-III gene expression level by RT-PCR, and Tenascin-C expression via immunofluorescent assay. TDSCs showed higher agility in tendon healing with better appearance density and well-organized longitudinal fibrous structure, though BMSCs also showed positive effects. Initially the ultimate failure load was considerably higher in TDSCs than other two study groups during the weeks 1 and 2, but at week 4 it attained an average or healthy tendon strength of 30.2 N. Similar higher tendency in Col-I/III gene expression level during weeks 1, 2, and 4 was observed in TDSCs treated group with an upregulation of 1.5-fold and 1.1-fold than the other two study groups. Immunofluorescent assay revealed higher expression of Tenascin-C in TDSCs at week 1, while both TDSCs and BMSCs treated groups showed detectable CM-Dil-labelled cells at week 4. Compared with BMSCs, TDSCs showed higher regenerative potential while treating ruptured Achilles tendons in rats. PMID:26339252

  3. Tissue Engineering of Tendons: A Comparison of Muscle-Derived Cells, Tenocytes, and Dermal Fibroblasts as Cell Sources.

    PubMed

    Chen, Bo; Ding, Jinping; Zhang, Wenjie; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Wang, Bin

    2016-03-01

    The rapid development of tendon tissue-engineering technology may offer an alternative graft for reconstruction of severe tendon losses. One critical factor for tendon tissue engineering is the optimization of seed cells. Little is known about the optimal cell source for engineered tendons. The aim of this study was to compare mouse muscle-derived cells, dermal fibroblasts, and tenocytes and determine the optimal cell source for tendon tissue engineering. Mouse muscle-derived cells, dermal fibroblasts, and tenocytes were isolated and cultured in vitro. At passage 1, cellular morphology, cell proliferation, and tenogenic marker expression were evaluated. After seeding on the polyglycolic acid scaffolds for 2 weeks in vitro and 12 weeks in vivo, histologic qualities, ultrastructure, and biomechanical characteristics were evaluated. Proliferation and cellular morphology were similar for dermal fibroblasts and tenocytes, whereas muscle-derived cells proliferated faster than the other two groups. With regard to the phenotype difference between them, muscle-derived cells and tenocytes shared the gene expression of SCX, TNMD, GDF-8, and Col-I, but with MyoD gene expression only in muscle-derived cells. In contrast to dermal fibroblast and tenocyte constructed tendons, neotendon with muscle-derived cells exhibited better aligned collagen fibers, more mature collagen fibril structure, and stronger mechanical properties, whereas no significant difference in the dermal fibroblast and tenocyte groups was observed. Although dermal fibroblasts are candidates for tendon tissue engineering because they are similar to tenocytes in proliferation and neotendon formation, muscle-derived cells appear to be the most suitable cells for further study and development of engineered tendon.

  4. In vitro discrimination of fluoroquinolones toxicity on tendon cells: involvement of oxidative stress.

    PubMed

    Pouzaud, F; Bernard-Beaubois, K; Thevenin, M; Warnet, J-M; Hayem, G; Rat, P

    2004-01-01

    Tendinopathy are classic side effects observed with fluoroquinolones antibiotics. A previously validated model based on a spontaneously immortalized rabbit tendon cell line (Teno cell line) was used to evaluate cellular responses to the fluoroquinolones pefloxacin (PEF), ofloxacin (OFX), levofloxacin (LVX), and ciprofloxacin (CIP), in various concentrations. Cell viability, redox status changes, reduced glutathione content, and reactive oxygen species production were assessed using neutral red, Alamar blue, monobromobimane and 2,7-dichlorofluorescindiacetate fluorescent probes, respectively. Living adherent tenocytes were analyzed using a cold light cytofluorometer adapted to 96-well microplates. All fluoroquinolones showed moderate cytotoxicity after 24 h and more severe, significant toxicity after 72 h on tendon cells. Moreover, two groups of fluoroquinolones may be differentiated: intrinsic toxicity for tendon cells was high with ciprofloxacin and pefloxacin [redox status decrease was 80 and 62% (*p < 0.05) for PEF and CIP at 1 mM for 72 h, respectively], but moderate with ofloxacin and levofloxacin LVX [redox status decrease was 30 and 22% (*p < 0.05) for OFX and LVX at 1 mM during 72 h, respectively]. Our model supports a role for early oxidative stress in the development of fluoroquinolone-induced tendinopathy. Moreover, our study indicates that intrinsic toxicity to tendon cells varies across fluoroquinolones. The Teno cell line may be a useful model for detecting and evaluating tendon toxicity of new fluoroquinolones and other drugs associated with tendinopathy.

  5. Tendon overload results in alterations in cell shape and increased markers of inflammation and matrix degradation.

    PubMed

    Thorpe, C T; Chaudhry, S; Lei, I I; Varone, A; Riley, G P; Birch, H L; Clegg, P D; Screen, H R C

    2015-08-01

    Tendon injury is thought to involve both damage accumulation within the matrix and an accompanying cell response. While several studies have characterized cell and matrix response in chronically injured tendons, few have assessed the initial response of tendon to overload-induced damage. In this study, we assessed cell response to cyclic loading. Fascicle bundles from the equine superficial digital flexor tendon were exposed to cyclic loading in vitro, designed to mimic a bout of high-intensity exercise. Changes in cell morphology and protein-level alterations in markers of matrix inflammation and degradation were investigated. Loading resulted in matrix damage, which was accompanied by cells becoming rounder. The inflammatory markers cyclooxygenase-2 and interleukin-6 were increased in loaded samples, as were matrix metalloproteinase-13 and the collagen degradation marker C1,2C. These results indicate upregulation of inflammatory and degradative pathways in response to overload-induced in vitro, which may be initiated by alterations in cell strain environment because of localized matrix damage. This provides important information regarding the initiation of tendinopathy, suggesting that inflammation may play an important role in the initial cell response to tendon damage. Full understanding of the early tenocyte response to matrix damage is critical in order to develop effective treatments for tendinopathy. © 2014 The Authors. Scandinavian Journal of Medicine & Science in Sports published by John Wiley & Sons Ltd.

  6. Release of Tensile Strain on Engineered Human Tendon Tissue Disturbs Cell Adhesions, Changes Matrix Architecture, and Induces an Inflammatory Phenotype

    PubMed Central

    Bayer, Monika L.; Schjerling, Peter; Herchenhan, Andreas; Zeltz, Cedric; Heinemeier, Katja M.; Christensen, Lise; Krogsgaard, Michael; Gullberg, Donald; Kjaer, Michael

    2014-01-01

    Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α11 mRNA and protein expression, and an increase in α2 integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced. PMID:24465881

  7. 3D Mimicry of Native-Tissue-Fiber Architecture Guides Tendon-Derived Cells and Adipose Stem Cells into Artificial Tendon Constructs.

    PubMed

    Laranjeira, Mariana; Domingues, Rui M A; Costa-Almeida, Raquel; Reis, Rui L; Gomes, Manuela E

    2017-08-01

    Tendon and ligament (T/L) function is intrinsically related with their unique hierarchically and anisotropically organized extracellular matrix. Their natural healing capacity is, however, limited. Here, continuous and aligned electrospun nanofiber threads (CANT) based on synthetic/natural polymer blends mechanically reinforced with cellulose nanocrystals are produced to replicate the nanoscale collagen fibrils grouped into microscale collagen fibers that compose the native T/L. CANT are then incrementally assembled into 3D hierarchical scaffolds, resulting in woven constructions, which simultaneously mimic T/L nano-to-macro architecture, nanotopography, and nonlinear biomechanical behavior. Biological performance is assessed using human-tendon-derived cells (hTDCs) and human adipose stem cells (hASCs). Scaffolds nanotopography and microstructure induce a high cytoskeleton elongation and anisotropic organization typical of tendon tissues. Moreover, the expression of tendon-related markers (Collagen types I and III, Tenascin-C, and Scleraxis) by both cell types, and the similarities observed on their expression patterns over time suggest that the developed scaffolds not only prevent the phenotypic drift of hTDCs, but also trigger tenogenic differentiation of hASCs. Overall, these results demonstrate a feasible approach for the scalable production of 3D hierarchical scaffolds that exhibit key structural and biomechanical properties, which can be advantageously explored in acellular and cellular T/L TE strategies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Therapeutic potential of mesenchymal stem cells to treat Achilles tendon injuries.

    PubMed

    Vieira, M H C; Oliveira, R J; Eça, L P M; Pereira, I S O; Hermeto, L C; Matuo, R; Fernandes, W S; Silva, R A; Antoniolli, A C M B

    2014-12-12

    Rupture of the Achilles tendon diminishes quality of life. The gold-standard therapy is a surgical suture, but this presents complications, including wound formation and inflammation. These complications spurred evaluation of the therapeutic potential of mesenchymal stem cells (MSCs) from adipose tissue. New Zealand rabbits were divided into 6 groups (three treatments with two time points each) evaluated at either 14 or 28 days after surgery: cross section of the Achilles tendon (CSAT); CSAT + Suture; and CSAT + MSC. A comparison between all groups at both time points showed a statistically significant increase in capillaries and in the structural organization of collagen in the healed tendon in the CSAT + Suture and CSAT + MSC groups at the 14-day assessment. Comparison between the two time points within the same group showed a statistically significant decrease in the inflammatory process and an increase in the structural organization of collagen in the CSAT and CSAT + MSC groups. A study of the genomic integrity of the cells suggested a linear correlation between an increase of injuries and culture time. Thus, MSC transplantation is a good alternative for treatment of Achilles tendon ruptures because it may be conducted without surgery and tendon suture and, therefore, has no risk of adverse effects resulting from the surgical wound or inflammation caused by nonabsorbable sutures. Furthermore, this alternative treatment exhibits a better capacity for wound healing and maintaining the original tendon architecture, depending on the arrangement of the collagen fibers, and has important therapeutic potential.

  9. Novel fiber-based pure chitosan scaffold for tendon augmentation: biomechanical and cell biological evaluation.

    PubMed

    Nowotny, J; Aibibu, D; Farack, J; Nimtschke, U; Hild, M; Gelinsky, M; Kasten, P; Cherif, Ch

    2016-07-01

    One possibility to improve the mechanical properties after tendon ruptures is augmentation with a scaffold. Based on wet spinning technology, chitosan fibres were processed to a novel pure high-grade multifilament yarn with reproducible quality. The fibres were braided to obtain a 3D tendon scaffold. The CS fibres and scaffolds were evaluated biomechanically and compared to human supraspinatus (SSP) tendons. For the cytobiological characterization, in vitro cell culture experiments with human mesenchymal stem cells (hMSC) were performed. Three types of 3D circular braided scaffolds were fabricated. Significantly, higher ultimate stress values were measured for scaffold with larger filament yarn, compared to scaffold with smaller filament yarn. During cultivation over 28 days, the cells showed in dependence of isolation method and/or donor a doubling or tripling of the cell number or even a six-fold increase on the CS scaffold, which was comparable to the control (polystyrene) or in the case of cells obtained from human biceps tendon even higher proliferation rates. After 14 days, the scaffold surface was covered homogeneously with a cell layer. In summary, the present work demonstrates that braided chitosan scaffolds constitute a straightforward approach for designing tendon analogues, maintaining important flexibility in scaffold design and providing favourable mechanical properties of the resulting construct.

  10. Characterization and comparison of post-natal rat Achilles tendon-derived stem cells at different development stages.

    PubMed

    Chen, Jialin; Zhang, Wei; Liu, Zeyu; Zhu, Ting; Shen, Weiliang; Ran, Jisheng; Tang, Qiaomei; Gong, Xiaonan; Backman, Ludvig J; Chen, Xiao; Chen, Xiaowen; Wen, Feiqiu; Ouyang, Hongwei

    2016-03-14

    Tendon stem/progenitor cells (TSPCs) are a potential cell source for tendon tissue engineering. The striking morphological and structural changes of tendon tissue during development indicate the complexity of TSPCs at different stages. This study aims to characterize and compare post-natal rat Achilles tendon tissue and TSPCs at different stages of development. The tendon tissue showed distinct differences during development: the tissue structure became denser and more regular, the nuclei became spindle-shaped and the cell number decreased with time. TSPCs derived from 7 day Achilles tendon tissue showed the highest self-renewal ability, cell proliferation, and differentiation potential towards mesenchymal lineage, compared to TSPCs derived from 1 day and 56 day tissue. Microarray data showed up-regulation of several groups of genes in TSPCs derived from 7 day Achilles tendon tissue, which may account for the unique cell characteristics during this specific stage of development. Our results indicate that TSPCs derived from 7 day Achilles tendon tissue is a superior cell source as compared to TSPCs derived from 1 day and 56 day tissue, demonstrating the importance of choosing a suitable stem cell source for effective tendon tissue engineering and regeneration.

  11. TGF-beta1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via PAI-1: Implications for Scarless Flexor Tendon Repair

    PubMed Central

    Farhat, Youssef M.; Al-Maliki, Alaa A.; Easa, Anas; O’Keefe, Regis J.; Schwarz, Edward M.; Awad, Hani A.

    2014-01-01

    Flexor tendon injuries caused by deep lacerations to the hands are a challenging problem as they often result in debilitating adhesions that prevent the movement of the afflicted fingers. Evidence exists that tendon adhesions as well as scarring throughout the body are largely precipitated by the pleiotropic growth factor, TGF-β1, but the effects of TGF-β1 are poorly understood in tendon healing. Using an in vitro model of tendon healing, we previously found that TGF-β1 causes gene expression changes in tenocytes that are consistent with scar tissue and adhesion formation, including upregulation of the anti-fibrinolytic protein, PAI-1. Therefore, we hypothesized that TGF-β1 contributes to scarring and adhesions by reducing the activity of proteases responsible for ECM degradation and remodeling, such as plasmin and MMPs, via upregulation of PAI-1. To test our hypothesis, we examined the effects of TGF-β1 on the protease activity of tendon cells. We found that flexor tendon tenocytes treated with TGF-β1 had significantly reduced levels of active MMP-2 and plasmin. Interestingly, the effects of TGF-β1 on protease activity were completely abolished in tendon cells from homozygous PAI-1 KO mice, which are unable to express PAI-1. Our findings support the hypothesis that TGF-β1 induces PAI-1, which suppresses plasmin and plasmin-mediated MMP activity, and provide evidence that PAI-1 may be a novel therapeutic target for preventing adhesions and promoting a scarless, regenerative repair of flexor tendon injuries. PMID:24962629

  12. Cracking open cell death in the Drosophila ovary

    PubMed Central

    Pritchett, Tracy L.; Tanner, Elizabeth A.; McCall, Kimberly

    2010-01-01

    The Drosophila melanogaster ovary is a powerful yet simple system with only a few cell types. Cell death in the ovary can be induced in response to multiple developmental and environmental signals. These cell deaths occur at distinct stages of oogenesis and involve unique mechanisms utilizing apoptotic, autophagic and perhaps necrotic processes. In this review, we summarize recent progress characterizing cell death mechanisms in the fly ovary. PMID:19533361

  13. Growth and Development Symposium: Stem cell therapy in equine tendon injury.

    PubMed

    Reed, S A; Leahy, E R

    2013-01-01

    Tendon injuries affect all levels of athletic horses and represent a significant loss to the equine industry. Accumulation of microdamage within the tendon architecture leads to formation of core lesions. Traditional approaches to tendon repair are based on an initial period of rest to limit the inflammatory process followed by a controlled reloading program designed to promote the maturation and linear arrangement of scar tissue within the lesion. However, these treatment protocols are inefficient, resulting in prolonged recovery periods and frequent recurrence. Current alternative therapies include the use of bone marrow-derived mesenchymal stem cells (BMSC) and a population of nucleated cells from adipose containing adipose-derived mesenchymal stem cells (AdMSC). Umbilical cord blood-derived stem cells (UCB) have recently received attention for their increased plasticity in vitro and potential as a therapeutic aid. Both BMSC and AdMSC require expansion in culture before implantation to obtain a pure stem cell population, limiting the time frame for implantation. Collected at parturition, UCB can be cryopreserved for future use. Furthermore, the low immunogenicity of the UCB population allows for allogeneic implantation. Current research indicates that BMSC, AdMSC, and UCB can differentiate into tenocyte-like cells in vitro, increasing expression of scleraxis, tenascin c, and extracellular matrix proteins. When implanted, BMSC and AdMSC engraft into the tendon and improve tendon architecture. However, treatment with these stem cells does not decrease recovery period. Furthermore, the resulting regeneration is not optimal, as the resulting tissue is still inferior to native tendon. Umbilical cord blood-derived stem cells may provide an alternate source of stem cells that promote improved regeneration of tendon tissue. A more naïve cell population, these cells may have a greater rate of engraftment as well as an increased ability to secrete bioactive factors and

  14. Engineered stem cell niche matrices for rotator cuff tendon regenerative engineering

    PubMed Central

    Peach, M. Sean; Ramos, Daisy M.; Morozowich, Nicole L.; Mazzocca, Augustus D.; Doty, Steven B.; Allcock, Harry R.; Kumbar, Sangamesh G.; Laurencin, Cato T.

    2017-01-01

    Rotator cuff (RC) tears represent a large proportion of musculoskeletal injuries attended to at the clinic and thereby make RC repair surgeries one of the most widely performed musculoskeletal procedures. Despite the high incidence rate of RC tears, operative treatments have provided minimal functional gains and suffer from high re-tear rates. The hypocellular nature of tendon tissue poses a limited capacity for regeneration. In recent years, great strides have been made in the area of tendonogenesis and differentiation towards tendon cells due to a greater understanding of the tendon stem cell niche, development of advanced materials, improved scaffold fabrication techniques, and delineation of the phenotype development process. Though in vitro models for tendonogenesis have shown promising results, in vivo models have been less successful. The present work investigates structured matrices mimicking the tendon microenvironment as cell delivery vehicles in a rat RC tear model. RC injuries augmented with a matrix delivering rat mesenchymal stem cells (rMSCs) showed enhanced regeneration over suture repair alone or repair with augmentation, at 6 and 12-weeks post-surgery. The local delivery of rMSCs led to increased mechanical properties and improved tissue morphology. We hypothesize that the mesenchymal stem cells function to modulate the local immune and bioactivity environment through autocrine/paracrine and/or cell homing mechanisms. This study provides evidence for improved tendon healing with biomimetic matrices and delivered MSCs with the potential for translation to larger, clinical animal models. The enhanced regenerative healing response with stem cell delivering biomimetic matrices may represent a new treatment paradigm for massive RC tendon tears. PMID:28369135

  15. Engineered stem cell niche matrices for rotator cuff tendon regenerative engineering.

    PubMed

    Peach, M Sean; Ramos, Daisy M; James, Roshan; Morozowich, Nicole L; Mazzocca, Augustus D; Doty, Steven B; Allcock, Harry R; Kumbar, Sangamesh G; Laurencin, Cato T

    2017-01-01

    Rotator cuff (RC) tears represent a large proportion of musculoskeletal injuries attended to at the clinic and thereby make RC repair surgeries one of the most widely performed musculoskeletal procedures. Despite the high incidence rate of RC tears, operative treatments have provided minimal functional gains and suffer from high re-tear rates. The hypocellular nature of tendon tissue poses a limited capacity for regeneration. In recent years, great strides have been made in the area of tendonogenesis and differentiation towards tendon cells due to a greater understanding of the tendon stem cell niche, development of advanced materials, improved scaffold fabrication techniques, and delineation of the phenotype development process. Though in vitro models for tendonogenesis have shown promising results, in vivo models have been less successful. The present work investigates structured matrices mimicking the tendon microenvironment as cell delivery vehicles in a rat RC tear model. RC injuries augmented with a matrix delivering rat mesenchymal stem cells (rMSCs) showed enhanced regeneration over suture repair alone or repair with augmentation, at 6 and 12-weeks post-surgery. The local delivery of rMSCs led to increased mechanical properties and improved tissue morphology. We hypothesize that the mesenchymal stem cells function to modulate the local immune and bioactivity environment through autocrine/paracrine and/or cell homing mechanisms. This study provides evidence for improved tendon healing with biomimetic matrices and delivered MSCs with the potential for translation to larger, clinical animal models. The enhanced regenerative healing response with stem cell delivering biomimetic matrices may represent a new treatment paradigm for massive RC tendon tears.

  16. Dissecting Social Cell Biology and Tumors Using Drosophila Genetics

    PubMed Central

    Pastor-Pareja, José Carlos; Xu, Tian

    2014-01-01

    Cancer was seen for a long time as a strictly cell-autonomous process in which oncogenes and tumor-suppressor mutations drive clonal cell expansions. Research in the past decade, however, paints a more integrative picture of communication and interplay between neighboring cells in tissues. It is increasingly clear as well that tumors, far from being homogenous lumps of cells, consist of different cell types that function together as complex tissue-level communities. The repertoire of interactive cell behaviors and the quantity of cellular players involved call for a social cell biology that investigates these interactions. Research into this social cell biology is critical for understanding development of normal and tumoral tissues. Such complex social cell biology interactions can be parsed in Drosophila. Techniques in Drosophila for analysis of gene function and clonal behavior allow us to generate tumors and dissect their complex interactive biology with cellular resolution. Here, we review recent Drosophila research aimed at understanding tissue-level biology and social cell interactions in tumors, highlighting the principles these studies reveal. PMID:23988119

  17. Treating Achilles tendon rupture in rats with bone-marrow-cell transplantation therapy.

    PubMed

    Okamoto, Naofumi; Kushida, Taketoshi; Oe, Kenichi; Umeda, Masayuki; Ikehara, Susumu; Iida, Hirokazu

    2010-12-01

    Bone marrow cells possess multipotentiality and have been used for several treatments. We hypothesized that bone marrow cells might differentiate into regenerated tendon and that several cytokines within bone marrow cells might accelerate tendon healing. Therefore, we treated Achilles tendon ruptures in a rat model with transplantation of whole bone marrow cells. Nine F344/Nslc (Fisher) rats were the source of bone marrow cells and mesenchymal stem cells as well as normal Achilles tendons. Eighty-seven Fisher rats were used for the experiments. The rats were divided into three groups: the BMC group (bone marrow cells injected around the tendon), the MSC group (mesenchymal stem cells injected around the tendon), and the non-treated control group (incision only). Outcome measures included mechanical testing, collagen immunohistochemistry, histological analysis, and reverse transcription-polymerase chain reaction to detect expression of transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF). The ultimate failure load in the BMC group was significantly greater than that in the non-treated or the MSC group at seven days after incision (3.8 N vs. 0.9 N or 2.1 N, p < 0.016) and at fourteen days after incision (10.2 N vs. 6.1 N or 8.2 N, p < 0.016). The ultimate failure load in the BMC group at twenty-eight days after incision (33.8 N) was the same as that of normal tendon (34.8 N). The BMC group demonstrated stronger staining for type-III collagen at seven days after incision and stronger staining for type-I collagen at twenty-eight days than did the MSC group. Expression of TGF-β and VEGF in the BMC group was significantly increased compared with that in the other groups at four days after incision (TGF-β: 1.6 vs. 1.3 or 0.6, p < 0.01; VEGF: 1.7 vs. 1.1 or 0.9, p < 0.01). Transplantation of whole bone marrow cells may be a better and more readily available treatment for Achilles tendon rupture than cultured mesenchymal stem cells.

  18. Measuring tendon properties in mdx mice: cell viability and viscoelastic characteristics.

    PubMed

    Rizzuto, E; Musarò, A; Catizone, A; Del Prete, Z

    2009-10-16

    Muscular dystrophy is a genetic disorder of skeletal muscle characterized by progressive muscle weakness. Here we assessed whether muscle wasting affects cell viability and mechanical properties of extensor digitorum longus (EDL) and of tibialis anterior (TA) tendons from mdx dystrophic mice compared to wild type (WT) mice. mdx mice represent the classical animal model for human Duchenne muscular dystrophy, and show several signs of the pathology, including a decrease in specific force and an increase of fibrotic index. Cell viability of tendons was evaluated by histological analysis, and viscoelastic properties have been assessed by a rapid measurement protocol that allowed us to compute, at the same time, tissue complex compliance for all the frequencies of interest. Confocal microscopy and mechanical properties measurements revealed that mdx tendons, compared to WT ones, have an increase in the number of dead cells and a significant reduction in tissue elasticity for all the frequencies that were tested. These findings indicate a reduced quality of the tissue. Moreover, mdx tendons have an increase in the viscous response, indicating that during dynamic loading, they dissipate more energy compared to WT. Our results demonstrate that muscular dystrophy involves not only muscle wasting, but also alteration in the viscoelastic properties of tendons, suggesting a paracrine effect of altered skeletal muscle on tendinous tissue.

  19. Disruption of Topoisomerase II Perturbs Pairing in Drosophila Cell Culture

    PubMed Central

    Williams, Benjamin R.; Bateman, Jack R.; Novikov, Natasha D.; Wu, C.-Ting

    2007-01-01

    Homolog pairing refers to the alignment and physical apposition of homologous chromosomal segments. Although commonly observed during meiosis, homolog pairing also occurs in nonmeiotic cells of several organisms, including humans and Drosophila. The mechanism underlying nonmeiotic pairing, however, remains largely unknown. Here, we explore the use of established Drosophila cell lines for the analysis of pairing in somatic cells. Using fluorescent in situ hybridization (FISH), we assayed pairing at nine regions scattered throughout the genome of Kc167 cells, observing high levels of homolog pairing at all six euchromatic regions assayed and variably lower levels in regions in or near centromeric heterochromatin. We have also observed extensive pairing in six additional cell lines representing different tissues of origin, different ploidies, and two different species, demonstrating homolog pairing in cell culture to be impervious to cell type or culture history. Furthermore, by sorting Kc167 cells into G1, S, and G2 subpopulations, we show that even progression through these stages of the cell cycle does not significantly change pairing levels. Finally, our data indicate that disrupting Drosophila topoisomerase II (Top2) gene function with RNAi and chemical inhibitors perturbs homolog pairing, suggesting Top2 to be a gene important for pairing. PMID:17890361

  20. Organizer activity of the polar cells during Drosophila oogenesis.

    PubMed

    Grammont, Muriel; Irvine, Kenneth D

    2002-11-01

    Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and are specified by the activation of Notch. We have investigated their role in follicle patterning by creating clones of cells mutant for the Notch modulator fringe. This genetic ablation of polar cells results in cell fate defects within surrounding follicle cells. At the anterior, the border cells, the immediately adjacent follicle cell fate, are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. Our observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis.

  1. Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos.

    PubMed

    Nonaka, Saori; Hori, Aki; Nakanishi, Yoshinobu; Kuraishi, Takayuki

    2017-08-03

    The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

  2. Sources of adult mesenchymal stem cells for ligament and tendon tissue engineering.

    PubMed

    Dhinsa, Baljinder S; Mahapatra, Anant N; Khan, Wasim S

    2015-01-01

    Tendon and ligament injuries are common, and repair slowly with reduced biomechanical properties. With increasing financial demands on the health service and patients to recover from tendon and ligament injuries faster, and with less morbidity, health professionals are exploring new treatment options. Tissue engineering may provide the answer, with its unlimited source of natural cells that in the correct environment may improve repair and regeneration of tendon and ligament tissue. Mesenchymal stem cells have demonstrated the ability to self renew and have multilineage differentiation potential. The use of bone marrow-derived mesenchymal stem cells has been reported, however significant in vitro culture expansion is required due to the low yield of cells, which has financial implications. Harvesting of bone marrow cells also has associated morbidity. Several studies have looked at alternative sources for mesenchymal stem cells. Reports in literature from animal studies have been encouraging, however further work is required. This review assesses the potential sources of mesenchymal stem cells for tissue engineering in tendons and ligaments.

  3. The development of germline stem cells in Drosophila.

    PubMed

    Dansereau, David A; Lasko, Paul

    2008-01-01

    Germline stem cells (GSCs) in Drosophila are a valuable model to explore of how adult stem cells are regulated in vivo. Genetic dissection of this system has shown that stem cell fate is determined and maintained by the stem cell's somatic microenvironment or niche. In Drosophila gonads, the stem cell niche -- the cap cell cluster in females and the hub in males -- acts as a signaling center to recruit GSCs from among a small population of undifferentiated primordial germ cells (PGCs). Short-range signals from the niche specify and regulate stem cell fate by maintaining the undifferentiated state of the PGCs next to the niche. Germline cells that do not receive the niche signals because of their location assume the default fate and differentiate. Once GSCs are specified, adherens junctions maintain close association between the stem cells and their niche and help to orient stem cell division so that one daughter is displaced from the niche and differentiates. In females, stem cell fate depends on bone morphogenetic protein (BMP) signals from the cap cells; in males, hub cells express the cytokine-like ligand Unpaired, which activates the Janus kinase-signal transducers and activators of transcription (Jak-Stat) pathway in stem cells. Although the signaling pathways operating between the niche and stem cells are different, there are common general features in both males and females, including the arrangement of cell types, many of the genes used, and the logic of the system that maintains stem cell fate.

  4. Microgrooved topographical surface directs tenogenic lineage specific differentiation of mouse tendon derived stem cells.

    PubMed

    Shi, Yuan; Zhou, Kaili; Zhang, Wenjie; Zhang, Zhiyong; Zhou, Guangdong; Cao, Yilin; Liu, Wei

    2017-01-10

    Tendon derived stem cells (TDSCs) are the endogenous cell source for tenocyte turnover and tendon functional maintenance. They are also the important cell source for tendon engineering and regeneration. In addition, TDSCs also play an important role in tendinopathy via their non-tenogenic lineage differentiation. It has been well demonstrated that cell shape could determine mesenchymal stem cell (MSC) lineage differentiation. In this study, a parallel microgrooved polydimethylsiloxane (PDMS) membrane (10 µm groove width and 3 µm depth) was employed to investigate the role of cell elongation via this particular topographic surface in directing murine TDSC (mTDSC) lineage differentiation. The results showed that elongated mTDSCs exhibited significantly enhanced the gene expression of tenogenic markers when compared to the spread cells that grew on smooth PDMS membrane including tenomodulin, scleraxis, collagens I, III, and VI, decorin and tenascin (p  <  0.05). Meanwhile, stemness related genes such as Nanog, Sox2 and Oct4 were significantly inhibited for their expression in elongated mTDSCs (p  <  0.05). When under tri-lineage induced differentiation, cell elongation significantly inhibited mTDSC differentiation towards chondrogenic and adipogenic lineages (p  <  0.05). Furthermore, cell elongation could significantly inhibit mTDSC osteogenic lineage differentiation (p  <  0.05) induced by BMP-2, a tendinopathy mimicking stimulant. In conclusion, simulation of native tendon structure via using parallel microgrooved topography can promote mTDSC differentiation specifically towards tenogenic lineage and prevent non-tenogenic lineage differentiation, providing an insight into the design of tendon regenerative materials.

  5. Isolation and characterization of 2 new human rotator cuff and long head of biceps tendon cells possessing stem cell-like self-renewal and multipotential differentiation capacity.

    PubMed

    Randelli, Pietro; Conforti, Erika; Piccoli, Marco; Ragone, Vincenza; Creo, Pasquale; Cirillo, Federica; Masuzzo, Pamela; Tringali, Cristina; Cabitza, Paolo; Tettamanti, Guido; Gagliano, Nicoletta; Anastasia, Luigi

    2013-07-01

    Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside. Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration. Controlled laboratory study. Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties. Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types--namely, osteoblasts, adipocytes, and skeletal muscle cells. This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status. Rotator cuff and long head of the biceps tendons contain a stem cell

  6. Transplantation of tendon-derived stem cells pre-treated with connective tissue growth factor and ascorbic acid in vitro promoted better tendon repair in a patellar tendon window injury rat model.

    PubMed

    Lui, Pauline Po Yee; Wong, On Tik; Lee, Yuk Wa

    2016-01-01

    Treatment of tendon-derived stem cells (TDSCs) with connective tissue growth factor (CTGF) and ascorbic acid promoted their tenogenic differentiation. We investigated the effects of TDSCs pre-treated with CTGF and ascorbic acid on tendon repair in a patellar tendon window injury rat model. Green fluorescent protein-TDSCs (GFP-TDSCs) were pre-treated with or without CTGF and ascorbic acid for 2 weeks before transplantation. The patellar tendons of rats were injured and divided into three groups: fibrin glue-only group (control group), untreated and treated TDSC group. The rats were followed up until week 16. The treated TDSCs accelerated and enhanced the quality of tendon repair compared with untreated TDSCs up to week 8, which was better than that in the controls up to week 16 as shown by histology, ultrasound imaging and biomechanical test. The fibrils in the treated TDSC group showed better alignment and larger size compared with those in the control group at week 8 (P = 0.004). There was lower risk of ectopic mineralization after transplantation of treated or untreated TDSCs (all P ≤ 0.050). The transplanted cells proliferated and could be detected in the window wound up to weeks 2 to 4 and week 8 for the untreated and treated TDSC groups, respectively. The transplantation of TDSCs promoted tendon repair up to week 16, with CTGF and ascorbic acid pre-treatment showing the best results up to week 8. Pre-treatment of TDSCs with CTGF and ascorbic acid may be used to further enhance the rate and quality of tendon repair after injury. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. Mesenchymal stem cells from a hypoxic culture improve and engraft Achilles tendon repair.

    PubMed

    Huang, Tung-Fu; Yew, Tu-Lai; Chiang, En-Rung; Ma, Hsiao-Li; Hsu, Chih-Yuan; Hsu, Shan-Hui; Hsu, Yuan-Tong; Hung, Shih-Chieh

    2013-05-01

    Bone marrow-derived mesenchymal stem cells (MSCs) from humans cultured under hypoxic conditions increase bone healing capacity. Rat MSCs cultured under hypoxic conditions increase the tendon healing potential after transplantation into injured Achilles tendons. Controlled laboratory study. Biomechanical testing, histological analysis, and bromodeoxyuridine (BrdU) labeling/collagen immunohistochemistry were performed to demonstrate that augmentation of an Achilles tendon rupture site with hypoxic MSCs increases healing capacity compared with normoxic MSCs and controls. Fifty Sprague-Dawley rats were used for the experiments, with 2 rats as the source of bone marrow MSCs. The cut Achilles tendons in the rats were equally divided into 3 groups: hypoxic MSC, normoxic MSC, and nontreated (vehicle control). The uncut tendons served as normal uncut controls. Outcome measures included mechanical testing in 24 rats, histological analysis, and BrdU labeling/collagen immunohistochemistry in another 24 rats. The ultimate failure load in the hypoxic MSC group was significantly greater than that in the nontreated or normoxic MSC group at 2 weeks after incision (2.1 N/mm(2) vs 1.1 N/mm(2) or 1.9 N/mm(2), respectively) and at 4 weeks after incision (5.5 N/mm(2) vs 1.7 N/mm(2) or 2.7 N/mm(2), respectively). The ultimate failure load in the hypoxic MSC group at 4 weeks after incision (5.5 N/mm(2)) was close to but still significantly less than that of the uncut tendon (7.2 N/mm(2)). Histological analysis as determined by the semiquantitative Bonar histopathological grading scale revealed that the hypoxic MSC group underwent a significant improvement in Achilles tendon healing both at 2 and 4 weeks when compared with the nontreated or normoxic MSC group via statistical analysis. Immunohistochemistry further demonstrated that the hypoxic and normoxic MSC groups had stronger immunostaining for type I and type III collagen than did the nontreated group both at 2 and 4 weeks after

  8. Scleraxis-overexpressed human embryonic stem cell-derived mesenchymal stem cells for tendon tissue engineering with knitted silk-collagen scaffold.

    PubMed

    Chen, Xiao; Yin, Zi; Chen, Jia-Lin; Liu, Huan-Huan; Shen, Wei-Liang; Fang, Zhi; Zhu, Ting; Ji, Junfeng; Ouyang, Hong-Wei; Zou, Xiao-Hui

    2014-06-01

    Despite our previous study that demonstrates that human embryonic stem cells (hESCs) can be used as seed cells for tendon tissue engineering after stepwise induction, suboptimal tendon regeneration implies that a new strategy needs to be developed for tendon repair. We investigated whether overexpression of the tendon-specific transcription factor scleraxis (SCX) in hESC-derived mesenchymal stem cells (hESC-MSCs) together with knitted silk-collagen sponge scaffold could promote tendon regeneration. hESCs were initially differentiated into MSCs and then engineered with scleraxis (SCX+hESC-MSCs). Engineered tendons were constructed with SCX+hESC-MSCs and a knitted silk-collagen sponge scaffold and then mechanical stress was applied. SCX elevated tendon gene expression in hESC-MSCs and concomitantly attenuated their adipogenic and chondrogenic potential. Mechanical stress further augmented the expression of tendon-specific genes in SCX+hESC-MSC-engineered tendon. Moreover, in vivo mechanical stimulation promoted the alignment of cells and increased the diameter of collagen fibers after ectopic transplantation. In the in vivo tendon repair model, the SCX+hESC-MSC-engineered tendon enhanced the regeneration process as shown by histological scores and superior mechanical performance compared with control cells, especially at early stages. Our study offers new evidence concerning the roles of SCX in tendon differentiation and regeneration. We demonstrated a novel strategy of combining hESCs, genetic engineering, and tissue-engineering principles for tendon regeneration, which are important for the future application of hESCs and silk scaffolds for tendon repair.

  9. Heterogeneous expression of Drosophila gustatory receptors in enteroendocrine cells.

    PubMed

    Park, Jeong-Ho; Kwon, Jae Young

    2011-01-01

    The gastrointestinal tract is emerging as a major site of chemosensation in mammalian studies. Enteroendocrine cells are chemosensory cells in the gut which produce regulatory peptides in response to luminal contents to regulate gut physiology, food intake, and glucose homeostasis, among other possible functions. Increasing evidence shows that mammalian taste receptors and taste signaling molecules are expressed in enteroendocrine cells in the gut. Invertebrate models such as Drosophila can provide a simple and genetically tractable system to study the chemosensory functions of enteroendocrine cells in vivo. To establish Drosophila enteroendocrine cells as a model for studying gut chemosensation, we used the GAL4/UAS system to examine the expression of all 68 Gustatory receptors (Grs) in the intestine. We find that 12 Gr-GAL4 drivers label subsets of enteroendocrine cells in the midgut, and examine colocalization of these drivers with the regulatory peptides neuropeptide F (NPF), locustatachykinin (LTK), and diuretic hormone 31 (DH31). RT-PCR analysis provides additional evidence for the presence of Gr transcripts in the gut. Our results suggest that the Drosophila Grs have chemosensory roles in the intestine to regulate physiological functions such as food uptake, nutrient absorption, or sugar homeostasis.

  10. A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes

    PubMed Central

    Mueller, A. J.; Tew, S. R.; Vasieva, O.; Clegg, P. D.; Canty-Laird, E. G.

    2016-01-01

    Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin. PMID:27670352

  11. Growth factor delivery vehicles for tendon injuries: Mesenchymal stem cells and Platelet Rich Plasma

    PubMed Central

    Guevara-Alvarez, Alberto; Schmitt, Andreas; Russell, Ryan P.; Imhoff, Andreas B.; Buchmann, Stefan

    2014-01-01

    Summary Background: tendon tissue shows limited regeneration potential with formation of scar tissue and inferior mechanical properties. The capacity of several growth factors to improve the healing response and decrease scar formation is described in different preclinical studies. Besides the application of isolated growth factors, current research focuses on two further strategies to improve the healing response in tendon injuries: platelet rich plasma (PRP) and mesenchymal stem cells (MSCs). Objective: the present review focuses on these two options and describes their potential to improve tendon healing. Results: in vitro experiments and animal studies showed promising results for the use of PRP, however clinical controlled studies have shown a tendency of reduced pain related symptoms but no significant differences in overall clinical scores. On the other hand MSCs are not totally arrived in clinical use so that there is still a lack of randomized controlled trials. In basic research experiments they show an extraordinary paracrine activity, anti-inflammatory effect and the possibility to differentiate in tenocytes when different activating-factors are added. Conclusion: preclinical studies have shown promising results in improving tendon remodeling but the comparability of current literature is difficult due to different compositions. PRP and MSCs can act as efficient growth factor vehicles, however further studies should be performed in order to adequate investigate their clinical benefits in different tendon pathologies. PMID:25489557

  12. Planar Cell Polarity Signaling in the Drosophila Eye

    PubMed Central

    Jenny, Andreas

    2017-01-01

    Planar cell polarity (PCP) signaling regulates the establishment of polarity within the plane of an epithelium and allows cells to obtain directional information. Its results are as diverse as the determination of cell fates, the generation of asymmetric but highly aligned structures (e.g., stereocilia in the human ear or hairs on a fly wing), or the directional migration of cells during convergent extension during vertebrate gastrulation. Aberrant PCP establishment can lead to human birth defects or kidney disease. PCP signaling is governed by the noncanonical Wnt or Fz/PCP pathway. Traditionally, PCP establishment has been best studied in Drosophila, mainly due to the versatility of the fly as a genetic model system. In Drosophila, PCP is essential for the orientation of wing and abdominal hairs, the orientation of the division axis of sensory organ precursors, and the polarization of ommatidia in the eye, the latter requiring a highly coordinated movement of groups of photoreceptor cells during the process of ommatidial rotation. Here, I review our current understanding of PCP signaling in the Drosophila eye and allude to parallels in vertebrates. PMID:20959167

  13. Planar cell polarity signaling in the Drosophila eye.

    PubMed

    Jenny, Andreas

    2010-01-01

    Planar cell polarity (PCP) signaling regulates the establishment of polarity within the plane of an epithelium and allows cells to obtain directional information. Its results are as diverse as the determination of cell fates, the generation of asymmetric but highly aligned structures (e.g., stereocilia in the human ear or hairs on a fly wing), or the directional migration of cells during convergent extension during vertebrate gastrulation. Aberrant PCP establishment can lead to human birth defects or kidney disease. PCP signaling is governed by the noncanonical Wnt or Fz/PCP pathway. Traditionally, PCP establishment has been best studied in Drosophila, mainly due to the versatility of the fly as a genetic model system. In Drosophila, PCP is essential for the orientation of wing and abdominal hairs, the orientation of the division axis of sensory organ precursors, and the polarization of ommatidia in the eye, the latter requiring a highly coordinated movement of groups of photoreceptor cells during the process of ommatidial rotation. Here, I review our current understanding of PCP signaling in the Drosophila eye and allude to parallels in vertebrates. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. Copper homoeostasis in Drosophila melanogaster S2 cells

    PubMed Central

    2004-01-01

    Copper homoeostasis was investigated in the Drosophila melanogaster S2 cell line to develop an insect model for the study of copper regulation. Real-time PCR studies have demonstrated expression in S2 cells of putative orthologues of human Cu regulatory genes involved in the uptake, transport, sequestration and efflux of Cu. Drosophila orthologues of the mammalian Cu chaperones, ATOX1 (a human orthologue of yeast ATX1), CCS (copper chaperone for superoxide dismutase), COX17 (a human orthologue of yeast COX17), and SCO1 and SCO2, did not significantly respond transcriptionally to increased Cu levels, whereas MtnA, MtnB and MtnD (Drosophila orthologues of human metallothioneins) were up-regulated by Cu in a time- and dose-dependent manner. To examine the effect on Cu homoeostasis, expression of several key copper homoeostasis genes was suppressed using double-stranded RNA interference. Suppression of the MTF-1 (metal-regulatory transcription factor 1), reduced both basal and Cu-induced gene expressions of MtnA, MtnB and MtnD, significantly reducing the tolerance of these cells to increased Cu. Suppression of either Ctr1A (a Drosophila orthologue of yeast CTR1) or Ctr1B significantly reduced Cu uptake from media, demonstrating that both these proteins function to transport Cu into S2 cells. Significantly, Cu induced Ctr1B gene expression, and this could be prevented by suppressing MTF-1, suggesting that Ctr1B might be involved in Cu detoxification. Suppression of DmATP7, the putative homologue of human Cu transporter genes ATP7A and ATP7B, significantly increased Cu accumulation, demonstrating that DmATP7 is essential for efflux of excess Cu. This work is consistent with previous studies in mammalian cells, validating S2 cells as a model system for studying Cu transport and identifying novel Cu regulatory mechanisms. PMID:15239669

  15. The utilization of decellularized tendon slices to provide an inductive microenvironment for the proliferation and tenogenic differentiation of stem cells.

    PubMed

    Ning, Liang-Ju; Zhang, Ya-Jing; Zhang, Yi; Qing, Quan; Jiang, Yan-Lin; Yang, Jie-Liang; Luo, Jing-Cong; Qin, Ting-Wu

    2015-06-01

    The extracellular matrix (ECM) microenvironment for the stem cell niches, including but not limited to the biochemical composition, matrix topography, and stiffness, is crucial to stem cell proliferation and differentiation. The purpose of this study was to explore the capacity of the decellularized tendon slices (DTSs) to induce stem cell proliferation and tenogenic differentiation. Rat adult stem cells, including tendon-derived stem cells (TDSCs) and bone marrow-derived stem cells (BMSCs), were identified to have universal stem cell characteristics. The DTSs were found to retain the native tendon ECM microenvironment cues, including the inherent surface topography, well-preserved tendon ECM biochemical composition and similar stiffness to native tendon. When the TDSCs and BMSCs were cultured on the DTSs respectively, the LIVE/DEAD assay, alamarBlue® assay, scanning electron microscopy examination and qRT-PCR analysis demonstrated that the DTSs have the capacity to support these stem cells homogeneous distribution, alignment, significant proliferation and tenogenic differentiation. Taken together, the findings of this study indicate that the DTSs can provide a naturally inductive microenvironment for the proliferation and tenogenic differentiation of TDSCs and BMSCs, supporting the use of decellularized tendon ECM as a promising and valuable approach for tendon repair/reconstruction. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Kakapo, a novel cytoskeletal-associated protein is essential for the restricted localization of the neuregulin-like factor, vein, at the muscle-tendon junction site.

    PubMed

    Strumpf, D; Volk, T

    1998-11-30

    In the Drosophila embryo, the correct association of muscles with their specific tendon cells is achieved through reciprocal interactions between these two distinct cell types. Tendon cell differentiation is initiated by activation of the EGF-receptor signaling pathway within these cells by Vein, a neuregulin-like factor secreted by the approaching myotube. Here, we describe the cloning and the molecular and genetic analyses of kakapo, a Drosophila gene, expressed in the tendons, that is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. A major defect in the kakapo mutant tendon cells is the failure of Vein to be localized at the muscle-tendon junctional site; instead, Vein is dispersed and its levels are reduced. This may lead to aberrant differentiation of tendon cells and consequently to the kakapo mutant deranged somatic muscle phenotype.

  17. Kakapo, a Novel Cytoskeletal-associated Protein Is Essential for the Restricted Localization of the Neuregulin-like Factor, Vein, at the Muscle–Tendon Junction Site

    PubMed Central

    Strumpf, Dan; Volk, Talila

    1998-01-01

    In the Drosophila embryo, the correct association of muscles with their specific tendon cells is achieved through reciprocal interactions between these two distinct cell types. Tendon cell differentiation is initiated by activation of the EGF-receptor signaling pathway within these cells by Vein, a neuregulin-like factor secreted by the approaching myotube. Here, we describe the cloning and the molecular and genetic analyses of kakapo, a Drosophila gene, expressed in the tendons, that is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. A major defect in the kakapo mutant tendon cells is the failure of Vein to be localized at the muscle–tendon junctional site; instead, Vein is dispersed and its levels are reduced. This may lead to aberrant differentiation of tendon cells and consequently to the kakapo mutant deranged somatic muscle phenotype. PMID:9832554

  18. Tendon Is Covered by a Basement Membrane Epithelium That Is Required for Cell Retention and the Prevention of Adhesion Formation

    PubMed Central

    Taylor, Susan H.; Al-Youha, Sarah; Van Agtmael, Tom; Lu, Yinhui; Wong, Jason; McGrouther, Duncan A.; Kadler, Karl E.

    2011-01-01

    The ability of tendons to glide smoothly during muscle contraction is impaired after injury by fibrous adhesions that form between the damaged tendon surface and surrounding tissues. To understand how adhesions form we incubated excised tendons in fibrin gels (to mimic the homeostatic environment at the injury site) and assessed cell migration. We noticed cells exiting the tendon from only the cut ends. Furthermore, treatment of the tendon with trypsin resulted in cell extravagation from the shaft of the tendons. Electron microscopy and immunolocalisation studies showed that the tendons are covered by a novel cell layer in which a collagen type IV/laminin basement membrane (BM) overlies a keratinised epithelium. PCR and western blot analyses confirmed the expression of laminin β1 in surface cells, only. To evaluate the cell retentive properties of the BM in vivo we examined the tendons of the Col4a1+/Svc mouse that is heterozygous for a G-to-A transition in the Col4a1 gene that produces a G1064D substitution in the α1(IV) chain of collagen IV. The flexor tendons had a discontinuous BM, developed fibrous adhesions with overlying tissues, and were acellular at sites of adhesion formation. In further experiments, tenotomy of wild-type mice resulted in expression of laminin throughout the adhesion. In conclusion, we show the existence of a novel tendon BM-epithelium that is required to prevent adhesion formation. The Col4a1+/Svc mouse is an effective animal model for studying adhesion formation because of the presence of a structurally-defective collagen type IV-containing BM. PMID:21298098

  19. Musculoskeletal diseases—tendon

    PubMed Central

    Sakabe, Tomoya; Sakai, Takao

    2011-01-01

    Introduction Tendons establish specific connections between muscles and the skeleton by transferring contraction forces from skeletal muscle to bone thereby allowing body movement. Tendon physiology and pathology are heavily dependent on mechanical stimuli. Tendon injuries clinically represent a serious and still unresolved problem since damaged tendon tissues heal very slowly and no surgical treatment can restore a damaged tendon to its normal structural integrity and mechanical strength. Understanding how mechanical stimuli regulate tendon tissue homeostasis and regeneration will improve the treatment of adult tendon injuries that still pose a great challenge in today's medicine. Source of data This review summarizes the current status of tendon treatment and discusses new directions from the point of view of cell-based therapy and regenerative medicine approach. We searched the available literature using PubMed for relevant original articles and reviews. Growing points Identification of tendon cell markers has enabled us to study precisely tendon healing and homeostasis. Clinically, tissue engineering for tendon injuries is an emerging technology comprising elements from the fields of cellular source, scaffold materials, growth factors/cytokines and gene delivering systems. Areas timely for developing research The clinical settings to establish appropriate microenvironment for injured tendons with the combination of these novel cellular- and molecular-based scaffolds will be critical for the treatment. PMID:21729872

  20. Biologics for tendon repair☆

    PubMed Central

    Docheva, Denitsa; Müller, Sebastian A.; Majewski, Martin; Evans, Christopher H.

    2015-01-01

    Tendon injuries are common and present a clinical challenge to orthopedic surgery mainly because these injuries often respond poorly to treatment and require prolonged rehabilitation. Therapeutic options used to repair ruptured tendons have consisted of suture, autografts, allografts, and synthetic prostheses. To date, none of these alternatives has provided a successful long-term solution, and often the restored tendons do not recover their complete strength and functionality. Unfortunately, our understanding of tendon biology lags far behind that of other musculoskeletal tissues, thus impeding the development of new treatment options for tendon conditions. Hence, in this review, after introducing the clinical significance of tendon diseases and the present understanding of tendon biology, we describe and critically assess the current strategies for enhancing tendon repair by biological means. These consist mainly of applying growth factors, stem cells, natural biomaterials and genes, alone or in combination, to the site of tendon damage. A deeper understanding of how tendon tissue and cells operate, combined with practical applications of modern molecular and cellular tools could provide the long awaited breakthrough in designing effective tendon-specific therapeutics and overall improvement of tendon disease management. PMID:25446135

  1. Isolation of Undifferentiated Female Germline Cells from Adult Drosophila Ovaries.

    PubMed

    Lim, Robyn Su May; Osato, Motomi; Kai, Toshie

    2015-08-03

    This unit describes a method for isolating undifferentiated, stem cell-like germline cells from adult Drosophila ovaries. Here, we demonstrate that this population of cells can be effectively purified from hand-dissected ovaries in considerably large quantities. Tumor ovaries with expanded populations of undifferentiated germline cells are first removed from fly abdomens and dissociated into a cell suspension with the aid of protease treatment. The target cells, which express Vasa-green fluorescent protein (GFP) fusion protein under the control of the germline-specific vasa promoter, are specifically selected from the suspension via fluorescence-activated cell sorting (FACS). These protocols can be adapted to isolate other cell types from fly ovaries, such as somatic follicle cells or escort cells, by driving GFP expression in the respective target cells.

  2. Autologous tendon-derived cell-seeded nanofibrous scaffolds improve rotator cuff repair in an age-dependent fashion.

    PubMed

    Huegel, Julianne; Kim, Dong Hwa; Cirone, James M; Pardes, Adam M; Morris, Tyler R; Nuss, Courtney A; Mauck, Robert L; Soslowsky, Louis J; Kuntz, Andrew F

    2017-06-01

    Rotator cuff tendon tears are one of the most common shoulder pathologies, especially in the aging population. Due to a poor healing response and degenerative changes associated with aging, rotator cuff repair failure remains common. Although cell-based therapies to augment rotator cuff repair appear promising, it is unknown whether the success of such a therapy is age-dependent. We hypothesized that autologous cell therapy would improve tendon-to-bone healing across age groups, with autologous juvenile cells realizing the greatest benefit. In this study, juvenile, adult, and aged rats underwent bilateral supraspinatus tendon repair with augmentation of one shoulder with autologous tendon-derived cell-seeded polycaprolactone scaffolds. At 8 weeks, shoulders treated with cells in both juvenile and aged animals exhibited increased cellularity, increased collagen organization, and improved mechanical properties. No changes between treated and control limbs were seen in adult rats. These findings suggest that cell delivery during supraspinatus repair initiates earlier matrix remodeling in juvenile and aged animals. This may be due to the relative "equilibrium" of adult tendon tissue with regards to catabolic and anabolic processes, contrasted with actively growing juvenile tendons and degenerative aged tendons. This study demonstrates the potential for autologous cell-seeded scaffolds to improve repairs in both the juvenile and aged population. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1250-1257, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  3. Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells

    PubMed Central

    Zhang, Jianying; Wang, James H-C.

    2015-01-01

    Aging is known to cause tendon degeneration whereas moderate exercise imparts beneficial effects on tendons. Since stem cells play a vital role in maintaining tissue integrity, in this study we aimed to define the effects of aging and moderate exercise on tendon stem/progenitor cells (TSCs) using in vitro and in vivo models. TSCs derived from aging mice (9 and 24 months) proliferated significantly slower than TSCs obtained from young mice (2.5 and 5 months). In addition, expression of the stem cell markers Oct-4, nucleostemin (NS), Sca-1 and SSEA-1 in TSCs decreased in an age-dependent manner. Interestingly, moderate mechanical stretching (4%) of aging TSCs in vitro significantly increased the expression of the stem cell marker, NS, but 8% stretching decreased NS expression. Similarly, 4% mechanical stretching increased the expression of Nanog, another stem cell marker, and the tenocyte-related genes, collagen I and tenomodulin. However, 8% stretching increased expression of the non-tenocyte-related genes, LPL, Sox-9 and Runx-2, while 4% stretching had minimal effects on the expression of these genes. In the in vivo study, moderate treadmill running (MTR) of aging mice (9 months) resulted in the increased proliferation rate of aging TSCs in culture, decreased lipid deposition, proteoglycan accumulation and calcification, and increased the expression of NS in the patellar tendons. These findings indicate that while aging impairs the proliferative ability of TSCs and reduces their stemness, moderate exercise can mitigate the deleterious effects of aging on TSCs and therefore may be responsible for decreased aging-induced tendon degeneration. PMID:26086850

  4. Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells.

    PubMed

    Zhang, Jianying; Wang, James H-C

    2015-01-01

    Aging is known to cause tendon degeneration whereas moderate exercise imparts beneficial effects on tendons. Since stem cells play a vital role in maintaining tissue integrity, in this study we aimed to define the effects of aging and moderate exercise on tendon stem/progenitor cells (TSCs) using in vitro and in vivo models. TSCs derived from aging mice (9 and 24 months) proliferated significantly slower than TSCs obtained from young mice (2.5 and 5 months). In addition, expression of the stem cell markers Oct-4, nucleostemin (NS), Sca-1 and SSEA-1 in TSCs decreased in an age-dependent manner. Interestingly, moderate mechanical stretching (4%) of aging TSCs in vitro significantly increased the expression of the stem cell marker, NS, but 8% stretching decreased NS expression. Similarly, 4% mechanical stretching increased the expression of Nanog, another stem cell marker, and the tenocyte-related genes, collagen I and tenomodulin. However, 8% stretching increased expression of the non-tenocyte-related genes, LPL, Sox-9 and Runx-2, while 4% stretching had minimal effects on the expression of these genes. In the in vivo study, moderate treadmill running (MTR) of aging mice (9 months) resulted in the increased proliferation rate of aging TSCs in culture, decreased lipid deposition, proteoglycan accumulation and calcification, and increased the expression of NS in the patellar tendons. These findings indicate that while aging impairs the proliferative ability of TSCs and reduces their stemness, moderate exercise can mitigate the deleterious effects of aging on TSCs and therefore may be responsible for decreased aging-induced tendon degeneration.

  5. In vitro characterization of stem/progenitor cells from semitendinosus and gracilis tendons as a possible new tool for cell-based therapy for tendon disorders.

    PubMed

    Stanco, Deborah; Viganò, Marco; Orfei, Carlotta Perucca; DI Giancamillo, Alessia; Thiebat, Gabriele; Peretti, Giuseppe; DE Girolamo, Laura

    2014-01-01

    this study was conducted to characterize tendon stem/progenitor cells (TSPCs) isolated from human semitendinosus and gracilis tendons in terms of stemness properties and multi-differentiation potential. TSPCs were isolated from waste portions of semitendinosus and gracilis tendons from three donors who underwent anterior cruciate ligament reconstruction. TSPCs were plated in culture until passage 4, when experiments to assess cell proliferation, viability and clonogenic ability were performed. The immunophenotype of TSPCs was evaluated by cytofluorimetric analysis. The in vitro osteogenic, chondrogenic, adipogenic and tenogenic potential was evaluated using biochemical, histological and gene expression analysis to detect specific differentiation markers. Statistical analysis was performed using Student's t-test. after a few passages in culture the cell populations showed a homogeneous fibroblast-like morphology typical of mesenchymal stem cells. The average doubling time of TSPCs increased from 52.4±4.8 at passage 2 to 100.8±23.4 hours at passage 4. The highest percentage of colonies was also found at passage 4 (4.7±2.3%). TSPCs showed the typical mesenchymal phenotype, with high expression of CD73, CD90 and CD105 and no expression of CD34 and CD45. Cells induced to differentiate toward osteogenic lineage showed significant upregulations of ALP activity (+189%, p<0.05) and calcified matrix deposition (+49%, p<0.05) compared with undifferentiated cells; culture in chondrogenic medium also provoked a significant increase in glycosaminoglycan levels (+108%, p<0.05). On the other hand, TSPCs were not able to respond to adipogenic stimuli. Scleraxis gene expression and decorin gene expression, considered tenogenic markers, were already very high in control cells, and culture in tenogenic medium further increased these values although not significantly. our data show that it is possible to isolate TSPCs from very small fragments of tissue and that they show the

  6. In vitro characterization of stem/progenitor cells from semitendinosus and gracilis tendons as a possible new tool for cell-based therapy for tendon disorders

    PubMed Central

    STANCO, DEBORAH; VIGANÒ, MARCO; PERUCCA ORFEI, CARLOTTA; DI GIANCAMILLO, ALESSIA; THIEBAT, GABRIELE; PERETTI, GIUSEPPE; de GIROLAMO, LAURA

    2014-01-01

    Purpose this study was conducted to characterize tendon stem/progenitor cells (TSPCs) isolated from human semitendinosus and gracilis tendons in terms of stemness properties and multi-differentiation potential. Methods TSPCs were isolated from waste portions of semitendinosus and gracilis tendons from three donors who underwent anterior cruciate ligament reconstruction. TSPCs were plated in culture until passage 4, when experiments to assess cell proliferation, viability and clonogenic ability were performed. The immunophenotype of TSPCs was evaluated by cytofluorimetric analysis. The in vitro osteogenic, chondrogenic, adipogenic and tenogenic potential was evaluated using biochemical, histological and gene expression analysis to detect specific differentiation markers. Statistical analysis was performed using Student’s t-test. Results after a few passages in culture the cell populations showed a homogeneous fibroblast-like morphology typical of mesenchymal stem cells. The average doubling time of TSPCs increased from 52.4±4.8 at passage 2 to 100.8±23.4 hours at passage 4. The highest percentage of colonies was also found at passage 4 (4.7±2.3%). TSPCs showed the typical mesenchymal phenotype, with high expression of CD73, CD90 and CD105 and no expression of CD34 and CD45. Cells induced to differentiate toward osteogenic lineage showed significant upregulations of ALP activity (+189%, p<0.05) and calcified matrix deposition (+49%, p<0.05) compared with undifferentiated cells; culture in chondrogenic medium also provoked a significant increase in glycosaminoglycan levels (+108%, p<0.05). On the other hand, TSPCs were not able to respond to adipogenic stimuli. Scleraxis gene expression and decorin gene expression, considered tenogenic markers, were already very high in control cells, and culture in tenogenic medium further increased these values although not significantly. Conclusions our data show that it is possible to isolate TSPCs from very small fragments

  7. Multifunctional glial support by Semper cells in the Drosophila retina

    PubMed Central

    Charlton-Perkins, Mark A.

    2017-01-01

    Glial cells play structural and functional roles central to the formation, activity and integrity of neurons throughout the nervous system. In the retina of vertebrates, the high energetic demand of photoreceptors is sustained in part by Müller glia, an intrinsic, atypical radial glia with features common to many glial subtypes. Accessory and support glial cells also exist in invertebrates, but which cells play this function in the insect retina is largely undefined. Using cell-restricted transcriptome analysis, here we show that the ommatidial cone cells (aka Semper cells) in the Drosophila compound eye are enriched for glial regulators and effectors, including signature characteristics of the vertebrate visual system. In addition, cone cell-targeted gene knockdowns demonstrate that such glia-associated factors are required to support the structural and functional integrity of neighboring photoreceptors. Specifically, we show that distinct support functions (neuronal activity, structural integrity and sustained neurotransmission) can be genetically separated in cone cells by down-regulating transcription factors associated with vertebrate gliogenesis (pros/Prox1, Pax2/5/8, and Oli/Olig1,2, respectively). Further, we find that specific factors critical for glial function in other species are also critical in cone cells to support Drosophila photoreceptor activity. These include ion-transport proteins (Na/K+-ATPase, Eaat1, and Kir4.1-related channels) and metabolic homeostatic factors (dLDH and Glut1). These data define genetically distinct glial signatures in cone/Semper cells that regulate their structural, functional and homeostatic interactions with photoreceptor neurons in the compound eye of Drosophila. In addition to providing a new high-throughput model to study neuron-glia interactions, the fly eye will further help elucidate glial conserved "support networks" between invertebrates and vertebrates. PMID:28562601

  8. Living nanofiber yarn-based woven biotextiles for tendon tissue engineering using cell tri-culture and mechanical stimulation.

    PubMed

    Wu, Shaohua; Wang, Ying; Streubel, Philipp N; Duan, Bin

    2017-10-15

    Non-woven nanofibrous scaffolds have been developed for tendon graft application by using electrospinning strategies. However, electrospun nanofibrous scaffolds face some obstacles and limitations, including suboptimal scaffold structure, weak tensile and suture-retention strengths, and compact structure for cell infiltration. In this work, a novel nanofibrous, woven biotextile, fabricated based on electrospun nanofiber yarns, was implemented as a tissue engineered tendon scaffold. Based on our modified electrospinning setup, polycaprolactone (PCL) nanofiber yarns were fabricated with reproducible quality, and were further processed into plain-weaving fabrics interlaced with polylactic acid (PLA) multifilaments. Nonwoven nanofibrous PCL meshes with random or aligned fiber structures were generated using typical electrospinning as comparative counterparts. The woven fabrics contained 3D aligned microstructures with significantly larger pore size and obviously enhanced tensile mechanical properties than their nonwoven counterparts. The biological results revealed that cell proliferation and infiltration, along with the expression of tendon-specific genes by human adipose derived mesenchymal stem cells (HADMSC) and human tenocytes (HT), were significantly enhanced on the woven fabrics compared with those on randomly-oriented or aligned nanofiber meshes. Co-cultures of HADMSC with HT or human umbilical vein endothelial cells (HUVEC) on woven fabrics significantly upregulated the functional expression of most tenogenic markers. HADMSC/HT/HUVEC tri-culture on woven fabrics showed the highest upregulation of most tendon-associated markers than all the other mono- and co-culture groups. Furthermore, we conditioned the tri-cultured constructs with dynamic conditioning and demonstrated that dynamic stretch promoted total collagen secretion and tenogenic differentiation. Our nanofiber yarn-based biotextiles have significant potential to be used as engineered scaffolds to

  9. Apoptosis in Drosophila: compensatory proliferation and undead cells.

    PubMed

    Martín, Francisco A; Peréz-Garijo, Ainhoa; Morata, Ginés

    2009-01-01

    Apoptosis (programmed cell death) is a conserved process in all animals, used to eliminate damaged or unwanted cells after stress events or during normal development to sculpt larval or adult structures. In Drosophila, it is known that stress events such as irradiation or heat shock give rise to high apoptotic levels which remove more than 50% of cells in imaginal discs. However, the surviving cells are able to restore normal size and pattern, indicating that they undergo additional proliferation. This compensatory proliferation is still poorly understood. One widely used method to study the properties of apoptotic cells is to keep them alive by expressing in them the baculoviral protein P35, which blocks the activity of the effector caspases. These "undead" cells acquire special features, such as the emission of the growth signals Dpp and Wg, changes in cellular morphology and induction of proliferation in neighbouring cells. Here, we review the various methods used in Drosophila to block apoptosis and its consequences, and focus on the generation and properties of undead cells in the wing imaginal disc. We describe their effects in epithelial architecture and growth in some detail, and discuss the possible relationship between undead cells and compensatory proliferation.

  10. fringe and Notch specify polar cell fate during Drosophila oogenesis.

    PubMed

    Grammont, M; Irvine, K D

    2001-06-01

    fringe encodes a glycosyltransferase that modulates the ability of the Notch receptor to be activated by its ligands. We describe studies of fringe function during early stages of Drosophila oogenesis. Animals mutant for hypomorphic alleles of fringe contain follicles with an incorrect number of germline cells, which are separated by abnormally long and disorganized stalks. Analysis of clones of somatic cells mutant for a null allele of fringe localizes the requirement for fringe in follicle formation to the polar cells, and demonstrates that fringe is required for polar cell fate. Clones of cells mutant for Notch also lack polar cells and the requirement for Notch in follicle formation appears to map to the polar cells. Ectopic expression of fringe or of an activated form of Notch can generate an extra polar cell. Our results indicate that fringe plays a key role in positioning Notch activation during early oogenesis, and establish a function for the polar cells in separating germline cysts into individual follicles.

  11. Electrophysiological Recordings from Lobula Plate Tangential Cells in Drosophila.

    PubMed

    Mauss, Alex S; Borst, Alexander

    2016-01-01

    Drosophila has emerged as an important model organism for the study of the neural basis of behavior. Its main asset is the experimental accessibility of identified neurons by genetic manipulation and physiological recordings. Drosophila therefore offers the opportunity to reach an integrative understanding of the development and neural underpinnings of behavior at all processing stages, from sensing to motor control, in a single species. Here, we will provide an account of the procedures involved in recording the electrical potential of individual neurons in the visual system of adult Drosophila using the whole-cell patch-clamp method. To this end, animals are fixed to a holder and mounted below a recording chamber. The head capsule is cut open and the glial sheath covering the brain is ruptured by a combination of shearing and enzymatic digest. Neuronal somata are thus exposed and targeted by low-resistance patch electrodes. After formation of a high resistance seal, electrical access to the cell is gained by small current pulses and suction. Stable recordings of large neurons are feasible for >1 h and can be combined with controlled visual stimulation as well as genetic and pharmacological manipulation of upstream circuit elements to infer circuit function in great detail.

  12. Wallenda regulates JNK-mediated cell death in Drosophila

    PubMed Central

    Ma, X; Xu, W; Zhang, D; Yang, Y; Li, W; Xue, L

    2015-01-01

    The c-Jun N-terminal kinase (JNK) pathway plays essential roles in regulating a variety of cellular processes including proliferation, migration and survival. Previous genetic studies in Drosophila have identified numerous cell death regulating genes, providing new insights into the mechanisms for related diseases. Despite the known role of the small GTPase Rac1 in regulating cell death, the downstream components and underlying mechanism remain largely elusive. Here, we show that Rac1 promotes JNK-dependent cell death through Wallenda (Wnd). In addition, we find that Wnd triggers JNK activation and cell death via its kinase domain. Moreover, we show that both MKK4 and Hep are critical for Wnd-induced cell death. Furthermore, Wnd is essential for ectopic Egr- or Rho1-induced JNK activation and cell death. Finally, Wnd is physiologically required for loss of scribble-induced JNK-dependent cell death. Thus, our data suggest that wnd encodes a novel essential cell death regulator in Drosophila. PMID:25950467

  13. The Development of Germline Stem Cells in Drosophila

    PubMed Central

    Dansereau, David A.; Lasko, Paul

    2009-01-01

    Summary Germline stem cells (GSCs) in Drosophila are a valuable model to explore of how adult stem cells are regulated in vivo. Genetic dissection of this system has shown that stem cell fate is determined and maintained by the stem cell’s somatic microenvironment or niche. In Drosophila gonads, the stem cell niche—the cap cell cluster in females and the hub in males—acts as a signaling center to recruit GSCs from among a small population of undifferentiated primordial germ cells (PGCs). Short-range signals from the niche specify and regulate stem cell fate by maintaining the undifferentiated state of the PGCs next to the niche. Germline cells that do not receive the niche signals because of their location assume the default fate and differentiate. Once GSCs are specified, adherens junctions maintain close association between the stem cells and their niche and help to orient stem cell division so that one daughter is displaced from the niche and differentiates. In females, stem cell fate depends on bone morphogenetic protein (BMP) signals from the cap cells; in males, hub cells express the cytokine-like ligand Unpaired, which activates the Janus kinase-signal transducers and activators of transcription (Jak-Stat) pathway in stem cells. Although the signaling pathways operating between the niche and stem cells are different, there are common general features in both males and females, including the arrangement of cell types, many of the genes used, and the logic of the system that maintains stem cell fate. PMID:18370048

  14. Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes☆

    PubMed Central

    Augusto, Lívia Maria Mendonça; Aguiar, Diego Pinheiro; Bonfim, Danielle Cabral; dos Santos Cavalcanti, Amanda; Casado, Priscila Ladeira; Duarte, Maria Eugênia Leite

    2016-01-01

    Objective This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. Methods Bovine tendons were used for preparation of the extract and were stored at −80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. Results The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. Conclusion Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes. PMID:26962503

  15. Bam and Bgcn in Drosophila germline stem cell differentiation.

    PubMed

    Perinthottathil, Sreejith; Kim, Changsoo

    2011-01-01

    The female Drosophila reproductive organ, the ovary, has provided researchers with an incisive genetic system with which principle regulation of stem cell maintenance and differentiation has been delineated. An environmental niche regulates a stem cell's asymmetric self-renewal division that produces a daughter stem cell and a differentiated daughter cell, which further differentiate into eggs. A number of extrinsic and intrinsic factors have been identified that are required either for stem cell maintenance or differentiation. Bam/Bgcn complex plays a pivotal role in promoting stem cell differentiation. Recent papers suggest that Bam/Bgcn complex regulates translation of important maintenance factors and is also involved in the regulation of microRNA-dependent translational repression. Here, we focus on Bam and Bgcn repression of stem cell maintenance factors in the differentiation of germline stem cells (GSCs).

  16. Mechanical Control of Myotendinous Junction Formation and Tendon Differentiation during Development

    PubMed Central

    Valdivia, Mauricio; Vega-Macaya, Franco; Olguín, Patricio

    2017-01-01

    The development of the musculoskeletal system is a great model to study the interplay between chemical and mechanical inter-tissue signaling in cell adhesion, tissue morphogenesis and differentiation. In both vertebrates and invertebrates (e.g., Drosophila melanogaster) the formation of muscle-tendon interaction generates mechanical forces which are required for myotendinous junction maturation and tissue differentiation. In addition, these forces must be withstood by muscles and tendons in order to prevent detachment from each other, deformation or even losing their integrity. Extracellular matrix remodeling at the myotendinous junction is key to resist mechanical load generated by muscle contraction. Recent evidences in vertebrates indicate that mechanical forces generated during junction formation regulate chemical signaling leading to extracellular matrix remodeling, however, the mechanotransduction mechanisms associated to this response remains elusive. In addition to extracellular matrix remodeling, the ability of Drosophila tendon-cells to bear mechanical load depends on rearrangement of tendon cell cytoskeleton, thus studying the molecular mechanisms involved in this process is critical to understand the contribution of mechanical forces to the development of the musculoskeletal system. Here, we review recent findings regarding the role of chemical and mechanical signaling in myotendinous junction formation and tendon differentiation, and discuss molecular mechanisms of mechanotransduction that may allow tendon cells to withstand mechanical load during development of the musculoskeletal system. PMID:28386542

  17. Mechanical Control of Myotendinous Junction Formation and Tendon Differentiation during Development.

    PubMed

    Valdivia, Mauricio; Vega-Macaya, Franco; Olguín, Patricio

    2017-01-01

    The development of the musculoskeletal system is a great model to study the interplay between chemical and mechanical inter-tissue signaling in cell adhesion, tissue morphogenesis and differentiation. In both vertebrates and invertebrates (e.g., Drosophila melanogaster) the formation of muscle-tendon interaction generates mechanical forces which are required for myotendinous junction maturation and tissue differentiation. In addition, these forces must be withstood by muscles and tendons in order to prevent detachment from each other, deformation or even losing their integrity. Extracellular matrix remodeling at the myotendinous junction is key to resist mechanical load generated by muscle contraction. Recent evidences in vertebrates indicate that mechanical forces generated during junction formation regulate chemical signaling leading to extracellular matrix remodeling, however, the mechanotransduction mechanisms associated to this response remains elusive. In addition to extracellular matrix remodeling, the ability of Drosophila tendon-cells to bear mechanical load depends on rearrangement of tendon cell cytoskeleton, thus studying the molecular mechanisms involved in this process is critical to understand the contribution of mechanical forces to the development of the musculoskeletal system. Here, we review recent findings regarding the role of chemical and mechanical signaling in myotendinous junction formation and tendon differentiation, and discuss molecular mechanisms of mechanotransduction that may allow tendon cells to withstand mechanical load during development of the musculoskeletal system.

  18. Drosophila dyskerin is required for somatic stem cell homeostasis.

    PubMed

    Vicidomini, Rosario; Petrizzo, Arianna; di Giovanni, Annamaria; Cassese, Laura; Lombardi, Antonella Anna; Pragliola, Caterina; Furia, Maria

    2017-03-23

    Drosophila represents an excellent model to dissect the roles played by the evolutionary conserved family of eukaryotic dyskerins. These multifunctional proteins are involved in the formation of H/ACA snoRNP and telomerase complexes, both involved in essential cellular tasks. Since fly telomere integrity is guaranteed by a different mechanism, we used this organism to investigate the specific role played by dyskerin in somatic stem cell maintenance. To this aim, we focussed on Drosophila midgut, a hierarchically organized and well characterized model for stemness analysis. Surprisingly, the ubiquitous loss of the protein uniquely affects the formation of the larval stem cell niches, without altering other midgut cell types. The number of adult midgut precursor stem cells is dramatically reduced, and this effect is not caused by premature differentiation and is cell-autonomous. Moreover, a few dispersed precursors found in the depleted midguts can maintain stem identity and the ability to divide asymmetrically, nor show cell-growth defects or undergo apoptosis. Instead, their loss is mainly specifically dependent on defective amplification. These studies establish a strict link between dyskerin and somatic stem cell maintenance in a telomerase-lacking organism, indicating that loss of stemness can be regarded as a conserved, telomerase-independent effect of dyskerin dysfunction.

  19. Adenomatous polyposis coli regulates Drosophila intestinal stem cell proliferation.

    PubMed

    Lee, Wen-Chih; Beebe, Katherine; Sudmeier, Lisa; Micchelli, Craig A

    2009-07-01

    Adult stem cells define a cellular reserve with the unique capacity to replenish differentiated cells of a tissue throughout an organism's lifetime. Previous analysis has demonstrated that the adult Drosophila midgut is maintained by a population of multipotent intestinal stem cells (ISCs) that resides in epithelial niches. Adenomatous polyposis coli (Apc), a tumor suppressor gene conserved in both invertebrates and vertebrates, is known to play a role in multiple developmental processes in Drosophila. Here, we examine the consequences of eliminating Apc function on adult midgut homeostasis. Our analysis shows that loss of Apc results in the disruption of midgut homeostasis and is associated with hyperplasia and multilayering of the midgut epithelium. A mosaic analysis of marked ISC cell lineages demonstrates that Apc is required specifically in ISCs to regulate proliferation, but is not required for ISC self-renewal or the specification of cell fate within the lineage. Cell autonomous activation of Wnt signaling in the ISC lineage phenocopied Apc loss and Apc mutants were suppressed in an allele-specific manner by abrogating Wnt signaling, suggesting that the effects of Apc are mediated in part by the Wnt pathway. Together, these data underscore the essential requirement of Apc in exerting regulatory control over stem cell activity, as well as the consequences that disrupting this regulation can have on tissue homeostasis.

  20. Application of stem cells derived from the periodontal ligament or gingival tissue sources for tendon tissue regeneration

    PubMed Central

    Moshaverinia, Alireza; Xu, Xingtian; Chen, Chider; Ansari, Sahar; Zadeh, Homayoun H.; Snead, Malcolm L.; Shi, Songtao

    2014-01-01

    Tendon injuries are often associated with significant dysfunction and disability due to tendinous tissue’s very limited self-repair capacity and propensity for scar formation. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material present an alternative therapeutic option for tendon repair/regeneration that may be advantageous compared to other current treatment modalities. The MSC delivery vehicle is the principal determinant for successful implementation of MSC-mediated regenerative therapies. In the current study, a co-delivery system based on TGF-β3-loaded RGD-coupled alginate microspheres was developed for encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs). The capacity of encapsulated dental MSCs to differentiate into tendon tissue was investigated in vitro and in vivo. Encapsulated dental-derived MSCs were transplanted subcutaneously into immunocompromised mice. Our results revealed that after 4 weeks of differentiation in vitro, PDLSCs and GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited high levels of mRNA expression for gene markers related to tendon regeneration (Scx, DCn, Tnmd, and Bgy) via qPCR measurement. In a corresponding in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs, as confirmed by histological and immunohistochemical staining for protein markers specific for tendons. Interestingly, in our quantitative PCR and in vivo histomorphometric analyses, PDLSCs showed significantly greater capacity for tendon regeneration than GMSCs or hBMMSCs (P<0.05). Altogether, these findings indicate that periodontal ligament and gingival tissues can be considered as suitable stem cell sources for tendon engineering. PDLSCs and GMSCs encapsulated in TGF-β3-loaded RGD-modified alginate microspheres are promising candidates for tendon regeneration. PMID

  1. Application of stem cells derived from the periodontal ligament or gingival tissue sources for tendon tissue regeneration.

    PubMed

    Moshaverinia, Alireza; Xu, Xingtian; Chen, Chider; Ansari, Sahar; Zadeh, Homayoun H; Snead, Malcolm L; Shi, Songtao

    2014-03-01

    Tendon injuries are often associated with significant dysfunction and disability due to tendinous tissue's very limited self-repair capacity and propensity for scar formation. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material present an alternative therapeutic option for tendon repair/regeneration that may be advantageous compared to other current treatment modalities. The MSC delivery vehicle is the principal determinant for successful implementation of MSC-mediated regenerative therapies. In the current study, a co-delivery system based on TGF-β3-loaded RGD-coupled alginate microspheres was developed for encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs). The capacity of encapsulated dental MSCs to differentiate into tendon tissue was investigated in vitro and in vivo. Encapsulated dental-derived MSCs were transplanted subcutaneously into immunocompromised mice. Our results revealed that after 4 weeks of differentiation in vitro, PDLSCs and GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited high levels of mRNA expression for gene markers related to tendon regeneration (Scx, DCn, Tnmd, and Bgy) via qPCR measurement. In a corresponding in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs, as confirmed by histological and immunohistochemical staining for protein markers specific for tendons. Interestingly, in our quantitative PCR and in vivo histomorphometric analyses, PDLSCs showed significantly greater capacity for tendon regeneration than GMSCs or hBMMSCs (P < 0.05). Altogether, these findings indicate that periodontal ligament and gingival tissues can be considered as suitable stem cell sources for tendon engineering. PDLSCs and GMSCs encapsulated in TGF-β3-loaded RGD-modified alginate microspheres are promising candidates for tendon regeneration. Copyright

  2. M1 and M2 macrophage recruitment during tendon regeneration induced by amniotic epithelial cell allotransplantation in ovine.

    PubMed

    Mauro, Annunziata; Russo, Valentina; Di Marcantonio, Lisa; Berardinelli, Paolo; Martelli, Alessandra; Muttini, Aurelio; Mattioli, Mauro; Barboni, Barbara

    2016-04-01

    Recently, we have demonstrated that ovine amniotic epithelial cells (oAECs) allotransplanted into experimentally induced tendon lesions are able to stimulate tissue regeneration also by reducing leukocyte infiltration. Amongst leukocytes, macrophages (Mφ) M1 and M2 phenotype cells are known to mediate inflammatory and repairing processes, respectively. In this research it was investigated if, during tendon regeneration induced by AECs allotransplantation, M1Mφ and M2Mφ phenotype cells are recruited and differently distributed within the lesion site. Ovine AECs treated and untreated (Ctr) tendons were explanted at 7, 14, and 28 days and tissue microarchitecture was analyzed together with the distribution and quantification of leukocytes (CD45 positive), Mφ (CD68 pan positive), and M1Mφ (CD86, and IL12b) and M2Mφ (CD206, YM1 and IL10) phenotype related markers. In oAEC transplanted tendons CD45 and CD68 positive cells were always reduced in the lesion site. At day 14, oAEC treated tendons began to recover their microarchitecture, contextually a reduction of M1Mφ markers, mainly distributed close to oAECs, and an increase of M2Mφ markers was evidenced. CD206 positive cells were distributed near the regenerating areas. At day 28 oAECs treated tendons acquired a healthy-like structure with a reduction of M2Mφ. Differently, Ctr tendons maintained a disorganized morphology throughout the experimental time and constantly showed high values of M1Mφ markers. These findings indicate that M2Mφ recruitment could be correlated to tendon regeneration induced by oAECs allotransplantation. Moreover, these results demonstrate oAECs immunomodulatory role also in vivo and support novel insights into their allogeneic use underlying the resolution of tendon fibrosis.

  3. Centrosome and microtubule instability in aging Drosophila cells

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Chakrabarti, A.; Hedrick, J.

    1999-01-01

    Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

  4. Centrosome and microtubule instability in aging Drosophila cells

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Chakrabarti, A.; Hedrick, J.

    1999-01-01

    Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

  5. Centrosome and microtubule instability in aging Drosophila cells.

    PubMed

    Schatten, H; Chakrabarti, A; Hedrick, J

    1999-08-01

    Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

  6. Prostaglandin E2 (PGE2) Exerts Biphasic Effects on Human Tendon Stem Cells

    PubMed Central

    Zhang, Jianying; Wang, James H-C.

    2014-01-01

    Prostaglandin E2 (PGE2) has been reported to exert different effects on tissues at low and high levels. In the present study, cell culture experiments were performed to determine the potential biphasic effects of PGE2 on human tendon stem/progenitor cells (hTSCs). After treatment with PGE2, hTSC proliferation, stemness, and differentiation were analyzed. We found that high concentrations of PGE2 (>1 ng/ml) decreased cell proliferation and induced non-tenocyte differentiation. However, at lower concentrations (<1 ng/ml), PGE2 markedly enhanced hTSC proliferation. The expression levels of stem cell marker genes, specifically SSEA-4 and Stro-1, were more extensive in hTSCs treated with low concentrations of PGE2 than in cells treated with high levels of PGE2. Moreover, high levels of PGE2 induced hTSCs to differentiate aberrantly into non-tenocytes, which was evident by the high levels of PPARγ, collagen type II, and osteocalcin expression in hTSCs treated with PGE2 at concentrations >1 ng/ml. The findings of this study reveal that PGE2 can exhibit biphasic effects on hTSCs, indicating that while high PGE2 concentrations may be detrimental to tendons, low levels of PGE2 may play a vital role in the maintenance of tendon homeostasis in vivo. PMID:24504456

  7. Ciprofloxacin up-regulates tendon cells to express matrix metalloproteinase-2 with degradation of type I collagen.

    PubMed

    Tsai, Wen-Chung; Hsu, Chih-Chin; Chen, Carl P C; Chang, Hsiang-Ning; Wong, Alice M K; Lin, Miao-Sui; Pang, Jong-Hwei S

    2011-01-01

    Ciprofloxacin-induced tendinopathy and tendon rupture have been previously described, principally affecting the Achilles tendon. This study was designed to investigate the effect of ciprofloxacin on expressions of matrix metalloproteinases (MMP)-2 and -9, tissue inhibitors of metalloproteinase (TIMP)-1 and -2 as well as type I collagen in tendon cells. Tendon cells intrinsic to rat Achilles tendon were treated with ciprofloxacin and then underwent MTT (tetrazolium) assay. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis were used, respectively, to evaluate the gene and protein expressions of type I collagen, and MMP-2. Gelatin zymography was used to evaluate the enzymatic activities of MMP-2 and -9. Reverse zymography was used to evaluate TIMP-1 and -2. Immunohistochemical staining for MMP-2 in ciprofloxacin-treated tendon explants was performed. Collagen degradation was evaluated by incubation of conditioned medium with collagen. The results revealed that ciprofloxacin up-regulated the expression of MMP-2 in tendon cells at the mRNA and protein levels. Immunohistochemistry also confirmed the increased expressions of MMP-2 in ciprofloxacin-treated tendon explants. The enzymatic activity of MMP-2 was up-regulated whereas that of MMP-9, TIMP-1 or TIMP-2 was unchanged. The amount of secreted type I collagen in the conditioned medium decreased and type I collagen was degraded after ciprofloxacin treatment. In conclusion, ciprofloxacin up-regulates the expressions of MMP-2 in tendon cells and thus degraded type I collagen. These findings suggest a possible mechanism of ciprofloxacin-associated tendinopathy. Copyright © 2010 Orthopaedic Research Society.

  8. Nak regulates Dlg basal localization in Drosophila salivary gland cells.

    PubMed

    Peng, Yu-Huei; Yang, Wei-Kan; Lin, Wei-Hsiang; Lai, Tzu-Ting; Chien, Cheng-Ting

    2009-04-24

    Protein trafficking is highly regulated in polarized cells. During development, how the trafficking of cell junctional proteins is regulated for cell specialization is largely unknown. In the maturation of Drosophila larval salivary glands (SGs), the Dlg protein is essential for septate junction formation. We show that Dlg was enriched in the apical membrane domain of proximal cells and localized basolaterally in distal mature cells. The transition of Dlg distribution was disrupted in nak mutants. Nak associated with the AP-2 subunit alpha-Ada and the AP-1 subunit AP-1gamma. In SG cells disrupting AP-1 and AP-2 activities, Dlg was enriched in the apical membrane. Therefore, Nak regulates the transition of Dlg distribution likely through endocytosis of Dlg from the apical membrane domain and transcytosis of Dlg to the basolateral membrane domain during the maturation of SGs development.

  9. Epigenetic stability increases extensively during Drosophila follicle stem cell differentiation.

    PubMed

    Skora, Andrew D; Spradling, Allan C

    2010-04-20

    Stem and embryonic cells facilitate programming toward multiple daughter cell fates, whereas differentiated cells resist reprogramming and oncogenic transformation. How alterations in the chromatin-based machinery of epigenetic inheritance contribute to these differences remains poorly known. We observed random, heritable changes in GAL4/UAS transgene programming during Drosophila ovarian follicle stem cell differentiation and used them to measure the stage-specific epigenetic stability of gene programming. The frequency of GAL4/UAS reprogramming declines more than 100-fold over the nine divisions comprising this stem cell lineage. Stabilization acts in cis, suggesting that it is chromatin-based, and correlates with increased S phase length. Our results suggest that stem/early progenitor cells cannot accurately transmit nongenetic information to their progeny; full epigenetic competence is acquired only gradually during early differentiation. Modulating epigenetic inheritance may be a critical process controlling transitions between the pleuripotent and differentiated states.

  10. Telomere loss in somatic cells of Drosophila causes cell cycle arrest and apoptosis.

    PubMed Central

    Ahmad, K; Golic, K G

    1999-01-01

    Checkpoint mechanisms that respond to DNA damage in the mitotic cell cycle are necessary to maintain the fidelity of chromosome transmission. These mechanisms must be able to distinguish the normal telomeres of linear chromosomes from double-strand break damage. However, on several occasions, Drosophila chromosomes that lack their normal telomeric DNA have been recovered, raising the issue of whether Drosophila is able to distinguish telomeric termini from nontelomeric breaks. We used site-specific recombination on a dispensable chromosome to induce the formation of a dicentric chromosome and an acentric, telomere-bearing, chromosome fragment in somatic cells of Drosophila melanogaster. The acentric fragment is lost when cells divide and the dicentric breaks, transmitting a chromosome that has lost a telomere to each daughter cell. In the eye imaginal disc, cells with a newly broken chromosome initially experience mitotic arrest and then undergo apoptosis when cells are induced to divide as the eye differentiates. Therefore, Drosophila cells can detect and respond to a single broken chromosome. It follows that transmissible chromosomes lacking normal telomeric DNA nonetheless must possess functional telomeres. We conclude that Drosophila telomeres can be established and maintained by a mechanism that does not rely on the terminal DNA sequence. PMID:10049921

  11. Stem cell therapy: a promising biological strategy for tendon-bone healing after anterior cruciate ligament reconstruction.

    PubMed

    Hao, Zi-Chen; Wang, Shan-Zheng; Zhang, Xue-Jun; Lu, Jun

    2016-04-01

    Tendon-bone healing after anterior cruciate ligament (ACL) reconstruction is a complex process, impacting significantly on patients' prognosis. Natural tendon-bone healing usually results in fibrous scar tissue, which is of inferior quality compared to native attachment. In addition, the early formed fibrous attachment after surgery is often not reliable to support functional rehabilitation, which may lead to graft failure or unsatisfied function of the knee joint. Thus, strategies to promote tendon-bone healing are crucial for prompt and satisfactory functional recovery. Recently, a variety of biological approaches, including active substances, gene transfer, tissue engineering and stem cells, have been proposed and applied to enhance tendon-bone healing. Among these, stem cell therapy has been shown to have promising prospects and draws increasing attention. From commonly investigated bone marrow-derived mesenchymal stem cells (bMSCs) to emerging ACL-derived CD34+ stem cells, multiple stem cell types have been proven to be effective in accelerating tendon-bone healing. This review describes the current understanding of tendon-bone healing and summarizes the current status of related stem cell therapy. Future limitations and perspectives are also discussed.

  12. Exoskeleton anchoring to tendon cells and muscles in molting isopod crustaceans

    PubMed Central

    Žnidaršič, Nada; Mrak, Polona; Tušek-Žnidarič, Magda; Štrus, Jasna

    2012-01-01

    Abstract Specialized mechanical connection between exoskeleton and underlying muscles in arthropods is a complex network of interconnected matrix constituents, junctions and associated cytoskeletal elements, which provides prominent mechanical attachment of the epidermis to the cuticle and transmits muscle tensions to the exoskeleton. This linkage involves anchoring of the complex extracellular matrix composing the cuticle to the apical membrane of tendon cells and linking of tendon cells to muscles basally. The ultrastructural arhitecture of these attachment complexes during molting is an important issue in relation to integument integrity maintenance in the course of cuticle replacement and in relation to movement ability. The aim of this work was to determine the ultrastructural organization of exoskeleton – muscles attachment complexes in the molting terrestrial isopod crustaceans, in the stage when integumental epithelium is covered by both, the newly forming cuticle and the old detached cuticle. We show that the old exoskeleton is extensively mechanically connected to the underlying epithelium in the regions of muscle attachment sites by massive arrays of fibers in adult premolt Ligia italica and in prehatching embryos and premolt marsupial mancas of Porcellio scaber. Fibers expand from the tendon cells, traverse the new cuticle and ecdysal space and protrude into the distal layers of the detached cuticle. They likely serve as final anchoring sites before exuviation and may be involved in animal movements in this stage. Tendon cells in the prehatching embryo and in marsupial mancas display a substantial apicobasally oriented transcellular arrays of microtubules, evidently engaged in myotendinous junctions and in apical anchoring of the cuticular matrix. The structural framework of musculoskeletal linkage is basically established in described intramarsupial developmental stages, suggesting its involvement in animal motility within the marsupium. PMID

  13. Giant cell tumor of the tendon sheath: a rare periungual location simulating myxoid cyst*

    PubMed Central

    Minotto, Renan; Rodrigues, Camila Britto; Grill, Aline Barcellos; Furian, Roque

    2017-01-01

    Giant cell tumor of the tendon sheath is a benign soft tissue tumor most frequent between the third and fifth decades of life. It can mimic and make differential diagnoses with several hand tumors. Definitive diagnosis and the treatment of choice are reached with complete resection and histopathological examination. Here we describe a case with clinical presentation similar to that of a myxoid cyst. PMID:28225971

  14. Live imaging of multicolor-labeled cells in Drosophila

    PubMed Central

    Boulina, Maria; Samarajeewa, Hasitha; Baker, James D.; Kim, Michael D.; Chiba, Akira

    2013-01-01

    We describe LOLLIbow, a Brainbow-based live imaging system with applications in developmental biology and neurobiology. The development of an animal, including the environmentally sensitive adaptation of its brain, is thought to proceed through continual orchestration among diverse cell types as they divide, migrate, transform and interact with one another within the body. To facilitate direct visualization of such dynamic morphogenesis by individual cells in vivo, we have modified the original Brainbow for Drosophila in which live imaging is practical during much of its development. Our system offers permanent fluorescent labels that reveal fine morphological details of individual cells without requiring dissection or fixation of the samples. It also features a non-invasive means to control the timing of stochastic tricolor cell labeling with a light pulse. We demonstrate applicability of the new system in a variety of settings that could benefit from direct imaging of the developing multicellular organism with single-cell resolution. PMID:23482495

  15. Tendonitis (image)

    MedlinePlus

    ... tendon. It can occur as a result of injury, overuse, or with aging as the tendon loses elasticity. Any action that places prolonged repetitive strain on the forearm muscles can cause tendonitis. The ...

  16. [Effect of cyclical stretch on matrix synthesis of human patellar tendon cells].

    PubMed

    Bosch, U; Zeichen, J; Skutek, M; Albers, I; van Griensven, M; Gässler, N

    2002-05-01

    The significance of mobilization and loading for healing ligaments and tendons is generally accepted today. Local deformation of cells thereby represents the key stimulus for the cellular response. Less is known, however, about the effects of cyclic strain on the cellular and molecular level. The aim of the in vitro investigation was to determine the effect of cyclic mechanical strain on collagen type I and III and on fibronectin formation in human patellar tendon derived fibroblasts. Human patellar tendon derived fibroblasts from 5 donors (mean age 29.2 years) were cultured under standard conditions. Monolayers of subconfluently grown 3rd passage cells were stretched in rectangular silicone dishes with cyclic movement along their longitudinal axes. Cyclic strain (5%, 1 Hz) was applied for 30 min and 60 min, respectively. Carboxyterminal procollagen type I propeptide (P-I-CP) and aminoterminal procollagen type III propeptide (P-III-NP) release was measured by a radio-immunoassay 6 h and 12 h after stretching. The release of fibronectin was measured by nephelometry following immunoreaction with a specific antiserum. Cells from each donor without any cyclic stretching served as controls. Compared with the controls, only cyclic stretching for 60 min resulted in a significantly increased release of P-I-CP and fibronectin after 6 h. The release of P-III-NP was significantly increased 12 h after 30 min of cyclic stretching as well as 6 h and 12 h after 60 min of cyclic stretching, respectively. We conclude that cyclic stretching causes a time-dependent, differential regulation of formation of fibronectin and collagen type I and III. This may effect the quality and thus the mechanical properties of healing tendon and ligament tissues. In order to improve current treatment protocols and to enlarge our knowledge of tissue healing, it is necessary to understand the cellular response to cyclic strain.

  17. Tendon Tissue Engineering: Mechanism and Effects of Human Tenocyte Coculture With Adipose-Derived Stem Cells.

    PubMed

    Long, Chao; Wang, Zhen; Legrand, Anais; Chattopadhyay, Arhana; Chang, James; Fox, Paige M

    2017-09-06

    Adipose-derived stem cells (ASCs) are a potential candidate for cell-based therapy targeting tendon injury; however, their therapeutic benefit relies on their ability to interact with native tenocytes. This study examines the mechanism and effects of coculturing human tenocytes and ASCs. Tenocytes (T) were directly cocultured with either ASCs (A) or fibroblasts (F) (negative control) in the following ratios: 50% T/50% A or F; 25% T/75% A or F; and 75% T/25% A or F. Cells were indirectly cocultured using a transwell insert that allowed for exchange of soluble factors only. Proliferation and collagen I production were measured and compared with monoculture controls. Synergy was quantified using the interaction index (II), which normalizes measured values by the expected values assuming no interaction (no synergy when II = 1). The ability of ASCs to elicit tenocyte migration was examined in vitro using a transwell migration assay and ex vivo using decellularized human flexor tendon explants. Compared with monoculture controls, II of proliferation was greater than 1 for all tenocyte and ASC direct coculture ratios, but not for tenocyte and fibroblast direct coculture ratios or for tenocyte and ASC indirect coculture. The ASCs elicited greater tenocyte migration in vitro and ex vivo. The II of collagen I production was greater than 1 for direct coculture groups with 25% T/75% A and 75% T/25% A. Direct coculture of ASCs and tenocytes demonstrated synergistic proliferation and collagen I production, and ASCs elicited tenocyte migration in vitro and ex vivo. These interactions play a key role in tendon healing and were absent when ASCs were replaced with fibroblasts, supporting the use of ASCs for cell-based therapy targeting tendon injuries. When ASCs are delivered for cell-based therapy, they directly interact with native tenocytes to increase cell proliferation, collagen I production, and tenocyte migration, which may enhance tendon healing. Copyright © 2017

  18. Biased DNA segregation in Drosophila male germline stem cells.

    PubMed

    Yamashita, Yukiko M

    2013-01-01

    The immortal strand hypothesis, which emerged four decades ago, proposes that certain cells retain a template copy of chromosomal DNA to protect against replication-induced mutations. As the interest in stem cells rose in recent years, researchers speculated that stem cells, which must maintain proliferative capacity throughout the life of the organism, may be the population that most needs the strong protection afforded by immortal strand segregation. Alternative hypotheses have also been proposed to explain observed non-random sister chromatid segregation. We recently found that Drosophila male germline stem cells segregate sister chromatids non-randomly, but such bias was limited to the sex chromosomes. Interestingly, the biased segregation does not lead to immortal strand segregation. We will discuss the implications of this observation and molecular mechanisms, which might be applicable to non-random sister chromatid segregation in other systems as well.

  19. Tissue landscape alters adjacent cell fates during Drosophila egg development

    PubMed Central

    Manning, Lathiena; Weideman, Ann Marie; Peercy, Bradford; Starz-Gaiano, Michelle

    2015-01-01

    Extracellular signaling molecules control many biological processes, but the influence of tissue architecture on the local concentrations of these factors is unclear. Here we examine this issue in the Drosophila egg chamber, where two anterior cells secrete Unpaired (Upd) to activate Signal Transducer and Activator of Transcription (STAT) signaling in the epithelium. High STAT signaling promotes cell motility. Genetic analysis shows that all cells near the Upd source can respond. However, using upright imaging, we show surprising asymmetries in STAT activation patterns, suggesting that some cells experience different Upd levels than predicted by their location. We develop a three-dimensional mathematical model to characterize the spatio-temporal distribution of the activator. Simulations show that irregular tissue domains can produce asymmetric distributions of Upd, consistent with results in vivo. Mutant analysis substantiates this idea. We conclude that cellular landscape can heavily influence the effect of diffusible activators and should be more widely considered. PMID:26082073

  20. IL-1β irreversibly inhibits tenogenic differentiation and alters metabolism in injured tendon-derived progenitor cells in vitro.

    PubMed

    Zhang, Kairui; Asai, Shuji; Yu, Bin; Enomoto-Iwamoto, Motomi

    2015-08-07

    Tendon injuries are common, and the damaged tendon often turns into scar tissue and never completely regains the original biomechanical properties. Previous studies have reported that the mRNA levels of inflammatory cytokines such as IL-1β are remarkably up-regulated in injured tendons. To examine how IL-1β impacts tendon repair process, we isolated the injured tendon-derived progenitor cells (inTPCs) from mouse injured Achilles tendons and studied the effects of IL-1β on the inTPCs in vitro. IL-1β treatment strongly reduced expression of tendon cell markers such as scleraxis and tenomodulin, and also down-regulated gene expression of collagen 1, collagen 3, biglycan and fibromodulin in inTPCs. Interestingly, IL-1β stimulated lactate production with increases in hexokinase II and lactate dehydrogenase expression and a decrease in pyruvate dehydrogenase. Inhibition of lactate production restored IL-1β-induced down-regulation of collagen1 and scleraxis expression. Furthermore, IL-1β significantly inhibited adipogenic, chondrogenic and osteogenic differentiation of inTPCs. Interestingly, inhibition of tenogenic and adipogenic differentiation was not recovered after removal of IL-1β while chondrogenic and osteogenic differentiation abilities were not affected. These findings indicate that IL-1β strongly and irreversibly impairs tenogenic potential and alters glucose metabolism in tendon progenitors appearing in injured tendons. Inhibition of IL-1β may be beneficial for maintaining function of tendon progenitor cells during the tendon repair process.

  1. Analysis of chromatin boundary activity in Drosophila cells

    PubMed Central

    Li, Mo; Belozerov, Vladimir E; Cai, Haini N

    2008-01-01

    Background Chromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to our limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared. Results We report here the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. We show that the activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are supported in these cells. We further show that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in our cell-based assay for probing insulator function. Conclusion The novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements. PMID:19077248

  2. The mechanobiological aetiopathogenesis of tendinopathy: is it the over-stimulation or the under-stimulation of tendon cells?

    PubMed Central

    Arnoczky, Steven P; Lavagnino, Michael; Egerbacher, Monika

    2007-01-01

    While there is a significant amount of information available on the clinical presentation(s) and pathological changes associated with tendinopathy, the precise aetiopathogenesis of this condition remains a topic of debate. Classically, the aetiology of tendinopathy has been linked to the performance of repetitive activities (so-called overuse injuries). This has led many investigators to suggest that it is the mechanobiologic over-stimulation of tendon cells that is the initial stimulus for the degradative processes which have been shown to accompany tendinopathy. Although several studies have been able to demonstrate that the in vitro over-stimulation of tendon cells in monolayer can result in a pattern(s) of gene expression seen in clinical cases of tendinopathy, the strain magnitudes and durations used in these in vitro studies, as well as the model systems, may not be clinically relevant. Using a rat tail tendon model, we have studied the in vitro mechanobiologic response of tendon cells in situ to various tensile loading regimes. These studies have led to the hypothesis that the aetiopathogenic stimulus for the degenerative cascade which precedes the overt pathologic development of tendinopathy is the catabolic response of tendon cells to mechanobiologic under-stimulation as a result of microscopic damage to the collagen fibres of the tendon. In this review, we examine the rationale for this hypothesis and provide evidence in support of this theory. PMID:17696902

  3. Visualization of adult stem cells within their niches using the Drosophila germline as a model system.

    PubMed

    König, Annekatrin; Shcherbata, Halyna R

    2013-01-01

    The germaria of the fruit fly Drosophila melanogaster present an excellent model to study germline stem cell-niche interactions. Two to three adult stem cells are surrounded by a number of somatic cells that form the niche. Here we describe how Drosophilae germaria can be dissected and specifically immuno-stained to allow for identification and analysis of both the adult stem cells and their somatic niche cells.

  4. Superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury.

    PubMed

    Scharf, Alexandra; Holmes, Shannon; Thoresen, Merrilee; Mumaw, Jennifer; Stumpf, Alaina; Peroni, John

    2015-01-01

    The goal of this study was to establish an SPIO-based cell-tracking method in an ovine model of tendonitis and to determine if this method may be useful for further study of cellular therapies in tendonitis in vivo. Functional assays were performed on labeled and unlabeled cells to ensure that no significant changes were induced by intracellular SPIOs. Following biosafety validation, tendon lesions were mechanically (n = 4) or chemically (n = 4) induced in four sheep and scanned ex vivo at 7 and 14 days to determine the presence and distribution of intralesional cells. Ovine MSCs labeled with 50 µg SPIOs/mL remained viable, proliferate, and undergo tri-lineage differentiation (p < 0.05). Labeled ovine MSCs remained detectable in vitro in concentrated cell numbers as low as 10 000 and in volumetric distributions as low as 100 000 cells/mL. Cells remained detectable by MRI at 7 days, as confirmed by correlative histology for dually labeled SPIO+/GFP+ cells. Histological evidence at 14 days suggested that SPIO particles remained embedded in tissue, providing MRI signal, although cells were no longer present. SPIO labeling has proven to be an effective method for cell tracking for a large animal model of tendon injury for up to 7 days post-injection. The data obtained in this study justify further investigation into the effects of MSC survival and migration on overall tendon healing and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.

  5. New Twists in Drosophila Cell Signaling.

    PubMed

    Shilo, Ben-Zion

    2016-04-08

    The discovery of a handful of conserved signaling pathways that dictate most aspects of embryonic and post-embryonic development of multicellular organisms has generated a universal view of animal development (Perrimon, N., Pitsouli, C., and Shilo, B. Z. (2012)Cold Spring Harb. Perspect. Biol.4, a005975). Although we have at hand most of the "hardware" elements that mediate cell communication events that dictate cell fate choices, we are still far from a comprehensive mechanistic understanding of these processes. One of the next challenges entails an analysis of developmental signaling pathways from the cell biology perspective. Where in the cell does signaling take place, and how do general cellular machineries and structures contribute to the regulation of developmental signaling? Another challenge is to examine these signaling pathways from a quantitative perspective, rather than as crude on/off switches. This requires more precise measurements, and incorporation of the time element to generate a dynamic sequence instead of frozen snapshots of the process. The quantitative outlook also brings up the issue of precision, and the unknown mechanisms that buffer variability in signaling between embryos, to produce a robust and reproducible output. Although these issues are universal to all multicellular organisms, they can be effectively tackled in theDrosophilamodel, by a combination of genetic manipulations, biochemical analyses, and a variety of imaging techniques. This review will present some of the recent advances that were accomplished by utilizing the versatility of theDrosophilasystem. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Development of a Drosophila cell-based error correction assay.

    PubMed

    Salemi, Jeffrey D; McGilvray, Philip T; Maresca, Thomas J

    2013-01-01

    Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT) interactions before anaphase onset results in chromosomal instability (CIN), which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F) is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5). Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due, in part, to kinesin-14 (Ncd) activity when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC). Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and CIN.

  7. Hypoxia inhibits primary cilia formation and reduces cell-mediated contraction in stress-deprived rat tail tendon fascicles

    PubMed Central

    Lavagnino, Michael; Oslapas, Anna N.; Gardner, Keri L.; Arnoczky, Steven P.

    2016-01-01

    Summary Background Hypoxia, which is associated with chronic tendinopathy, has recently been shown to decrease the mechanosensitivity of some cells. Therefore, the purpose of this study was to determine the effect of hypoxia on the formation of elongated primary cilia (a mechanosensing organelle of tendon cells) in vitro and to determine the effect of hypoxia on cell-mediated contraction of stress-deprived rat tail tendon fascicles (RTTfs). Methods Tendon cells isolated from RTTfs were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 hours. The cells were then stained for tubulin and the number of cells with elongated cilia counted. RTTfs from 1-month-old male Sprague-Dawley rats were also cultured under hypoxic and normoxic conditions for three days and tendon length measured daily. Results A significant (p=0.002) decrease in the percent of elongated cilia was found in cells maintained in hypoxic conditions (54.1%±12.2) when compared in normoxic conditions (71.7%±6.32). RTTfs in hypoxia showed a significant decrease in the amount of contraction compared to RTTfs in normoxia after two (p=0.007) and three (p=0.001) days. Conclusion The decreased incidence of elongated primary cilia in a hypoxic environment, as well as the decreased mechanoresponsiveness of tendon cells under these conditions may relate to the inability of some cases of chronic tendinopathy to respond to strain-based rehabilitation modalities (i.e. eccentric loading). PMID:27900292

  8. The Dynamics in Epithelial Cell Intercalation in Drosophila Morphogenesis

    NASA Astrophysics Data System (ADS)

    Wolf, Fred; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Eule, Stephan; Metzger, Jakob; Großhans, Jörg

    2015-03-01

    Epithelial cell rearrangement is important for many processes in morphogenesis. During germband extension in early gastrulation of Drosophila embryos, exchange of neighbors is achieved by junction remodeling that follows a topological T1 process. Its first step is the constriction of dorsal-ventral junctions and fusion of two 3x vertices into a 4x vertex a process believed to be junction autonomous. We established a high throughput imaging pipeline, by which we recorded, segmented and analysed more than 1000 neighbor exchanges in drosophila embryos. Characterizing the dynamics of junction lengths we find that the constriction of cell contacts follows intriguingly simple quantitative laws. (1) The mean contact length decreases approximately as a square root of time to collapse. (2) The time dependent variance of contact lengths is proportional to the square of the mean. (3) The time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations are sufficient to derive a stochastic differential equation for contact length that captures the non-equilibrium statistical mechanics of contact collapse. Supported by the German Research Foundation.

  9. Scleraxis-Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon.

    PubMed

    Agarwal, Shailesh; Loder, Shawn J; Cholok, David; Peterson, Joshua; Li, John; Breuler, Christopher; Cameron Brownley, R; Hsin Sung, Hsiao; Chung, Michael T; Kamiya, Nobuhiro; Li, Shuli; Zhao, Bin; Kaartinen, Vesa; Davis, Thomas A; Qureshi, Ammar T; Schipani, Ernestina; Mishina, Yuji; Levi, Benjamin

    2017-03-01

    The pathologic development of heterotopic ossification (HO) is well described in patients with extensive trauma or with hyperactivating mutations of the bone morphogenetic protein (BMP) receptor ACVR1. However, identification of progenitor cells contributing to this process remains elusive. Here we show that connective tissue cells contribute to a substantial amount of HO anlagen caused by trauma using postnatal, tamoxifen-inducible, scleraxis-lineage restricted reporter mice (Scx-creERT2/tdTomato(fl/fl) ). When the scleraxis-lineage is restricted specifically to adults prior to injury marked cells contribute to each stage of the developing HO anlagen and coexpress markers of endochondral ossification (Osterix, SOX9). Furthermore, these adult preinjury restricted cells coexpressed mesenchymal stem cell markers including PDGFRα, Sca1, and S100A4 in HO. When constitutively active ACVR1 (caACVR1) was expressed in scx-cre cells in the absence of injury (Scx-cre/caACVR1(fl/fl) ), tendons and joints formed HO. Postnatal lineage-restricted, tamoxifen-inducible caACVR1 expression (Scx-creERT2/caACVR1(fl/fl) ) was sufficient to form HO after directed cardiotoxin-induced muscle injury. These findings suggest that cells expressing scleraxis within muscle or tendon contribute to HO in the setting of both trauma or hyperactive BMP receptor (e.g., caACVR1) activity. Stem Cells 2017;35:705-710.

  10. Transcriptional control of stem cell maintenance in the Drosophila intestine.

    PubMed

    Bardin, Allison J; Perdigoto, Carolina N; Southall, Tony D; Brand, Andrea H; Schweisguth, François

    2010-03-01

    Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. Here we find that transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance, and identify genes of the Enhancer of split complex [E(spl)-C] as the major targets of this repression. In addition, we find that the bHLH transcription factor Daughterless is essential to maintain ISC identity and that bHLH binding sites promote ISC-specific enhancer activity. We propose that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation.

  11. Photoencapsulation of bone morphogenetic protein-2 and periosteal progenitor cells improve tendon graft healing in a bone tunnel.

    PubMed

    Chen, Chih-Hwa; Liu, Hsia-Wei; Tsai, Ching-Lin; Yu, Chung-Ming; Lin, I-Hsuan; Hsiue, Ging-Ho

    2008-03-01

    Tissue-engineered solutions for promoting the tendon graft incorporation within the bone tunnel appear to be promising. To determine the feasibility that conjugation of hyaluronic acid-tethered bone morphogenetic protein-2 can be used to stimulate periosteal progenitor cells direct fibrocartilagenous attachment and new bone formation in an extra-articular tendon-bone healing model. Controlled laboratory study. A total of 42 mature New Zealand White rabbits were used. The long digitorum extensor tendon was transplanted into a bone tunnel of the proximal tibia. The tendon was pulled through a drill hole in the proximal tibia and attached to the medial aspect of the tibia. Photopolymerizable hydrogel based on poly (ethylene glycol) diacrylate with hyaluronic acid-tethered bone morphogenetic protein-2 was injected and photogelated in a bone tunnel. Histological and biomechanical examination of the tendon-bone interface was evaluated at postoperative weeks 3 and 6. Histological analysis showed an interface fibrocartilage and new bone formed by photoencapsulation of bone morphogenetic protein-2 and periosteal progenitor cells at 6 weeks. Biomechanical testing revealed higher maximum pullout strength and stiffness in experimental groups with a statistically significant difference at 3 and 6 weeks after tendon transplantation. The healing tendon-bone interface undergoes a gradual remodeling process; it appears that photoencapsulation of bone morphogenetic protein-2 and periosteal progenitor cells possesses a powerful inductive ability between the tendon and the bone to incorporate the healing in a rabbit model. Novel technologies, such as those described in this study, including photopolymerization and tissue engineering, may provide minimally invasive therapeutic procedures via arthroscopy to enhance biological healing after reconstruction of the anterior cruciate ligament.

  12. Regulation of tendon differentiation by scleraxis distinguishes force-transmitting tendons from muscle-anchoring tendons.

    PubMed

    Murchison, Nicholas D; Price, Brian A; Conner, David A; Keene, Douglas R; Olson, Eric N; Tabin, Clifford J; Schweitzer, Ronen

    2007-07-01

    The scleraxis (Scx) gene, encoding a bHLH transcription factor, is expressed in the progenitors and cells of all tendon tissues. To determine Scx function, we produced a mutant null allele. Scx-/- mice were viable, but showed severe tendon defects, which manifested in a drastically limited use of all paws and back muscles and a complete inability to move the tail. Interestingly, although the differentiation of all force-transmitting and intermuscular tendons was disrupted, other categories of tendons, the function of which is mainly to anchor muscles to the skeleton, were less affected and remained functional, enabling the viability of Scx-/- mutants. The force-transmitting tendons of the limbs and tail varied in the severity to which they were affected, ranging from dramatic failure of progenitor differentiation resulting in the loss of segments or complete tendons, to the formation of small and poorly organized tendons. Tendon progenitors appeared normal in Scx-/- embryos and a phenotype resulting from a failure in the condensation of tendon progenitors to give rise to distinct tendons was first detected at embryonic day (E)13.5. In the tendons that persisted in Scx-/- mutants, we found a reduced and less organized tendon matrix and disorganization at the cellular level that led to intermixing of tenocytes and endotenon cells. The phenotype of Scx-/- mutants emphasizes the diversity of tendon tissues and represents the first molecular insight into the important process of tendon differentiation.

  13. Steroid signaling promotes stem cell maintenance in the Drosophila testis.

    PubMed

    Li, Yijie; Ma, Qing; Cherry, Christopher M; Matunis, Erika L

    2014-10-01

    Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). Here, we show that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which we previously showed maintains CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance.

  14. Male germline stem cell division and spermatocyte growth require insulin signaling in Drosophila.

    PubMed

    Ueishi, Satoru; Shimizu, Hanako; H Inoue, Yoshihiro

    2009-01-01

    Spermatogenesis in Drosophila commences with cell division of germline stem cells (GSCs) to produce male germline cells at the tip of the testis. However, molecular mechanisms inducing division of male GSCs have not been reported. Insulin-like peptides are known to play an essential role in stimulation of proliferation and growth of somatic cells, and it has recently been reported that such peptides promote cell division in female Drosophila GSCs. However, their effects on male germline cells have not been characterized. We found that inhibition of insulin production and insulin signaling mutations resulted in decreased numbers of germline cells in Drosophila testes. GSC numbers were maintained in young mutant males, with a gradual decrease in abundance of GSCs with age. Furthermore, in mutants, fewer germline cysts originated from GSCs and a lower frequency of GSC division was seen. Insulin signaling was found to promote cell cycle progression of the male GSCs at the G(2)/M phase. The cell volume of spermatocytes increases up to 25 times before initiation of meiosis in Drosophila. We examined whether insulin signaling extrinsically induces the greatest cell growth in Drosophila diploid cells and found that spermatocyte growth was affected in mutants. The results indicate that in addition to its function in somatic cells, insulin signaling plays an essential role in cell proliferation and growth during male Drosophila gametogenesis and that sperm production is regulated by hormonal control via insulin-like peptides.

  15. Nucleostemin- and Oct 3/4-positive stem/progenitor cells exhibit disparate anatomical and temporal expression during rat Achilles tendon healing.

    PubMed

    Runesson, Eva; Ackermann, Paul; Karlsson, Jón; Eriksson, Bengt I

    2015-08-20

    The recent discovery of residing tendon stem/progenitor cells has triggered a growing interest in stem cells as a useful tool in tendon repair. Our knowledge of their involvement in naturally healing tendons is, however, sparse. The aim of this study was to identify and determine stem/progenitor cells in relation to different healing phases and regions in a rat model of Achilles tendon rupture. Surgery was performed to create a mid-tendon rupture on the right Achilles tendon of 24 rats, whereas the left tendon was used as a control. Tendons were harvested at one, two, eight and 17 weeks post-rupture and stained with antibodies specific to stem/progenitor cells (Octamer-binding transcription factor 3/4 (Oct 3/4) and nucleostemin), migrating cells (Dynamin 2 (Dyn 2)) and leukocytes (CD45). A histological examination was performed on sections stained with Alcian blue. At one and two weeks post-rupture, a large number of stem/progenitor cells were discovered throughout the tendon. Most of these cells were nucleostemin positive, whereas only a few Oct 3/4-positive cells were found, mainly situated inside the injury region (I region). At eight and 17 weeks, the increment in stem/progenitor cells had diminished to equal that in the control tendons. At all time points, Oct 3/4-positive cells were also found in the connective tissue surrounding the tendon and at the muscle-tendon junction in both ruptured and control tendons and were often seen at the same location as the migration marker, Dyn 2. The whole length of the Achilles tendon is infiltrated by stem/progenitor cells at early time points after a mid-tendon rupture. However, different stem/progenitor cell populations exhibit varying anatomical and temporal expressions during Achilles tendon healing, suggesting distinct reparative implications. Oct 3/4 may thus act as a more local, migrating stem/progenitor cell involved in injury-site-specific regenerative effects, as compared to the more general proliferative role

  16. Life span extension and neuronal cell protection by Drosophila nicotinamidase.

    PubMed

    Balan, Vitaly; Miller, Gregory S; Kaplun, Ludmila; Balan, Karina; Chong, Zhao-Zhong; Li, Faqi; Kaplun, Alexander; VanBerkum, Mark F A; Arking, Robert; Freeman, D Carl; Maiese, Kenneth; Tzivion, Guri

    2008-10-10

    The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases. In Caenorhabditis elegans, knockdown of a pnc1 homolog was shown recently to shorten the worm life span, whereas its overexpression increased survival under conditions of oxidative stress. The function and regulation of nicotinamidases in higher organisms has not been determined. Here, we report the identification and biochemical characterization of the Drosophila nicotinamidase, D-NAAM, and demonstrate that its overexpression significantly increases median and maximal fly life span. The life span extension was reversed in Sir2 mutant flies, suggesting Sir2 dependence. Testing for physiological effectors of D-NAAM in Drosophila S2 cells, we identified oxidative stress as a primary regulator, both at the transcription level and protein activity. In contrast to the yeast model, stress factors such as high osmolarity and heat shock, calorie restriction, or inhibitors of TOR and phosphatidylinositol 3-kinase pathways do not appear to regulate D-NAAM in S2 cells. Interestingly, the expression of D-NAAM in human neuronal cells conferred protection from oxidative stress-induced cell death in a sirtuin-dependent manner. Together, our findings establish a life span extending the ability of nicotinamidase in flies and offer a role for nicotinamide-modulating genes in oxidative stress regulated pathways influencing longevity and neuronal cell survival.

  17. Neuralized functions cell autonomously to regulate Drosophila sense organ development.

    PubMed

    Yeh, E; Zhou, L; Rudzik, N; Boulianne, G L

    2000-09-01

    Neurogenic genes, including Notch and Delta, are thought to play important roles in regulating cell-cell interactions required for Drosophila sense organ development. To define the requirement of the neurogenic gene neuralized (neu) in this process, two independent neu alleles were used to generate mutant clones. We find that neu is required for determination of cell fates within the proneural cluster and that cells mutant for neu autonomously adopt neural fates when adjacent to wild-type cells. Furthermore, neu is required within the sense organ lineage to determine the fates of daughter cells and accessory cells. To gain insight into the mechanism by which neu functions, we used the GAL4/UAS system to express wild-type and epitope-tagged neu constructs. We show that Neu protein is localized primarily at the plasma membrane. We propose that the function of neu in sense organ development is to affect the ability of cells to receive Notch-Delta signals and thus modulate neurogenic activity that allows for the specification of non-neuronal cell fates in the sense organ.

  18. No donor age effect of human serum on collagen synthesis signaling and cell proliferation of human tendon fibroblasts.

    PubMed

    Bayer, Monika L; Schjerling, Peter; Biskup, Edyta; Herchenhan, Andreas; Heinemeier, Katja M; Doessing, Simon; Krogsgaard, Michael; Kjaer, Michael

    2012-05-01

    The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-β1 (TGF-β1), which is presumed to be reduced systemically with advanced age. The aim of this study was to investigate whether human serum from elderly donors would have an inhibiting effect on the expression of collagen and collagen-related genes as well as on cell proliferative capacity in tendon cells from young individuals. There was no difference in systemic TGF-β1 levels in serum obtained from young and elderly donors, and we found no difference in collagen expression when cells were subjected to human serum from elderly versus young donors. In addition, tendon cell proliferation was similar when culture medium was supplemented with serum of different donor age. These findings suggest that factors such as the cell intrinsic capacity or the tissue-specific environment rather than systemic circulating factors are important for functional capacity throughout life in human tendon cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  19. Directing the Differentiation of Parthenogenetic Stem Cells into Tenocytes for Tissue‐Engineered Tendon Regeneration

    PubMed Central

    Liu, Wei; Yin, Lu; Yan, Xingrong; Cui, Jihong; Liu, Wenguang; Rao, Yang; Sun, Mei; Wei, Qi

    2016-01-01

    Abstract Uniparental parthenogenesis yields pluripotent stem cells without the political and ethical concerns surrounding the use of embryonic stem cells (ESCs) for biomedical applications. In the current study, we hypothesized that parthenogenetic stem cells (pSCs) could be directed to differentiate into tenocytes and applied for tissue‐engineered tendon. We showed that pSCs displayed fundamental properties similar to those of ESCs, including pluripotency, clonogenicity, and self‐renewal capacity. pSCs spontaneously differentiated into parthenogenetic mesenchymal stem cells (pMSCs), which were positive for mesenchymal stem cell surface markers and possessed osteogenic, chondrogenic, and adipogenic potential. Then, mechanical stretch was applied to improve the tenogenic differentiation of pMSCs, as indicated by the expression of tenogenic‐specific markers and an increasing COL1A1:3A1 ratio. The pSC‐derived tenocytes could proliferate and secrete extracellular matrix on the surface of poly(lactic‐co‐glycolic) acid scaffolds. Finally, engineered tendon‐like tissue was successfully generated after in vivo heterotopic implantation of a tenocyte‐scaffold composite. In conclusion, our experiment introduced an effective and practical strategy for applying pSCs for tendon regeneration. Stem Cells Translational Medicine 2017;6:196–208 PMID:28170171

  20. No evidence for the use of stem cell therapy for tendon disorders: a systematic review.

    PubMed

    Pas, Haiko I M F L; Moen, Maarten H; Haisma, Hidde J; Winters, Marinus

    2017-07-01

    Stem cells have emerged as a new treatment option for tendon disorders. We systematically reviewed the current evidence for stem cell therapy in tendon disorders. Randomised and non-randomised controlled trials, cohort studies and case series with a minimum of 5 cases were searched in MEDLINE, CENTRAL, EMBASE, CINAHL, PEDro and SPORTDiscus. In addition, we searched grey literature databases and trial registers. Only human studies were included and no time or language restrictions were applied to our search. All references of included trials were checked for possibly eligible trials. Risk of bias assessment was performed using the Cochrane risk of bias tool for controlled trials and the Newcastle-Ottawa scale for case series. Levels of evidence were assigned according to the Oxford levels of evidence. 4 published and three unpublished/pending trials were found with a total of 79 patients. No unpublished data were available. Two trials evaluated bone marrow-derived stem cells in rotator cuff repair surgery and found lower retear rates compared with historical controls or the literature. One trial used allogenic adipose-derived stem cells to treat lateral epicondylar tendinopathy. Improved Mayo Elbow Performance Index, Visual Analogue Pain scale and ultrasound findings after 1-year follow-up compared with baseline were found. Bone marrow-derived stem cell-treated patellar tendinopathy showed improved International Knee Documentation Committee, Knee injury and Osteoarthritis Outcome Score subscales and Tegner scores after 5-year follow-up. One trial reported adverse events and found them to be mild (eg, swelling, effusion). All trials were at high risk of bias and only level 4 evidence was available. No evidence (level 4) was found for the therapeutic use of stem cells for tendon disorders. The use of stem cell therapy for tendon disorders in clinical practice is currently not advised. Published by the BMJ Publishing Group Limited. For permission to use (where not

  1. Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

    PubMed Central

    Gelfand, Vladimir I.

    2013-01-01

    Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport. PMID:24300413

  2. Organelle transport in cultured Drosophila cells: S2 cell line and primary neurons.

    PubMed

    Lu, Wen; Del Castillo, Urko; Gelfand, Vladimir I

    2013-11-20

    Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

  3. Mechanisms of programmed cell death during oogenesis in Drosophila virilis.

    PubMed

    Velentzas, Athanassios D; Nezis, Ioannis P; Stravopodis, Dimitrios J; Papassideri, Issidora S; Margaritis, Lukas H

    2007-02-01

    We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers.

  4. Volumetric Measurements of Amnioserosa Cells in Developing Drosophila

    NASA Astrophysics Data System (ADS)

    Mashburn, David; Jayasinghe, Aroshan; Hutson, Shane

    2013-03-01

    The behavior of cells in tissue in developing Drosophila melanogaster has become increasingly clearer over the past few decades, in large part due to advances in imaging techniques, genetic markers, predictive modeling, and micromanipulation (notably laser microsurgery). We now know apical contractions in amnioserosa cells are a significant factor in large scale processes like germ band retraction and dorsal closure. Also, laser microsurgery induces cellular recoil that strongly mimics a 2D elastic sheet. Still, what we know about these processes comes entirely from the apical surface where the standard fluorescent markers like cadherin are located, but many open questions exist concerning the remaining ``dark'' portion of cells. Does cell volume remain constant during contraction or do cells leak? Also, what shape changes do cells undergo? Do they bulge, wedge, contract prismatically, or something else? By using a marker that labels the entire membrane of amnioserosa cells (Resille, 117) and adapting our watershed segmentation routines for 4D datasets, we have been able to quantify the entire volumetric region of cells in tissue through time and compare changes in apical area and volume. Preliminary results suggest a fairly constant volume over the course of a contraction cycle.

  5. Cell type–specific genomics of Drosophila neurons

    PubMed Central

    Henry, Gilbert L.; Davis, Fred P.; Picard, Serge; Eddy, Sean R.

    2012-01-01

    Many tools are available to analyse genomes but are often challenging to use in a cell type–specific context. We have developed a method similar to the isolation of nuclei tagged in a specific cell type (INTACT) technique [Deal,R.B. and Henikoff,S. (2010) A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell, 18, 1030–1040; Steiner,F.A., Talbert,P.B., Kasinathan,S., Deal,R.B. and Henikoff,S. (2012) Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. Genome Res., doi:10.1101/gr.131748.111], first developed in plants, for use in Drosophila neurons. We profile gene expression and histone modifications in Kenyon cells and octopaminergic neurons in the adult brain. In addition to recovering known gene expression differences, we also observe significant cell type–specific chromatin modifications. In particular, a small subset of differentially expressed genes exhibits a striking anti-correlation between repressive and activating histone modifications. These genes are enriched for transcription factors, recovering those known to regulate mushroom body identity and predicting analogous regulators of octopaminergic neurons. Our results suggest that applying INTACT to specific neuronal populations can illuminate the transcriptional regulatory networks that underlie neuronal cell identity. PMID:22855560

  6. A Cell-Level Biomechanical Model of Drosophila Dorsal Closure

    PubMed Central

    Wang, Qiming; Feng, James J.; Pismen, Len M.

    2012-01-01

    We report a model describing the various stages of dorsal closure of Drosophila. Inspired by experimental observations, we represent the amnioserosa by 81 hexagonal cells that are coupled mechanically through the position of the nodes and the elastic forces on the edges. In addition, each cell has radial spokes representing actin filaments on which myosin motors can attach and exert contractile forces on the nodes, the attachment being controlled by a signaling molecule. Thus, the model couples dissipative cell and tissue motion with kinetic equations describing the myosin and signal dynamics. In the early phase, amnioserosa cells oscillate as a result of coupling among the chemical signaling, myosin attachment/detachment, and mechanical deformation of neighboring cells. In the slow phase, we test two ratcheting mechanisms suggested by experiments: an internal ratchet by the apical and junctional myosin condensates, and an external one by the supracellular actin cables encircling the amnioserosa. Within the range of parameters tested, the model predictions suggest the former as the main contributor to cell and tissue area reduction in this stage. In the fast phase of dorsal closure, cell pulsation is arrested, and the cell and tissue areas contract consistently. This is realized in the model by gradually shrinking the resting length of the spokes. Overall, the model captures the key features of dorsal closure through the three distinct phases, and its predictions are in good agreement with observations. PMID:23283225

  7. Polyphosphazene functionalized polyester fiber matrices for tendon tissue engineering: in vitro evaluation with human mesenchymal stem cells.

    PubMed

    Peach, M Sean; James, Roshan; Toti, Udaya S; Deng, Meng; Morozowich, Nicole L; Allcock, Harry R; Laurencin, Cato T; Kumbar, Sangamesh G

    2012-08-01

    Poly[(ethyl alanato)(1)(p-methyl phenoxy)(1)] phosphazene (PNEA-mPh) was used to modify the surface of electrospun poly(ε-caprolactone) (PCL) nanofiber matrices having an average fiber diameter of 3000 ± 1700 nm for the purpose of tendon tissue engineering and augmentation. This study reports the effect of polyphosphazene surface functionalization on human mesenchymal stem cell (hMSC) adhesion, cell-construct infiltration, proliferation and tendon differentiation, as well as long term cellular construct mechanical properties. PCL fiber matrices functionalized with PNEA-mPh acquired a rougher surface morphology and led to enhanced cell adhesion as well as superior cell-construct infiltration when compared to smooth PCL fiber matrices. Long-term in vitro hMSC cultures on both fiber matrices were able to produce clinically relevant moduli. Both fibrous constructs expressed scleraxis, an early tendon differentiation marker, and a bimodal peak in expression of the late tendon differentiation marker tenomodulin, a pattern that was not observed in PCL thin film controls. Functionalized matrices achieved a more prominent tenogenic differentiation, possessing greater tenomodulin expression and superior phenotypic maturity according to the ratio of collagen I to collagen III expression. These findings indicate that PNEA-mPh functionalization is an efficient method for improving cell interactions with electrospun PCL matrices for the purpose of tendon repair.

  8. Effects of the pulsed electromagnetic field PST® on human tendon stem cells: a controlled laboratory study.

    PubMed

    Randelli, Pietro; Menon, Alessandra; Ragone, Vincenza; Creo, Pasquale; Alfieri Montrasio, Umberto; Perucca Orfei, Carlotta; Banfi, Giuseppe; Cabitza, Paolo; Tettamanti, Guido; Anastasia, Luigi

    2016-08-18

    Current clinical procedures for rotator cuff tears need to be improved, as a high rate of failure is still observed. Therefore, new approaches have been attempted to stimulate self-regeneration, including biophysical stimulation modalities, such as low-frequency pulsed electromagnetic fields, which are alternative and non-invasive methods that seem to produce satisfying therapeutic effects. While little is known about their mechanism of action, it has been speculated that they may act on resident stem cells. Thus, the purpose of this study was to evaluate the effects of a pulsed electromagnetic field (PST®) on human tendon stem cells (hTSCs) in order to elucidate the possible mechanism of the observed therapeutic effects. hTSCs from the rotator cuff were isolated from tendon biopsies and cultured in vitro. Then, cells were exposed to a 1-h PST® treatment and compared to control untreated cells in terms of cell morphology, proliferation, viability, migration, and stem cell marker expression. Exposure of hTSCs to PST® did not cause any significant changes in proliferation, viability, migration, and morphology. Instead, while stem cell marker expression significantly decreased in control cells during cell culturing, PST®-treated cells did not have a significant reduction of the same markers. While PST® did not have significant effects on hTSCs proliferation, the treatment had beneficial effects on stem cell marker expression, as treated cells maintained a higher expression of these markers during culturing. These results support the notion that PST® treatment may increase the patient stem cell regenerative potential.

  9. The adult Drosophila malphigian tubules are maintained by multipotent stem cells | Center for Cancer Research

    Cancer.gov

    All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable and no stem cells have been identified.

  10. In situ cell-matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials.

    PubMed

    Duncan, Neil A; Bruehlmann, Sabina B; Hunter, Christopher J; Shao, Xinxin; Kelly, Elizabeth J

    2014-01-01

    Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell-matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell-matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (∼2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell-matrix boundary with visible shear across cell nuclei of >1 μm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.

  11. Sex, stem cells and tumors in the Drosophila ovary.

    PubMed

    Salz, Helen K

    2013-01-01

    The Drosophila Sex-lethal (Sxl) gene encodes a female-specific RNA binding protein that in somatic cells globally regulates all aspects of female-specific development and behavior. Sxl also has a critical, but less well understood, role in female germ cells. Germ cells without Sxl protein can adopt a stem cell fate when housed in a normal ovary, but fail to successfully execute the self-renewal differentiation fate switch. The failure to differentiate is accompanied by the inappropriate expression of a set of male specific markers, continued proliferation, and formation of a tumor. The findings in Chau et al., (2012) identify the germline stem cell maintenance factor nanos as one of its target genes, and suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional downregulation of nanos expression. These studies provide the basis for a new model in which Sxl directly couples sexual identity with the self-renewal differentiation decision and raises several interesting questions about the genesis of the tumor phenotype.

  12. Efficacy of supraspinatus tendon repair using mesenchymal stem cells along with a collagen I scaffold.

    PubMed

    Tornero-Esteban, Pilar; Hoyas, José Antonio; Villafuertes, Esther; Rodríguez-Bobada, Cruz; López-Gordillo, Yamila; Rojo, Francisco J; Guinea, Gustavo V; Paleczny, Anna; Lópiz-Morales, Yaiza; Rodriguez-Rodriguez, Luis; Marco, Fernando; Fernández-Gutiérrez, Benjamín

    2015-08-14

    Our main objective was to biologically improve rotator cuff healing in an elderly rat model using mesenchymal stem cells (MSCs) in combination with a collagen membrane and compared against other current techniques. A chronic rotator cuff tear injury model was developed by unilaterally detaching the supraspinatus (SP) tendons of Sprague-Dawley rats. At 1 month postinjury, the tears were repaired using one of the following techniques: (a) classical surgery using sutures (n = 12), (b) type I collagen membranes (n = 15), and (c) type I collagen membranes + 1 × 106 allogeneic MSCs (n = 14). Lesion restoration was evaluated at 1, 2, and 3 months postinjury based on biomechanical criteria. Continuous variables were described using mean and standard deviation (SD). To analyse the effect of the different surgical treatments in the repaired tendons' biomechanical capabilities (maximum load, stiffness, and deformity), a two-way ANOVA model was used, introducing an interaction between such factor and time (1, 2, and 3 months postinjury). With regard to maximum load, we observed an almost significant interaction between treatment and time (F = 2.62, df = 4, p = 0.053). When we analysed how this biomechanical capability changed with time for each treatment, we observed that repair with OrthADAPT and MSCs was associated with a significant increase in maximum load (p = 0.04) between months 1 and 3. On the other hand, when we compared the different treatments among themselves at different time points, we observed that the repair with OrthADAPT and MSCs has associated with a significant higher maximum load, when compared with the use of suture, but only at 3 months (p = 0.014). With regard to stiffness and deformity, no significant interaction was observed (F = 1.68, df = 4, p = 0.18; F = 0.40, df = 4, p = 0.81; respectively). The implantation of MSCs along with a collagen I scaffold into surgically created tendon

  13. PROGRAMMED CELL DEATH IN EXTRAOCULAR MUSCLE TENDON/SCLERA PRECURSORS

    EPA Science Inventory

    Abstract

    Purpose: This study was designed to examine the occurrence of natural cell death in the periocular mesenchyme of mouse embryos.

    Methods: Vital staining with LysoTracker Red and Nile blue sulphate as well as terminal nick end labeling (TUNEL) were utiliz...

  14. PROGRAMMED CELL DEATH IN EXTRAOCULAR MUSCLE TENDON/SCLERA PRECURSORS

    EPA Science Inventory

    Abstract

    Purpose: This study was designed to examine the occurrence of natural cell death in the periocular mesenchyme of mouse embryos.

    Methods: Vital staining with LysoTracker Red and Nile blue sulphate as well as terminal nick end labeling (TUNEL) were utiliz...

  15. Epithelial neoplasia in Drosophila entails switch to primitive cell states

    PubMed Central

    Khan, Sumbul J.; Bajpai, Anjali; Alam, Mohammad Atif; Gupta, Ram P.; Harsh, Sneh; Pandey, Ravi K.; Goel-Bhattacharya, Surbhi; Nigam, Aditi; Mishra, Arati; Sinha, Pradip

    2013-01-01

    Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl− clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl− clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl− clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl− clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl− clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of “cells-of-origin” in epithelial cancers, namely their propensity for switch to primitive cell states. PMID:23708122

  16. Epithelial neoplasia in Drosophila entails switch to primitive cell states.

    PubMed

    Khan, Sumbul J; Bajpai, Anjali; Alam, Mohammad Atif; Gupta, Ram P; Harsh, Sneh; Pandey, Ravi K; Goel-Bhattacharya, Surbhi; Nigam, Aditi; Mishra, Arati; Sinha, Pradip

    2013-06-11

    Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl(-) clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl(-) clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl(-) clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl(-) clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl(-) clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of "cells-of-origin" in epithelial cancers, namely their propensity for switch to primitive cell states.

  17. Tissue growth and tumorigenesis in Drosophila: cell polarity and the Hippo pathway.

    PubMed

    Richardson, Helena E; Portela, Marta

    2017-03-28

    Cell polarity regulation is critical for defining membrane domains required for the establishment and maintenance of the apical-basal axis in epithelial cells (apico-basal polarity), asymmetric cell divisions, planar organization of tissues (planar cell polarity), and the formation of the front-rear axis in cell migration (front-rear polarity). In the vinegar fly, Drosophila melanogaster, cell polarity regulators also interact with the Hippo tissue growth control signaling pathway. In this review we survey the recent Drosophila literature linking cell polarity regulators with the Hippo pathway in epithelial tissue growth, neural stem cell asymmetric divisions and in cell migration in physiological and tumorigenic settings.

  18. Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems.

    PubMed

    James, R; Kumbar, S G; Laurencin, C T; Balian, G; Chhabra, A B

    2011-04-01

    Tendon tissue engineering with a biomaterial scaffold that mimics the tendon extracellular matrix (ECM) and is biomechanically suitable, and when combined with readily available autologous cells, may provide successful regeneration of defects in tendon. Current repair strategies using suitable autografts and freeze-dried allografts lead to a slow repair process that is sub-optimal and fails to restore function, particularly in difficult clinical situations such as zone II flexor tendon injuries of the hand. We have investigated the effect of GDF-5 on cell proliferation and gene expression by primary rat adipose-derived stem cells (ADSCs) that were cultured on a poly(DL-lactide-co-glycolide) PLAGA fiber scaffold and compared to a PLAGA 2D film scaffold. The electrospun scaffold mimics the collagen fiber bundles present in native tendon tissue, and supports the adhesion and proliferation of multipotent ADSCs. Gene expression of scleraxis, the neotendon marker, was upregulated seven- to eightfold at 1 week with GDF-5 treatment when cultured on a 3D electrospun scaffold, and was significantly higher at 2 weeks compared to 2D films with or without GDF-5 treatment. Expression of the genes that encode the major tendon ECM protein, collagen type I, was increased by fourfold starting at 1 week on treatment with 100 ng mL(-1) GDF-5, and at all time points the expression was significantly higher compared to 2D films irrespective of GDF-5 treatment. Thus stimulation with GDF-5 can modulate primary ADSCs on a PLAGA fiber scaffold to produce a soft, collagenous musculoskeletal tissue that fulfills the need for tendon regeneration.

  19. Formation and specification of a Drosophila dopaminergic precursor cell.

    PubMed

    Watson, Joseph D; Crews, Stephen T

    2012-09-01

    Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb(+)/gsb-n(+) MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons.

  20. Formation and specification of a Drosophila dopaminergic precursor cell

    PubMed Central

    Watson, Joseph D.; Crews, Stephen T.

    2012-01-01

    Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb+/gsb-n+ MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons. PMID:22874915

  1. Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila.

    PubMed

    Biteau, Benoît; Jasper, Heinrich

    2014-06-26

    In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. Here, we identify a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. We find that EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, we show that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. Our work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. Our results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage.

  2. In vitro functional response of human tendon cells to different dosages of low-frequency pulsed electromagnetic field.

    PubMed

    de Girolamo, L; Viganò, M; Galliera, E; Stanco, D; Setti, S; Marazzi, M G; Thiebat, G; Corsi Romanelli, M M; Sansone, V

    2015-11-01

    Chronic tendinopathy is a degenerative process causing pain and disability. Current treatments include biophysical therapies, such as pulsed electromagnetic fields (PEMF). The aim of this study was to compare, for the first time, the functional in vitro response of human tendon cells to different dosages of PEMF, varying in field intensity and duration and number of exposures. Tendon cells, isolated from human semitendinosus and gracilis tendons (hTCs; n = 6), were exposed to different PEMF treatments (1.5 or 3 mT for 8 or 12 h, single or repeated treatments). Scleraxis (SCX), COL1A1, COL3A1 and vascular endothelial growth factor-A (VEGF-A) expression and cytokine production were assessed. None of the different dosages provoked apoptotic events. Proliferation of hTCs was enhanced by all treatments, whereas only 3 mT-PEMF treatment increased cell viability. However, the single 1.5 mT-PEMF treatment elicited the highest up-regulation of SCX, VEGF-A and COL1A1 expression, and it significantly reduced COL3A1 expression with respect to untreated cells. The treated hTCs showed a significantly higher release of IL-1β, IL-6, IL-10 and TGF-β. Interestingly, the repeated 1.5 mT-PEMF significantly further increased IL-10 production. 1.5 mT-PEMF treatment was able to give the best results in in vitro healthy human tendon cell culture. Although the clinical relevance is not direct, this investigation should be considered an attempt to clarify the effect of different PEMF protocols on tendon cells, in particular focusing on the potential applicability of this cell source for regenerative medicine purpose, both in surgical and in conservative treatment for tendon disorders.

  3. Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development.

    PubMed

    Bello, Bruno C; Izergina, Natalya; Caussinus, Emmanuel; Reichert, Heinrich

    2008-02-19

    In the mammalian brain, neural stem cells divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. Here we investigate whether specific neural stem cell-like neuroblasts in the brain of Drosophila might also amplify neuronal proliferation by generating symmetrically dividing intermediate progenitors. Cell lineage-tracing and genetic marker analysis show that remarkably large neuroblast lineages exist in the dorsomedial larval brain of Drosophila. These lineages are generated by brain neuroblasts that divide asymmetrically to self renew but, unlike other brain neuroblasts, do not segregate the differentiating cell fate determinant Prospero to their smaller daughter cells. These daughter cells continue to express neuroblast-specific molecular markers and divide repeatedly to produce neural progeny, demonstrating that they are proliferating intermediate progenitors. The proliferative divisions of these intermediate progenitors have novel cellular and molecular features; they are morphologically symmetrical, but molecularly asymmetrical in that key differentiating cell fate determinants are segregated into only one of the two daughter cells. Our findings provide cellular and molecular evidence for a new mode of neurogenesis in the larval brain of Drosophila that involves the amplification of neuroblast proliferation through intermediate progenitors. This type of neurogenesis bears remarkable similarities to neurogenesis in the mammalian brain, where neural stem cells as primary progenitors amplify the number of progeny they generate through generation of secondary progenitors. This suggests that key aspects of neural stem cell biology might be conserved in brain development of insects and mammals.

  4. Planar cell polarity: the Dachsous/Fat system contributes differently to the embryonic and larval stages of Drosophila

    PubMed Central

    Saavedra, Pedro; Brittle, Amy; Palacios, Isabel M.; Strutt, David; Casal, José; Lawrence, Peter A.

    2016-01-01

    ABSTRACT The epidermal patterns of all three larval instars (L1–L3) of Drosophila are made by one unchanging set of cells. The seven rows of cuticular denticles of all larval stages are consistently planar polarised, some pointing forwards, others backwards. In L1 all the predenticles originate at the back of the cells but, in L2 and L3, they form at the front or the back of the cell depending on the polarity of the forthcoming denticles. We find that, to polarise all rows, the Dachsous/Fat system is differentially utilised; in L1 it is active in the placement of the actin-based predenticles but is not crucial for the final orientation of the cuticular denticles, in L2 and L3 it is needed for placement and polarity. We find Four-jointed to be strongly expressed in the tendon cells and show how this might explain the orientation of all seven rows. Unexpectedly, we find that L3 that lack Dachsous differ from larvae lacking Fat and we present evidence that this is due to differently mislocalised Dachs. We make some progress in understanding how Dachs contributes to phenotypes of wildtype and mutant larvae and adults. PMID:26935392

  5. Induction temperature of human heat shock factor is reprogrammed in a Drosophila cell environment

    NASA Astrophysics Data System (ADS)

    Clos, Joachim; Rabindran, Sridhar; Wisniewski, Jan; Wu, Carl

    1993-07-01

    HEAT shock factor (HSF)1,2, the transcriptional activator of eukaryotic heat shock genes, is induced to bind DNA by a monomer to trimer transition involving leucine zipper interactions3,4. Although this mode of regulation is shared among many eukaryotic species, there is variation in the temperature at which HSF binding activity is induced. We investigated the basis of this variation by analysing the response of a human HSF expressed in Drosophila cells and Drosophila HSF expressed in human cells. We report here that the temperature that induces DNA binding and trimerization of human HSF in Drosophila was decreased by ~10 °C to the induction temperature for the host cell, whereas Drosophila HSF expressed in human cells was constitutively active. The results indicate that the activity of HSF in vivo is not a simple function of the absolute environmental temperature.

  6. Low frequency pulsed electromagnetic field affects proliferation, tissue-specific gene expression, and cytokines release of human tendon cells.

    PubMed

    de Girolamo, L; Stanco, D; Galliera, E; Viganò, M; Colombini, A; Setti, S; Vianello, E; Corsi Romanelli, M M; Sansone, V

    2013-07-01

    Low frequency pulsed electromagnetic field (PEMF) has proven to be effective in the modulation of bone and cartilage tissue functional responsiveness, but its effect on tendon tissue and tendon cells (TCs) is still underinvestigated. PEMF treatment (1.5 mT, 75 Hz) was assessed on primary TCs, harvested from semitendinosus and gracilis tendons of eight patients, under different experimental conditions (4, 8, 12 h). Quantitative PCR analyses were conducted to identify the possible effect of PEMF on tendon-specific gene transcription (scleraxis, SCX and type I collagen, COL1A1); the release of pro- and anti-inflammatory cytokines and of vascular endothelial growth factor (VEGF) was also assessed. Our findings show that PEMF exposure is not cytotoxic and is able to stimulate TCs' proliferation. The increase of SCX and COL1A1 in PEMF-treated cells was positively correlated to the treatment length. The release of anti-inflammatory cytokines in TCs treated with PEMF for 8 and 12 h was significantly higher in comparison with untreated cells, while the production of pro-inflammatory cytokines was not affected. A dramatically higher increase of VEGF-A mRNA transcription and of its related protein was observed after PEMF exposure. Our data demonstrated that PEMF positively influence, in a dose-dependent manner, the proliferation, tendon-specific marker expression, and release of anti-inflammatory cytokines and angiogenic factor in a healthy human TCs culture model.

  7. Glial cell development and function in the Drosophila visual system

    PubMed Central

    CHOTARD, CAROLE; SALECKER, IRIS

    2008-01-01

    In the developing nervous system, building a functional neuronal network relies on coordinating the formation, specification and survival to diverse neuronal and glial cell subtypes. The establishment of neuronal connections further depends on sequential neuron–neuron and neuron–glia interactions that regulate cell-migration patterns and axon guidance. The visual system of Drosophila has a highly regular, retinotopic organization into reiterated interconnected synaptic circuits. It is therefore an excellent invertebrate model to investigate basic cellular strategies and molecular determinants regulating the different developmental processes that lead to network formation. Studies in the visual system have provided important insights into the mechanisms by which photoreceptor axons connect with their synaptic partners within the optic lobe. In this review, we highlight that this system is also well suited for uncovering general principles that underlie glial cell biology. We describe the glial cell subtypes in the visual system and discuss recent findings about their development and migration. Finally, we outline the pivotal roles of glial cells in mediating neural circuit assembly, boundary formation, neural proliferation and survival, as well as synaptic function. PMID:18333286

  8. Mapping signaling pathway cross-talk in Drosophila cells

    PubMed Central

    Ammeux, Noemie; Housden, Benjamin E.; Georgiadis, Andrew; Hu, Yanhui; Perrimon, Norbert

    2016-01-01

    During development and homeostasis, cells integrate multiple signals originating either from neighboring cells or systemically. In turn, responding cells can produce signals that act in an autocrine, paracrine, or endocrine manner. Although the nature of the signals and pathways used in cell–cell communication are well characterized, we lack, in most cases, an integrative view of signaling describing the spatial and temporal interactions between pathways (e.g., whether the signals are processed sequentially or concomitantly when two pathways are required for a specific outcome). To address the extent of cross-talk between the major metazoan signaling pathways, we characterized immediate transcriptional responses to either single- or multiple pathway stimulations in homogeneous Drosophila cell lines. Our study, focusing on seven core pathways, epidermal growth factor receptor (EGFR), bone morphogenetic protein (BMP), Jun kinase (JNK), JAK/STAT, Notch, Insulin, and Wnt, revealed that many ligands and receptors are primary targets of signaling pathways, highlighting that transcriptional regulation of genes encoding pathway components is a major level of signaling cross-talk. In addition, we found that ligands and receptors can integrate multiple pathway activities and adjust their transcriptional responses accordingly. PMID:27528688

  9. Enhancement of tendon graft osteointegration using mesenchymal stem cells in a rabbit model of anterior cruciate ligament reconstruction.

    PubMed

    Lim, Jit-Kheng; Hui, James; Li, Li; Thambyah, Ashvin; Goh, James; Lee, Eng-Hin

    2004-11-01

    To study the effect of coating tendon grafts with mesenchymal stem cells (MSCs) on the rate and quality of graft osteointegration in anterior cruciate ligament (ACL) reconstruction. Animal model. Bilateral ACL reconstructions using hamstring tendon autografts were performed on 48 adult rabbits. Grafts were coated with MSCs in a fibrin glue carrier in one limb, and fibrin glue only in the other. Assessment was done at 2, 4, and 8 weeks. Histologic analysis was carried out using standard and immunohistochemical stains. Biomechanical testing of force and stiffness during loading to ultimate failure was performed. Control reconstructions showed mature scar tissue with some Sharpey's-like fibers spanning the tendon-bone interface at 8 weeks. The MSC-enhanced reconstructions had large areas of cartilage cells at the tendon-bone junction at 2 weeks. By 8 weeks, a mature zone of cartilage was seen gradually blending from bone into the tendon grafts. This zone stained strongly for type II collagen and showed histologic characteristics similar to normal rabbit ACL insertions. Biomechanically, there was no statistical difference between limbs at 2 and 4 weeks. At 8 weeks, the MSC-enhanced grafts had significantly higher failure load and stiffness. Coating of tendon grafts with MSCs results in healing by an intervening zone of cartilage resembling the chondral enthesis of normal ACL insertions rather than collagen fibers and scar tissue. MSC-enhanced ACL reconstructions perform significantly better than controls on biomechanical testing. Enhancement of tendon graft osteointegration with MSCs is a novel method offering the potential for more physiologic and biomechanically stronger ligament reconstructions.

  10. Anterior cruciate ligament- and hamstring tendon-derived cells: in vitro differential properties of cells involved in ACL reconstruction.

    PubMed

    Ghebes, Corina Adriana; Kelder, Cindy; Schot, Thomas; Renard, Auke J; Pakvis, Dean F M; Fernandes, Hugo; Saris, Daniel B

    2015-03-11

    Anterior cruciate ligament (ACL) reconstruction involves the replacement of the torn ligament with a new graft, often a hamstring tendon (HT). Described as similar, the ACL and HT have intrinsic differences related to their distinct anatomical locations. From a cellular perspective, identifying these differences represents a step forward in the search for new cues that enhance recovery after the reconstruction. The purpose of this study was to characterize the phenotype and multilineage potential of ACL- and HT-derived cells. ACL- and HT-derived cells were isolated from tissue harvest from patients undergoing total knee arthroplasty (TKA) or ACL reconstruction. In total, three ACL and three HT donors were investigated. Cell morphology, self-renewal potential (CFU-F), surface marker profiling, expression of tendon/ligament-related markers (PCR) and multilineage potential were analysed for both cell types; both had fibroblast-like morphology and low self-renewal potential. No differences in the expression of tendon/ligament-related genes or a selected set of surface markers were observed between the two cell types. However, differences in their multilineage potential were observed: while ACL-derived cells showed a high potential to differentiate into chondrocytes and adipocytes, but not osteoblasts, HT-derived cells showed poor potential to form adipocytes, chondrocytes and osteoblasts. Our results demonstrated that HT-derived cells have low multilineage potential compared to ACL-derived cells, further highlighting the need for extrinsic signals to fully restore the function of the ACL upon reconstruction. Copyright © 2015 John Wiley & Sons, Ltd.

  11. Embryonic Mechanical and Soluble Cues Regulate Tendon Progenitor Cell Gene Expression as a Function of Developmental Stage and Anatomical Origin

    PubMed Central

    Brown, Jeffrey P; Finley, Violet G; Kuo, Catherine K

    2014-01-01

    Stem cell-based engineering strategies for tendons have yet to yield a normal functional tissue, due in part to a need for tenogenic factors. Additionally, the ability to evaluate differentiation has been challenged by a lack of markers for differentiation. We propose to inform tendon regeneration with developmental cues involved in normal tissue formation and with phenotypic markers that are characteristic of differentiating tendon progenitor cells (TPCs). Mechanical forces, fibroblast growth factor (FGF)-4 and transforming growth factor (TGF)-β2 are implicated in embryonic tendon development, yet the isolated effects of these factors on differentiating TPCs are unknown. Additionally, developmental mechanisms vary between limb and axial tendons, suggesting the respective cell types are programmed to respond uniquely to exogenous factors. To characterize developmental cues and benchmarks for differentiation toward limb vs. axial phenotypes, we dynamically loaded and treated TPCs with growth factors and assessed gene expression profiles as a function of developmental stage and anatomical origin. Based on scleraxis expression, TGFβ2 was tenogenic for TPCs at all stages, while loading was for late-stage cells only, and FGF4 had no effect despite regulation of other genes. When factors were combined, TGF 2 continued to be tenogenic, while FGF4 appeared anti-tenogenic. Various treatments elicited distinct responses by axial vs. limb TPCs of specific stages. These results identified tenogenic factors, suggest tendon engineering strategies should be customized for tissues by anatomical origin, and provide stage-specific gene expression profiles of limb and axial TPCs as benchmarks with which to monitor tenogenic differentiation of stem cells. PMID:24231248

  12. Specification of regional intestinal stem cell identity during Drosophila metamorphosis

    PubMed Central

    Driver, Ian; Ohlstein, Benjamin

    2014-01-01

    In the adult Drosophila midgut the bone morphogenetic protein (BMP) signaling pathway is required to specify and maintain the acid-secreting region of the midgut known as the copper cell region (CCR). BMP signaling is also involved in the modulation of intestinal stem cell (ISC) proliferation in response to injury. How ISCs are able to respond to the same signaling pathway in a regionally different manner is currently unknown. Here, we show that dual use of the BMP signaling pathway in the midgut is possible because BMP signals are only capable of transforming ISC and enterocyte identity during a defined window of metamorphosis. ISC heterogeneity is established prior to adulthood and then maintained in cooperation with regional signals from surrounding tissue. Our data provide a conceptual framework for how other tissues maintained by regional stem cells might be patterned and establishes the pupal and adult midgut as a novel genetic platform for identifying genes necessary for regional stem cell specification and maintenance. PMID:24700821

  13. Two Drosophila retrotransposon gypsy subfamilies differ in ability to produce new DNA copies via reverse transcription in Drosophila cultured cells.

    PubMed Central

    Lyubomirskaya, N V; Avedisov, S N; Surkov, S A; Ilyin, Y V

    1993-01-01

    Plasmid DNA constructs containing 5' end truncated retrotransposon gypsy were introduced into Drosophila cultured cells. Appearance of new complete DNA copies with reconstructed via reverse transcription 5'LTR were detected by PCR after transient expression and by Southern blot analysis of genome DNA of stably transformed cells. Two gypsy subfamilies supposed to be different in transpositional activity were analyzed in terms of their ability to produce new DNA copies via reverse transcription in D. hydei cultured cells. It was demonstrated that both gypsy variants undergo retrotransposition but with different efficiency. Images PMID:7688116

  14. Radial shock waves effectively introduced NF-kappa B decoy into rat achilles tendon cells in vitro.

    PubMed

    Sugioka, Kaori; Nakagawa, Koichi; Murata, Ryo; Ochiai, Nobuyasu; Sasho, Takahisa; Arai, Momoko; Tsuruoka, Hiroaki; Ohtori, Seiji; Saisu, Takashi; Gemba, Takefumi; Takahashi, Kazuhisa

    2010-08-01

    The purpose of this study was to test if radial shock waves could enhance the introduction of nuclear factor-kappa B (NF-kappaB) decoy oligodeoxynucleotides, which is reported to markedly inhibit NF-kappaB activation and suppress pro-inflammatory cytokine gene expression, using rat Achilles tendon cells. In the presence of NF-kappaB decoy labeled with or without fluorescein isothiocyanate (FITC) in culture media, radial shock waves were applied to the tendon cells in variable conditions and cultivated for 24 h. The transfection rate was assessed by counting FITC-positive cells, and IL-1-induced NF-kappaB activation in the cells was assessed. Radial shock waves significantly enhanced introduction of NF-kappaB decoy-FITC into the tendon cells. IL-1-induced NF-kappaB activation was significantly inhibited by pretreatment of the cells with NF-kappaB decoy combined with radial shock wave exposure. The present study demonstrated the effectiveness of radial shock waves on introduction of NF-kappaB decoy into tendon cells. Radial shock wave treatment combined with local NF-kappaB decoy administration could be a novel therapeutic strategy for chronic tendinopathy.

  15. The behavior of neuronal cells on tendon-derived collagen sheets as potential substrates for nerve regeneration.

    PubMed

    Alberti, Kyle A; Hopkins, Amy M; Tang-Schomer, Min D; Kaplan, David L; Xu, Qiaobing

    2014-04-01

    Peripheral nervous system injuries result in a decreased quality of life, and generally require surgical intervention for repair. Currently, the gold standard of nerve autografting, based on the use of host tissue such as sensory nerves is suboptimal as it results in donor-site loss of function and requires a secondary surgery. Nerve guidance conduits fabricated from natural polymers such as collagen are a common alternative to bridge nerve defects. In the present work, tendon sections derived through a process named bioskiving were studied for their potential for use as a substrate to fabricate nerve guidance conduits. We show that cells such as rat Schwann cells adhere, proliferate, and align along the fibrous tendon substrate which has been shown to result in a more mature phenotype. Additionally we demonstrate that chick dorsal root ganglia explants cultured on the tendon grow to similar lengths compared to dorsal root ganglia cultured on collagen gels, but also grow in a more oriented manner on the tendon sections. These results show that tendon sections produced through bioskiving can support directional nerve growth and may be of use as a substrate for the fabrication of nerve guidance conduits. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Preclinical regulatory validation of an engineered diaphragmatic tendon made with amniotic mesenchymal stem cells.

    PubMed

    Turner, Christopher G; Klein, Justin D; Steigman, Shaun A; Armant, Myriam; Nicksa, Grace A; Zurakowski, David; Ritz, Jerome; Fauza, Dario O

    2011-01-01

    Under a Food and Drug Administration directive, we examined definite long-term safety and efficacy aspects of an engineered diaphragmatic tendon graft as a regulatory prerequisite for clinical trials. Newborn lambs (N = 27) underwent partial diaphragmatic replacement with a Teflon patch, a composite acellular bioprosthesis, or the same bioprosthesis seeded with autologous amniotic mesenchymal stem cells processed under Good Manufacturing Practice guidelines. Multiple safety and efficacy analyses were performed at different time points up to 14 months of age (ovine adulthood). There was no mortality. None of the blood tests or full body autopsy specimens showed any abnormality. There was a significantly higher failure rate in animals that received an acellular bioprosthetic graft vs an engineered graft, with no significant differences between Teflon and acellular bioprosthetic implants. Tensile strength and total collagen levels were significantly higher in engineered grafts than in acellular bioprosthetic grafts. On histology, lysozyme and myeloperoxidase stainings were unremarkable in all grafts. Diaphragmatic repair with a clinically viable autologous tendon engineered with amniotic mesenchymal stem cells leads to improved outcomes when compared with an equivalent acellular bioprosthesis, with no local or systemic adverse effects. Clinical trials of engineered diaphragmatic repair appear practicable within regulatory guidelines. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Dexamethasone inhibits the differentiation of rat tendon stem cells into tenocytes by targeting the scleraxis gene.

    PubMed

    Chen, Wan; Tang, Hong; Zhou, Mei; Hu, Chao; Zhang, Jiqiang; Tang, Kanglai

    2015-08-01

    Glucocorticoid-induced tendon rupture is very common in clinical practice, and the overall outcome of surgical suture repair is rather poor. The mechanism remains unclear, and effective treatments are still lacking. In the present study, we investigated the effect of dexamethasone on the differentiation of rat tendon stem cells (TSCs) to tenocytes and the underlying molecular mechanisms and found that dexamethasone inhibits the differentiation of TSCs to tenocytes by analyzing the development of long, spindle-shaped cells and detecting the expression of tenocyte markers type I collagen and tenomodulin (TNMD) at both the mRNA and protein levels. We also discovered that after treatment with dexamethasone, the scleraxis expression level is downregulated in vitro and in human specimen. Chromatin immunoprecipitation (ChIP)-PCR showed that dexamethasone promotes glucocorticoid receptor interacted with the TGGAAGCC sequence located between -734 and -726 base pairs (bp) upstream of the start codon of the scleraxis gene. Furthermore, TSCs were transfected with scleraxis knockdown or overexpression plasmids, and the results indicated that scleraxis plays a pivotal role in the differentiation of TSCs to tenocytes. In conclusion, dexamethasone inhibits the differentiation of TSCs to tenocytes by inhibiting the scleraxis gene.

  18. Mechanical Actuation Systems for the Phenotype Commitment of Stem Cell-Based Tendon and Ligament Tissue Substitutes.

    PubMed

    Govoni, Marco; Muscari, Claudio; Lovecchio, Joseph; Guarnieri, Carlo; Giordano, Emanuele

    2016-04-01

    High tensile forces transmitted by tendons and ligaments make them susceptible to tearing or complete rupture. The present standard reparative technique is the surgical implantation of auto- or allografts, which often undergo failure.Currently, different cell types and biomaterials are used to design tissue engineered substitutes. Mechanical stimulation driven by dedicated devices can precondition these constructs to a remarkable degree, mimicking the local in vivo environment. A large number of dynamic culture instruments have been developed and many appealing results collected. Of the cells that have been used, tendon stem cells are the most promising for a reliable stretch-induced tenogenesis, but their reduced availability represents a serious limitation to upscaled production. Biomaterials used for scaffold fabrication include both biological molecules and synthetic polymers, the latter being improved by nanotechnologies which reproduce the architecture of native tendons. In addition to cell type and scaffold material, other variables which must be defined in mechanostimulation protocols are the amplitude, frequency, duration and direction of the applied strain. The ideal conditions seem to be those producing intermittent tension rather than continuous loading. In any case, all physical parameters must be adapted to the specific response of the cells used and the tensile properties of the scaffold. Tendon/ligament grafts in animals usually have the advantage of mechanical preconditioning, especially when uniaxial cyclic forces are applied to cells engineered into natural or decellularized scaffolds. However, due to the scarcity of in vivo research, standard protocols still need to be defined for clinical applications.

  19. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

    DOE PAGES

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; ...

    2016-01-15

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest suchmore » atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. In conclusion, this atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.« less

  20. Haemocytes control stem cell activity in the Drosophila intestine.

    PubMed

    Ayyaz, Arshad; Li, Hongjie; Jasper, Heinrich

    2015-06-01

    Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. Here we show that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. We propose that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans.

  1. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

    PubMed Central

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; Brown, Ben; Cherbas, Peter

    2016-01-01

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly. PMID:26772746

  2. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

    SciTech Connect

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; Brown, Ben; Cherbas, Peter

    2016-01-15

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. In conclusion, this atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.

  3. Stonewalling Drosophila stem cell differentiation by epigenetic controls.

    PubMed

    Maines, Jean Z; Park, Joseph K; Williams, Meredith; McKearin, Dennis M

    2007-04-01

    During Drosophila oogenesis, germline stem cell (GSC) identity is maintained largely by preventing the expression of factors that promote differentiation. This is accomplished via the activity of several genes acting either in the GSC or in its niche. The translational repressors Nanos and Pumilio act in GSCs to prevent differentiation, probably by inhibiting the translation of early differentiation factors, whereas niche signals prevent differentiation by silencing transcription of the differentiation factor Bam. We have found that the DNA-associated protein Stonewall (Stwl) is also required for GSC maintenance. stwl is required cell-autonomously; clones of stwl(-) germ cells were lost by differentiation, and ectopic Stwl caused an expansion of GSCs. stwl mutants acted as Suppressors of variegation, indicating that stwl normally acts in chromatin-dependent gene repression. In contrast to several previously described GSC maintenance factors, Stwl probably functions epigenetically to prevent GSC differentiation. Stwl-dependent transcriptional repression does not target bam, but rather Stwl represses the expression of many genes, including those that may be targeted by Nanos and Pumilio translational inhibition.

  4. Acentrosomal Drosophila epithelial cells exhibit abnormal cell division, leading to cell death and compensatory proliferation

    PubMed Central

    Poulton, John S; Cuningham, John C; Peifer, Mark

    2014-01-01

    Summary Mitotic spindles are critical for accurate chromosome segregation. Centrosomes, the primary microtubule nucleating centers of animal cells, play key roles in forming and orienting mitotic spindles. However, the survival of Drosophila without centrosomes suggested they are dispensable in somatic cells, challenging the canonical view. We used fly wing disc epithelia as a model to resolve these conflicting hypotheses, revealing that centrosomes play vital roles in spindle assembly, function, and orientation. Many acentrosomal cells exhibit prolonged spindle assembly, chromosome mis-segregation, DNA damage, misoriented divisions, and eventual apoptosis. We found that multiple mechanisms buffer the effects of centrosome loss, including alternative microtubule nucleation pathways and the Spindle Assembly Checkpoint. Apoptosis of acentrosomal cells is mediated by JNK signaling, which also drives compensatory proliferation to maintain tissue integrity and viability. These data reveal the importance of centrosomes in fly epithelia, but also demonstrate the robust compensatory mechanisms at the cellular and organismal level. PMID:25241934

  5. Regeneration of Full-Thickness Rotator Cuff Tendon Tear After Ultrasound-Guided Injection With Umbilical Cord Blood-Derived Mesenchymal Stem Cells in a Rabbit Model.

    PubMed

    Park, Gi-Young; Kwon, Dong Rak; Lee, Sang Chul

    2015-11-01

    Rotator cuff tendon tear is one of the most common causes of chronic shoulder pain and disability. In this study, we investigated the therapeutic effects of ultrasound-guided human umbilical cord blood (UCB)-derived mesenchymal stem cell (MSC) injection to regenerate a full-thickness subscapularis tendon tear in a rabbit model by evaluating the gross morphology and histology of the injected tendon and motion analysis of the rabbit's activity. At 4 weeks after ultrasound-guided UCB-derived MSC injection, 7 of the 10 full-thickness subscapularis tendon tears were only partial-thickness tears, and 3 remained full-thickness tendon tears. The tendon tear size and walking capacity at 4 weeks after UCB-derived MSC injection under ultrasound guidance were significantly improved compared with the same parameters immediately after tendon tear. UCB-derived MSC injection under ultrasound guidance without surgical repair or bioscaffold resulted in the partial healing of full-thickness rotator cuff tendon tears in a rabbit model. Histology revealed that UCB-derived MSCs induced regeneration of rotator cuff tendon tear and that the regenerated tissue was predominantly composed of type I collagens. In this study, ultrasound-guided injection of human UCB-derived MSCs contributed to regeneration of the full-thickness rotator cuff tendon tear without surgical repair. The results demonstrate the effectiveness of local injection of MSCs into the rotator cuff tendon. The results of this study suggest that ultrasound-guided umbilical cord blood-derived mesenchymal stem cell injection may be a useful conservative treatment for full-thickness rotator cuff tendon tear repair. ©AlphaMed Press.

  6. Regeneration of Full-Thickness Rotator Cuff Tendon Tear After Ultrasound-Guided Injection With Umbilical Cord Blood-Derived Mesenchymal Stem Cells in a Rabbit Model

    PubMed Central

    Park, Gi-Young; Lee, Sang Chul

    2015-01-01

    Rotator cuff tendon tear is one of the most common causes of chronic shoulder pain and disability. In this study, we investigated the therapeutic effects of ultrasound-guided human umbilical cord blood (UCB)-derived mesenchymal stem cell (MSC) injection to regenerate a full-thickness subscapularis tendon tear in a rabbit model by evaluating the gross morphology and histology of the injected tendon and motion analysis of the rabbit’s activity. At 4 weeks after ultrasound-guided UCB-derived MSC injection, 7 of the 10 full-thickness subscapularis tendon tears were only partial-thickness tears, and 3 remained full-thickness tendon tears. The tendon tear size and walking capacity at 4 weeks after UCB-derived MSC injection under ultrasound guidance were significantly improved compared with the same parameters immediately after tendon tear. UCB-derived MSC injection under ultrasound guidance without surgical repair or bioscaffold resulted in the partial healing of full-thickness rotator cuff tendon tears in a rabbit model. Histology revealed that UCB-derived MSCs induced regeneration of rotator cuff tendon tear and that the regenerated tissue was predominantly composed of type I collagens. In this study, ultrasound-guided injection of human UCB-derived MSCs contributed to regeneration of the full-thickness rotator cuff tendon tear without surgical repair. The results demonstrate the effectiveness of local injection of MSCs into the rotator cuff tendon. Significance The results of this study suggest that ultrasound-guided umbilical cord blood-derived mesenchymal stem cell injection may be a useful conservative treatment for full-thickness rotator cuff tendon tear repair. PMID:26371340

  7. (*) Comparison of Autologous, Allogeneic, and Cell-Free Scaffold Approaches for Engineered Tendon Repair in a Rabbit Model-A Pilot Study.

    PubMed

    Wang, Wenbo; Deng, Dan; Wang, Bin; Zhou, Guangdong; Zhang, WenJie; Cao, Yilin; Zhang, Peihua; Liu, Wei

    2017-08-01

    Tendons are subjected to high strength dynamic mechanical forces in vivo. Mechanical strength is an essential requirement for tendon scaffold materials. A composite scaffold was used in this study to provide mechanical strength, which was composed of an inter part of nonwoven polyglycolic acid (PGA) fibers and an outer part of the net knitted with PGA and polylactic acid (PLA) fibers in a ratio of 4:2. This study compared three different approaches for in vivo tendon engineering, that is, cell-free scaffold and allogeneic and autologous cell seeded scaffolds, using a rabbit Achilles tendon repair model. Dermal fibroblasts were, respectively, isolated from the dermis of regular rabbits or green fluorescence protein transgenic rabbits as the autologous and the allogeneic cell sources, respectively. The cell scaffolds and cell-free scaffolds were implanted to bridge a partial segmental defect of rabbit Achilles tendon. The engineered tendons were harvested at 7 and 13 months postsurgery for various examinations. The results showed that all three groups could achieve in vivo tendon regeneration similarly with slightly better tissue formation in autologous group than in other two groups, including better scaffold degradation and relatively thicker collagen fibrils. There were no statistically significant differences in mechanical parameters among three groups. This work demonstrated that allogeneic fibroblasts and scaffold alone are likely to be used for tendon tissue engineering.

  8. Molecular Evolution of Drosophila Germline Stem Cell and Neural Stem Cell Regulating Genes.

    PubMed

    Choi, Jae Young; Aquadro, Charles F

    2015-10-27

    Here, we study the molecular evolution of a near complete set of genes that had functional evidence in the regulation of the Drosophila germline and neural stem cell. Some of these genes have previously been shown to be rapidly evolving by positive selection raising the possibility that stem cell genes as a group have elevated signatures of positive selection. Using recent Drosophila comparative genome sequences and population genomic sequences of Drosophila melanogaster, we have investigated both long- and short-term evolution occurring across these two different stem cell systems, and compared them with a carefully chosen random set of genes to represent the background rate of evolution. Our results showed an excess of genes with evidence of a recent selective sweep in both germline and neural stem cells in D. melanogaster. However compared with their control genes, both stem cell systems had no significant excess of genes with long-term recurrent positive selection in D. melanogaster, or across orthologous sequences from the melanogaster group. The evidence of long-term positive selection was limited to a subset of genes with specific functions in both the germline and neural stem cell system.

  9. Achilles Tendonitis

    MedlinePlus

    ... You Prevent Achilles Tendonitis? Take these steps to reduce your risk of Achilles tendonitis: Stay in good shape year-round and try to keep your muscles as strong as they can be. Strong, flexible muscles work more efficiently and put less stress on your tendon. Increase the intensity and length ...

  10. Human Adipose Stem Cells Differentiated on Braided Polylactide Scaffolds Is a Potential Approach for Tendon Tissue Engineering.

    PubMed

    Vuornos, Kaisa; Björninen, Miina; Talvitie, Elina; Paakinaho, Kaarlo; Kellomäki, Minna; Huhtala, Heini; Miettinen, Susanna; Seppänen-Kaijansinkko, Riitta; Haimi, Suvi

    2016-03-01

    Growing number of musculoskeletal defects increases the demand for engineered tendon. Our aim was to find an efficient strategy to produce tendon-like matrix in vitro. To allow efficient differentiation of human adipose stem cells (hASCs) toward tendon tissue, we tested different medium compositions, biomaterials, and scaffold structures in preliminary tests. This is the first study to report that medium supplementation with 50 ng/mL of growth and differentiation factor-5 (GDF-5) and 280 μM l-ascorbic acid are essential for tenogenic differentiation of hASCs. Tenogenic medium (TM) was shown to significantly enhance tendon-like matrix production of hASCs compared to other tested media groups. Cell adhesion, proliferation, and tenogenic differentiation of hASCs were supported on braided poly(l/d)lactide (PLA) 96l/4d copolymer filament scaffolds in TM condition compared to foamed poly(l-lactide-co-ɛ-caprolactone) (PLCL) 70L/30CL scaffolds. A uniform cell layer formed on braided PLA 96/4 scaffolds when hASCs were cultured in TM compared to maintenance medium (MM) condition after 14 days of culture. Furthermore, total collagen content and gene expression of tenogenic marker genes were significantly higher in TM condition after 2 weeks of culture. The elastic modulus of PLA 96/4 scaffold was more similar to the elastic modulus reported for native Achilles tendon. Our study showed that the optimized TM is needed for efficient and rapid in vitro tenogenic extracellular matrix production of hASCs. PLA 96/4 scaffolds together with TM significantly stimulated hASCs, thus demonstrating the potential clinical relevance of this novel and emerging approach to tendon injury treatments in the future.

  11. A possible link between loading, inflammation and healing: Immune cell populations during tendon healing in the rat

    PubMed Central

    Blomgran, Parmis; Blomgran, Robert; Ernerudh, Jan; Aspenberg, Per

    2016-01-01

    Loading influences tendon healing, and so does inflammation. We hypothesized that the two are connected. 48 rats underwent Achilles tendon transection. Half of the rats received Botox injections into calf muscles to reduce mechanical loading. Cells from the regenerating tissue were analyzed by flow cytometry. In the loaded group, the regenerating tissue contained 83% leukocytes (CD45+) day 1, and 23% day 10. The M1/M2 macrophage ratio (CCR7/CD206) peaked at day 3, while T helper (CD3+CD4+) and Treg cells (CD25+ Foxp3+) increased over time. With Botox, markers associated with down-regulation of inflammation were more common day 5 (CD163, CD206, CD25, Foxp3), and M1 or M2 macrophages and Treg cells were virtually absent day 10, while still present with full loading. The primary variable, CCR7/CD206 ratio day 5, was higher with full loading (p = 0.001) and the Treg cell fraction was lower (p < 0.001). Free cage activity loading is known to increase size and strength of the tendon in this model compared to Botox. Loading now appeared to delay the switch to an M2 type of inflammation with more Treg cells. It seems a prolonged M1 phase due to loading might make the tendon regenerate bigger. PMID:27405922

  12. Assessing in vivo microRNA function in the germline stem cells of the Drosophila ovary.

    PubMed

    Chan, Kin; Ruohola-Baker, Hannele

    2010-01-01

    A more complete understanding of the biology of adult stem cells could yield important insights toward devising effective cell-based regenerative therapies to treat disease. The germline stem cells (GSCs) in the fruit fly Drosophila melanogaster are an excellent in vivo model for the study of adult stem cell biology. There is increasing evidence from a growing field that microRNAs (miRNAs) play important roles in controlling many aspects of stem-cell biology. Using straightforward genetic manipulations combined with well-established cell biological analysis techniques, we and others have found that the miRNA pathway regulates the cell division rate of Drosophila GSCs as well as the maintenance of the GSCs in their niche. In this chapter, we offer a detailed, self-contained description of a general method to assess the in vivo functions of miRNAs in the GSCs of the Drosophila ovary.

  13. Giant Cell Tumor of the Tendon Sheath With Discordant Metabolism as a False Positive on Staging of Mantle Cell Lymphoma.

    PubMed

    Rezaee, Alireza; Chen, Wengen; Dilsizian, Vasken; Chen, Qing; Kimball, Amy S

    2015-10-01

    A baseline F-FDG PET/CT scan in a patient with mantle cell lymphoma showed diffuse minimally FDG-avid lymphadenopathy and splenomegaly. There was also a focus of uptake in the left subscapularis muscle without a CT correlate. A post-chemotherapy scan showed interval decrease in size, and resolution of FDG uptake, of the lymph nodes and spleen. Persistent activity was seen in the subscapularis muscle. Posttreatment biopsy of the FDG-avid lesion showed a benign giant cell tumor of tendon sheath. This case illustrates that a lesion with a markedly discordant SUV should raise suspicion for a second process.

  14. Strategies to engineer tendon/ligament-to-bone interface: Biomaterials, cells and growth factors.

    PubMed

    Font Tellado, Sonia; Balmayor, Elizabeth R; Van Griensven, Martijn

    2015-11-01

    Integration between tendon/ligament and bone occurs through a specialized tissue interface called enthesis. The complex and heterogeneous structure of the enthesis is essential to ensure smooth mechanical stress transfer between bone and soft tissues. Following injury, the interface is not regenerated, resulting in high rupture recurrence rates. Tissue engineering is a promising strategy for the regeneration of a functional enthesis. However, the complex structural and cellular composition of the native interface makes enthesis tissue engineering particularly challenging. Thus, it is likely that a combination of biomaterials and cells stimulated with appropriate biochemical and mechanical cues will be needed. The objective of this review is to describe the current state-of-the-art, challenges and future directions in the field of enthesis tissue engineering focusing on four key parameters: (1) scaffold and biomaterials, (2) cells, (3) growth factors and (4) mechanical stimuli.

  15. Sequoia establishes tip-cell number in Drosophila trachea by regulating FGF levels.

    PubMed

    Araújo, Sofia J; Casanova, Jordi

    2011-07-15

    Competition and determination of leading and trailing cells during collective cell migration is a widespread phenomenon in development, wound healing and tumour invasion. Here, we analyse this issue during in vivo ganglionic branch cell migration in the Drosophila tracheal system. We identify Sequoia (Seq) as a negative transcriptional regulator of Branchless (Bnl), a Drosophila FGF homologue, and observe that modulation of Bnl levels determines how many cells will lead this migrating cluster, regardless of Notch lateral inhibition. Our results show that becoming a tip cell does not prevent others in the branch taking the same position, suggesting that leader choice does not depend only on sensing relative amounts of FGF receptor activity.

  16. Reduced levels of mesenchymal stem cells at the tendon-bone interface tuberosity in patients with symptomatic rotator cuff tear.

    PubMed

    Hernigou, Philippe; Merouse, Guillaume; Duffiet, Pascal; Chevalier, Nathalie; Rouard, Helene

    2015-06-01

    While the use of bone marrow concentrate (BMC) has been described in the treatment of rotator cuff tears, the impact of a rotator cuff injury on the mesenchymal stem cells (MSCs) content present in the human shoulder has not been determined, especially with regard to changes in the levels of MSCs at the tendon-bone interface. With the hypothesis that there was a decreased level of MSCs at the tendon-bone interface tuberosity in patients with rotator cuff tear, we assessed the level of MSCs in the tuberosity of the shoulder of patients undergoing a rotator cuff repair. We analysed the data of 125 patients with symptomatic rotator cuff tears and of 75 control patients without rotator cuff injury. We recorded the following data: size of tear, number of torn tendons, aetiology of the tear, lag time between onset of shoulder symptoms/injury and repair, and also fatty infiltration of muscles. Mesenchymal stem cell content at the tendon-bone interface tuberosity was evaluated by bone marrow aspiration collected in the humeral tuberosities of patients at the beginning of surgery. A significant reduction in MSC content (from moderate, 30-50 %, to severe >70 %) at the tendon-bone interface tuberosity relative to the MSC content of the control was observed in all rotator cuff repair study patients. Severity of the decrease was statistically correlated to a number of factors, including the delay between onset of symptoms and surgery, number of involved tendons, fatty infiltration stage and increasing patient age. This study demonstrates that the level of MSCs present in the greater tuberosity of patients with a rotator cuff tear decreases as a function of a number of clinical factors, including lag time from tear onset to treatment, tear size, number of tears and stage of fatty infiltration, among others. This information may help the practices in using biologic augmentation of a rotator cuff repair.

  17. Rotator cuff repair augmentation in a rat model that combines a multilayer xenograft tendon scaffold with bone marrow stromal cells.

    PubMed

    Omi, Rei; Gingery, Anne; Steinmann, Scott P; Amadio, Peter C; An, Kai-Nan; Zhao, Chunfeng

    2016-03-01

    A composite of multilayer tendon slices (COMTS) seeded with bone marrow stromal cells (BMSCs) may impart mechanical and biologic augmentation effects on supraspinatus tendon repair under tension, thereby improving the healing process after surgery in rats. Adult female Lewis rats (n = 39) underwent transection of the supraspinatus tendon and a 2-mm tendon resection at the distal end, followed by immediate repair to its bony insertion site under tension. Animals received 1 of 3 treatments at the repair site: (1) no augmentation, (2) COMTS augmentation alone, or (3) BMSC-seeded COMTS augmentation. BMSCs were labeled with a fluorescent cell marker. Animals were euthanized 6 weeks after surgery, and the extent of healing of the repaired supraspinatus tendon was evaluated with biomechanical testing and histologic analysis. Histologic analysis showed gap formation between the repaired tendon and bone in all specimens, regardless of treatment. Robust fibrous tissue was observed in rats with BMSC-seeded COMTS augmentation; however, fibrous tissue was scarce within the gap in rats with no augmentation or COMTS-only augmentation. Labeled transplanted BMSCs were observed throughout the repair site. Biomechanical analysis showed that the repairs augmented with BMSC-seeded COMTS had significantly greater ultimate load to failure and stiffness compared with other treatments. However, baseline (time 0) data showed that COMTS-only augmentation did not increase mechanical strength of the repair site. Although the COMTS scaffold did not increase the initial repair strength, the BMSC-seeded scaffold increased healing strength and stiffness 6 weeks after rotator cuff repair in a rat model. Copyright © 2016 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

  18. Rotator cuff repair augmentation in a rat model that combines a multilayer xenograft tendon scaffold with bone marrow stromal cells

    PubMed Central

    Omi, Rei; Gingery, Anne; Steinmann, Scott P.; Amadio, Peter C.; An, Kai-Nan; Zhao, Chunfeng

    2016-01-01

    Hypothesis A composite of multilayer tendon slices (COMTS) seeded with bone marrow stromal cells (BMSCs) may impart mechanical and biologic augmentation effects on supraspinatus tendon repair under tension, thereby improving the healing process after surgery in rats. Methods Adult female Lewis rats (n = 39) underwent transection of the supraspinatus tendon and a 2-mm tendon resection at the distal end, followed by immediate repair to its bony insertion site under tension. Animals received 1 of 3 treatments at the repair site: (1) no augmentation, (2) COMTS augmentation alone, or (3) BMSC-seeded COMTS augmentation. BMSCs were labeled with a fluorescent cell marker. Animals were euthanized 6 weeks after surgery, and the extent of healing of the repaired supraspinatus tendon was evaluated with biomechanical testing and histologic analysis. Results Histologic analysis showed gap formation between the repaired tendon and bone in all specimens, regardless of treatment. Robust fibrous tissue was observed in rats with BMSC-seeded COMTS augmentation; however, fibrous tissue was scarce within the gap in rats with no augmentation or COMTS-only augmentation. Labeled transplanted BMSCs were observed throughout the repair site. Biomechanical analysis showed that the repairs augmented with BMSC-seeded COMTS had significantly greater ultimate load to failure and stiffness compared with other treatments. However, baseline (time 0) data showed that COMTS-only augmentation did not increase mechanical strength of the repair site. Conclusion Although the COMTS scaffold did not increase the initial repair strength, the BMSC-seeded scaffold increased healing strength and stiffness 6 weeks after rotator cuff repair in a rat model. Level of evidence Basic Science Study, Animal Model. PMID:26387915

  19. Bcl-2 proteins and autophagy regulate mitochondrial dynamics during programmed cell death in the Drosophila ovary

    PubMed Central

    Tanner, Elizabeth A.; Blute, Todd A.; Brachmann, Carrie Baker; McCall, Kimberly

    2011-01-01

    The Bcl-2 family has been shown to regulate mitochondrial dynamics during cell death in mammals and C. elegans, but evidence for this in Drosophila has been elusive. Here, we investigate the regulation of mitochondrial dynamics during germline cell death in the Drosophila melanogaster ovary. We find that mitochondria undergo a series of events during the progression of cell death, with remodeling, cluster formation and uptake of clusters by somatic follicle cells. These mitochondrial dynamics are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery, and the autophagy machinery. Furthermore, Bcl-2 family mutants show a striking defect in cell death in the ovary. These data indicate that a mitochondrial pathway is a major mechanism for activation of cell death in Drosophila oogenesis. PMID:21177345

  20. A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins.

    PubMed

    Baron, Matthew N; Klinger, Christen M; Rachubinski, Richard A; Simmonds, Andrew J

    2016-05-01

    Peroxisomes are membrane-bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence-based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope-tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well-developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Preparation of Drosophila S2 cells for Light Microscopy

    PubMed Central

    Buster, Daniel W.; Nye, Jonathan; Klebba, Joseph E.; Rogers, Gregory C.

    2010-01-01

    The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells. S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24°C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks. Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is

  2. Preparation of Drosophila S2 cells for light microscopy.

    PubMed

    Buster, Daniel W; Nye, Jonathan; Klebba, Joseph E; Rogers, Gregory C

    2010-06-03

    The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells. S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media. In addition, they grow optimally and quickly at 21-24 degrees C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks. Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA). S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of

  3. Molecular organization of Drosophila neuroendocrine cells by Dimmed.

    PubMed

    Park, Dongkook; Hadžić, Tarik; Yin, Ping; Rusch, Jannette; Abruzzi, Katharine; Rosbash, Michael; Skeath, James B; Panda, Satchidananda; Sweedler, Jonathan V; Taghert, Paul H

    2011-09-27

    In Drosophila, the basic-helix-loop-helix protein DIMM coordinates the molecular and cellular properties of all major neuroendocrine cells, irrespective of the secretory peptides they produce. When expressed by nonneuroendocrine neurons, DIMM confers the major properties of the regulated secretory pathway and converts such cells away from fast neurotransmission and toward a neuroendocrine state. We first identified 134 transcripts upregulated by DIMM in embryos and then evaluated them systematically using diverse assays (including embryo in situ hybridization, in vivo chromatin immunoprecipitation, and cell-based transactivation assays). We conclude that of eleven strong candidates, six are strongly and directly controlled by DIMM in vivo. The six targets include several large dense-core vesicle (LDCV) proteins, but also proteins in non-LDCV compartments such as the RNA-associated protein Maelstrom. In addition, a functional in vivo assay, combining transgenic RNA interference with MS-based peptidomics, revealed that three DIMM targets are especially critical for its action. These include two well-established LDCV proteins, the amidation enzyme PHM and the ascorbate-regenerating electron transporter cytochrome b(561-1). The third key DIMM target, CAT-4 (CG13248), has not previously been associated with peptide neurosecretion-it encodes a putative cationic amino acid transporter, closely related to the Slimfast arginine transporter. Finally, we compared transcripts upregulated by DIMM with those normally enriched in DIMM neurons of the adult brain and found an intersection of 18 DIMM-regulated genes, which included all six direct DIMM targets. The results provide a rigorous molecular framework with which to describe the fundamental regulatory organization of diverse neuroendocrine cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. MOLECULAR ORGANIZATION OF DROSOPHILA NEUROENDOCRINE CELLS BY DIMMED

    PubMed Central

    Park, Dongkook; Hadžić, Tarik; Yin, Ping; Rusch, Jannette; Abruzzi, Katharine; Rosbash, Michael; Skeath, James B.; Panda, Satchidananda; Sweedler, Jonathan V.; Taghert, Paul H.

    2011-01-01

    SUMMARY Background In Drosophila the bHLH protein DIMM coordinates the molecular and cellular properties of all major neuroendocrine cells, irrespective of the secretory peptides they produce. When expressed by non-neuroendocrine neurons, DIMM confers the major properties of the Regulated Secretory Pathway and converts such cells away from fast neurotransmission and towards a neuroendocrine state. Results We first identified 134 transcripts upregulated by DIMM in embryos, then evaluated them systematically using diverse assays (including embryo in situ hybridization, in vivo ChIP, and cell-based transactivation assays). We conclude that of 11 strong candidates, six are strongly and directly controlled by DIMM in vivo. The six targets include several large dense-core vesicle (LDCV) proteins, but also proteins in non-LDCV compartments such as the RNA-associated protein MAELSTROM. In addition, a functional in vivo assay, combining transgenic RNAi with MS-based peptidomics, revealed that three DIMM targets are especially critical for its action: These include two well-established LDCV proteins, the amidation enzyme PHM and the ascorbate-regenerating electron transporter Cytochrome-b561-1. The third key DIMM target, CAT-4 (CG13248), has not previously been associated with peptide neurosecretion – it encodes a putative cationic amino acid transporter, closely related to the SLIMFAST Arginine transporter. Finally, we compared transcripts upregulated by DIMM with those normally enriched in DIMM neurons of the adult brain and found an intersection of 18 DIMM-regulated genes, which included all six direct DIMM targets. Conclusions The results provide a rigorous molecular framework with which to describe the fundamental regulatory organization of diverse neuroendocrine cells. PMID:21885285

  5. Comparison of Morphology, Orientation, and Migration of Tendon Derived Fibroblasts and Bone Marrow Stromal Cells on Electrochemically Aligned Collagen Constructs

    PubMed Central

    Gurkan, Umut Atakan; Cheng, Xingguo; Kishore, Vipuil; Uquillas, Jorge Alfredo; Akkus, Ozan

    2010-01-01

    There are approximately 33 million injuries involving musculoskeletal tissues (including tendons and ligaments) every year in the United States. In certain cases the tendons and ligaments are damaged irreversibly and require replacements that possess the natural functional properties of these tissues. As a biomaterial, collagen has been a key ingredient in tissue engineering scaffolds. The application range of collagen in tissue engineering would be greatly broadened if the assembly process could be better controlled to facilitate the synthesis of dense, oriented tissue-like constructs. An electrochemical method has recently been developed in our laboratory to form highly oriented and densely packed collagen bundles with mechanical strength approaching that of tendons. However, there is limited information whether this electrochemically aligned collagen bundle (ELAC) presents advantages over randomly oriented bundles in terms of cell response. Therefore, the current study aimed to assess the biocompatibility of the collagen bundles in vitro, and compare tendon derived fibroblasts (TDFs) and bone marrow stromal cells (MSCs) in terms of their ability to populate and migrate on the single and braided ELAC bundles. The results indicated that the ELAC was not cytotoxic; both cell types were able to populate and migrate on the ELAC bundles more efficiently than that observed for random collagen bundles. The braided ELAC constructs were efficiently populated by both TDFs and MSCs in vitro. Therefore, both TDFs and MSCs can be used with the ELAC bundles for tissue engineering purposes. PMID:20694974

  6. CORR® ORS Richard A. Brand Award for Outstanding Orthopaedic Research: Engineering flexor tendon repair with lubricant, cells, and cytokines in a canine model.

    PubMed

    Zhao, Chunfeng; Ozasa, Yasuhiro; Reisdorf, Ramona L; Thoreson, Andrew R; Jay, Gregory D; An, Kai-Nan; Amadio, Peter C

    2014-09-01

    Adhesions and poor healing are complications of flexor tendon repair. The purpose of this study was to investigate a tissue engineering approach to improve functional outcomes after flexor tendon repair in a canine model. Flexor digitorum profundus tendons were lacerated and repaired in 60 dogs that were followed for 10, 21, or 42 days. One randomly selected repair from either the second or fifth digit in one paw in each dog was treated with carbodiimide-derivatized hyaluronic acid, gelatin, and lubricin plus autologous bone marrow stromal cells stimulated with growth and differentiation factor 5; control repair tendons were not treated. Digits were analyzed by adhesion score, work of flexion, tendon-pulley friction, failure force, and histology. In the control group, 35 of 52 control tendons had adhesions, whereas 19 of 49 treated tendons had adhesions. The number of repaired tendons with adhesions in the control group was greater than the number in the treated group at all three times (p = 0.005). The normalized work of flexion in treated tendons was 0.28 (± 0.08), 0.29 (± 0.19), and 0.32 (± 0.22) N/mm/° at Day 10, Day 21, and Day 42 respectively, compared with the untreated tendons of 0.46 (± 0.19) at Day 10 (effect size, 1.5; p = 0.01), 0.77 (± 0.49) at Day 21 (effect size, 1.4; p < 0.001), and 1.17 (± 0.82) N/mm/° at Day 42 (effect size, 1.6; p < 0.001). The friction data were comparable to the work of flexion data at all times. The repaired tendon failure force in the untreated group at 42 days was 70.2 N (± 8.77), which was greater than the treated tendons 44.7 N (± 8.53) (effect size, 1.9; p < 0.001). Histologically, treated repairs had a smooth surface with intrinsic healing, whereas control repairs had surface adhesions and extrinsic healing. Our study provides evidence that tissue engineering coupled with restoration of tendon gliding can improve the quality of tendon healing in a large animal in vivo model. Tissue engineering may enhance

  7. Rho1-Wnd signaling regulates loss-of-cell polarity-induced cell invasion in Drosophila.

    PubMed

    Ma, X; Chen, Y; Zhang, S; Xu, W; Shao, Y; Yang, Y; Li, W; Li, M; Xue, L

    2016-02-18

    Both cell polarity and c-Jun N-terminal kinase (JNK) activity are essential to the maintenance of tissue homeostasis, and disruption of either is commonly seen in cancer progression. Despite the established connection between loss-of-cell polarity and JNK activation, much less is known about the molecular mechanism by which aberrant cell polarity induces JNK-mediated cell migration and tumor invasion. Here we show results from a genetic screen using an in vivo invasion model via knocking down cell polarity gene in Drosophila wing discs, and identify Rho1-Wnd signaling as an important molecular link that mediates loss-of-cell polarity-triggered JNK activation and cell invasion. We show that Wallenda (Wnd), a protein kinase of the mitogen-activated protein kinase kinase kinase family, by forming a complex with the GTPase Rho1, is both necessary and sufficient for Rho1-induced JNK-dependent cell invasion, MMP1 activation and epithelial-mesenchymal transition. Furthermore, Wnd promotes cell proliferation and tissue growth through wingless production when apoptosis is inhibited by p35. Finally, Wnd shows oncogenic cooperation with Ras(V12) to trigger tumor growth in eye discs and causes invasion into the ventral nerve cord. Together, our data not only provides a novel mechanistic insight on how cell polarity loss contributes to cell invasion, but also highlights the value of the Drosophila model system to explore human cancer biology.

  8. Identification of a putative azadirachtin-binding complex from Drosophila Kc167 cells.

    PubMed

    Robertson, Susan L; Ni, Weiting; Dhadialla, Tarlochan S; Nisbet, Alasdair J; McCusker, Catherine; Ley, Steven V; Mordue, William; Mordue 'Luntz', A Jennifer

    2007-04-01

    Cell-proliferation in Drosophila Kc167 cells was inhibited by 50% when cell cultures contained 1.7 x 10(-7) M azadirachtin for 48 h (a tertranortriterpenoid from the neem tree Azadirachta indica). Drosophila Kc167 cells exhibited direct nuclear damage within 6-h exposure to azadirachtin (5 x 10(-7) M and above) or within 24 h when lower concentrations were used (1 x 10(-9) M). Fractionation of an extract of Drosophila Kc167 cells combined with ligand overlay technique resulted in the identification of a putative azadirachtin binding complex. Identification of the members of this complex by Peptide Mass Fingerprinting (PMF) and N-terminal sequencing identified heat shock protein 60 (hsp60) as one of its components.

  9. A spindle-independent cleavage pathway controls germ cell formation in Drosophila

    PubMed Central

    Cinalli, Ryan M.; Lehmann, Ruth

    2013-01-01

    The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis. While the process of somatic cell formation has been studied in detail, the mechanics of PGC formation are poorly understood. Here, using 4D multi-photon imaging combined with genetic and pharmacological manipulations, we find that PGC formation requires an anaphase spindle-independent cleavage pathway. In addition to utilizing core regulators of cleavage, including the small GTPase RhoA (Drosophila Rho) and the Rho associated kinase, ROCK (Drosophila Rok), we show that this pathway requires Germ cell-less (Gcl), a conserved BTB-domain protein not previously implicated in cleavage mechanics. This alternate form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage. PMID:23728423

  10. Stereological quantification of immune-competent cells in baseline biopsy specimens from achilles tendons: results from patients with chronic tendinopathy followed for more than 4 years.

    PubMed

    Kragsnaes, Maja Skov; Fredberg, Ulrich; Stribolt, Katrine; Kjaer, Soren Geill; Bendix, Knud; Ellingsen, Torkell

    2014-10-01

    Limited data exist on the presence and function of immune-competent cells in chronic tendinopathic tendons and their potential role in inflammation and tissue healing as well as in predicting long-term outcome. To quantify subtypes of immune-competent cells in biopsy specimens from nonruptured chronic tendinopathic Achilles tendons and healthy control tendons. In addition, to examine whether findings in baseline cell biopsy specimens can predict the long-term presence of Achilles tendon symptoms. Cross-sectional and case-control study; Level of evidence, 3. Fifty patients with nonruptured chronic Achilles tendinopathy and 15 healthy participants were included. At time of inclusion, an ultrasound examination was performed immediately before an ultrasound-guided Achilles tendon biopsy specimen was obtained. Tissue samples were evaluated immunohistochemically by quantifying the presence of macrophages (CD68-PGM1(+), CD68-KP1(+)), hemosiderophages (Perls blue), T lymphocytes (CD2(+), CD3(+), CD4(+), CD7(+), CD8(+)), B lymphocytes (CD20(+)), natural killer cells (CD56(+)), mast cells (NaSDCl(+)), Schwann cells (S100(+)), and endothelial cells (CD34(+)) using a stereological technique. A follow-up examination was conducted more than 4 years (range, 4-9 years) after the biopsy procedure to evaluate the long-term presence of Achilles tendon symptoms. Macrophages, T lymphocytes, mast cells, and natural killer cells were observed in the majority (range, 52%-96%) of biopsy specimens from nonruptured chronic tendinopathic Achilles tendons. CD68-KP1(+) macrophages (0.29% vs 0; P = .005) and CD34(+) endothelial cells (3% vs 0.97%; P = .04) were significantly more numerous in tendinopathic tendons compared with healthy tendons. The presence of iron(+) hemosiderophages was more frequently observed in biopsy specimens obtained from the group who was asymptomatic at follow-up compared with the symptomatic group (42% vs 5%; P = .02). This study provides evidence for the presence of

  11. Lineage tracing quantification reveals symmetric stem cell division in Drosophila male germline stem cells.

    PubMed

    Salzmann, Viktoria; Inaba, Mayu; Cheng, Jun; Yamashita, Yukiko M

    2013-12-01

    In the homeostatic state, adult stem cells divide either symmetrically to increase the stem cell number to compensate stem cell loss, or asymmetrically to maintain the population while producing differentiated cells. We have investigated the mode of stem cell division in the testes of Drosophila melanogaster by lineage tracing and confirm the presence of symmetric stem cell division in this system. We found that the rate of symmetric division is limited to 1-2% of total germline stem cell (GSC) divisions, but it increases with expression of a cell adhesion molecule, E-cadherin, or a regulator of the actin cytoskeleton, Moesin, which may modulate adhesiveness of germ cells to the stem cell niche. Our results indicate that the decision regarding asymmetric vs. symmetric division is a dynamically regulated process that contributes to tissue homeostasis, responding to the needs of the tissue.

  12. Dying Cells Protect Survivors from Radiation-Induced Cell Death in Drosophila

    PubMed Central

    Bilak, Amber; Uyetake, Lyle; Su, Tin Tin

    2014-01-01

    We report a phenomenon wherein induction of cell death by a variety of means in wing imaginal discs of Drosophila larvae resulted in the activation of an anti-apoptotic microRNA, bantam. Cells in the vicinity of dying cells also become harder to kill by ionizing radiation (IR)-induced apoptosis. Both ban activation and increased protection from IR required receptor tyrosine kinase Tie, which we identified in a genetic screen for modifiers of ban. tie mutants were hypersensitive to radiation, and radiation sensitivity of tie mutants was rescued by increased ban gene dosage. We propose that dying cells activate ban in surviving cells through Tie to make the latter cells harder to kill, thereby preserving tissues and ensuring organism survival. The protective effect we report differs from classical radiation bystander effect in which neighbors of irradiated cells become more prone to death. The protective effect also differs from the previously described effect of dying cells that results in proliferation of nearby cells in Drosophila larval discs. If conserved in mammals, a phenomenon in which dying cells make the rest harder to kill by IR could have implications for treatments that involve the sequential use of cytotoxic agents and radiation therapy. PMID:24675716

  13. Dying cells protect survivors from radiation-induced cell death in Drosophila.

    PubMed

    Bilak, Amber; Uyetake, Lyle; Su, Tin Tin

    2014-03-01

    We report a phenomenon wherein induction of cell death by a variety of means in wing imaginal discs of Drosophila larvae resulted in the activation of an anti-apoptotic microRNA, bantam. Cells in the vicinity of dying cells also become harder to kill by ionizing radiation (IR)-induced apoptosis. Both ban activation and increased protection from IR required receptor tyrosine kinase Tie, which we identified in a genetic screen for modifiers of ban. tie mutants were hypersensitive to radiation, and radiation sensitivity of tie mutants was rescued by increased ban gene dosage. We propose that dying cells activate ban in surviving cells through Tie to make the latter cells harder to kill, thereby preserving tissues and ensuring organism survival. The protective effect we report differs from classical radiation bystander effect in which neighbors of irradiated cells become more prone to death. The protective effect also differs from the previously described effect of dying cells that results in proliferation of nearby cells in Drosophila larval discs. If conserved in mammals, a phenomenon in which dying cells make the rest harder to kill by IR could have implications for treatments that involve the sequential use of cytotoxic agents and radiation therapy.

  14. Extracorporeal Shock Wave Treatment (ESWT) enhances the in vitro-induced differentiation of human tendon-derived stem/progenitor cells (hTSPCs).

    PubMed

    Leone, Laura; Raffa, Salvatore; Vetrano, Mario; Ranieri, Danilo; Malisan, Florence; Scrofani, Cristina; Vulpiani, Maria Chiara; Ferretti, Andrea; Torrisi, Maria Rosaria; Visco, Vincenzo

    2016-02-09

    Extracorporeal shock wave therapy (ESWT) is a non-invasive and innovative technology for the management of specific tendinopathies. In order to elucidate the ESWT-mediated clinical benefits, human Tendon-derived Stem/Progenitor cells (hTSPCs) explanted from 5 healthy semitendinosus (ST) and 5 ruptured Achilles (AT) tendons were established. While hTSPCs from the two groups showed similar proliferation rates and stem cell surface marker profiles, we found that the clonogenic potential was maintained only in cells derived from healthy donors. Interestingly, ESWT significantly accelerated hTSPCs differentiation, suggesting that the clinical benefits of ESWT may be ascribed to increased efficiency of tendon repair after injury.

  15. How Obesity Affects Tendons?

    PubMed

    Abate, Michele; Salini, Vincenzo; Andia, Isabel

    Several epidemiological and clinical observations have definitely demonstrated that obesity has harmful effects on tendons. The pathogenesis of tendon damage is multi-factorial. In addition to overload, attributable to the increased body weight, which significantly affects load-bearing tendons, systemic factors play a relevant role. Several bioactive peptides (chemerin, leptin, adiponectin and others) are released by adipocytes, and influence tendon structure by means of negative activities on mesenchymal cells. The ensuing systemic state of chronic, sub-clinic, low-grade inflammation can damage tendon structure. Metabolic disorders (diabetes, impaired glucose tolerance, and dislipidemia), frequently associated with visceral adiposity, are concurrent pathogenetic factors. Indeed, high glucose levels increase the formation of Advanced Glycation End-products, which in turn form stable covalent cross-links within collagen fibers, modifying their structure and functionality.Sport activities, so useful for preventing important cardiovascular complications, may be detrimental for tendons if they are submitted to intense acute or chronic overload. Therefore, two caution rules are mandatory: first, to engage in personalized soft training program, and secondly to follow regular check-up for tendon pathology.

  16. Regional Cell Specific RNA Expression Profiling of FACS Isolated Drosophila Intestinal Cell Populations.

    PubMed

    Dutta, Devanjali; Buchon, Nicolas; Xiang, Jinyi; Edgar, Bruce A

    2015-08-03

    The adult Drosophila midgut is built of five distinct cell types, including stem cells, enteroblasts, enterocytes, enteroendocrine cells, and visceral muscles, and is divided into five major regions (R1 to R5), which are morphologically and functionally distinct from each other. This unit describes a protocol for the isolation of Drosophila intestinal cell populations for the purpose of cell type-specific transcriptome profiling from the five different regions. A method to select a cell type of interest labeled with green or yellow fluorescent protein (GFP, YFP) by making use of the GAL4-UAS bipartite system and fluorescent-activated cell sorting (FACS) is presented. Total RNA is isolated from the sorted cells of each region, and linear RNA amplification is used to obtain sufficient amounts of high-quality RNA for analysis by microarray, RT-PCR, or RNA sequencing. This method will be useful for quantitative transcriptome comparison across intestinal cell types in the different regions under normal and various experimental conditions. Copyright © 2015 John Wiley & Sons, Inc.

  17. Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

    PubMed Central

    Guo, Yongfeng; Flegel, Kerry; Kumar, Jayashree; McKay, Daniel J.

    2016-01-01

    ABSTRACT During development, cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. Here we examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. We find that a cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa wing. We show that in the wing, ecdysone signaling at the larva-to-puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva-to-puparium pulse during early metamorphosis, Broad expression plummets, allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing. PMID:27737823

  18. Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila

    PubMed Central

    2012-01-01

    In Drosophila, most neurogenetic research is carried out in vivo. Mammalian research demonstrates that primary cell culture techniques provide a powerful model to address cell autonomous and non-autonomous processes outside their endogenous environment. We developed a cell culture system in Drosophila using wildtype and genetically manipulated primary neural tissue for long-term observations. We assessed the molecular identity of distinct neural cell types by immunolabeling and genetically expressed fluorescent cell markers. We monitored mitotic activity of cell cultures derived from wildtype and tumorous larval brains. Our system provides a powerful approach to unveil developmental processes in the nervous system and to complement studies in vivo. PMID:22554060

  19. A Systematic Analysis of Drosophila Regulatory Peptide Expression in Enteroendocrine Cells.

    PubMed

    Chen, Ji; Kim, Seol-Min; Kwon, Jae Young

    2016-04-30

    The digestive system is gaining interest as a major regulator of various functions including immune defense, nutrient accumulation, and regulation of feeding behavior, aside from its conventional function as a digestive organ. The Drosophila midgut epithelium is completely renewed every 1-2 weeks due to differentiation of pluripotent intestinal stem cells in the midgut. Intestinal stem cells constantly divide and differentiate into enterocytes that secrete digestive enzymes and absorb nutrients, or enteroendocrine cells that secrete regulatory peptides. Regulatory peptides have important roles in development and metabolism, but study has mainly focused on expression and functions in the nervous system, and not much is known about the roles in endocrine functions of enteroendocrine cells. We systemically examined the expression of 45 regulatory peptide genes in the Drosophila midgut, and verified that at least 10 genes are expressed in the midgut enteroendocrine cells through RT-PCR, in situ hybridization, antisera, and 25 regulatory peptide-GAL transgenes. The Drosophila midgut is highly compartmentalized, and individual peptides in enteroendocrine cells were observed to express in specific regions of the midgut. We also confirmed that some peptides expressed in the same region of the midgut are expressed in mutually exclusive enteroendocrine cells. These results indicate that the midgut enteroendocrine cells are functionally differentiated into different subgroups. Through this study, we have established a basis to study regulatory peptide functions in enteroendocrine cells as well as the complex organization of enteroendocrine cells in the Drosophila midgut.

  20. Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation

    PubMed Central

    Su, Tin Tin

    2016-01-01

    Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue. PMID:27584613

  1. The use of cultured Drosophila cells for studying the microtubule cytoskeleton.

    PubMed

    Nye, Jonathan; Buster, Daniel W; Rogers, Gregory C

    2014-01-01

    Cultured Drosophila cell lines have been developed into a powerful tool for studying a wide variety of cellular processes. Their ability to be easily and cheaply cultured as well as their susceptibility to protein knockdown via double-stranded RNA-mediated interference (RNAi) has made them the model system of choice for many researchers in the fields of cell biology and functional genomics. Here we describe basic techniques for gene knockdown, transgene expression, preparation for fluorescence microscopy, and centrosome enrichment using cultured Drosophila cells with an emphasis on studying the microtubule cytoskeleton.

  2. Genetic Mosaic Analysis of Stem Cell Lineages in the Drosophila Ovary.

    PubMed

    Laws, Kaitlin M; Drummond-Barbosa, Daniela

    2015-01-01

    Genetic mosaic analyses represent an invaluable approach for the study of stem cell lineages in the Drosophila ovary. The generation of readily identifiable, homozygous mutant cells in the context of wild-type ovarian tissues within intact organisms allows the pinpointing of cellular requirements for gene function, which is particularly important for understanding the physiological control of stem cells and their progeny. Here, we provide a step-by-step guide to the generation and analysis of genetically mosaic ovaries using flippase (FLP)/FLP recognition target (FRT)-mediated recombination in adult Drosophila melanogaster, with a focus on the processes of oogenesis that are controlled by diet-dependent factors.

  3. Tendon's ultrastructure.

    PubMed

    Tresoldi, Ilaria; Oliva, Francesco; Benvenuto, Monica; Fantini, Massimo; Masuelli, Laura; Bei, Roberto; Modesti, Andrea

    2013-01-01

    The structure of a tendon is an important example of complexity of ECM three-dimensional organization. The extracellular matrix (ECM) is a macromolecular network with both structural and regulatory functions. ECM components belong to four major types of macromolecules: the collagens, elastin, proteoglycans, and noncollagenous glycoproteins. Tendons are made by a fibrous, compact connective tissue that connect muscle to bone designed to transmit forces and withstand tension during muscle contraction. Here we show the ultrastructural features of tendon's components.

  4. Gcn5 determines the fate of Drosophila germline stem cells through degradation of Cyclin A.

    PubMed

    Liu, Tianqi; Wang, Qi; Li, Wenqing; Mao, Feiyu; Yue, Shanshan; Liu, Sun; Liu, Xiaona; Xiao, Shan; Xia, Laixin

    2017-02-10

    The fluctuating CDK-CYCLIN complex plays a general role in cell-cycle control. Many types of stem cells use unique features of the cell cycle to facilitate asymmetric division. However, the manner in which these features are established remains poorly understood. The cell cycle of Drosophila female germline stem cells (GSCs) is characterized by short G1 and very long G2 phases, making it an excellent model for the study of cell cycle control in stem cell fate determination. Using a Drosophila female GSCs model, we found Gcn5, the first discovered histone acetyltransferase, to maintain germline stem cells in Drosophila ovaries. Results showed that Gcn5 is dispensable for the transcriptional silencing of bam, but interacts with Cyclin A to facilitate proper turnover in GSCs. Results also showed that Gcn5 promotes Cyclin A ubiquitination, which is dependent on its acetylating activity. Finally, results showed that knockdown of Cyclin A rescued the GSC-loss phenotype caused by lack of Gcn5. Collectively, these findings support the conclusion that Gcn5 acts through acetylation to facilitate Cyclin A ubiquitination and proper turnover, thereby determining the fate of GSCs.-Liu, T., Wang, Q., Li, W., Mao, F., Yue, S., Liu, S., Liu, X., Xiao, S., Xia, L. Gcn5 determines the fate of Drosophila germline stem cells through degradation of Cyclin A.

  5. Gilgamesh is required for the maintenance of germline stem cells in Drosophila testis.

    PubMed

    Chen, Dongsheng; Zhu, Xiangxiang; Zhou, Lijuan; Wang, Jian; Tao, Xiaoqian; Wang, Shuang; Sun, Fuling; Kan, Xianzhao; Han, Zhengqi; Gu, Yuelin

    2017-07-18

    Emerging evidence supports that stem cells are regulated by both intrinsic and extrinsic mechanisms. However, factors that determine the fate of stem cells remain incompletely understood. The Drosophila testis provides an exclusive powerful model in searching for potential important regulatory factors and their underlying mechanisms for controlling the fate of germline stem cells (GSCs). In this study, we have found that Drosophila gilgamesh (gish), which encodes a homologue of human CK1-γ (casein kinase 1-gamma), is required intrinsically for GSC maintenance. Our genetic analyses indicate gish is not required for Dpp/Gbb signaling silencing of bam and is dispensable for Dpp/Gbb signaling-dependent Dad expression. Finally, we show that overexpression of gish fail to dramatically increase the number of GSCs. These findings demonstrate that gish controls the fate of GSCs in Drosophila testis by a novel Dpp/Gbb signaling-independent pathway.

  6. Distinct modes of centromere protein dynamics during cell cycle progression in Drosophila S2R+ cells.

    PubMed

    Lidsky, Peter V; Sprenger, Frank; Lehner, Christian F

    2013-10-15

    Centromeres are specified epigenetically in animal cells. Therefore, faithful chromosome inheritance requires accurate maintenance of epigenetic centromere marks during progression through the cell cycle. Clarification of the mechanisms that control centromere protein behavior during the cell cycle should profit from the relatively simple protein composition of Drosophila centromeres. Thus we have analyzed the dynamics of the three key players Cid/Cenp-A, Cenp-C and Cal1 in S2R+ cells using quantitative microscopy and fluorescence recovery after photobleaching, in combination with novel fluorescent cell cycle markers. As revealed by the observed protein abundances and mobilities, centromeres proceed through at least five distinct states during the cell cycle, distinguished in part by unexpected Cid behavior. In addition to the predominant Cid loading onto centromeres during G1, a considerable but transient increase was detected during early mitosis. A low level of Cid loading was detected in late S and G2, starting at the reported time of centromere DNA replication. Our results reveal the complexities of Drosophila centromere protein dynamics and its intricate coordination with cell cycle progression.

  7. Guidance signalling regulates leading edge behaviour during collective cell migration of cardiac cells in Drosophila.

    PubMed

    Raza, Qanber; Jacobs, J Roger

    2016-11-15

    Collective cell migration is the coordinated movement of cells, which organize tissues during morphogenesis, repair and some cancers. The motile cell membrane of the advancing front in collective cell migration is termed the Leading Edge. The embryonic development of the vertebrate and Drosophila hearts are both characterized by the coordinated medial migration of a bilateral cluster of mesodermal cells. In Drosophila, the cardioblasts form cohesive bilateral rows that migrate collectively as a unit towards the dorsal midline to form the dorsal vessel. We have characterized the collective cell migration of cardioblasts as an in vivo quantitative model to study the behaviour of the Leading Edge. We investigated whether guidance signalling through Slit and Netrin pathways plays a role in cell migration during heart development. Through time-lapse imaging and quantitative assessment of migratory behaviour of the cardioblasts in loss-of-function mutants, we demonstrate that both Slit and Netrin mediated signals are autonomously and concomitantly required to maximize migration velocity, filopodial and lamellipodial activities. Additionally, we show that another Slit and Netrin receptor, Dscam1, the role of which during heart development was previously unknown, is required for both normal migration of cardioblasts and luminal expansion. Leading edge behaviour analysis revealed a dosage dependent genetic interaction between Slit and Netrin receptors suggesting that downstream signalling through these receptors converge on a common output that increases leading edge activity of the cardioblasts. Finally, we found that guidance signalling maintains the balance between epithelial and mesenchymal characteristics of the migrating cardioblasts.

  8. Larval cells become imaginal cells under the control of homothorax prior to metamorphosis in the Drosophila tracheal system.

    PubMed

    Sato, Makoto; Kitada, Yusuke; Tabata, Tetsuya

    2008-06-15

    In Drosophila melanogaster, one of the most derived species among holometabolous insects, undifferentiated imaginal cells that are set-aside during larval development are thought to proliferate and replace terminally differentiated larval cells to constitute adult structures. Essentially all tissues that undergo extensive proliferation and drastic morphological changes during metamorphosis are thought to derive from these imaginal cells and not from differentiated larval cells. The results of studies on metamorphosis of the Drosophila tracheal system suggested that large larval tracheal cells that are thought to be terminally differentiated may be eliminated via apoptosis and rapidly replaced by small imaginal cells that go on to form the adult tracheal system. However, the origin of the small imaginal tracheal cells has not been clear. Here, we show that large larval cells in tracheal metamere 2 (Tr2) divide and produce small imaginal cells prior to metamorphosis. In the absence of homothorax gene activity, larval cells in Tr2 become non-proliferative and small imaginal cells are not produced, indicating that homothorax is necessary for proliferation of Tr2 larval cells. These unexpected results suggest that larval cells can become imaginal cells and directly contribute to the adult tissue in the Drosophila tracheal system. During metamorphosis of less derived species of holometabolous insects, adult structures are known to be formed via cells constituting larval structures. Thus, the Drosophila tracheal system may utilize ancestral mode of metamorphosis.

  9. [Value of clusterin expression in pathologic diagnosis and histogenesis of giant cell tumor of tendon sheath].

    PubMed

    Tang, Li; Zhou, Jun; Jiang, Zhi-ming; Zhang, Hui-zhen; Liu, Liang; Chen, Jie

    2012-03-01

    Analyze the immunophenotype of the different cells in the various subtypes of giant cell tumor of tendon sheath (GCTS) and investigate the value of clusterin in pathological diagnosis and histogenesis of giant cell tumor of tendon sheath. A total of 104 cases of GCTS from the surgical pathology files of Shanghai Jiaotong university affiliated the sixth people's hospital were identified. Immunohistochemistry (IHC) for clusterin, desmin, CD163, CD68, p63, p53, Ki-67 and CD35 was performed on all cases, using EnVision technique. All cases of GCTS were researched, including 44 cases of localized type (L-GCTS), 32 cases of diffused type (D-GCTS), 26 cases of pigmented villonodular synovitis (PVNS) and 2 cases of malignant type. There was a slight female predominance in all these subtypes, and the male to female ratio was about 38:66. L-GCTS usually occured within the small joints (90.9%, 40/44), while D-GCTS, PVNS and M-GCTS commonly occured within the large weight-bearing joints [68.8% (22/32), 100% (26/26) and 2/2 respectively]. Of 74 cases with follow-up, the recurrence rates of L-GCTS, D-GCTS, PVNS and M-GCTS respectively were 30.3% (10/33), 30.4% (7/23), 18.8% (3/16) and 2/2. The different subtypes of GCTS had the same cell components, including the large synovial-like mononuclear cells, the small histiocytoid cells, foamy histiocytes cells, inflammatory cells, fibroblasts and the osteoclast-like multinucleated giant cells. There were obvious differences among immunophenotype of the various cell components in GCTS: the large synovial-like mononuclear cells were strong positive for clusterin, partly positive for desmin and Ki-67, and negative for CD163. The small histiocytoid cells were strong positive for CD163 but negative for clusterin and desmin. The osteoclast-like multinucleated giant cells were strong positive for CD68 but negative for clusterin, CD163 and desmin. Normal synoviocytes were strong positive for clusterin, partly positive for desmin. The number

  10. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    SciTech Connect

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2014-07-25

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

  11. Long-Term Live Cell Imaging and Automated 4D Analysis of Drosophila Neuroblast Lineages

    PubMed Central

    Berger, Christian; Lendl, Thomas; Knoblich, Juergen A.

    2013-01-01

    The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain. PMID:24260257

  12. Wolbachia enhance Drosophila stem cell proliferation and target the germline stem cell niche.

    PubMed

    Fast, Eva M; Toomey, Michelle E; Panaram, Kanchana; Desjardins, Danielle; Kolaczyk, Eric D; Frydman, Horacio M

    2011-11-18

    Wolbachia are widespread maternally transmitted intracellular bacteria that infect most insect species and are able to alter the reproduction of innumerous hosts. The cellular bases of these alterations remain largely unknown. Here, we report that Drosophila mauritiana infected with a native Wolbachia wMau strain produces about four times more eggs than the noninfected counterpart. Wolbachia infection leads to an increase in the mitotic activity of germline stem cells (GSCs), as well as a decrease in programmed cell death in the germarium. Our results suggest that up-regulation of GSC division is mediated by a tropism of Wolbachia for the GSC niche, the cellular microenvironment that supports GSCs.

  13. Evaluation of the Asymmetric Division of Drosophila Male Germline Stem Cells.

    PubMed

    Inaba, Mayu; Yamashita, Yukiko M

    2017-01-01

    Asymmetric cell division (ACD) is utilized in many stem cell systems to produce two daughter cells with different cell fates. Despite the fundamental importance of ACD during development and tissue homeostasis, the nature of ACD is far from being fully understood. Step-by-step observation of events during ACD allows us to understand processes that lead to ACD. Here we describe examples of how we evaluate ACD in vivo using the Drosophila male germline stem cell system.

  14. Drosophila germline stem cells for in vitro analyses of PIWI-mediated RNAi.

    PubMed

    Niki, Yuzo; Sato, Takuya; Yamaguchi, Takafumi; Saisho, Ayaka; Uetake, Hiroshi; Watanabe, Hidenori

    2014-01-01

    The Drosophila piwi gene has multiple functions in soma and germ cells. An in vitro system provides a powerful tool for elucidating PIWI function in each cell type using stable cell lines originating from germline stem cells (GSCs) and ovarian soma of adult ovaries. We have described methods for the maintenance and expansion of GSCs in an established cell line (fGS/OSS) and an in situ hybridization method for analyzing piwi.

  15. Cdk4 functions in multiple cell types to control Drosophila intestinal stem cell proliferation and differentiation

    PubMed Central

    Adlesic, Mojca; Frei, Christian; Frew, Ian J.

    2016-01-01

    ABSTRACT The proliferation of intestinal stem cells (ISCs) and differentiation of enteroblasts to form mature enteroendocrine cells and enterocytes in the Drosophila intestinal epithelium must be tightly regulated to maintain homeostasis. We show that genetic modulation of CyclinD/Cdk4 activity or mTOR-dependent signalling cell-autonomously regulates enterocyte growth, which influences ISC proliferation and enteroblast differentiation. Increased enterocyte growth results in higher numbers of ISCs and defective enterocyte growth reduces ISC abundance and proliferation in the midgut. Adult midguts deficient for Cdk4 show severe disruption of intestinal homeostasis characterised by decreased ISC self-renewal, enteroblast differentiation defects and low enteroendocrine cell and enterocyte numbers. The ISC/enteroblast phenotypes result from a combination of cell autonomous and non-autonomous requirements for Cdk4 function. One non-autonomous consequence of Cdk4-dependent deficient enterocyte growth is high expression of Delta in ISCs and Delta retention in enteroblasts. We postulate that aberrant activation of the Delta–Notch pathway is a possible partial cause of lost ISC stemness. These results support the idea that enterocytes contribute to a putative stem cell niche that maintains intestinal homeostasis in the Drosophila anterior midgut. PMID:26879465

  16. Discovery of progenitor cell signatures by time-series synexpression analysis during Drosophila embryonic cell immortalization

    PubMed Central

    Dequéant, Mary-Lee; Fagegaltier, Delphine; Hu, Yanhui; Spirohn, Kerstin; Simcox, Amanda; Hannon, Gregory J.; Perrimon, Norbert

    2015-01-01

    The use of time series profiling to identify groups of functionally related genes (synexpression groups) is a powerful approach for the discovery of gene function. Here we apply this strategy during RasV12 immortalization of Drosophila embryonic cells, a phenomenon not well characterized. Using high-resolution transcriptional time-series datasets, we generated a gene network based on temporal expression profile similarities. This analysis revealed that common immortalized cells are related to adult muscle precursors (AMPs), a stem cell-like population contributing to adult muscles and sharing properties with vertebrate satellite cells. Remarkably, the immortalized cells retained the capacity for myogenic differentiation when treated with the steroid hormone ecdysone. Further, we validated in vivo the transcription factor CG9650, the ortholog of mammalian Bcl11a/b, as a regulator of AMP proliferation predicted by our analysis. Our study demonstrates the power of time series synexpression analysis to characterize Drosophila embryonic progenitor lines and identify stem/progenitor cell regulators. PMID:26438832

  17. Immunostaining of germline stem cells and the niche in Drosophila ovaries.

    PubMed

    Luo, Lichao; Chai, Phing Chian; Cai, Yu

    2013-01-01

    Stem cells have the ability to switch between proliferative (self-renewal) and differentiation modes. The Drosophila germarium is a well-established in vivo model for the study of communication between stem cells and their niche. One commonly used technique for such study is immunostaining that allows examination of protein localization at a fixed time point. This chapter provides a detailed protocol for immunofluorescence staining of Drosophila ovaries. This protocol has been optimized to enable explicit visualization of the niche structure, as well as to maximize the degree of multiplexing for protein labeling and detection.

  18. Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells

    SciTech Connect

    Mason, J.M.; Zeiger, E.; Haworth, S.; Ivett, J.; Valencia, R.

    1987-01-01

    The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatic exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.

  19. Soft-focused extracorporeal shock waves increase the expression of tendon-specific markers and the release of anti-inflammatory cytokines in an adherent culture model of primary human tendon cells.

    PubMed

    de Girolamo, Laura; Stanco, Deborah; Galliera, Emanuela; Viganò, Marco; Lovati, Arianna Barbara; Marazzi, Monica Gioia; Romeo, Pietro; Sansone, Valerio

    2014-06-01

    Focused extracorporeal shock waves have been found to upregulate the expression of collagen and to initiate cell proliferation in healthy tenocytes and to positively affect the metabolism of tendons, promoting the healing process. Recently, soft-focused extracorporeal shock waves have also been found to have a significant effect on tissue regeneration. However, very few in vitro reports have dealt with the application of this type of shock wave to cells, and in particular, no previous studies have investigated the response of tendon cells to this impulse. We devised an original model to investigate the in vitro effects of soft-focused shock waves on a heterogeneous population of human resident tendon cells in adherent monolayer culture. Our results indicate that soft-focused extracorporeal shock wave treatment (0.17 mJ/mm(2)) is able to induce positive modulation of cell viability, proliferation and tendon-specific marker expression, as well as release of anti-inflammatory cytokines. This could prefigure a new rationale for routine employment of soft-focused shock waves to treat the failed healing status that distinguishes tendinopathies. Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  20. Achilles Tendonitis

    MedlinePlus

    ... up. Tight calf muscles or muscles that lack flexibility decrease a person's range of motion and put an extra strain on the tendon. Running or exercising on a hard or uneven surface or doing lunges or plyometrics without adequate training. A traumatic injury to the Achilles tendon. How ...

  1. Mmp1 and Mmp2 cooperatively induce Drosophila fat body cell dissociation with distinct roles.

    PubMed

    Jia, Qiangqiang; Liu, Yang; Liu, Hanhan; Li, Sheng

    2014-12-18

    During Drosophila metamorphosis, the single-cell layer of fat body tissues gradually dissociates into individual cells. Via a fat body-specific RNAi screen in this study, we found that two matrix metalloproteinases (MMPs), Mmp1 and Mmp2, are both required for fat body cell dissociation. As revealed through a series of cellular, biochemical, molecular, and genetic experiments, Mmp1 preferentially cleaves DE-cadherin-mediated cell-cell junctions, while Mmp2 preferentially degrades basement membrane (BM) components and thus destroy cell-BM junctions, resulting in the complete dissociation of the entire fat body tissues into individual cells. Moreover, several genetic interaction experiments demonstrated that the roles of Mmp1 and Mmp2 in this developmental process are cooperative. In conclusion, Mmp1 and Mmp2 induce fat body cell dissociation during Drosophila metamorphosis in a cooperative yet distinct manner, a finding that sheds light on the general mechanisms by which MMPs regulate tissue remodeling in animals.

  2. The role of microvesicles derived from mesenchymal stem cells in tissue regeneration; a dream for tendon repair?

    PubMed Central

    Tetta, Ciro; Consiglio, Anna Lange; Bruno, Stefania; Tetta, Emanuele; Gatti, Emanuele; Dobreva, Miryana; Cremonesi, Fausto; Camussi, Giovanni

    2012-01-01

    Summary Tendon injuries represent even today a challenge as repair may be exceedingly slow and incomplete. Regenerative medicine and stem cell technology have shown to be of great promise. Here, we will review the current knowledge on the mechanisms of the regenerative potential of mesenchymal stem cells (MSCs) obtained from different sources (bone marrow, fat, cord blood, placenta). More specifically, we will devote attention to the current use of MSCs that have been used experimentally and in limited numbers of clinical cases for the surgical treatment of subchondral-bone cysts, bone-fracture repair and cartilage repair. Based on the recently emerging role in regenerative mechanisms of soluble factors and of extracellular vesicles, we will discuss the potential of non-cellular therapies in horse tendon injuries. PMID:23738299

  3. Scaffolds in Tendon Tissue Engineering

    PubMed Central

    Longo, Umile Giuseppe; Lamberti, Alfredo; Petrillo, Stefano; Maffulli, Nicola; Denaro, Vincenzo

    2012-01-01

    Tissue engineering techniques using novel scaffold materials offer potential alternatives for managing tendon disorders. Tissue engineering strategies to improve tendon repair healing include the use of scaffolds, growth factors, cell seeding, or a combination of these approaches. Scaffolds have been the most common strategy investigated to date. Available scaffolds for tendon repair include both biological scaffolds, obtained from mammalian tissues, and synthetic scaffolds, manufactured from chemical compounds. Preliminary studies support the idea that scaffolds can provide an alternative for tendon augmentation with an enormous therapeutic potential. However, available data are lacking to allow definitive conclusion on the use of scaffolds for tendon augmentation. We review the current basic science and clinical understanding in the field of scaffolds and tissue engineering for tendon repair. PMID:22190961

  4. An ABC transporter controls export of a Drosophila germ cell attractant.

    PubMed

    Ricardo, Sara; Lehmann, Ruth

    2009-02-13

    Directed cell migration, which is critical for embryonic development, leukocyte trafficking, and cell metastasis, depends on chemoattraction. 3-hydroxy-3-methylglutaryl coenzyme A reductase regulates the production of an attractant for Drosophila germ cells that may itself be geranylated. Chemoattractants are commonly secreted through a classical, signal peptide-dependent pathway, but a geranyl-modified attractant would require an alternative pathway. In budding yeast, pheromones produced by a-cells are farnesylated and secreted in a signal peptide-independent manner, requiring the adenosine triphosphate-binding cassette (ABC) transporter Ste6p. Here we show that Drosophila germ cell migration uses a similar pathway, demonstrating that invertebrate germ cells, like yeast cells, are attracted to lipid-modified peptides. Components of this unconventional export pathway are highly conserved, suggesting that this pathway may control the production of similarly modified chemoattractants in organisms ranging from yeast to humans.

  5. High-pressure paint-gun injury of the finger simulating giant cell tumor of tendon sheath.

    PubMed

    Stefanato, Catherine M; Turner, Matthew S; Bhawan, Jag

    2005-02-01

    High-pressure paint guns deliver paint at approximately 3000 pounds per square inch. At this pressure, paint will penetrate the skin and spread quickly through fascial planes and tendon sheaths. The present case is that of a lesion from the finger of a 35-year-old white male in whom a history was initially unavailable. Histologic examination revealed diffuse fibrohistiocytic proliferation and giant cells, with numerous darkly pigmented, uniformly small-sized particles throughout the lesion. The initial impression was that of a giant cell tumor of tendon sheath. However, the pigment particles were negative for Perls stain, and polariscopic examination revealed clear refractile fragments. These findings raised the possibility that the lesion was the result of a traumatic event. On further inquiry, it was revealed that the patient had sustained a high-pressure paint-gun injury 1 year earlier. The simulation, histopathologically, of a giant cell tumor of tendon sheath by a high-pressure paint-gun injury has not, to our knowledge, been reported previously, nor has the histologic finding of small, uniformly sized pigment particles and polarizable refractile fragments in this particular type of injury.

  6. Longitudinal Cell Tracking and Simultaneous Monitoring of Tissue Regeneration after Cell Treatment of Natural Tendon Disease by Low-Field Magnetic Resonance Imaging

    PubMed Central

    Berner, Dagmar; Brehm, Walter; Gerlach, Kerstin; Gittel, Claudia; Offhaus, Julia; Paebst, Felicitas; Scharner, Doreen; Burk, Janina

    2016-01-01

    Treatment of tendon disease with multipotent mesenchymal stromal cells (MSC) is a promising option to improve tissue regeneration. To elucidate the mechanisms by which MSC support regeneration, longitudinal tracking of MSC labelled with superparamagnetic iron oxide (SPIO) by magnetic resonance imaging (MRI) could provide important insight. Nine equine patients suffering from tendon disease were treated with SPIO-labelled or nonlabelled allogeneic umbilical cord-derived MSC by local injection. Labelling of MSC was confirmed by microscopy and MRI. All animals were subjected to clinical, ultrasonographical, and low-field MRI examinations before and directly after MSC application as well as 2, 4, and 8 weeks after MSC application. Hypointense artefacts with characteristically low signal intensity were identified at the site of injection of SPIO-MSC in T1- and T2∗-weighted gradient echo MRI sequences. They were visible in all 7 cases treated with SPIO-MSC directly after injection, but not in the control cases treated with nonlabelled MSC. Furthermore, hypointense artefacts remained traceable within the damaged tendon tissue during the whole follow-up period in 5 out of 7 cases. Tendon healing could be monitored at the same time. Clinical and ultrasonographical findings as well as T2-weighted MRI series indicated a gradual improvement of tendon function and structure. PMID:26880932

  7. Longitudinal Cell Tracking and Simultaneous Monitoring of Tissue Regeneration after Cell Treatment of Natural Tendon Disease by Low-Field Magnetic Resonance Imaging.

    PubMed

    Berner, Dagmar; Brehm, Walter; Gerlach, Kerstin; Gittel, Claudia; Offhaus, Julia; Paebst, Felicitas; Scharner, Doreen; Burk, Janina

    2016-01-01

    Treatment of tendon disease with multipotent mesenchymal stromal cells (MSC) is a promising option to improve tissue regeneration. To elucidate the mechanisms by which MSC support regeneration, longitudinal tracking of MSC labelled with superparamagnetic iron oxide (SPIO) by magnetic resonance imaging (MRI) could provide important insight. Nine equine patients suffering from tendon disease were treated with SPIO-labelled or nonlabelled allogeneic umbilical cord-derived MSC by local injection. Labelling of MSC was confirmed by microscopy and MRI. All animals were subjected to clinical, ultrasonographical, and low-field MRI examinations before and directly after MSC application as well as 2, 4, and 8 weeks after MSC application. Hypointense artefacts with characteristically low signal intensity were identified at the site of injection of SPIO-MSC in T1- and T2 (∗) -weighted gradient echo MRI sequences. They were visible in all 7 cases treated with SPIO-MSC directly after injection, but not in the control cases treated with nonlabelled MSC. Furthermore, hypointense artefacts remained traceable within the damaged tendon tissue during the whole follow-up period in 5 out of 7 cases. Tendon healing could be monitored at the same time. Clinical and ultrasonographical findings as well as T2-weighted MRI series indicated a gradual improvement of tendon function and structure.

  8. Automated cell tracking identifies mechanically oriented cell divisions during Drosophila axis elongation.

    PubMed

    Wang, Michael F Z; Hunter, Miranda V; Wang, Gang; McFaul, Christopher; Yip, Christopher M; Fernandez-Gonzalez, Rodrigo

    2017-04-01

    Embryos extend their anterior-posterior (AP) axis in a conserved process known as axis elongation. Drosophila axis elongation occurs in an epithelial monolayer, the germband, and is driven by cell intercalation, cell shape changes, and oriented cell divisions at the posterior germband. Anterior germband cells also divide during axis elongation. We developed image analysis and pattern-recognition methods to track dividing cells from confocal microscopy movies in a generally applicable approach. Mesectoderm cells, forming the ventral midline, divided parallel to the AP axis, while lateral cells displayed a uniform distribution of division orientations. Mesectoderm cells did not intercalate and sustained increased AP strain before cell division. After division, mesectoderm cell density increased along the AP axis, thus relieving strain. We used laser ablation to isolate mesectoderm cells from the influence of other tissues. Uncoupling the mesectoderm from intercalating cells did not affect cell division orientation. Conversely, separating the mesectoderm from the anterior and posterior poles of the embryo resulted in uniformly oriented divisions. Our data suggest that mesectoderm cells align their division angle to reduce strain caused by mechanical forces along the AP axis of the embryo.

  9. Bleomycin-induced double-strand breaks in mitochondrial DNA of Drosophila cells are repaired.

    PubMed

    Morel, Frederic; Renoux, Monique; Lachaume, Philippe; Alziari, Serge

    2008-01-01

    Mitochondrial DNA lesions cause numerous human diseases, and it is therefore important to identify the mechanisms whereby the mitochondrion repairs the damage. We have studied in cultured Drosophila cells the repair of bleomycin-induced double-strand breaks (DSBs) in mitochondrial DNA. Our results show that DSBs are repaired as rapidly and effectively in the mitochondria as in the nucleus. DNA repair is complete within 2h following bleomycin treatment, showing that Drosophila mitochondria have an effective system of DSB repair. The mechanism and mitochondrial proteins involved remain to be identified.

  10. Mechanisms of Horizontal Cell-to-Cell Transfer of Wolbachia spp. in Drosophila melanogaster.

    PubMed

    White, Pamela M; Pietri, Jose E; Debec, Alain; Russell, Shelbi; Patel, Bhavin; Sullivan, William

    2017-04-01

    Wolbachia is an intracellular endosymbiont present in most arthropod and filarial nematode species. Transmission between hosts is primarily vertical, taking place exclusively through the female germ line, although horizontal transmission has also been documented. The results of several studies indicate that Wolbachia spp. can undergo transfer between somatic and germ line cells during nematode development and in adult flies. However, the mechanisms underlying horizontal cell-to-cell transfer remain largely unexplored. Here, we establish a tractable system for probing horizontal transfer of Wolbachia cells between Drosophila melanogaster cells in culture using fluorescence in situ hybridization (FISH). First, we show that horizontal transfer is independent of cell-to-cell contact and can efficiently take place through the culture medium within hours. Further, we demonstrate that efficient transfer utilizes host cell phagocytic and clathrin/dynamin-dependent endocytic machinery. Lastly, we provide evidence that this process is conserved between species, showing that horizontal transfer from mosquito to Drosophila cells takes place in a similar fashion. Altogether, our results indicate that Wolbachia utilizes host internalization machinery during infection, and this mechanism is conserved across insect species.IMPORTANCE Our work has broad implications for the control and treatment of tropical diseases. Wolbachia can confer resistance against a variety of human pathogens in mosquito vectors. Elucidating the mechanisms of horizontal transfer will be useful for efforts to more efficiently infect nonnatural insect hosts with Wolbachia as a biological control agent. Further, as Wolbachia is essential for the survival of filarial nematodes, understanding horizontal transfer might provide new approaches to treating human infections by targeting Wolbachia Finally, this work provides a key first step toward the genetic manipulation of Wolbachia.

  11. Distinct P-element excision products in somatic and germline cells of Drosophila melanogaster.

    PubMed Central

    Gloor, G B; Moretti, J; Mouyal, J; Keeler, K J

    2000-01-01

    The footprints remaining following somatic P-element excision from the Drosophila white locus were recovered and characterized. Two different types of footprints were observed. Over 75% of the footprints were short, composed of 4 or 7 nucleotides of the P-element inverted terminal repeat, and were similar to those found in a previously described plasmid excision assay. The remaining footprints were composed of 14-18 nucleotides of both inverted terminal repeats. These large footprints were indistinguishable from those recovered following germline P-element excision. Enhanced expression of the Drosophila homologue of the Ku70 protein did not affect the structure of the somatic footprints. Therefore, this protein is not a limiting factor for double-strand break repair by nonhomologous end-joining in Drosophila somatic cells. PMID:10924477

  12. Mechanisms of Regulating Tissue Elongation in Drosophila Wing: Impact of Oriented Cell Divisions, Oriented Mechanical Forces, and Reduced Cell Size

    PubMed Central

    Li, Yingzi; Naveed, Hammad; Kachalo, Sema; Xu, Lisa X.; Liang, Jie

    2014-01-01

    Regulation of cell growth and cell division plays fundamental roles in tissue morphogenesis. However, the mechanisms of regulating tissue elongation through cell growth and cell division are still not well understood. The wing imaginal disc of Drosophila provides a model system that has been widely used to study tissue morphogenesis. Here we use a recently developed two-dimensional cellular model to study the mechanisms of regulating tissue elongation in Drosophila wing. We simulate the effects of directional cues on tissue elongation. We also computationally analyze the role of reduced cell size. Our simulation results indicate that oriented cell divisions, oriented mechanical forces, and reduced cell size can all mediate tissue elongation, but they function differently. We show that oriented cell divisions and oriented mechanical forces act as directional cues during tissue elongation. Between these two directional cues, oriented mechanical forces have a stronger influence than oriented cell divisions. In addition, we raise the novel hypothesis that reduced cell size may significantly promote tissue elongation. We find that reduced cell size alone cannot drive tissue elongation. However, when combined with directional cues, such as oriented cell divisions or oriented mechanical forces, reduced cell size can significantly enhance tissue elongation in Drosophila wing. Furthermore, our simulation results suggest that reduced cell size has a short-term effect on cell topology by decreasing the frequency of hexagonal cells, which is consistent with experimental observations. Our simulation results suggest that cell divisions without cell growth play essential roles in tissue elongation. PMID:24504016

  13. Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper.

    PubMed

    Tung, Tran Thanh; Nagaosa, Kaz; Fujita, Yu; Kita, Asana; Mori, Hiroki; Okada, Ryo; Nonaka, Saori; Nakanishi, Yoshinobu

    2013-05-01

    The membrane phospholipid phosphatidylserine is exposed on the cell surface during apoptosis and acts as an eat-me signal in the phagocytosis of apoptotic cells in mammals and nematodes. However, whether this is also true in insects was unclear. When milk fat globule-epidermal growth factor 8, a phosphatidylserine-binding protein of mammals, was ectopically expressed in Drosophila, the level of phagocytosis was reduced, whereas this was not the case for the same protein lacking a domain responsible for the binding to phosphatidylserine. We found that the extracellular region of Draper, an engulfment receptor of Drosophila, binds to phosphatidylserine in an enzyme-linked immunosorbent assay-like solid-phase assay and in an assay for surface plasmon resonance. A portion of Draper containing domains EMI and NIM located close to the N-terminus was required for binding to phosphatidylserine, and a Draper protein lacking this region was not active in Drosophila. Finally, the level of tyrosine-phosphorylated Draper, indicative of the activation of Draper, in a hemocyte-derived cell line was increased after treatment with phosphatidylserine-containing liposome. These results indicated that phosphatidylserine serves as an eat-me signal in the phagocytic removal of apoptotic cells in Drosophila and that Draper is a phosphatidylserine-binding receptor for phagocytosis.

  14. Odd-skipped maintains prohemocyte potency and blocks blood cell development in Drosophila.

    PubMed

    Gao, Hongjuan; Wu, Xiaorong; Fossett, Nancy

    2011-03-01

    Studies using Drosophila have contributed significantly to our understanding of regulatory mechanisms that control stem cell fate choice. The Drosophila blood cell progenitor or prohemocyte shares important characteristics with mammalian hematopoietic stem cells, including quiescence, niche dependence, and the capacity to form all three fly blood cell types. This report extends our understanding of prohemocyte fate choice by showing that the zinc-finger protein Odd-skipped promotes multipotency and blocks differentiation. Odd-skipped was expressed in prohemocytes and downregulated in terminally differentiated plasmatocytes. Furthermore, Odd-skipped maintained the prohemocyte population and blocked differentiation of plasmatocytes and lamellocytes but not crystal cells. A previous study showed that Odd-skipped expression is downregulated by Decapentaplegic signaling. This report provides a functional basis for this regulator/target pair by suggesting that Decapentaplegic signaling limits Odd-skipped expression to promote prohemocyte differentiation. Overall, these studies are the basis for a gene regulatory model of prohemocyte cell fate choice.

  15. Generation of cell diversity and segmental pattern in the embryonic central nervous system of Drosophila.

    PubMed

    Technau, Gerhard M; Berger, Christian; Urbach, Rolf

    2006-04-01

    Development of the central nervous system (CNS) involves the transformation of a two-dimensional epithelial sheet of uniform ectodermal cells, the neuroectoderm, into a highly complex three-dimensional structure consisting of a huge variety of different neural cell types. Characteristic numbers of each cell type become arranged in reproducible spatial patterns, which is a prerequisite for the establishment of specific functional contacts. The fruitfly Drosophila is a suitable model to approach the mechanisms controlling the generation of cell diversity and pattern in the developing CNS, as it allows linking of gene function to individually identifiable cells. This review addresses aspects of the formation and specification of neural stem cells (neuroblasts) in Drosophila in the light of recent studies on their segmental diversification.

  16. [Role of GAGA Factor in Drosophila Primordial Germ Cell Migration and Gonad Development].

    PubMed

    Dorogova, N V; Khrushcheva, A S; Fedorova, E V; Ogienko, A A; Baricheva, E M

    2016-01-01

    The GAGA protein of drosophila is a factor involved in epigenetic transcription regulation of a large gene group controlling developmental processes. In this paper, the role of GAGA factor in germ cell migration is demonstrated as well as its effect on the gonad development in drosophila embryogenesis. Mutations in the Trl gene, encoding GAGA factor, prematurely induces the active migration program and relocation of the primordial cells inward the embryo before the beginning of gastrulation. The germ cells that prematurely separated from the main group migrate ectopically, lose orientation, and stay out of gonad development. Expression pattern of the Trl gene suggests its activity in epithelial cells of the embryonic blastoderm, part of which contact primordial cells. Thus, GAGA factor influences migration of these cells in an indirect manner via their somatic environment.

  17. Germ cell comparative Drosophila mutagenesis: sensitivity and mutation pattern in chemically treated stem cells

    SciTech Connect

    Abrahamson, S.; Houtchens, K.; Li Jia, X.; Foureman, P.

    1983-01-01

    Mutagenesis studies on Drosophila oogonial cells with methylnitrosourea, dimethylnitrosamine, and diethylnitrosamine revealed unexpectedly high rates of sex-linked recessive lethals relative to other male and female germ cell stages. Indeed, the oogonial mutation rates with chemicals are higher than with massive x-ray or neutron exposures of oogonia. Analysis of the distribution of lethals per treated female suggests most of the mutations recovered are of independent origin, with very small levels of clustering of identical mutations. In the male stem cell population (spermatogonia) on the other hand, the distribution of lethals is primarily nonrandom and highly clustered. The nature of the mutational endpoint and the different pattern of germ cell development in the two sexes are the probable causes of this difference. The oogonial sensitivity to chemical mutagens may have important bearing on strategies for assessing human hazard.

  18. Multiple signalling pathways establish cell fate and cell number in Drosophila malpighian tubules.

    PubMed

    Wan, S; Cato, A M; Skaer, H

    2000-01-01

    A unique cell, the tip mother cell, arises in the primordium of each Drosophila Malpighian tubule by lateral inhibition within a cluster of achaete-expressing cells. This cell maintains achaete expression and divides to produce daughters of equivalent potential, of which only one, the tip cell, adopts the primary fate and continues to express achaete, while in the other, the sibling cell, achaete expression is lost (M. Hoch et al., 1994, Development 120, 3439-3450). In this paper we chart the mechanisms by which achaete expression is differentially maintained in the tip cell lineage to stabilise cell fate. First, wingless is required to maintain the expression of achaete in the tubule primordium so that wingless mutants lack tip cells. Conversely, increasing wingless expression results in the persistence of achaete expression in the cell cluster. Second, Notch signalling is restricted by the asymmetric segregation of Numb, as the tip mother cell divides, so that achaete expression is maintained only in the tip cell. In embryos mutant for Notch tip cells segregate at the expense of sibling cells, whereas in numb neither daughter cell adopts the tip cell fate resulting in tubules with two sibling cells. Conversely, when numb is overexpressed two tip cells segregate and tubules have no sibling cells. Analysis of cell proliferation in the developing tubules of embryos lacking Wingless after the critical period for tip cell allocation reveals an additional requirement for wingless for the promotion of cell division. In contrast, alteration in the expression of numb has no effect on the final tubule cell number.

  19. Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells*

    PubMed Central

    Herchenhan, Andreas; Uhlenbrock, Franziska; Eliasson, Pernilla; Weis, MaryAnn; Eyre, David; Kadler, Karl E.; Magnusson, S. Peter; Kjaer, Michael

    2015-01-01

    Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation. PMID:25979340

  20. Male and female Drosophila germline stem cells: two versions of immortality.

    PubMed

    Fuller, Margaret T; Spradling, Allan C

    2007-04-20

    Drosophila male and female germline stem cells (GSCs) are sustained by niches and regulatory pathways whose common principles serve as models for understanding mammalian stem cells. Despite striking cellular and genetic similarities that suggest a common evolutionary origin, however, male and female GSCs also display important differences. Comparing these two stem cells and their niches in detail is likely to reveal how a common heritage has been adapted to the differing requirements of male and female gamete production.

  1. Escargot restricts niche cell to stem cell conversion in the Drosophila testis.

    PubMed

    Voog, Justin; Sandall, Sharsti L; Hime, Gary R; Resende, Luís Pedro F; Loza-Coll, Mariano; Aslanian, Aaron; Yates, John R; Hunter, Tony; Fuller, Margaret T; Jones, D Leanne

    2014-05-08

    Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. We identified Esg-interacting proteins and demonstrate an interaction between Esg and the corepressor C-terminal binding protein (CtBP), which was also required for maintenance of hub cell fate. Our results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo.

  2. Escargot restricts niche cell to stem cell conversion in the Drosophila testis

    PubMed Central

    Voog, Justin; Sandall, Sharsti L.; Hime, Gary R.; Resende, Luís Pedro F.; Loza-Coll, Mariano; Aslanian, Aaron; Yates, John R.; Hunter, Tony; Fuller, Margaret T.; Jones, D. Leanne

    2014-01-01

    Summary Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behaviour. Somatic hub cells in the Drosophila testis regulate the behaviour of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually, CySCs and GSCs, similar to the shof mutant phenotype. We identified Esg-interacting proteins and demonstrate an interaction between Esg and the co-repressor C-terminal binding protein (CtBP), which was also required for maintenance of hub cell fate. Our results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo. PMID:24794442

  3. Joint regulation of cell size and cell number in the wing blade of Drosophila melanogaster.

    PubMed

    McCabe, J; French, V; Partridge, L

    1997-02-01

    We used Drosophila melanogaster to test for compensatory control of cell area and cell number in the regulation of total wing area. In two random bred wild-type base stocks collected from different geographic locations we found a negative association between the area and the number of cells in the wing blade. Three replicate lines were selected for increased or decreased wing area, with cell area maintained at the same level as in the three controls. After eight generations of selection, despite a large and highly significant difference in wing area between the large, control and small selection lines, cell area did not differ significantly between them. Rather, the difference in wing area between selection regimes was attributable to differences in cell number. Over the course of selection, the initially significant negative correlation between cell area and cell number in the wing increased, providing evidence for compensatory regulation of cell area and cell number. As a result of the increasingly negative association between the two traits, the variance in wing area declined as selection proceeded. It will be important to discover the mechanisms underlying the compensatory regulation of cell area and cell number.

  4. Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila.

    PubMed

    Bischoff, Marcus; Cseresnyés, Zoltán

    2009-07-01

    During morphogenesis, cell movements, cell divisions and cell death work together to form complex patterns and to shape organs. These events are the outcome of decisions made by many individual cells, but how these decisions are controlled and coordinated is elusive. The adult abdominal epidermis of Drosophila is formed during metamorphosis by divisions and extensive cell migrations of the diploid histoblasts, which replace the polyploid larval cells. Using in vivo 4D microscopy, we have studied the behaviour of the histoblasts and analysed in detail how they reach their final position and to what extent they rearrange during their spreading. Tracking individual cells, we show that the cells migrate in two phases that differ in speed, direction and amount of cellular rearrangement. Cells of the anterior (A) and posterior (P) compartments differ in their behaviour. Cells near the A/P border are more likely to change their neighbours during migration. The mitoses do not show any preferential orientation. After mitosis, the sisters become preferentially aligned with the direction of movement. Thus, in the abdomen, it is the extensive cell migrations that appear to contribute most to morphogenesis. This contrasts with other developing epithelia, such as the wing imaginal disc and the embryonic germband in Drosophila, where oriented mitoses and local cell rearrangements appear to direct morphogenesis. Furthermore, our results suggest that an active force created by the histoblasts contributes to the formation of the adult epidermis. Finally, we show that histoblasts occasionally undergo apoptosis.

  5. Drosophila as a model for the two myeloid blood cell systems in vertebrates

    PubMed Central

    Gold, Katrina S.; Brückner, Katja

    2016-01-01

    Fish, mice and men rely on two coexisting myeloid blood cell systems. One is sustained by hematopoietic progenitor cells, which reside in specialized microenvironments in hematopoietic organs and give rise to cells of the monocyte lineage. The other system corresponds to the independent lineage of self-renewing tissue macrophages, which colonize organs during embryonic development and are maintained during later life by proliferation in local tissue microenvironments. However, little is known about the nature of these microenvironments and their regulation. Moreover, many vertebrate tissues contain a mix of both tissue-resident and monocyte-derived macrophages, posing a challenge to the study of lineage-specific regulatory mechanisms and function. This review highlights how research in the simple model organism Drosophila melanogaster can address many of these outstanding questions in the field. Drawing parallels between hematopoiesis in Drosophila and vertebrates, we illustrate the evolutionary conservation of the two myeloid systems across animal phyla. Much like vertebrates, Drosophila possesses a lineage of self-renewing tissue-resident macrophages, as well as a ‘definitive’ lineage of macrophages that derive from hematopoiesis in the progenitor-based lymph gland. We summarize key findings from Drosophila hematopoiesis that illustrate how local microenvironments, systemic signals, immune challenges and nervous inputs regulate adaptive responses of tissue-resident macrophages and progenitor-based hematopoiesis to achieve optimal fitness of the animal. PMID:24946019

  6. Bällchen is required for self-renewal of germline stem cells in Drosophila melanogaster.

    PubMed

    Herzig, Bettina; Yakulov, Toma A; Klinge, Kathrin; Günesdogan, Ufuk; Jäckle, Herbert; Herzig, Alf

    2014-05-29

    Self-renewing stem cells are pools of undifferentiated cells, which are maintained in cellular niche environments by distinct tissue-specific signalling pathways. In Drosophila melanogaster, female germline stem cells (GSCs) are maintained in a somatic niche of the gonads by BMP signalling. Here we report a novel function of the Drosophila kinase Bällchen (BALL), showing that its cell autonomous role is to maintain the self-renewing capacity of female GSCs independent of BMP signalling. ball mutant GSCs are eliminated from the niche and subsequently differentiate into mature eggs, indicating that BALL is largely dispensable for differentiation. Similar to female GSCs, BALL is required to maintain self-renewal of male GSCs, suggesting a tissue independent requirement of BALL for self-renewal of germline stem cells.

  7. During Drosophila disc regeneration, JAK/STAT coordinates cell proliferation with Dilp8-mediated developmental delay

    PubMed Central

    Katsuyama, Tomonori; Comoglio, Federico; Seimiya, Makiko; Cabuy, Erik; Paro, Renato

    2015-01-01

    Regeneration of fragmented Drosophila imaginal discs occurs in an epimorphic manner involving local cell proliferation at the wound site. After disc fragmentation, cells at the wound site activate a restoration program through wound healing, regenerative cell proliferation, and repatterning of the tissue. However, the interplay of signaling cascades driving these early reprogramming steps is not well-understood. Here, we profiled the transcriptome of regenerating cells in the early phase within 24 h after wounding. We found that JAK/STAT signaling becomes activated at the wound site and promotes regenerative cell proliferation in cooperation with Wingless (Wg) signaling. In addition, we showed that the expression of Drosophila insulin-like peptide 8 (dilp8), which encodes a paracrine peptide to delay the onset of pupariation, is controlled by JAK/STAT signaling in early regenerating discs. Our findings suggest that JAK/STAT signaling plays a pivotal role in coordinating regenerative disc growth with organismal developmental timing. PMID:25902518

  8. During Drosophila disc regeneration, JAK/STAT coordinates cell proliferation with Dilp8-mediated developmental delay.

    PubMed

    Katsuyama, Tomonori; Comoglio, Federico; Seimiya, Makiko; Cabuy, Erik; Paro, Renato

    2015-05-05

    Regeneration of fragmented Drosophila imaginal discs occurs in an epimorphic manner involving local cell proliferation at the wound site. After disc fragmentation, cells at the wound site activate a restoration program through wound healing, regenerative cell proliferation, and repatterning of the tissue. However, the interplay of signaling cascades driving these early reprogramming steps is not well-understood. Here, we profiled the transcriptome of regenerating cells in the early phase within 24 h after wounding. We found that JAK/STAT signaling becomes activated at the wound site and promotes regenerative cell proliferation in cooperation with Wingless (Wg) signaling. In addition, we showed that the expression of Drosophila insulin-like peptide 8 (dilp8), which encodes a paracrine peptide to delay the onset of pupariation, is controlled by JAK/STAT signaling in early regenerating discs. Our findings suggest that JAK/STAT signaling plays a pivotal role in coordinating regenerative disc growth with organismal developmental timing.

  9. Weft-knitted silk-poly(lactide-co-glycolide) mesh scaffold combined with collagen matrix and seeded with mesenchymal stem cells for rabbit Achilles tendon repair.

    PubMed

    Zhang, Wenyuan; Yang, Yadong; Zhang, Keji; Li, Ying; Fang, Guojian

    2015-02-01

    Natural silk fibroin fiber scaffolds have excellent mechanical properties, but degrade slowly. In this study, we used poly(lactide-co-glycolide) (PLGA, 10:90) fibers to adjust the overall degradation rate of the scaffolds and filled them with collagen to reserve space for cell growth. Silk fibroin-PLGA (36:64) mesh scaffolds were prepared using weft-knitting, filled with type I collagen, and incubated with rabbit autologous bone marrow-derived mesenchymal stem cells (MSCs). These scaffold-cells composites were implanted into rabbit Achilles tendon defects. At 16 weeks after implantation, morphological and histological observations showed formation of tendon-like tissues that expressed type I collagen mRNA and a uniformly dense distribution of collagen fibers. The maximum load of the regenerated Achilles tendon was 58.32% of normal Achilles tendon, which was significantly higher than control group without MSCs. These findings suggest that it is feasible to construct tissue engineered tendon using weft-knitted silk fibroin-PLGA fiber mesh/collagen matrix seeded with MSCs for rabbit Achilles tendon defect repair.

  10. Non-cell autonomous cell death caused by transmission of Huntingtin aggregates in Drosophila.

    PubMed

    Babcock, Daniel T; Ganetzky, Barry

    2015-01-01

    Recent evidence indicates that protein aggregates can spread between neurons in several neurodegenerative diseases but much remains unknown regarding the underlying mechanisms responsible for this spreading and its role in disease progression. We recently demonstrated that mutant Huntingtin aggregates spread between cells within the Drosophila brain resulting in non-cell autonomous loss of a pair of large neurons in the posterior protocerebrum. However, the full extent of neuronal loss throughout the brain was not determined. Here we examine the effects of driving expression of mutant Huntingtin in Olfactory Receptor Neurons (ORNs) by using a marker for cleaved caspase activity to monitor neuronal apoptosis as a function of age. We find widespread caspase activity in various brain regions over time, demonstrating that non-cell autonomous damage is widespread. Improved understanding of which neurons are most vulnerable and why should be useful in developing treatment strategies for neurodegenerative diseases that involve transcellular spreading of aggregates.

  11. The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling.

    PubMed

    Takashima, Shigeo; Mkrtchyan, Marianna; Younossi-Hartenstein, Amelia; Merriam, John R; Hartenstein, Volker

    2008-07-31

    The intestinal tract maintains proper function by replacing aged cells with freshly produced cells that arise from a population of self-renewing intestinal stem cells (ISCs). In the mammalian intestine, ISC self renewal, amplification and differentiation take place along the crypt-villus axis, and are controlled by the Wnt and hedgehog (Hh) signalling pathways. However, little is known about the mechanisms that specify ISCs within the developing intestinal epithelium, or about the signalling centres that help maintain them in their self-renewing stem cell state. Here we show that in adult Drosophila melanogaster, ISCs of the posterior intestine (hindgut) are confined to an anterior narrow segment, which we name the hindgut proliferation zone (HPZ). Within the HPZ, self renewal of ISCs, as well as subsequent proliferation and differentiation of ISC descendants, are controlled by locally emanating Wingless (Wg, a Drosophila Wnt homologue) and Hh signals. The anteriorly restricted expression of Wg in the HPZ acts as a niche signal that maintains cells in a slow-cycling, self-renewing mode. As cells divide and move posteriorly away from the Wg source, they enter a phase of rapid proliferation. During this phase, Hh signal is required for exiting the cell cycle and the onset of differentiation. The HPZ, with its characteristic proliferation dynamics and signalling properties, is set up during the embryonic phase and becomes active in the larva, where it generates all adult hindgut cells including ISCs. The mechanism and genetic control of cell renewal in the Drosophila HPZ exhibits a large degree of similarity with what is seen in the mammalian intestine. Our analysis of the Drosophila HPZ provides an insight into the specification and control of stem cells, highlighting the way in which the spatial pattern of signals that promote self renewal, growth and differentiation is set up within a genetically tractable model system.

  12. Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5.

    PubMed

    Ye, Anna A; Maresca, Thomas J

    2016-01-20

    Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

  13. Three-dimensional structure of Drosophila testis tip: the spatial relation between dividing cells.

    PubMed

    Lee, Kyung Eun; Han, Sung Sik; Jeon, Hyesung

    2013-08-01

    At the apical tip of Drosophila testis, there is a stem cell niche known as the proliferation center, where the stem cells are maintained by hub cell cluster for the regulation of differentiation and proliferation. Germline stem cells go through mitosis four times from one primary spermatogonial cell to the 16-cell stage before the maturation. The cells derived from the same germline stem cell are located within one cyst, an enclosed system by two cyst cells, and they are connected by the intercellular bridges called ring canals. In this study, the three-dimensional (3D) structure of Drosophila testis tip was reconstructed from serial sections. The size of cells at each stage was compared in volume from the 3D structure. The stages of cells in a cyst could be distinguishable exactly by counting the cells linked with intercellular bridges in 3D-reconstructed structure. The cysts containing the same stage cells appeared in the horizontal plane. Both the germline stem cell directly attached to the hub cell and the spermatogonial cells detached from the hub cell were divided at the almost perpendicular direction to the spermatogonial cell layers. The dividing phase in one cyst was delayed gradually through the cytoplasmic region of intercellular bridge.

  14. Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila.

    PubMed

    Ma, Meifang; Zhao, Hang; Zhao, Hanfei; Binari, Richard; Perrimon, Norbert; Li, Zhouhua

    2016-03-15

    Adult stem cells or residential progenitor cells are critical to maintain the structure and function of adult tissues (homeostasis) throughout the lifetime of an individual. Mis-regulation of stem cell proliferation and differentiation often leads to diseases including cancer, however, how wildtype adult stem cells and cancer cells respond to cellular damages remains unclear. We find that in the adult Drosophila midgut, intestinal stem cells (ISCs), unlike tumor intestinal cells, are resistant to various cellular damages. Tumor intestinal cells, unlike wildtype ISCs, are easily eliminated by apoptosis. Further, their proliferation is inhibited upon autophagy induction, and autophagy-mediated tumor inhibition is independent of caspase-dependent apoptosis. Interestingly, inhibition of tumorigenesis by autophagy is likely through the sequestration and degradation of mitochondria, as compromising mitochondria activity in these tumor models mimics the induction of autophagy and increasing the production of mitochondria alleviates the tumor-suppression capacity of autophagy. Together, these data demonstrate that wildtype adult stem cells and tumor cells show dramatic differences in sensitivity to cellular damages, thus providing potential therapeutic implications targeting tumorigenesis.

  15. Modulation of cell functions of human tendon fibroblasts by different repetitive cyclic mechanical stress patterns.

    PubMed

    Barkhausen, Tanja; van Griensven, Martijn; Zeichen, Johannes; Bosch, Ulrich

    2003-09-01

    Mechanical stress is a factor that is thought to play an essential role in tissue generation and reparation processes. The aim of the present study was to investigate the influence of different repetitive cyclic longitudinal stress patterns on proliferation, apoptosis and expression of heat shock protein (HSP) 72. To perform this study, human tendon fibroblasts were seeded on flexible silicone dishes. After adherence to the dish, cells were longitudinally stressed with three different repetitive stress patterns having a frequency of 1 Hz and an amplitude of 5%. The proliferation and apoptosis rates were investigated 0, 6, 12 and 24 hours after application of cyclic mechanical longitudinal strain. Expression of HSP 72 was tested after 0, 2, 4 and 8 hours. Control cells were also grown on silicone dishes, but did not receive any stress. Stress patterns applied during one day resulted in a significant increase in proliferation and a slight increase in apoptosis. HSP 72 expression was rather unchanged. A stress pattern applied during two days resulted in a reduced proliferation and apoptosis rate whereas the expression of HSP 72 showed a significant increase. This study shows that different stress patterns result in different cellular reactions dependent on the strength of applied stress. Repetitive stress applied during one day stimulated proliferation and apoptosis in contrast to an extended stress duration. The latter induced an inhibition of proliferation and apoptosis probably through an increased HSP 72 activity. This may be related to an excess of applied stress. Our results may implicate future modulation techniques for tissue reparation and tissue engineering.

  16. CalpB modulates border cell migration in Drosophila egg chambers

    PubMed Central

    2012-01-01

    Background Calpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration. Using this genetically pliable model we can investigate the physiological role of calpains in cell motility. Results We demonstrate at the whole organism level that CalpB is implicated in cell migration, while the structurally related CalpA paralog can not fulfill the same function. The downregulation of the CalpB gene by mutations or RNA interference results in a delayed migration of the border cells in Drosophila egg chambers. This phenotype is significantly enhanced when the focal adhesion complex genes encoding for α-PS2 integrin ( if), β-PS integrin ( mys) and talin ( rhea) are silenced. The reduction of CalpB activity diminishes the release of integrins from the rear end of the border cells. The delayed migration and the reduced integrin release phenotypes can be suppressed by expressing wild-type talin-head in the border cells but not talin-headR367A, a mutant form which is not able to bind β-PS integrin. CalpB can cleave talin in vitro, and the two proteins coimmunoprecipitate from Drosophila extracts. Conclusions The physiological function of CalpB in border cell motility has been demonstrated in vivo. The genetic interaction between the CalpB and the if, mys, as well as rhea genes, the involvement of active talin head-domains in the process, and the fact that CalpB and talin interact with each other collectively suggest that the limited proteolytic cleavage of talin is one of the possible mechanisms through which CalpB regulates cell migration. PMID:22827336

  17. Runx2-Modified Adipose-Derived Stem Cells Promote Tendon Graft Integration in Anterior Cruciate Ligament Reconstruction.

    PubMed

    Zhang, Xin; Ma, Yong; Fu, Xin; Liu, Qiang; Shao, Zhenxing; Dai, Linghui; Pi, Yanbin; Hu, Xiaoqing; Zhang, Jiying; Duan, Xiaoning; Chen, Wenqing; Chen, Ping; Zhou, Chunyan; Ao, Yingfang

    2016-01-08

    Runx2 is a powerful osteo-inductive factor and adipose-derived stem cells (ADSCs) are multipotent. However, it is unknown whether Runx2-overexpressing ADSCs (Runx2-ADSCs) could promote anterior cruciate ligament (ACL) reconstruction. We evaluated the effect of Runx2-ADSCs on ACL reconstruction in vitro and in vivo. mRNA expressions of osteocalcin (OCN), bone sialoprotein (BSP) and collagen I (COLI) increased over time in Runx2-ADSCs. Runx2 overexpression inhibited LPL and PPARγ mRNA expressions. Runx2 induced alkaline phosphatase activity markedly. In nude mice injected with Runx2-ADSCs, promoted bone formation was detected by X-rays 8 weeks after injection. The healing of tendon-to-bone in a rabbit model of ACL reconstruction treated with Runx2-ADSCs, fibrin glue only and an RNAi targeting Runx2, was evaluated with CT 3D reconstruction, histological analysis and biomechanical methods. CT showed a greater degree of new bone formation around the bone tunnel in the group treated with Runx2-ADSCs compared with the fibrin glue group and RNAi Runx2 group. Histology showed that treatment with Runx2-ADSCs led to a rapid and significant increase at the tendon-to-bone compared with the control groups. Biomechanical tests demonstrated higher tendon pullout strength in the Runx2-ADSCs group at early time points. The healing of the attachment in ACL reconstruction was enhanced by Runx2-ADSCs.

  18. Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition.

    PubMed

    Casas-Tintó, Sergio; Lolo, Fidel-Nicolás; Moreno, Eduardo

    2015-12-11

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue.

  19. Tendon phenotype should dictate tissue engineering modality in tendon repair: a review.

    PubMed

    Gott, Michael; Ast, Michael; Lane, Lewis B; Schwartz, John A; Catanzano, Anthony; Razzano, Pasquale; Grande, Daniel A

    2011-07-01

    Advancements in the technical aspects of tendon repair have significantly improved the treatment of tendon injuries. Arthroscopic techniques, suture material, and improved rehabilitation have all been contributing factors. Biological augmentation and tissue engineering appear to have the potential to improve clinical outcomes as well. After review of the physiology of tendon repair, three critical components of tissue engineering can be discerned: the cellular component, the carrier vehicle (matrix or scaffold), and the bioactive component (growth factors, platelet rich plasma). These three components are discussed with regard to each of three tendon types: Intra-synovial (flexor tendon), extra-synovial (Achilles tendon), and extra-synovial tendon under compression (rotator cuff). Scaffolds, biologically enhanced scaffolds, growth factors, platelet rich plasma, gene therapy, mesenchymal stem cells, and local environment factors in combination or alone may contribute to tendon healing. In the future it may be beneficial to differentiate these modes of healing augmentation with regard to tendon subtype.

  20. The Hippo signalling pathway maintains quiescence in Drosophila neural stem cells

    PubMed Central

    Ding, Rouven; Weynans, Kevin; Bossing, Torsten; Barros, Claudia S.; Berger, Christian

    2016-01-01

    Stem cells control their mitotic activity to decide whether to proliferate or to stay in quiescence. Drosophila neural stem cells (NSCs) are quiescent at early larval stages, when they are reactivated in response to metabolic changes. Here we report that cell-contact inhibition of growth through the canonical Hippo signalling pathway maintains NSC quiescence. Loss of the core kinases hippo or warts leads to premature nuclear localization of the transcriptional co-activator Yorkie and initiation of growth and proliferation in NSCs. Yorkie is necessary and sufficient for NSC reactivation, growth and proliferation. The Hippo pathway activity is modulated via inter-cellular transmembrane proteins Crumbs and Echinoid that are both expressed in a nutrient-dependent way in niche glial cells and NSCs. Loss of crumbs or echinoid in the niche only is sufficient to reactivate NSCs. Finally, we provide evidence that the Hippo pathway activity discriminates quiescent from non-quiescent NSCs in the Drosophila nervous system. PMID:26821647

  1. The Hippo signalling pathway maintains quiescence in Drosophila neural stem cells.

    PubMed

    Ding, Rouven; Weynans, Kevin; Bossing, Torsten; Barros, Claudia S; Berger, Christian

    2016-01-29

    Stem cells control their mitotic activity to decide whether to proliferate or to stay in quiescence. Drosophila neural stem cells (NSCs) are quiescent at early larval stages, when they are reactivated in response to metabolic changes. Here we report that cell-contact inhibition of growth through the canonical Hippo signalling pathway maintains NSC quiescence. Loss of the core kinases hippo or warts leads to premature nuclear localization of the transcriptional co-activator Yorkie and initiation of growth and proliferation in NSCs. Yorkie is necessary and sufficient for NSC reactivation, growth and proliferation. The Hippo pathway activity is modulated via inter-cellular transmembrane proteins Crumbs and Echinoid that are both expressed in a nutrient-dependent way in niche glial cells and NSCs. Loss of crumbs or echinoid in the niche only is sufficient to reactivate NSCs. Finally, we provide evidence that the Hippo pathway activity discriminates quiescent from non-quiescent NSCs in the Drosophila nervous system.

  2. Programmed cell death acts at different stages of Drosophila neurodevelopment to shape the central nervous system

    PubMed Central

    Desplan, Claude

    2016-01-01

    Nervous system development is a process that integrates cell proliferation, differentiation and programmed cell death (PCD). PCD is an evolutionary conserved mechanism and a fundamental developmental process by which the final cell number in a nervous system is established. In vertebrates and invertebrates, PCD can be determined intrinsically by cell lineage and age, as well as extrinsically by nutritional, metabolic and hormonal states. Drosophila has been an instrumental model for understanding how this mechanism is regulated. We review the role of PCD in Drosophila central nervous system development from neural progenitors to neurons, its molecular mechanism and function, how it is regulated and implemented, and how it ultimately shapes the fly central nervous system from the embryo to the adult. Finally, we discuss ideas that emerge while integrating this information. PMID:27404003

  3. A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult

    PubMed Central

    Strauss, Alexandra L.; Kawasaki, Fumiko; Ordway, Richard W.

    2015-01-01

    Previous studies of Drosophila flight muscle neuromuscular synapses have revealed their tripartite architecture and established an attractive experimental model for genetic analysis of glial function in synaptic transmission. Here we extend these findings by defining a new Drosophila glial cell type, designated peripheral perisynaptic glia (PPG), which resides in the periphery and interacts specifically with fine motor axon branches forming neuromuscular synapses. Identification and specific labeling of PPG was achieved through cell type-specific RNAi-mediated knockdown (KD) of a glial marker, Glutamine Synthetase 2 (GS2). In addition, comparison among different Drosophila neuromuscular synapse models from adult and larval developmental stages indicated the presence of tripartite synapses on several different muscle types in the adult. In contrast, PPG appear to be absent from larval body wall neuromuscular synapses, which do not exhibit a tripartite architecture but rather are imbedded in the muscle plasma membrane. Evolutionary conservation of tripartite synapse architecture and peripheral perisynaptic glia in vertebrates and Drosophila suggests ancient and conserved roles for glia-synapse interactions in synaptic transmission. PMID:26053860

  4. A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult.

    PubMed

    Strauss, Alexandra L; Kawasaki, Fumiko; Ordway, Richard W

    2015-01-01

    Previous studies of Drosophila flight muscle neuromuscular synapses have revealed their tripartite architecture and established an attractive experimental model for genetic analysis of glial function in synaptic transmission. Here we extend these findings by defining a new Drosophila glial cell type, designated peripheral perisynaptic glia (PPG), which resides in the periphery and interacts specifically with fine motor axon branches forming neuromuscular synapses. Identification and specific labeling of PPG was achieved through cell type-specific RNAi-mediated knockdown (KD) of a glial marker, Glutamine Synthetase 2 (GS2). In addition, comparison among different Drosophila neuromuscular synapse models from adult and larval developmental stages indicated the presence of tripartite synapses on several different muscle types in the adult. In contrast, PPG appear to be absent from larval body wall neuromuscular synapses, which do not exhibit a tripartite architecture but rather are imbedded in the muscle plasma membrane. Evolutionary conservation of tripartite synapse architecture and peripheral perisynaptic glia in vertebrates and Drosophila suggests ancient and conserved roles for glia-synapse interactions in synaptic transmission.

  5. MR appearance of clear cell sarcoma of tendons and aponeuroses (malignant melanoma of soft parts): radiologic-pathologic correlation.

    PubMed

    Hourani, Mukbil; Khoury, Nabil; Mourany, Bassem; Shabb, Nina Salem

    2005-09-01

    Clear cell sarcoma of tendons and aponeuroses (CCSTA) is a rare aggressive soft tissue tumor that frequently produces melanin. Its MR findings are rarely described in the literature. We report the case of a previously healthy 54-year-old man with clear cell sarcoma of the thigh who presented with a large painless mass of 1 year's duration. MR imaging showed the tumor to be of high signal intensity on fast spin-echo and STIR images. Both fine needle aspiration and excisional biopsy showed abundant melanin pigments. Histologic diagnosis was compatible with CCSTA.

  6. JAK-STAT signaling in stem cells and their niches in Drosophila

    PubMed Central

    Bausek, Nina

    2013-01-01

    JAK-STAT signaling is a highly conserved regulator of stem cells and their niches. Aberrant activation in hematopoietic stem cells is the underlying cause of a majority of myeloproliferative diseases. This review will focus on the roles of JAK-STAT activity in three different adult stem cell systems in Drosophila. Tightly controlled levels of JAK-STAT signaling are required for stem cell maintenance and self-renewal, as hyperactivation of the pathway is associated with stem cell overproliferation. JAK-STAT activity is further essential for anchoring the stem cells in their respective niches by regulating different adhesion molecules. PMID:24069566

  7. Histological and Immunohistochemical Evaluation of Autologous Cultured Bone Marrow Mesenchymal Stem Cells and Bone Marrow Mononucleated Cells in Collagenase-Induced Tendinitis of Equine Superficial Digital Flexor Tendon

    PubMed Central

    Crovace, Antonio; Lacitignola, Luca; Rossi, Giacomo; Francioso, Edda

    2010-01-01

    The aim of this study was to compare treatment with cultured bone marrow stromal cells (cBMSCs), bone marrow Mononucleated Cells (BMMNCs), and placebo to repair collagenase-induced tendinitis in horses. In six adult Standardbred horses, 4000 IU of collagenase were injected in the superficial digital flexor tendon (SDFT). Three weeks after collagenase treatment, an average of either 5.5 × 106 cBMSCs or 1.2 × 108 BMMNCs, fibrin glue, and saline solution was injected intralesionally in random order. In cBMSC- and BMMNCS-treated tendons, a high expression of cartilage oligomeric matrix protein (COMP) and type I collagen, but low levels of type III collagen were revealed by immunohistochemistry, with a normal longitudinally oriented fiber pattern. Placebo-treated tendons expressed very low quantities of COMP and type I collagen but large numbers of randomly oriented type III collagen fibers. Both cBMSC and BMMNCS grafts resulted in a qualitatively similar heling improvement of tendon extracellular matrix, in terms of the type I/III collagen ratio, fiber orientation, and COMP expression. PMID:20445779

  8. Aging-related viscoelasticity variation of tendon stem cells (TSCs) characterized by quartz thickness shear mode (TSM) resonators

    PubMed Central

    Wu, Huiyan; Zhao, Guangyi; Zu, Hongfei; Wang, James H.-C.; Wang, Qing-Ming

    2015-01-01

    Aging not only affects the whole body performance but also alters cellular biological properties, including cell proliferation and differentiation. This study was designed to determine the effect of aging on the mechanical properties of tendon stem cells (TSCs), a newly discovered stem cell type in tendons, using quartz thickness shear mode (TSM) resonators. TSCs were isolated from both old and young rats, and allowed to grow to confluency on the surface of TSM resonators. The admittance spectrums of TSM with TSC monolayer were acquired, and a series of complex shear modulus G′ + jG″ as well as average thickness hTSC were calculated based on a two-layer-loading transmission line model (TLM) for TSM resonator sensor. The results showed an overall increase in G′, G″ and hTSC during aging process. Specifically, the storage modulus G′ of aging TSCs was over ten times than that of young, revealing an important increase in stiffness of aging TSCs. Additionally, through phase-contrast and scanning electronic microscopy, it was shown that aging TSCs were large, flat and heterogeneous in morphologies while young TSCs were uniformly elongated. Increased cell size and irregular cell shape might be associated with the dense cytoskeleton organization, which could lead to an increase in both stiffness and viscosity. These results are in agreement with previously published data using different measurement methods, indicating TSM resonator sensor as a promising tool to measure the mechanical properties of cells. PMID:26251564

  9. Aging-related viscoelasticity variation of tendon stem cells (TSCs) characterized by quartz thickness shear mode (TSM) resonators.

    PubMed

    Wu, Huiyan; Zhao, Guangyi; Zu, Hongfei; Wang, James H-C; Wang, Qing-Ming

    2015-04-01

    Aging not only affects the whole body performance but also alters cellular biological properties, including cell proliferation and differentiation. This study was designed to determine the effect of aging on the mechanical properties of tendon stem cells (TSCs), a newly discovered stem cell type in tendons, using quartz thickness shear mode (TSM) resonators. TSCs were isolated from both old and young rats, and allowed to grow to confluency on the surface of TSM resonators. The admittance spectrums of TSM with TSC monolayer were acquired, and a series of complex shear modulus G' + jG″ as well as average thickness hTSC were calculated based on a two-layer-loading transmission line model (TLM) for TSM resonator sensor. The results showed an overall increase in G', G″ and hTSC during aging process. Specifically, the storage modulus G' of aging TSCs was over ten times than that of young, revealing an important increase in stiffness of aging TSCs. Additionally, through phase-contrast and scanning electronic microscopy, it was shown that aging TSCs were large, flat and heterogeneous in morphologies while young TSCs were uniformly elongated. Increased cell size and irregular cell shape might be associated with the dense cytoskeleton organization, which could lead to an increase in both stiffness and viscosity. These results are in agreement with previously published data using different measurement methods, indicating TSM resonator sensor as a promising tool to measure the mechanical properties of cells.

  10. Drosophila Low Temperature Viability Protein 1 (LTV1) Is Required for Ribosome Biogenesis and Cell Growth Downstream of Drosophila Myc (dMyc)*

    PubMed Central

    Kim, Wonho; Kim, Hag Dong; Jung, Youjin; Kim, Joon; Chung, Jongkyeong

    2015-01-01

    During animal development, various signaling pathways converge to regulate cell growth. In this study, we identified LTV1 as a novel cell growth regulator in Drosophila. LTV1 mutant larvae exhibited developmental delays and lethality at the second larval stage. Using biochemical studies, we discovered that LTV1 interacted with ribosomal protein S3 and co-purified with free 40S ribosome subunits. We further demonstrated that LTV1 is crucial for ribosome biogenesis through 40S ribosome subunit synthesis and preribosomal RNA processing, suggesting that LTV1 is required for cell growth by regulating protein synthesis. We also demonstrated that Drosophila Myc (dMyc) directly regulates LTV1 transcription and requires LTV1 to stimulate ribosome biogenesis. Importantly, the loss of LTV1 blocked the cell growth and endoreplication induced by dMyc. Combined, these results suggest that LTV1 is a key downstream factor of dMyc-induced cell growth by properly maintaining ribosome biogenesis. PMID:25858587

  11. Reduction of Cullin-2 in somatic cells disrupts differentiation of germline stem cells in the Drosophila ovary.

    PubMed

    Ayyub, Champakali; Banerjee, Kushal Kr; Joti, Prakash

    2015-09-15

    Signaling from a niche consisting of somatic cells is essential for maintenance of germline stem cells (GSCs) in the ovary of Drosophila. Decapentaplegic (Dpp), a type of bone morphogenetic protein (BMP) signal, emanating from the niche, is the most important signal for this process. Cullin proteins constitute the core of a multiprotein E3-ligase important for their functions viz. degradation or modification of proteins necessary for different cellular processes. We have found that a Cullin protein called Cullin-2 (Cul-2) expresses in both somatic and germline cells of the Drosophila ovary. Reduction of Cul-2 in somatic cells causes upregulation of Dpp signal and produces accumulation of extra GSC-like cells inside germarium, the anteriormost structure of the ovary. Our results suggest that Cullin-2 protein present in the somatic cells is involved in a non cell-autonomous regulation of the extent of Dpp signaling and thus controls the differentiation of GSCs to cystoblasts (CBs).

  12. Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

    SciTech Connect

    Igaki, Tatsushi; Suzuki, Yasuyuki; Tokushige, Naoko; Aonuma, Hiroka; Takahashi, Ryosuke . E-mail: ryosuket@kuhp.kyoto-u.ac.jp; Miura, Masayuki . E-mail: miura@mol.f.u-tokyo.ac.jp

    2007-05-18

    Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution.

  13. Transcription factor EGR1 directs tendon differentiation and promotes tendon repair

    PubMed Central

    Guerquin, Marie-Justine; Charvet, Benjamin; Nourissat, Geoffroy; Havis, Emmanuelle; Ronsin, Olivier; Bonnin, Marie-Ange; Ruggiu, Mathilde; Olivera-Martinez, Isabel; Robert, Nicolas; Lu, Yinhui; Kadler, Karl E.; Baumberger, Tristan; Doursounian, Levon; Berenbaum, Francis; Duprez, Delphine

    2013-01-01

    Tendon formation and repair rely on specific combinations of transcription factors, growth factors, and mechanical parameters that regulate the production and spatial organization of type I collagen. Here, we investigated the function of the zinc finger transcription factor EGR1 in tendon formation, healing, and repair using rodent animal models and mesenchymal stem cells (MSCs). Adult tendons of Egr1–/– mice displayed a deficiency in the expression of tendon genes, including Scx, Col1a1, and Col1a2, and were mechanically weaker compared with their WT littermates. EGR1 was recruited to the Col1a1 and Col2a1 promoters in postnatal mouse tendons in vivo. Egr1 was required for the normal gene response following tendon injury in a mouse model of Achilles tendon healing. Forced Egr1 expression programmed MSCs toward the tendon lineage and promoted the formation of in vitro–engineered tendons from MSCs. The application of EGR1-producing MSCs increased the formation of tendon-like tissues in a rat model of Achilles tendon injury. We provide evidence that the ability of EGR1 to promote tendon differentiation is partially mediated by TGF-β2. This study demonstrates EGR1 involvement in adult tendon formation, healing, and repair and identifies Egr1 as a putative target in tendon repair strategies. PMID:23863709

  14. Plasticity of both planar cell polarity and cell identity during the development of Drosophila.

    PubMed

    Saavedra, Pedro; Vincent, Jean-Paul; Palacios, Isabel M; Lawrence, Peter A; Casal, José

    2014-02-11

    Drosophila has helped us understand the genetic mechanisms of pattern formation. Particularly useful have been those organs in which different cell identities and polarities are displayed cell by cell in the cuticle and epidermis (Lawrence, 1992; Bejsovec and Wieschaus, 1993; Freeman, 1997). Here we use the pattern of larval denticles and muscle attachments and ask how this pattern is maintained and renewed over the larval moult cycles. During larval growth each epidermal cell increases manyfold in size but neither divides nor dies. We follow individuals from moult to moult, tracking marked cells and find that, as cells are repositioned and alter their neighbours, their identities change to compensate and the pattern is conserved. Single cells adopting a new fate may even acquire a new polarity: an identified cell that makes a forward-pointing denticle in the first larval stage may make a backward-pointing denticle in the second and third larval stages. DOI: http://dx.doi.org/10.7554/eLife.01569.001.

  15. Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia

    PubMed Central

    Rolls, Melissa M.; Albertson, Roger; Shih, Hsin-Pei; Lee, Cheng-Yu; Doe, Chris Q.

    2003-01-01

    Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Here, we show that Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, we show that aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype, and that reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes. PMID:14657233

  16. Mechanism of osteogenic and adipogenic differentiation of tendon stem cells induced by sirtuin 1.

    PubMed

    Liu, Junpeng; Han, Weifeng; Chen, Lei; Tang, Kanglai

    2016-08-01

    The aim of the present study was to assess the expression of sirtuin (Sirt)1 in tendon stem cells (TSCs) and to elucidate its association with osteogenic and adipogenic differentiation of TSCs. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analyses were performed to detect Sirt1 mRNA and protein levels in TSCs, respectively. TSCs were positive for Sirt1 expression, which was elevated by Sirt1 activator SRT1720 in a time- and concentration- dependent manner, and decreased by Sirt1 inhibitor EX527. TSCs were treated with SRT1720 and EX527 for various time periods and resulting changes in osteogenic and adipogenic protein markers were analyzed using alizarin red and oil red O staining. According to RT-qPCR and western blot analyses, the associated factors β‑catenin, Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 were elevated following increases of Sirt1 levels, while CCAAT/enhancer binding protein (CEBP)α and peroxisome proliferator-activated receptor (PPAR)γ were decreased. These results suggested that osteogenic differentiation capacity was enhanced, while adipogenic differentiation capacity declined. Further mechanistic study revealed that phosphoinositide‑3 kinase (PI3K) and AKT were decreased following activation of Sirt1. In conclusion, the present study suggested that Sirt1 promotes the osteogenic differentiation of TSCs through upregulating β‑catenin and Runx2 and inhibits the adipogenic differentiation of TSCs through the PI3K/AKT pathway with downregulation of CEBPα and PPARγ.

  17. Loss of Tenomodulin Results in Reduced Self-Renewal and Augmented Senescence of Tendon Stem/Progenitor Cells

    PubMed Central

    Alberton, Paolo; Dex, Sarah; Popov, Cvetan; Shukunami, Chisa; Schieker, Matthias

    2015-01-01

    Tenomodulin (Tnmd) is a well-known gene marker for the tendon and ligament lineage, but its exact functions in these tissues still remain elusive. In this study, we investigated Tnmd loss of function in mouse tendon stem/progenitor cells (mTSPC) by implicating a previously established Tnmd knockout (KO) mouse model. mTSPC were isolated from control and Tnmd KO tail tendons and their stemness features, such as gene marker profile, multipotential, and self-renewal, were compared. Immunofluorescence and reverse transcriptase-polymerase chain reaction analyses for stem cell-, tenogenic-, osteogenic-, and chondrogenic-related genes confirmed their stemness and lineage specificity and demonstrated no profound differences between the two genotypes. Multipotential was not significantly affected since both cell types differentiated successfully into adipogenic, osteogenic, and chondrogenic lineages. In contrast, self-renewal assays validated that Tnmd KO TSPC exhibit significantly reduced proliferative potential, which was also reflected in lower Cyclin D1 levels. When analyzing possible cellular mechanisms behind the observed decreased self-renewability of Tnmd KO TSPC, we found that cellular senescence plays a major role, starting earlier and cumulating more in Tnmd KO compared with control TSPC. This was accompanied with augmented expression of the cell cycle inhibitor p53. Finally, the proliferative effect of Tnmd in TSPC was confirmed with transient transfection of Tnmd cDNA into Tnmd KO TSPC, which rescued their proliferative deficit. Taken together, we can report that loss of Tnmd affects significantly the self-renewal and senescence properties, but not the multipotential of TSPC. PMID:25351164

  18. Effect of Adipose-Derived Stromal Cells and BMP12 on Intrasynovial Tendon Repair: A Biomechanical, Biochemical, and Proteomics Study

    PubMed Central

    Gelberman, Richard H.; Shen, Hua; Kormpakis, Ioannis; Rothrauff, Benjamin; Yang, Guang; Tuan, Rocky S.; Xia, Younan; Sakiyama-Elbert, Shelly; Silva, Matthew J.; Thomopoulos, Stavros

    2016-01-01

    The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor- and cell-based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin-based scaffold. Control groups consisted of repair-only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC+BMP12 treatment for range of motion or tensile properties outcomes versus repair-only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC+BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC+BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC+BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions. PMID:26445383

  19. Stable Drosophila Cell Lines: An Alternative Approach to Exogenous Protein Expression.

    PubMed

    Backovic, Marija; Krey, Thomas

    2016-01-01

    Recombinant protein production has become an indispensable tool for various research directions and biotechnological applications in the past decades. Among the numerous reported expression systems, insect cells provide the possibility to produce complex target proteins that require posttranslational modifications. Stable expression in Drosophila S2 cells represents an attractive alternative to the widely used baculovirus expression system, offering important advantages in particular for difficult-to-express proteins, e.g., membrane proteins or heavily glycosylated multi-domain proteins that are stabilized by a complex disulfide pattern. Here we present the methodology that is required for the generation of stable Drosophila S2 cell transfectants and for production of recombinant proteins using those transfectants.

  20. A Drosophila melanogaster cell line tested for the presence of active NORs by silver staining.

    PubMed

    Privitera, E

    1980-01-01

    Silver staining was used to detect active NORs in a Drosophila melanogaster cell line (C1 82) characterized by dimorphic X chromosomes (XXL), one of the two Xs showing a marked increase in heterochromatin where the nucleolar organizer (NO) is located. The Q-banding technique was used to determine the karyotype characteristics of the line. Ag-positive NORs appeared only on structurally changed X chromosomes (XL), both in diploid and tetraploid cells, indicating that rRNA genes of XL are more active or numerous than those on normal homologues. A possible relationship between NOR stainability, the presence of an increased heterochromatic portion and the selective advantage of XXL cells, recurrent in numerous Drosophila female lines, is discussed.

  1. Platelet-rich plasma increases proliferation of tendon cells by modulating Stat3 and p27 to up-regulate expression of cyclins and cyclin-dependent kinases.

    PubMed

    Yu, T-Y; Pang, J-H S; Wu, K P-H; Lin, L-P; Tseng, W-C; Tsai, W-C

    2015-08-01

    To investigate effects of platelet-rich plasma on tendon cell proliferation and the underlying molecular mechanisms. Platelet-rich plasma was prepared manually by two-step centrifugation. Proliferation was evaluated in cultured rat tendon cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Cell cycle progression was assessed by flow cytometry. Messenger RNA expression of proliferating cell nuclear antigen (PCNA), cyclin E1, A2 and B1, and cyclin-dependent kinases (Cdks) 1 and 2 was assessed by real-time polymerase chain reaction. Protein expression of the above cyclins and Cdks and of signal transducer and activator of transcription (Stat) 3 and p27 was evaluated by western blotting. Platelet-rich plasma used in the present study had concentrations of platelets, TGF-β1 and PDGF over 3-fold higher than normal whole blood. Platelet-rich plasma enhanced tendon cell proliferation (P = 0.008) by promoting G1 /S phase transition in the cell cycle, and increased expression of PCNA, cyclin E1, A2 and B1, Cdks1 and 2, and phosphorylated Stat3, while inhibiting p27 expression. Platelet-rich plasma contains high concentrations of TGF-β1 and PDGF that increase tendon cell proliferation by modulating Stat3/p27(Kip1), which enhances expression of cyclin-Cdk complexes that promote cell cycle progression. These results provide molecular evidence for positive effects of platelet-rich plasma on tendon cell proliferation, which can be useful in clinical applications of tendon injury. © 2015 John Wiley & Sons Ltd.

  2. Silver nanoparticles disrupt germline stem cell maintenance in the Drosophila testis

    NASA Astrophysics Data System (ADS)

    Ong, Cynthia; Lee, Qian Ying; Cai, Yu; Liu, Xiaoli; Ding, Jun; Yung, Lin-Yue Lanry; Bay, Boon-Huat; Baeg, Gyeong-Hun

    2016-02-01

    Silver nanoparticles (AgNPs), one of the most popular nanomaterials, are commonly used in consumer products and biomedical devices, despite their potential toxicity. Recently, AgNP exposure was reported to be associated with male reproductive toxicity in mammalian models. However, there is still a limited understanding of the effects of AgNPs on spermatogenesis. The fruit fly Drosophila testis is an excellent in vivo model to elucidate the mechanisms underlying AgNP-induced defects in spermatogenesis, as germ lineages can be easily identified and imaged. In this study, we evaluated AgNP-mediated toxicity on spermatogenesis by feeding Drosophila with AgNPs at various concentrations. We first observed a dose-dependent uptake of AgNPs in vivo. Concomitantly, AgNP exposure caused a significant decrease in the viability and delay in the development of Drosophila in a dose-dependent manner. Furthermore, AgNP-treated male flies showed a reduction in fecundity, and the resulting testes contained a decreased number of germline stem cells (GSCs) compared to controls. Interestingly, testes exposed to AgNPs exhibited a dramatic increase in reactive oxygen species levels and showed precocious GSC differentiation. Taken together, our study suggests that AgNP exposure may increase ROS levels in the Drosophila testis, leading to a reduction of GSC number by promoting premature GSC differentiation.

  3. Silver nanoparticles disrupt germline stem cell maintenance in the Drosophila testis.

    PubMed

    Ong, Cynthia; Lee, Qian Ying; Cai, Yu; Liu, Xiaoli; Ding, Jun; Yung, Lin-Yue Lanry; Bay, Boon-Huat; Baeg, Gyeong-Hun

    2016-02-05

    Silver nanoparticles (AgNPs), one of the most popular nanomaterials, are commonly used in consumer products and biomedical devices, despite their potential toxicity. Recently, AgNP exposure was reported to be associated with male reproductive toxicity in mammalian models. However, there is still a limited understanding of the effects of AgNPs on spermatogenesis. The fruit fly Drosophila testis is an excellent in vivo model to elucidate the mechanisms underlying AgNP-induced defects in spermatogenesis, as germ lineages can be easily identified and imaged. In this study, we evaluated AgNP-mediated toxicity on spermatogenesis by feeding Drosophila with AgNPs at various concentrations. We first observed a dose-dependent uptake of AgNPs in vivo. Concomitantly, AgNP exposure caused a significant decrease in the viability and delay in the development of Drosophila in a dose-dependent manner. Furthermore, AgNP-treated male flies showed a reduction in fecundity, and the resulting testes contained a decreased number of germline stem cells (GSCs) compared to controls. Interestingly, testes exposed to AgNPs exhibited a dramatic increase in reactive oxygen species levels and showed precocious GSC differentiation. Taken together, our study suggests that AgNP exposure may increase ROS levels in the Drosophila testis, leading to a reduction of GSC number by promoting premature GSC differentiation.

  4. Trafficking through COPII Stabilises Cell Polarity and Drives Secretion during Drosophila Epidermal Differentiation

    PubMed Central

    Norum, Michaela; Tång, Erika; Chavoshi, Tina; Schwarz, Heinz; Linke, Dirk; Uv, Anne; Moussian, Bernard

    2010-01-01

    Background The differentiation of an extracellular matrix (ECM) at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation. Principal Findings We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus. Conclusion Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes. PMID:20520821

  5. Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

    PubMed Central

    Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele

    2015-01-01

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIA-BLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer. PMID:25959206

  6. Informing tendon tissue engineering with embryonic development

    PubMed Central

    Glass, Zachary A.; Schiele, Nathan R.; Kuo, Catherine K.

    2014-01-01

    Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering. PMID:24484642

  7. Informing tendon tissue engineering with embryonic development.

    PubMed

    Glass, Zachary A; Schiele, Nathan R; Kuo, Catherine K

    2014-06-27

    Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering.

  8. Synthetic Lethality of Retinoblastoma Mutant Cells in the Drosophila Eye by Mutation of a Novel Peptidyl Prolyl Isomerase Gene

    PubMed Central

    Edgar, Kyle A.; Belvin, Marcia; Parks, Annette L.; Whittaker, Kellie; Mahoney, Matt B.; Nicoll, Monique; Park, Christopher C.; Winter, Christopher G.; Chen, Feng; Lickteig, Kim; Ahmad, Ferhad; Esengil, Hanife; Lorenzi, Matthew V.; Norton, Amanda; Rupnow, Brent A.; Shayesteh, Laleh; Tabios, Mariano; Young, Lynn M.; Carroll, Pamela M.; Kopczynski, Casey; Plowman, Gregory D.; Friedman, Lori S.; Francis-Lang, Helen L.

    2005-01-01

    Mutations that inactivate the retinoblastoma (Rb) pathway are common in human tumors. Such mutations promote tumor growth by deregulating the G1 cell cycle checkpoint. However, uncontrolled cell cycle progression can also produce new liabilities for cell survival. To uncover such liabilities in Rb mutant cells, we performed a clonal screen in the Drosophila eye to identify second-site mutations that eliminate Rbf− cells, but allow Rbf+ cells to survive. Here we report the identification of a mutation in a novel highly conserved peptidyl prolyl isomerase (PPIase) that selectively eliminates Rbf− cells from the Drosophila eye. PMID:15744054

  9. Genotoxicity of two arsenic compounds in germ cells and somatic cells of Drosophila melanogaster

    SciTech Connect

    Ramos-Morales, P.; Rodriguez-Arnaiz, R.

    1995-12-31

    Two arsenic compounds, sodium arsenite (NaAsO{sup 2}) and sodium arsenate (Na{sub 2}HasO{sub 4}), were tested for their possible genotoxicity in germinal and somatic cells of Drosophila melanagaster. For germinal cells, the sex-linked recessive lethal test (SLRLT) and the sea chromosome loss test (SCLT) were used. In both tests, a broad scheme of 2-3-3 days was employed. Two routes of administration were used for the SLRLT: adult male injection (0.38, 0.77 mM used for Sodium arsenite; and 0.01, 0.02 mM for sodium arsenate). The the SCLT the compounds were injected into males. Controls were treated with a solution of 5% sucrose which was employed as solvent. The somatic mutation and recombination test (SMART) was run in the w{sup +}/w eye assay as well as in the mwh +/+ flr{sup 3} wing test, employing the standard and insecticide-resistant strains. In both tests, third instar larvae were treated for 6 hr with sodium arsenite (0.38, 0.77, 1.15 mM), and sodium arsenate (0.54, 1.34, 2.69 mM). In the SLRLT, both compounds were positive, but they were negative in the SCLT. The genotoxicity of both compounds was localized mainly in somatic cells, in agreement with reports on the carcinogenic potential of arsenical compounds Solium and arsenite was an order of magnitude more toxic and mutagenic than sodium arsenate. This study confirms the reliability of the Drosophila in vivo system to test the genotoxicity of environmental compounds. 75 refs., 4 figs., 4 tabs.

  10. Comparison of the early period effects of bone marrow-derived mesenchymal stem cells and platelet-rich plasma on the Achilles tendon ruptures in rats.

    PubMed

    Yuksel, Serdar; Guleç, M Akif; Gultekin, M Zeki; Adanır, Oktay; Caglar, Aysel; Beytemur, Ozan; Onur Küçükyıldırım, B; Avcı, Ali; Subaşı, Cansu; İnci, Çiğdem; Karaoz, Erdal

    2016-09-01

    This study aims to histopathologically, biomechanically, and immunohistochemically compare the fourth-week efficiencies of local platelet-rich plasma (PRP) and bone marrow-derived mesenchymal stem cell (rBM-MSC) treatments of the Achilles tendon ruptures created surgically in rats. The study included 35 12-month-old male Sprague Dawley rats, with an average weight of 400-500 g. Five rats were used as donors for MSC and PRP, and 30 rats were separated into MSC, PRP, and control groups (n = 10). The Achilles tendons of the rats were cut transversely, the MSC from bone marrow was administered to the MSC group, the PRP group received PRP, and the control group received physiological saline to create the same surgical effect. In previous studies, it was shown that this physiological saline does not have any effect on tendon recovery. Thirty days after the treatment, the rats were sacrificed and their Achilles tendons were examined histopathologically, immunohistochemically, and biomechanically. The use of rBM-MSC and PRP in the Achilles tendon ruptures when the tendon is in its weakest phase positively affected the recovery of the tendon in histopathologic, immunohistochemical, and biomechanical manners compared to the control group (p < 0.05). While the levels of pro-inflammatory cytokines TNF-α, IFNγ, and IL 1β were significantly low, the levels of anti-inflammatory cytokines and growth factors playing key roles in tendon recovery, such as IL2, VEGF, transforming growth factor-beta, and HGF, were significantly higher in the MSC group than those of the PRP and control groups (p < 0.05). In the MSC group, the [Formula: see text] (mm) value was significantly higher (p ˂ 0.05) than that in the PRP and control groups. rBM-MSC and PRP promote the recovery of the tendon and increase its structural strength. The use of PRP and MSC provides hope for the treatment of the Achilles tendon ruptures that limit human beings' functionalities and quality of life, particularly for

  11. Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary

    PubMed Central

    Timmons, Allison K.; Mondragon, Albert A.; Schenkel, Claire E.; Yalonetskaya, Alla; Taylor, Jeffrey D.; Moynihan, Katherine E.; Etchegaray, Jon Iker; Meehan, Tracy L.; McCall, Kimberly

    2016-01-01

    Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line–derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms. PMID:26884181

  12. EGFR and Notch signaling respectively regulate proliferative activity and multiple cell lineage differentiation of Drosophila gastric stem cells.

    PubMed

    Wang, Chenhui; Guo, Xingting; Xi, Rongwen

    2014-05-01

    Quiescent, multipotent gastric stem cells (GSSCs) in the copper cell region of adult Drosophila midgut can produce all epithelial cell lineages found in the region, including acid-secreting copper cells, interstitial cells and enteroendocrine cells, but mechanisms controlling their quiescence and the ternary lineage differentiation are unknown. By using cell ablation or damage-induced regeneration assays combined with cell lineage tracing and genetic analysis, here we demonstrate that Delta (Dl)-expressing cells in the copper cell region are the authentic GSSCs that can self-renew and continuously regenerate the gastric epithelium after a sustained damage. Lineage tracing analysis reveals that the committed GSSC daughter with activated Notch will invariably differentiate into either a copper cell or an interstitial cell, but not the enteroendocrine cell lineage, and loss-of-function and gain-of-function studies revealed that Notch signaling is both necessary and sufficient for copper cell/interstitial cell differentiation. We also demonstrate that elevated epidermal growth factor receptor (EGFR) signaling, which is achieved by the activation of ligand Vein from the surrounding muscle cells and ligand Spitz from progenitor cells, mediates the regenerative proliferation of GSSCs following damage. Taken together, we demonstrate that Dl is a specific marker for Drosophila GSSCs, whose cell cycle status is dependent on the levels of EGFR signaling activity, and the Notch signaling has a central role in controlling cell lineage differentiation from GSSCs by separating copper/interstitial cell lineage from enteroendocrine cell lineage.

  13. Investigating tendon mineralisation in the avian hindlimb: a model for tendon ageing, injury and disease

    PubMed Central

    Agabalyan, Natacha A; Evans, Darrell J R; Stanley, Rachael L

    2013-01-01

    Mineralisation of the tendon tissue has been described in various models of injury, ageing and disease. Often resulting in painful and debilitating conditions, the processes underlying this mechanism are poorly understood. To elucidate the progression from healthy tendon to mineralised tendon, an appropriate model is required. In this study, we describe the spontaneous and non-pathological ossification and calcification of tendons of the hindlimb of the domestic chicken (Gallus gallus domesticus). The appearance of the ossified avian tendon has been described previously, although there have been no studies investigating the developmental processes and underlying mechanisms leading to the ossified avian tendon. The tissue and cells from three tendons – the ossifying extensor and flexor digitorum longus tendons and the non-ossifying Achilles tendon – were analysed for markers of ageing and mineralisation using histology, immunohistochemistry, cytochemistry and molecular analysis. Histologically, the adult tissue showed a loss of healthy tendon crimp morphology as well as markers of calcium deposits and mineralisation. The tissue showed a lowered expression of collagens inherent to the tendon extracellular matrix and presented proteins expressed by bone. The cells from the ossified tendons showed a chondrogenic and osteogenic phenotype as well as tenogenic phenotype and expressed the same markers of ossification and calcification as the tissue. A molecular analysis of the gene expression of the cells confirmed these results. Tendon ossification within the ossified avian tendon seems to be the result of an endochondral process driven by its cells, although the roles of the different cell populations have yet to be elucidated. Understanding the role of the tenocyte within this tissue and the process behind tendon ossification may help us prevent or treat ossification that occurs in injured, ageing or diseased tendon. PMID:23826786

  14. Investigating tendon mineralisation in the avian hindlimb: a model for tendon ageing, injury and disease.

    PubMed

    Agabalyan, Natacha A; Evans, Darrell J R; Stanley, Rachael L

    2013-09-01

    Mineralisation of the tendon tissue has been described in various models of injury, ageing and disease. Often resulting in painful and debilitating conditions, the processes underlying this mechanism are poorly understood. To elucidate the progression from healthy tendon to mineralised tendon, an appropriate model is required. In this study, we describe the spontaneous and non-pathological ossification and calcification of tendons of the hindlimb of the domestic chicken (Gallus gallus domesticus). The appearance of the ossified avian tendon has been described previously, although there have been no studies investigating the developmental processes and underlying mechanisms leading to the ossified avian tendon. The tissue and cells from three tendons - the ossifying extensor and flexor digitorum longus tendons and the non-ossifying Achilles tendon - were analysed for markers of ageing and mineralisation using histology, immunohistochemistry, cytochemistry and molecular analysis. Histologically, the adult tissue showed a loss of healthy tendon crimp morphology as well as markers of calcium deposits and mineralisation. The tissue showed a lowered expression of collagens inherent to the tendon extracellular matrix and presented proteins expressed by bone. The cells from the ossified tendons showed a chondrogenic and osteogenic phenotype as well as tenogenic phenotype and expressed the same markers of ossification and calcification as the tissue. A molecular analysis of the gene expression of the cells confirmed these results. Tendon ossification within the ossified avian tendon seems to be the result of an endochondral process driven by its cells, although the roles of the different cell populations have yet to be elucidated. Understanding the role of the tenocyte within this tissue and the process behind tendon ossification may help us prevent or treat ossification that occurs in injured, ageing or diseased tendon. © 2013 Anatomical Society.

  15. Do patient age and sex influence tendon cell biology and clinical/radiographic outcomes after rotator cuff repair?

    PubMed

    Pauly, Stephan; Stahnke, Katharina; Klatte-Schulz, Franka; Wildemann, Britt; Scheibel, Markus; Greiner, Stefan

    2015-03-01

    Many clinical and radiographic studies suggest that patient age and sex have an influence on rotator cuff (RC) repair outcomes. However, these findings result from retrospective statistical analyses and cannot provide a causal answer. To analyze whether age and sex influence the biological potential at the time of RC repair or midterm clinical and radiographic outcomes. Also assessed was the effect of the biological potential on intraindividual clinical/radiographic results. Cohort study; Level of evidence, 2. A total of 40 patients underwent arthroscopic RC repair. At the time of surgery (t = 0), supraspinatus tendon biopsy specimens were obtained, cultivated, and assessed for their biological potential, particularly (1) cell growth and (2) collagen type I production. After a follow-up at 24 months (t = 1), all patients were assessed by clinical scores (Constant score, subjective shoulder value, American Shoulder and Elbow Surgeons [ASES] score, and Western Ontario Rotator Cuff Index [WORC] score) and underwent magnetic resonance imaging to determine RC integrity. The data were examined for age- and sex-related differences and to identify the correlation between biological potential (t = 0) and clinical/radiographic outcome (t = 1). The follow-up rate for the imaging and clinical evaluation was 100%. Age, but not sex, influenced the biological tendon cell parameters at t = 0. However, there was no effect of age or sex on the clinical and radiographic results at t = 1. Furthermore, no correlation was observed between the initial biological parameters and later clinical outcomes or radiographic RC integrity. Finally, there was no significant difference between intact and nonhealed repairs in terms of the respective clinical scores. Age, but not sex, was found to have a negative effect on RC tendon cell biology. However, neither sex nor, in particular, a higher age influenced repair outcomes after 24 months. © 2015 The Author(s).

  16. Insulin-independent role of adiponectin receptor signaling in Drosophila germline stem cell maintenance.

    PubMed

    Laws, Kaitlin M; Sampson, Leesa L; Drummond-Barbosa, Daniela

    2015-03-15

    Adipocytes have key endocrine roles, mediated in large part by secreted protein hormones termed adipokines. The adipokine adiponectin is well known for its role in sensitizing peripheral tissues to insulin, and several lines of evidence suggest that adiponectin might also modulate stem cells/precursors. It remains unclear, however, how adiponectin signaling controls stem cells and whether this role is secondary to its insulin-sensitizing effects or distinct. Drosophila adipocytes also function as an endocrine organ and, although no obvious adiponectin homolog has been identified, Drosophila AdipoR encodes a well-conserved homolog of mammalian adiponectin receptors. Here, we generate a null AdipoR allele and use clonal analysis to demonstrate an intrinsic requirement for AdipoR in germline stem cell (GSC) maintenance in the Drosophila ovary. AdipoR null GSCs are not fully responsive to bone morphogenetic protein ligands from the niche and have a slight reduction in E-cadherin levels at the GSC-niche junction. Conversely, germline-specific overexpression of AdipoR inhibits natural GSC loss, suggesting that reduction in adiponectin signaling might contribute to the normal decline in GSC numbers observed over time in wild-type females. Surprisingly, AdipoR is not required for insulin sensitization of the germline, leading us to speculate that insulin sensitization is a more recently acquired function than stem cell regulation in the evolutionary history of adiponectin signaling. Our findings establish Drosophila female GSCs as a new system for future studies addressing the molecular mechanisms whereby adiponectin receptor signaling modulates stem cell fate.

  17. Quantitative Evaluation of Signaling Events in Drosophila S2 Cells

    PubMed Central

    2008-01-01

    Drosophila activates a robust defense response to gram-negative bacteria through the Immune deficiency (Imd) pathway. Imd signaling proceeds through c-Jun N-terminal Kinase (JNK), NF-kB and caspase modules. The individual signaling modules act in a highly coordinated manner to yield a stereotypical response to infection. While considerable attention has focused on NF-kB-mediated antimicrobial activities, more recent studies have highlighted the involvement of JNK signaling in the Imd pathway response. JNK signaling occurs in a transitory burst and drives the expression of a number of gene products through the AP-1 transcription factor. In this report, we describe a simple method for the quantification of JNK activation by Western blot analysis or directly in tissue culture plates. PMID:18385808

  18. In Vivo Evaluation of Adipose-Derived Stromal Cells Delivered with a Nanofiber Scaffold for Tendon-to-Bone Repair

    PubMed Central

    Lipner, Justin; Shen, Hua; Cavinatto, Leonardo; Liu, Wenying; Havlioglu, Necat; Xia, Younan; Galatz, Leesa M.

    2015-01-01

    Rotator cuff tears are common and cause a great deal of lost productivity, pain, and disability. Tears are typically repaired by suturing the tendon back to its bony attachment. Unfortunately, the structural (e.g., aligned collagen) and compositional (e.g., a gradient in mineral) elements that produce a robust attachment in the healthy tissue are not regenerated during healing, and the repair is prone to failure. Two features of the failed healing response are deposition of poorly aligned scar tissue and loss of bone at the repair site. Therefore, the objective of the current study was to improve tendon-to-bone healing by promoting aligned collagen deposition and increased bone formation using a biomimetic scaffold seeded with pluripotent cells. An aligned nanofibrous poly(lactic-co-glycolic acid) scaffold with a gradient in mineral content was seeded with adipose-derived stromal cells (ASCs) and implanted at the repair site of a rat rotator cuff model. In one group, cells were transduced with the osteogenic factor bone morphogenetic protein 2 (BMP2). The healing response was examined in four groups (suture only, acellular scaffold, cellular scaffold, and cellular BMP2 scaffold) using histologic, bone morphology, and biomechanical outcomes at 14, 28, and 56 days. Histologically, the healing interface was dominated by a fibrovascular scar response in all groups. The acellular scaffold group showed a delayed healing response compared to the other groups. When examining bone morphology parameters, bone loss was evident in the cellular BMP2 group compared to other groups at 28 days. When examining repair-site mechanical properties, strength and modulus were decreased in the cellular BMP2 groups compared to other groups at 28 and 56 days. These results indicated that tendon-to-bone healing in this animal model was dominated by scar formation, preventing any positive effects of the implanted biomimetic scaffold. Furthermore, cells transduced with the osteogenic factor

  19. Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells.

    PubMed

    Boone, Jason Q; Doe, Chris Q

    2008-08-01

    Mammalian neural stem cells generate transit amplifying progenitors that expand the neuronal population, but these type of progenitors have not been studied in Drosophila. The Drosophila larval brain contains approximately 100 neural stem cells (neuroblasts) per brain lobe, which are thought to bud off smaller ganglion mother cells (GMCs) that each produce two post-mitotic neurons. Here, we use molecular markers and clonal analysis to identify a novel neuroblast cell lineage containing "transit amplifying GMCs" (TA-GMCs). TA-GMCs differ from canonical GMCs in several ways: each TA-GMC has nuclear Deadpan, cytoplasmic Prospero, forms Prospero crescents at mitosis, and generates up to 10 neurons; canonical GMCs lack Deadpan, have nuclear Prospero, lack Prospero crescents at mitosis, and generate two neurons. We conclude that there are at least two types of neuroblast lineages: a Type I lineage where GMCs generate two neurons, and a type II lineage where TA-GMCs have longer lineages. Type II lineages allow more neurons to be produced faster than Type I lineages, which may be advantageous in a rapidly developing organism like Drosophila. (c) 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008.

  20. Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells

    PubMed Central

    Boone, Jason Q.; Doe, Chris Q.

    2009-01-01

    Mammalian neural stem cells generate transit amplifying progenitors that expand the neuronal population, but these type of progenitors have not been studied in Drosophila. The Drosophila larval brain contains ~100 neural stem cells (neuroblasts) per brain lobe, which are thought to bud off smaller ganglion mother cells (GMCs) that each produce two post-mitotic neurons. Here we use molecular markers and clonal analysis to identify a novel neuroblast cell lineage containing "transit amplifying GMCs" (TA-GMCs). TA-GMCs differ from canonical GMCs in several ways: each TA-GMC has nuclear Deadpan, cytoplasmic Prospero, forms Prospero crescents at mitosis, and generates up to 10 neurons; canonical GMCs lack Deadpan, have nuclear Prospero, lack Prospero crescents at mitosis, and generate two neurons. We conclude that there are at least two types of neuroblast lineages: a type I lineage where GMCs generate two neurons, and a type II lineage where TA-GMCs have longer lineages. Type II lineages allow more neurons to be produced faster than type I lineages, which may be advantageous in a rapidly developing organism like Drosophila. PMID:18548484

  1. Fipronil induces apoptosis through caspase-dependent mitochondrial pathways in Drosophila S2 cells.

    PubMed

    Zhang, Baoyan; Xu, Zhiping; Zhang, Yixi; Shao, Xusheng; Xu, Xiaoyong; Cheng, Jiaogao; Li, Zhong

    2015-03-01

    Fipronil is the first phenylpyrazole insecticide widely used in controlling pests, including pyrethroid, organophosphate and carbamate insecticides. It is generally accepted that fipronil elicits neurotoxicity via interactions with GABA and glutamate receptors, although alternative mechanisms have recently been proposed. This study evaluates the genotoxicity of fipronil and its likely mode of action in Drosophila S2 cells, as an in vitro model. Fipronil administrated the concentration- and time-dependent S2 cell proliferation. Intracellular biochemical assays showed that fipronil-induced S2 cell apoptosis coincided with a decrease in the mitochondrial membrane potential and an increase reactive oxygen species generation, a significant decrease of Bcl-2 and DIAP1, and a marked augmentation of Cyt c and caspase-3. Because caspase-3 is the major executioner caspase downstream of caspase-9 in Drosophila, enzyme activity assays were used to determine the activities of caspase-3 and caspase-9. Our results indicated that fipronil effectively induced apoptosis in Drosophila S2 cells through caspase-dependent mitochondrial pathways.

  2. Germline self-renewal requires cyst stem cells and stat regulates niche adhesion in Drosophila testes.

    PubMed

    Leatherman, Judith L; Dinardo, Stephen

    2010-08-01

    Adults maintain tissue-specific stem cells through niche signals. A model for niche function is the Drosophila melanogaster testis, where a small cluster of cells called the hub produce locally available signals that allow only adjacent cells to self-renew. We show here that the principal signalling pathway implicated in this niche, chemokine activation of STAT, does not primarily regulate self-renewal of germline stem cells (GSCs), but rather governs GSC adhesion to hub cells. In fact, GSC renewal does not require hub cell contact, as GSCs can be renewed solely by contact with the second resident stem cell population, somatic cyst stem cells (CySCs), and this involves BMP signalling. These data suggest a modified paradigm whereby the hub cells function as architects of the stem cell environment, drawing into proximity cellular components necessary for niche function. Self-renewal functions are shared by the hub cells and the CySCs. This work also reconciles key differences in GSC renewal between Drosophila testis and ovary niches, and highlights how a niche can coordinate the production of distinct lineages by having one stem cell type rely on a second.

  3. Pattern reorganization occurs independently of cell division during Drosophila wing disc regeneration in situ.

    PubMed

    Díaz-García, Sandra; Baonza, Antonio

    2013-08-06

    One of the most intriguing problems in developmental biology is how an organism can replace missing organs or portions of its body after injury. This capacity, known as regeneration, is conserved across different phyla. The imaginal discs of Drosophila melanogaster provide a particularly well-characterized model for analyzing regeneration. We have developed a unique method to study organ regeneration under physiological conditions using the imaginal discs of Drosophila. Using this method, we revisited different aspects of organ regeneration. The results presented in this report suggest that during the initial stages of regeneration, different processes occur, including wound healing, a temporary loss of markers of cell-fate commitment, and pattern reorganization. We present evidence indicating that all of these processes occur even when cell division has been arrested. Our data also suggested that Wingless is not required during the early stages of disc regeneration.

  4. Loss of PI3K blocks cell-cycle progression in a Drosophila tumor model.

    PubMed

    Willecke, M; Toggweiler, J; Basler, K

    2011-09-29

    Tumorigenesis is a complex process, which requires alterations in several tumor suppressor or oncogenes. Here, we use a Drosophila tumor model to identify genes, which are specifically required for tumor growth. We found that reduction of phosphoinositide 3-kinase (PI3K) activity resulted in very small tumors while only slightly affecting growth of wild-type tissue. The observed inhibition on tumor growth occurred at the level of cell-cycle progression. We conclude that tumor cells become dependent on PI3K function and that reduction of PI3K activity synthetically interferes with tumor growth. The results presented here broaden our insights into the intricate mechanisms underling tumorigenesis and illustrate the power of Drosophila genetics in revealing weak points of tumor progression.

  5. Pattern reorganization occurs independently of cell division during Drosophila wing disc regeneration in situ

    PubMed Central

    Díaz-García, Sandra; Baonza, Antonio

    2013-01-01

    One of the most intriguing problems in developmental biology is how an organism can replace missing organs or portions of its body after injury. This capacity, known as regeneration, is conserved across different phyla. The imaginal discs of Drosophila melanogaster provide a particularly well-characterized model for analyzing regeneration. We have developed a unique method to study organ regeneration under physiological conditions using the imaginal discs of Drosophila. Using this method, we revisited different aspects of organ regeneration. The results presented in this report suggest that during the initial stages of regeneration, different processes occur, including wound healing, a temporary loss of markers of cell-fate commitment, and pattern reorganization. We present evidence indicating that all of these processes occur even when cell division has been arrested. Our data also suggested that Wingless is not required during the early stages of disc regeneration. PMID:23878228

  6. Role of cell death in the formation of sexual dimorphism in the Drosophila central nervous system.

    PubMed

    Kimura, Ken-Ichi

    2011-02-01

    Currently, sex differences in behavior are believed to result from sexually dimorphic neural circuits in the central nervous system (CNS). Drosophila melanogaster is a common model organism for studying the relationship between brain structure, behavior, and genes. Recent studies of sex-specific reproductive behaviors in D. melanogaster have addressed the contribution of sexual differences in the CNS to the control of sex-specific behaviors and the development of sexual dimorphism. For example, sexually dimorphic regions of the CNS are involved in the initiation of male courtship behavior, the generation of the courtship song, and the induction of male-specific muscles in D. melanogaster. In this review, I discuss recent findings about the contribution of cell death to the formation of sexually dimorphic neural circuitry and the regulation of sex-specific cell death by two sex determination factors, Fruitless and Doublesex, in Drosophila.

  7. Lipid phosphate phosphatase activity regulates dispersal and bilateral sorting of embryonic germ cells in Drosophila

    PubMed Central

    Renault, Andrew D.; Kunwar, Prabhat S.; Lehmann, Ruth

    2010-01-01

    In Drosophila, germ cell survival and directionality of migration are controlled by two lipid phosphate phosphatases (LPP), wunen (wun) and wunen-2 (wun2). wun wun2 double mutant analysis reveals that the two genes, hereafter collectively called wunens, act redundantly in primordial germ cells. We find that wunens mediate germ cell-germ cell repulsion and that this repulsion is necessary for germ cell dispersal and proper transepithelial migration at the onset of migration and for the equal sorting of the germ cells between the two embryonic gonads during their migration. We propose that this dispersal function optimizes adult fecundity by assuring maximal germ cell occupancy of both gonads. Furthermore, we find that the requirement for wunens in germ cell survival can be eliminated by blocking germ cell migration. We suggest that this essential function of Wunen is needed to maintain cell integrity in actively migrating germ cells. PMID:20431117

  8. Piwi is required in multiple cell types to control germline stem cell lineage development in the Drosophila ovary.

    PubMed

    Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

    2014-01-01

    The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.

  9. Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

    PubMed Central

    Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

    2014-01-01

    The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary. PMID:24658126

  10. Extracorporeal Shock Wave Treatment (ESWT) enhances the in vitro-induced differentiation of human tendon-derived stem/progenitor cells (hTSPCs)

    PubMed Central

    Leone, Laura; Raffa, Salvatore; Vetrano, Mario; Ranieri, Danilo; Malisan, Florence; Scrofani, Cristina; Vulpiani, Maria Chiara; Ferretti, Andrea; Torrisi, Maria Rosaria; Visco, Vincenzo

    2016-01-01

    Extracorporeal shock wave therapy (ESWT) is a non-invasive and innovative technology for the management of specific tendinopathies. In order to elucidate the ESWT-mediated clinical benefits, human Tendon-derived Stem/Progenitor cells (hTSPCs) explanted from 5 healthy semitendinosus (ST) and 5 ruptured Achilles (AT) tendons were established. While hTSPCs from the two groups showed similar proliferation rates and stem cell surface marker profiles, we found that the clonogenic potential was maintained only in cells derived from healthy donors. Interestingly, ESWT significantly accelerated hTSPCs differentiation, suggesting that the clinical benefits of ESWT may be ascribed to increased efficiency of tendon repair after injury. PMID:26843618

  11. DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis.

    PubMed

    Mitra, Kasturi; Rikhy, Richa; Lilly, Mary; Lippincott-Schwartz, Jennifer

    2012-05-14

    Exit from the cell cycle is essential for cells to initiate a terminal differentiation program during development, but what controls this transition is incompletely understood. In this paper, we demonstrate a regulatory link between mitochondrial fission activity and cell cycle exit in follicle cell layer development during Drosophila melanogaster oogenesis. Posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 had mitochondrial elements extensively fused instead of being dispersed. These cells did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf-1 in posterior-localized DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf-1-depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Thus, DRP1-dependent mitochondrial fission activity is a novel regulator of the onset of follicle cell differentiation during Drosophila oogenesis.

  12. The mitochondria targeted antioxidant MitoQ protects against fluoroquinolone-induced oxidative stress and mitochondrial membrane damage in human Achilles tendon cells.

    PubMed

    Lowes, Damon A; Wallace, Carol; Murphy, Michael P; Webster, Nigel R; Galley, Helen F

    2009-04-01

    Tendinitis and tendon rupture during treatment with fluoroquinolone antibiotics is thought to be mediated via oxidative stress. This study investigated whether ciprofloxacin and moxifloxacin cause oxidative stress and mitochondrial damage in cultured normal human Achilles' tendon cells and whether an antioxidant targeted to mitochondria (MitoQ) would protect against such damage better than a non-mitochondria targeted antioxidant. Human tendon cells from normal Achilles' tendons were exposed to 0-0.3 mM antibiotic for 24 h and 7 days in the presence of 1 microM MitoQ or an untargeted form, idebenone. Both moxifloxacin and ciprofloxacin resulted in up to a 3-fold increase in the rate of oxidation of dichlorodihydrofluorescein, a marker of general oxidative stress in tenocytes (p<0.0001) and loss of mitochondrial membrane permeability (p<0.001). In cells treated with MitoQ the oxidative stress was less and mitochondrial membrane potential was maintained. Mitochondrial damage to tenocytes during fluoroquinolone treatment may be involved in tendinitis and tendon rupture.

  13. High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

    PubMed

    Shim, Hee Jin; Lee, Eun-Mi; Nguyen, Long Duy; Shim, Jaekyung; Song, Young-Han

    2014-01-01

    Ionizing radiation (IR) treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs) generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis.

  14. Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells.

    PubMed

    Grönholm, Juha; Kaustio, Meri; Myllymäki, Henna; Kallio, Jenni; Saarikettu, Juha; Kronhamn, Jesper; Valanne, Susanna; Silvennoinen, Olli; Rämet, Mika

    2012-03-01

    The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-γ- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.

  15. Medium from γ-irradiated Escherichia coli bacteria stimulates a unique immune response in Drosophila cells.

    PubMed

    Lindberg, Bo G; Oldenvi, Sandra; Steiner, Håkan

    2014-10-01

    It is well known that γ-irradiated, non-dividing bacteria can elicit potent immune responses in mammals. Compared to traditional heat or chemical inactivation of microbes, γ-irradiation likely preserves metabolic activity and antigenic features to a larger extent. We have previously shown that antimicrobial peptides are induced in Drosophila by peptidoglycan fragments secreted into the medium of exponentially growing bacterial cultures. In this study, we γ-irradiated Escherichiacoli cells at a dose that halted cell division. The temporal synthesis and release of peptidoglycan fragments were followed as well as the potential of bacterial supernatants to induce immune responses in Drosophila S2 cells. We demonstrate that peptidoglycan synthesis continues for several days post irradiation and that monomeric peptidoglycan is shed into the medium. Whole transcriptome analysis revealed a strong immune response against the bacterial medium. The response to medium taken directly post irradiation shows a large overlap to that of peptidoglycan. Medium from prolonged bacterial incubation does, however, stimulate a selective set of immune genes. A shift towards a stress response was instead observed with a striking induction of several heat shock proteins. Our findings suggest that γ-irradiated bacteria release elicitors that stimulate a novel response in Drosophila.

  16. Trehalase Regulates Neuroepithelial Stem Cell Maintenance and Differentiation in the Drosophila Optic Lobe

    PubMed Central

    Wang, Hongbin; Luo, Hong

    2014-01-01

    As one of the major hydrolases in Drosophila, trehalase (Treh) catalyzes the hydrolysis of trehalose into glucose providing energy for flight muscle activity. Treh is highly conserved from bacteria to humans, but little is known about its function during animal development. Here, we analyze the function of Treh in Drosophila optic lobe development. In the optic lobe, neuroepithelial cells (NEs) first divide symmetrically to expand the stem cell pool and then differentiate into neuroblasts, which divide asymmetrically to generate medulla neurons. We find that the knockdown of Treh leads to a loss of the lamina and a smaller medulla. Analyses of Treh RNAi-expressing clones and loss-of-function mutants indicate that the lamina and medulla phenotypes result from neuroepithelial disintegration and premature differentiation into medulla neuroblasts. Although the principal role of Treh is to generate glucose, the Treh loss-of-function phenotype cannot be rescued by exogenous glucose. Thus, our results indicate that in addition to being a hydrolase, Treh plays a role in neuroepithelial stem cell maintenance and differentiation during Drosophila optic lobe development. PMID:25003205

  17. PI4KIIIα is required for cortical integrity and cell polarity during Drosophila oogenesis.

    PubMed

    Tan, Julie; Oh, Karen; Burgess, Jason; Hipfner, David R; Brill, Julie A

    2014-03-01

    Phosphoinositides regulate myriad cellular processes, acting as potent signaling molecules in conserved signaling pathways and as organelle gatekeepers that recruit effector proteins to membranes. Phosphoinositide-generating enzymes have been studied extensively in yeast and cultured cells, yet their roles in animal development are not well understood. Here, we analyze Drosophila melanogaster phosphatidylinositol 4-kinase IIIα (PI4KIIIα) during oogenesis. We demonstrate that PI4KIIIα is required for production of plasma membrane PtdIns4P and PtdIns(4,5)P2 and is crucial for actin organization, membrane trafficking and cell polarity. Female germ cells mutant for PI4KIIIα exhibit defects in cortical integrity associated with failure to recruit the cytoskeletal-membrane crosslinker Moesin and the exocyst subunit Sec5. These effects reflect a unique requirement for PI4KIIIα, as egg chambers from flies mutant for either of the other Drosophila PI4Ks, fwd or PI4KII, show Golgi but not plasma membrane phenotypes. Thus, PI4KIIIα is a vital regulator of a functionally distinct pool of PtdIns4P that is essential for PtdIns(4,5)P2-dependent processes in Drosophila development.

  18. Uncovering the link between malfunctions in Drosophila neuroblast asymmetric cell division and tumorigenesis

    PubMed Central

    2012-01-01

    Asymmetric cell division is a developmental process utilized by several organisms. On the most basic level, an asymmetric division produces two daughter cells, each possessing a different identity or fate. Drosophila melanogaster progenitor cells, referred to as neuroblasts, undergo asymmetric division to produce a daughter neuroblast and another cell known as a ganglion mother cell (GMC). There are several features of asymmetric division in Drosophila that make it a very complex process, and these aspects will be discussed at length. The cell fate determinants that play a role in specifying daughter cell fate, as well as the mechanisms behind setting up cortical polarity within neuroblasts, have proved to be essential to ensuring that neurogenesis occurs properly. The role that mitotic spindle orientation plays in coordinating asymmetric division, as well as how cell cycle regulators influence asymmetric division machinery, will also be addressed. Most significantly, malfunctions during asymmetric cell division have shown to be causally linked with neoplastic growth and tumor formation. Therefore, it is imperative that the developmental repercussions as a result of asymmetric cell division gone awry be understood. PMID:23151376

  19. Tendon repair

    MedlinePlus

    ... the area to see if there are any injuries to nerves and blood vessels. When the repair is complete, the wound is closed. If the tendon damage is too severe, the repair and reconstruction ... to repair part of the injury. Another surgery will be done at a later ...

  20. Modeling Monogenic Human Nephrotic Syndrome in the Drosophila Garland Cell Nephrocyte.

    PubMed

    Hermle, Tobias; Braun, Daniela A; Helmstädter, Martin; Huber, Tobias B; Hildebrandt, Friedhelm

    2017-05-01

    Steroid-resistant nephrotic syndrome is characterized by podocyte dysfunction. Drosophila garland cell nephrocytes are podocyte-like cells and thus provide a potential in vivo model in which to study the pathogenesis of nephrotic syndrome. However, relevant pathomechanisms of nephrotic syndrome have not been studied in nephrocytes. Here, we discovered that two Drosophila slit diaphragm proteins, orthologs of the human genes encoding nephrin and nephrin-like protein 1, colocalize within a fingerprint-like staining pattern that correlates with ultrastructural morphology. Using RNAi and conditional CRISPR/Cas9 in nephrocytes, we found this pattern depends on the expression of both orthologs. Tracer endocytosis by nephrocytes required Cubilin and reflected size selectivity analogous to that of glomerular function. Using RNAi and tracer endocytosis as a functional read-out, we screened Drosophila orthologs of human monogenic causes of nephrotic syndrome and observed conservation of the central pathogenetic alterations. We focused on the coenzyme Q10 (CoQ10) biosynthesis gene Coq2, the silencing of which disrupted slit diaphragm morphology. Restoration of CoQ10 synthesis by vanillic acid partially rescued the phenotypic and functional alterations induced by Coq2-RNAi. Notably, Coq2 colocalized with mitochondria, and Coq2 silencing increased the formation of reactive oxygen species (ROS). Silencing of ND75, a subunit of the mitochondrial respiratory chain that controls ROS formation independently of CoQ10, phenocopied the effect of Coq2-RNAi. Moreover, the ROS scavenger glutathione partially rescued the effects of Coq2-RNAi. In conclusion, Drosophila garland cell nephrocytes provide a model with which to study the pathogenesis of nephrotic syndrome, and ROS formation may be a pathomechanism of COQ2-nephropathy. Copyright © 2017 by the American Society of Nephrology.

  1. Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting.

    PubMed

    Centanin, Lázaro; Dekanty, Andrés; Romero, Nuria; Irisarri, Maximiliano; Gorr, Thomas A; Wappner, Pablo

    2008-04-01

    Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. Here, we show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-alpha homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. We propose that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting.

  2. Bioreactor Design for Tendon/Ligament Engineering

    PubMed Central

    Wang, Tao; Gardiner, Bruce S.; Lin, Zhen; Rubenson, Jonas; Kirk, Thomas B.; Wang, Allan; Xu, Jiake

    2013-01-01

    Tendon and ligament injury is a worldwide health problem, but the treatment options remain limited. Tendon and ligament engineering might provide an alternative tissue source for the surgical replacement of injured tendon. A bioreactor provides a controllable environment enabling the systematic study of specific biological, biochemical, and biomechanical requirements to design and manufacture engineered tendon/ligament tissue. Furthermore, the tendon/ligament bioreactor system can provide a suitable culture environment, which mimics the dynamics of the in vivo environment for tendon/ligament maturation. For clinical settings, bioreactors also have the advantages of less-contamination risk, high reproducibility of cell propagation by minimizing manual operation, and a consistent end product. In this review, we identify the key components, design preferences, and criteria that are required for the development of an ideal bioreactor for engineering tendons and ligaments. PMID:23072472

  3. Bioreactor design for tendon/ligament engineering.

    PubMed

    Wang, Tao; Gardiner, Bruce S; Lin, Zhen; Rubenson, Jonas; Kirk, Thomas B; Wang, Allan; Xu, Jiake; Smith, David W; Lloyd, David G; Zheng, Ming H

    2013-04-01

    Tendon and ligament injury is a worldwide health problem, but the treatment options remain limited. Tendon and ligament engineering might provide an alternative tissue source for the surgical replacement of injured tendon. A bioreactor provides a controllable environment enabling the systematic study of specific biological, biochemical, and biomechanical requirements to design and manufacture engineered tendon/ligament tissue. Furthermore, the tendon/ligament bioreactor system can provide a suitable culture environment, which mimics the dynamics of the in vivo environment for tendon/ligament maturation. For clinical settings, bioreactors also have the advantages of less-contamination risk, high reproducibility of cell propagation by minimizing manual operation, and a consistent end product. In this review, we identify the key components, design preferences, and criteria that are required for the development of an ideal bioreactor for engineering tendons and ligaments.

  4. MRI-Based Assessment of Intralesional Delivery of Bone Marrow-Derived Mesenchymal Stem Cells in a Model of Equine Tendonitis

    PubMed Central

    Scharf, Alexandra; Holmes, Shannon P.; Thoresen, Merrilee; Mumaw, Jennifer; Stumpf, Alaina

    2016-01-01

    Ultrasound-guided intralesional injection of mesenchymal stem cells (MSCs) is held as the benchmark for cell delivery in tendonitis. The primary objective of this study was to investigate the immediate cell distribution following intralesional injection of MSCs. Unilateral superficial digital flexor tendon (SDFT) lesions were created in the forelimb of six horses and injected with 10 × 106 MSCs labeled with superparamagnetic iron oxide nanoparticles (SPIOs) under ultrasound guidance. Assays were performed to confirm that there were no significant changes in cell viability, proliferation, migration, or trilineage differentiation due to the presence of SPIOs. Limbs were imaged on a 1.5-tesla clinical MRI scanner postmortem before and after injection to determine the extent of tendonitis and detect SPIO MSCs. Clusters of labeled cells were visible as signal voids in 6/6 subjects. Coalescing regions of signal void were diffusely present in the peritendinous tissues. Although previous reports have determined that local injury retains cells within a small radius of the site of injection, our study shows greater than expected delocalization and relatively few cells retained within collagenous tendon compared to surrounding fascia. Further work is needed if this is a reality in vivo and to determine if directed intralesional delivery of MSCs is as critical as presently thought. PMID:27746821

  5. Drosophila male germline stem cells do not asymmetrically segregate chromosome strands.

    PubMed

    Yadlapalli, Swathi; Cheng, Jun; Yamashita, Yukiko M

    2011-03-15

    Adult stem cells continuously supply differentiated cells throughout the life of organisms. This increases the risk of replicative senescence or neoplastic transformation due to mutations that accumulate over many rounds of DNA replication. The immortal strand hypothesis proposes that stem cells reduce the accumulation of replication-induced mutations by retaining the older template DNA strands. Other models have also been proposed in which stem cells asymmetrically segregate chromosome strands for other reasons, such as retention of epigenetic memories. Recently, the idea has emerged that the mother centrosome, which is stereotypically retained within some asymmetrically dividing stem cells, might be utilized as a means of asymmetrically segregating chromosome strands. We have tested this hypothesis in germline stem cells (GSCs) from Drosophila melanogaster testis, which undergo asymmetric divisions marked by the asymmetric segregation of centrosomes and the acquisition of distinct daughter cell fates (stem cell self-renewal versus differentiation). Using 5-bromo-2-deoxyuridine labeling combined with direct visualization of GSC-gonialblast (differentiating daughter) pairs, we directly scored the outcome of chromosome strand segregation. Our data show that, in male GSCs in the Drosophila testis, chromosome strands are not asymmetrically segregated, despite asymmetrically segregating centrosomes. Our data demonstrate that asymmetric centrosome segregation in stem cells does not necessarily lead to asymmetric chromosome strand segregation.

  6. Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

    PubMed Central

    Tamada, Masako; Zallen, Jennifer A.

    2015-01-01

    Summary Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. Here we identify a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand, Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation. PMID:26506305

  7. Dynamics of cell polarity in tissue morphogenesis: a comparative view from Drosophila and Ciona.<